IN SEARCH OF A PROTEIN NUCLEATOR OF HYDROXYAPATITE IN BONE

Carmel O Domeni cucci

A rhesis subrnitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistq University of Toronto

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The author retallis ownership of the L'auteur conserve la propriété du copyright in ths thesis. Neither the droit d'auteur qui protège cette thése. thesis nor substantial extracts &om it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Yn Search of a Protein Nucleator of Hydroxyapatite in Bone" Doctor of Philosophy. 1997 Carme10 Domenicucci Department of Biochemistry University of Toronto

The formation of mineralized conneciive tissues is characterized by the nucleation of hydroxyapatite crystals that are generated initiaily within the gap region of collagen fibrils. However, the mechanisrn of mineral nuckation has not been resolved. It is believed that a heterogenous (epitactic) nucleator is likely required to provide a tempiate for the cqstal lanice. Consistent with the location of the minerai crystals a nucleator is envisaged as king a collagen-binding protein with the ability to bind to calcium and hydroxyapatite. Thus, i t is hypodiesized that by isolating proteins from newky-forming bone tissues according to their affinit): for collapn and hydroxyapatite the subsequent identification and characterization of the mineral nucleator could be facilitateci. To test this hypothesis an extraction procedure was developed in which guanidine hydrochloride (GuHCI) and ethylenediaminetetra-=tic acid (EDTA) were used to sequentially solubilize proteins [rom fetai porcine bone. Initial extractions of the bone with 4 M GuHCl released proteins that were associated with the osteoidsoft tissue matrix. Subsequent extractions with 0.5 M

EDT A demineralized the bone and released mineral-bound proteins, included osteonectin, decorin, osteopontin, bone sialoprotein, small collagenous apatite-binding proteins and a novel chondroitin sulfate proteogiycan (CS-PGIII), which were punfied for funher characterization. Initial studies were focussed on osteonectin since it had ken proposed as a potential nucleator. However, studies of osteonectin biosynthesis, tissue distribution and physicochemical characteristics revealed properties that were inconsistent with a bomfide nucleator. Consequently, studies were then directed at proteins dissociatively extracted from the de-mineralized collagen matrix with 4 M GuHCl. Two apparently unique 32 kDa and 24 kDa proteins were puri fied and identified as 1ysyl oxidase and tyrosine rich acidic matrix protein (TRAMP). Since these proteins did not have the chmctensûcs of a nucleator. the tissue residue was digested with CNBr to identify proteins tightly bound to the demineralized collagen matrix Although a protein nucleator was not identified in these studies, the development of a selective extraction procedure and protein purification protocds facilitated the chamcterization of the major proteins in fetd porcine bone. Man. of these proteins are likely io be involved in the formation, growth and stabilization of h ydrox yapati te crystals. This thesis is dedicated to Kei th, Kendra and Parnela- I would like to thank the rnernbers of the Medical Research Council in Penodontal Physiology for their help and suppon, and express rny thanks to Hmey Goldberg. Qi Zhang, Kam-Ling Yao. Harq Moe, Fumiyuuk, Kuwata. Masao Maeno, Paul Zung, Pierre Tung, Safia Wasi, Jukka Salonen, Theo Hofmann, David Isenman, Anders Bennick, Luc Malaval, Elisa Knssîias, Ana Vanek and especiall y Jaro Sodek. STATEMENT OF CONTRIBUTIONS TO THIS THESIS

Many of the studies described in ths thesis have ken published in collaborations with a nurnber of graduate students and post-doctoral fellows working in the ~aboratory. My contri butions fonn the basis of this thesis. Specificall y, the contri butions in each chapter are as follows:

Chapkr II: 1 was responsible for developing the protocols used to extract and analyze proteins from the different mineralized tissues. Although. the same basic procedure was used in a nurnber of studies subsequently, I generated dl the data reporied in the thesis.

Chapter HIA: 1 performed al1 the studies described in the isolation. purification and characterization of SPARC with assistance in the purification procedures from Drs. Goldberg and Wasi and advtce on the circular dichroism studies by Dr. Eisenman and on protein analysis by Dr. Hofmann.

Chapter iIIB: The biosynthetic studies in rat bone and dentine were primarilp done by a summer student (P. Zung), with mp assistance and under the supenision of myself and Dr. Wasi. The ceIl free protein synthesis studies were from a more comprehensive studg pnrnanly conducted by Dr. Kuwata, in whch 1 assisted. Only selected data have ken used in the thesis and were from experiments in which I was directly involved. The immunosiaining studies were perlormed by Drs. Tung and Salonen for which 1 prepared the affinity- purified antibodies and assisted in the immunostaining of some of the sections and preparauon of data for publication.

Chapter IIIC: The procedures for purifiying the the proteoglycans and sidoproteins were developed in joint studies with Dr. Goldberg and Dr. Zhang. While 1 also assisted in the characterization, I have utilized only results of basic characterktics, and where relevant, indicate dara that was prirnady generated by others. Chapter IIID: I generated al1 data on the characterization of the pN-propeptides in bone and serum. in which 1 compare the propenies of each form. Some of the data reporteci in the thesis were used in a publication describing the bone pN-propeptides in which the majonty of the studies were perforrned by Dr. Goldberg.

Chapter IV: The purification of the G2-exmt proteins was done w i th Dr. Goldberg w ho studied the proteoglycans and the collagns. Dr. Zhang assisted in the work in\roiving the G2-derived bone sialoprotein protein. Dr. Overall performed the enzymography experiments with the bone-extracts that 1 provided. All the data presented in the thesis including the purification and charactenzation of 1 ysyl oxidase and TRAMP were from experiments that 1 conducted.

Chapter V: 1 carried out the experiments analyzing noncollagenous proteins in the collagenous rnatrix remaining after the dissociative extractions and demineralization.

vii TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION ...... 1 1) MINERALIZED TISSUES ...... 1 2) EXTRACELLULAR MATRIX OF MINERALIZED TISSUES ... 6 A) MINERAL PHASE ...... 6 B) ORGAMC CONSTITUENTS ...... 7 1 . COLLAGENOUS PROTEINS: ...... 7 II . NON-COLLAGENOUSPROTEINS ...... 9 3) MINERALIZATION MECHAMSMS ...... 18 I . THEORES OF MINERAL FORMATION ...... 19 11a. COLLAGENASANIMTIATOROFMlNERAL DEPOSITS ...... 31 II b . THE ROLE OF NON-COLLAGENOUS PRCrrEINS IN THE MINERALIZATION OF HARD CONNECTIVE TISSUES ...... 23 II1 . FEATURES OF A NUCLEATOR ...... 24

OBJECTIVES: ...... 17

CHAPTER II: THE DEVELOPh4ENT OF A PROCEDURE TO EXTRACT PROTEINS FROM MlNERALlZED CONNECTI VE TISSUES ...... 28

INTRODUCTION ...... 28 MATWALS AND METHODS ...... 29 RESULTS ...... 31 DISCUSSION ...... 33

viii CHAPTER III A: ISOLATION.PURIFICATION AND CHARACTERIUTION OF OSTEONECTIN EXTRACIED FROM FETAL PORCINE CALVARIAF- SOME COMPAEUSONS WITH PURIFiED PORCINE DENTINEi OSTEONECTIN...... 39

INTRODUCTION ...... 39 MATERIALS AND METHODS ...... 40 EESULTS ...... 48 DISCUSSION ...... 52

CHAPTER III B: BIOSY NTHESIS AND IMMUNOLOCALIZATION OF OSTEONECTIN/SPARC . 65

INTRODUCTION ...... 65 MATERIALS AND METHODS ...... 65 RESULTS ...... 68 DISCUSSION ...... 73

CHAPTER III C: PURIFICATION OF PROTEOGLYCANS:CS-PG II (DECORIN) ALWCS-PG III AND SIALOPROTEINS:OPN AND BSP FROM E-EXTRACTS ...... 85

INTRODUCTION ...... 85 MATERIALS AND METHODS ...... 86 RESULTS ...... 89 DISCUSSION ...... 93

CHAPTER III D: PN-PROPEPTIDESASSOCIATED WITH THE DIFFERENT COMPARTMENTS OF BONE: COMPARISONS WITH SERUM-DERIVED PN-PROPEPTIDE .... 103

INTRODUCïION ...... 103 MATERIALS AND METHODS ...... 104 RESULTS ...... 107 DISCUSSiON ...... 111 CHAPTER IV: IDENTIFICATION. PURIFICATION AND CHARACTERIZATION OF THE MAJOR PROTEINS FROM THE G2 EXTRACTS OF MINERALIZED CONNECTIVE TISSUES ...... 134

INTRODUCTION ...... 134 MATEEUALS AND METHODS ...... 124 RESULTS ...... 130 DISCUSSION ...... 134

CHAPTER V: PRELIMINARY ANALYSIS OF PROTEINS COVALENTLY BOWTO THE COUGEN MATRIX OF BONE ...... 151

INTRODUCTION ...... 151 MATERIALS AND MErHODS ...... 152 RESULTS ...... 153 DISCUSSION ...... 154

CHAPTER VI: DISCUSSION ...... 157

REFERENCES ...... 170

Figures 9.1-6: Imrnunohistochemical Localization of OsteonectinJSPARC and Type 1 Collagen in Adult Rat Dental Tissues ...... 83 Figure 9.7-. 12 Immunohistochemical Localization of OsteonectinJSPARC and Type III Collagen in Rat Dental Tissues...... 84

CHAPTER III C Figure 1: Chromatography of the EDTA Extract and Isolation of hoteogi ycans .... % Figure 2: Further Purification of Fetal Porcine Calvarial Proteoglycans by Hydroxyapati te chromatography...... 97 Figure 3: SDS-polyacrylarnide Gel electrophoresis of Bone Proteoglycans ...... 98 Figure 4: Purification of Fetal Porcine Bone Sialoproteins by FPLC ...... 99 Figure 5: Amino-terminal Arnino Acid Sequence Analysis of OPN and BSP ..... 100

CHAPTER III D Figure 1: SDS-PAGE Analysis of Small Collagenous Proteins Extracted from Bone . 1 14 Figure 7: Purification of the Small Collagenous Proteins from E- and G?-extracts of Bone on Hydroxyapatite ...... 115 Figure 3: Characterization of the Purified Small Collagenous Proteins ...... 116 Figure 4 Reparatory Fractionation of Fetal Porcine Serum by Ion Eschange Chromatography on a QAE Filter Cartridge...... 117 Figure 5: Chromatograrn of Partially Fractionated Fetal Porcine Semm on CL-6B Sepharose...... 117 Figure 6: SDS-PAGE Analysis for Serum pN-propeptides Separated by Chromatography on Sepharose CL-6B under the Denaturing Conditions of 4MGuHCl ...... 118 Figure 7: FPLC and HPLC Chromatography of Serum pN-propeptide Pooled after CL-6BGel Filtration ...... 119 Figure 8: SDS-PAGEAnalyses Demonstraiing Purity of pN-propeptide ...... 120 Figure 9: Composite Elution Profile of SCAB 3a. SCAB 3b. G3-28 kDa Protein and Semm pN-propeptide run on FPLC Mono Q Chromatography ...... 120 Figure 10: SDS-PAGE And ysis of pN.propeptides ...... 131 Figure 1 1: Collagenase Sensi tivi ty of pN.propeptides ...... 131 Figure 12 Susceptibility of pN-propeptides to CNBr ...... 122 FigureI3: ImmunoreactivityofpN~propeptides...... 132 CXAPTER IV Figure 1 A: Gel filtration Profile of Fetal Porcine Calvarid G2-1 extract Protein Run on Sepharose CL-68 Resin...... 137 Figure 1 B: SDS-PAGEAnalysis of Fetal Porcine Calvarial G2-exuact Roteins in Fractions from Gel Filtration with Sepharose CLdB Resin...... 138 Figure 2: SDS-PAGEof the Major Roteins Comprising the G3-extract of Dentine. 139 Figure 3: FPLC Chromatographie Profiles for the Partial Punfication of 33 kDa and 24 Daproteins...... 140 Figure 4: HPLC Reversed Phase Chromatography for the Purification of 33 kDa

and 24 kDa Proteins...... , ...... 142 Figure 5 A, B and C: Purification of the 32 kDa Protein from the Contaminant 24 kDa Protein from a Mixture of 32/24 kDa Proteins ...... 143 Figure 6: CNBr Cleavage Sensi tivi ty of Bone-derived 32 kDa Protein and Electrophoretic Transfer Roperties of Peptides onto PVDF Sequencing Membrane...... 145 Figure 7: Identification of the 32 kDa Protein of Porcine Bone and Dentine as LysyI Oxidase by Sequence Analysis...... 146 Figure 8: Roperties of the 24 kDa Roteins Isolated from Porcine Bone...... 147 Figure 9: Identification of the 74 kDa Proteins 1solated from Adult Porcine Dentine as TRAMP by Immunoblotting ...... 148 FIgure10: DepolymeraseActivity ...... 149 Figure 11: Gelatinase Activity of Bone Protein Extracts ...... 149

CHAPTER V Figure 1: F?LC Chromatography of Acetic Acid Insoluble Proteins...... 1%

APPENDIX A Figure lA, B, C: FPLC Purification of CS-PG II and BSP from Fetal Porcine G2-extracts...... 167

APPENDIX 82 FPLCIHPLC Chromatograms for Proteins Purified from the G3-extract ...... 169 LIST OF TABLES:

CHAPTER 111 A Table I: Amino Acid Composition of Calvarial Osteonectin Compared with Proteins from other Sources ...... 64

CHAPTER III C Table I: Amino Acid Analysis of Porcine Bone Roteoglycans ...... 101 Table 11: Amino Acid Analysis of Porcine Bone Sialoproteins: OPN and BSP Comparisons with Compositions for the Human Proteins ...... 102

CHAPTER III D Table I: Comparisons of Amino Acid Compositions Between Bone and Slan pN-propeptides ......

CHAPTER IV Table I: Cornpanson of Amino Acid Compositions between 24 kDa Protein from Bone and the 24 kDa Proteins from Dentine with that of TRAMP Isolated from Vanous Tissues...... I50

CHAPTER VI Table I: Proteinsof Bone Matrix ...... 166

APPENDIX B1 Table I: Purification Scheme Tor LOK Molecular Weight Proteins from the G2-extract...... 168

xiv ABBREVIATIONS

ACP arnorphous calcium phosphate BAG-75 bone acidic glycoprotein, 75 kDa BGP bone gla protein, ostdcin BMP bone morphogenic protein BSA bovine serum al burnin BSP bone sialoprotein CBB R-250 Cmmassie Bril lian t Blue R-250 CS chondroitin sui fate CS-PG 1, II, III chondroi tin sulfate proteogl ycans 1, II, and III DCPD dicalcium phosphate di hydrate Dmpl dentine matrix protein DS dermatan sulfate DSP dentine sialoprotein E-buf fer 0.5 M EDTA, 50 mM-TnsIHC1, pH 7.4 EmA ethylenediarnine telraa~eticacid GE-buffer 4 M GuHCl, 0.5 M EDTA, M mM-TrislHCl, pH 7.4 GEextrac t the resulting estract obtained by treatment of mineralized tissues with a combination demineraliùng (EDTA) and dissociati\.e (4 M GuHCl) containing solution 4 M GuHC1,50 mM-TrislHCl, pH 7.4 extract obtained after treatment of tissue with 4 M GuHCl 4 M GuHCl eatract obtained pnor to demineralization of mineral ized tissues 4 M GuHCl eatract obtained subsequent to demineralization of mineralized tissues gl ycosaminoglycan gigabequerel y-carboxyglutamic h ydrox yapati te high molecular weight low molecular weight magnesium molecular weight N- terminus amino terminus OCP octacalcium phosphate OPN osteopon tin (SPP- 1 ) (pp69) (7s) osteonec tin SPARC,'culture shock protein', BM-40, PBS phosphate buffered saline pl-propep tide (1 1(I)pC-propeptide PG proteoglycan Pl protease inhi bitors pN- propepti de a 1 (1)pN-propeptide pp69 69 Daphosphoprotein (osteopontin) PP phosphophoryn SCAB mal1 collagenous apatite-binding SDS-PAGE sodium dodecyl sulfate - polpcrylamide gel electrophoresis Ser(F043-) phosphoserine SPARC secreted protein acidic rich in cgsteine (os~eonectin) SPP-1 secreted phosphoprotein 1 (osteopontin) TGF-fi transforming growth factor-p ThT(Pû$-) phosphothreoni ne TI MP tissue inhi bitor of rnetalloproreinases 2ar os teopn ti n

Tyr@04) tyrosine sulfate ul traviolet-circular dichroism

xvi CHAPTER 1

INTRODUCTION

1) MINERALiZED TISSUES Bone is the principal mineraiized tissue in vertebrate organisms where i t foms the skeietal structures required for support, locomotion and protection of internai organs. Other minerdized tissues include cementum, dentine and enamel which together wi th the aheolar bone of the jaw form the teeth and their supporting structures. In addition, cartilage tissue will mineralize at sites of cartilage to bone transition in the growth plates of endochondral bones. With the exception of enamel, the mineralized tissues are hghly specialized connective tissues in which the interstitial, type I collagen haa principal role in deterrnining their structure and biophysical properties. Al though mineral izing comective tissues have a similar composition with respect to the organic and inorganic components of the exüaceI1ula.r mairix, and share many characteristics associated with their function and development, they display distinct differences in their structure as revealed in the following description of the individuai tissues.

BONE As reviewed by Wheater et al. ( 1979),Cormack ( 19'759, Tdfit ( 1980) and Ten Cate (1989) the various foms of bony structures which contnbute a significant portion of the body mass are characterized by the mineralized tissue component Despite the importance of their structural integrity bones are dpamic organs that undergo continuous remodelling, such that the equivalent of the totai bone mass is replaced over a 7 year pend in mature primates, including man. The functions of bone can be divided into three major areas: mechanical, minerai storage and haematopoiesis. Mechankail y, bone acts as a support system in w hic h the organic and the inorganic components provide the tissue integrity and resilience; properties that are important in movement, locomotion. the protection of organs and in tooth support. Bone also acts as an important reservoir for minerals and regulates Caz+/P043- homeostasis, the organic components playing an essentid role in the precipitation, stabilization and dissolution of these ions. Bone also provides a protective environment for the development of marrow tissues which are responsible for the production of the cellular elements of blood, including the red Mood cells that function in respiratory processes, and the leukocytes, lymphocytes and macrophages of the immune system. The cells in bone are generally of four types: osteoblasts, osteocytes, lining cells and osteoclasts. The first three types are derived from osteoprogenitor cells, which are of the suornai stem ceIl lineage. The osteoclasts are derived from progeni tor cells, derived frorn the myeloid stem ceIl lineage, and develop in the bone marrow cavities. Osteobiasts are responsible for the production of the organic components of the extracellular matnx of bone called osteoid which subsequently mineralizes to fonn mineraiized bone. Osteoblasts. whch becorne trapped wi thm the mineralized bone matrix, are called osteocytes, and are involved in the subsequent maintenance of the bone maîrix. Other osteoblasts become quiescent &ter bone formation. change from a cuboidal tc~a flattened morphology and form lining cells, which cover and pmtect the bone suk.Osteoclasts are multinucleated cells that fom from the fusion of monocytic precursors and are involved in the resorptlve process associated wi th the continuous remodelling of bone. Bone can fonn by an 'endochondral ' or 'membranous* pathway. Intramem branous ossification involves a process of direct bone deposition within primitive fibrous mesenchymal condensates that differentiate into osteoblasts. The bone of the vault of the skull, the clavicles, the mavillae and most of the mandibles are formed in this fashion. Endochondrd ossification, a more comples pathway, involves chondrogenesis and resorption of calcified cartilage, followed by osteogenesis. The long bones. vertebrae. pelvis, and bones of the base of the skull are formed in this manner. Bone on exist in two morphologicdIy distinct foms, woven and lamellar bone. Woven bone is described as 'immature' bone and is characterized by i ts rapid fonnation and irregularly organized infrastnicture. I t is found in the Sones of the developing fetus and serves as a temporary scaffolding for support. Lameliar bone is formed more slowly and often replaces woven bone. 1t is composed of successive rnineralized tissue layers. each of which has a highly organized infrastructure. Lamellar bone predominates in the adul t skeleton where i t may form a mas of mineralizd tissue that is described as compact bone. Such compact bone is found in the outer cortex of long bones. Al tematively. larnellar bone may be formed as a spongy mas, described as cancellous or trabecular bone whch is found as a reticular network in the central medullary cavities of long bones or calvarial bones. Woven bone is remodelled by resorption followed by apposi tional growth, in which there is an increase in bone mass following the deposition of new matris on the surface of older bone by adjacent surface cells. to fom mature adult lamellar bone. The presence of woven bone in the adul t skeleton generall y reflects a pathologie condition such as cancerous growth, infection, fracture or as a respnse to a necrotic focus. DENTINE As reviewed by Linde ( l984a), Johnson ( l986), Ten Cate ( 1989).Linde and Goldberg ( 1993) dentine comprises the principal component of teeth and. following its formation, is a relatively quiescent tissue which is not remodelled. Coronall y, dentine underlies the enamel and because of i ts resilient quality it provides both flexibility and support and protects against enamel fracture. Apically, dentine is associaîed with the cementum of teeth and is called root dentine. Root dentine provides a surface upon which cementum is formed. A thin fibrous membrane, the periodontal ligament, then serves as an attachment apparatus between the tooth root and the associated alveolar bone. Intemally, dentine ovedies a central pulp chamber within the tooth. Although dentine is a hard connective tissue and the pulp is a soft connective tissue the): share cornmon embryological origins and are interdependent dunng development. Thus dentine is considered the hard tissue portion of what is known as the dentine-pulp cornplex. The cells responsi ble for dentine formation are called odontoblasts. Odontoblasts differentiate from ectornesenchymal cells of the dental papilla, under the infiuence of the dental epithelium. The odontoblasts initiate tooth formation by deposition of an organic matrix between the odontoblastic and ameloblastic layers; mineralization of this matris then induces enamel formation by cells called ameloblasts. Dentine formation then proceeds by the continuai deposition of dentine matnx and its subsequent mineralization. Each odontoblast leaves behind a slender cytoplasmic process, the odontoblastic process, within a fine den tinal tubule. When dentine formation is complete, the dentine is permeated by parallel odontoblastic processes radiating [rom the odontoblast layer on the dentinal surface of the dentai pulp to its periphery. After tooth formation is complete, a small amount of les organized secondq dentine continues to be laid dom resul ting in the progressive obliteration of'the pulp cavi ty wi th advancing age. Redentine is a layer of variable thickness ( 10-50 pm) that lines the pulpal portion of dentine. It represents unmineralized dentine matrix and is Ihicker where active dentinogenesis is occuning. Its presence is important in maintaining the integnty of dentine since in its absence, exposed dentine is vulnerable to resorption by odontoclasts, cells which are morphologicall y identical to osteoclasts, but are involveci in the removal of mineralized tooth tissues. Dentine exists in a number of specialized forms called mande, circumpulpal, secondary, intratubular and reparative dentine. Ci rcumpul pal dentine, also know n as primaiy dentine or intertubulx dentine, constinites the bulk of tooth dentine, the organic components of the tissue matrix king formed exclusivelp by the odontoblasts . Chamcteristically the collagen fibrils are small (50-200 nm in diameter), closely packed and intenvoven, and aligned at 90 degrees to the dentinal tubules. CEMENTUM As reviewed by Linde ( 1984a). Johnson ( l986),Ten Cate ( 1gag), Linde and Goldberg (1993) cernentum is a highly speciaiiled rnineralized connective tissue that invests the roots of teeth where coronally it begins as a thin layer (20-50 w) that progressi vel y becomes thicker as i t approaches the root apex ( 1SMûû pm). Cemen tum consists of a dense, mineralized organic material similar to the matrk of bone. However, unlike bone, it is avascular, does not have the ability to remodel, and is more resistant to resorption. By providing for the insertion of the dense fibers of the periodontal ligament, the cementum, together wi th the penodontal ligament anchors the tooth wi thin the alveolus of the jaw bone. Cementoblasts, which originate from mesenchymal cells of the dental follicle, are responsi ble for the production of cementoid, the unmineralized organic matris of cementum. Cementoblasts, once in apposition to the newly-formed root surface, begm to deposit cementoid in successive lamellae, which are then mineralized. Mineraiization of cementoid occurs in a manner similar to mantle dentine in that the deposition of apatite crystals are first observed in association with rnatrix vesicles in relation to collagen fibiils which are pamllel to the ceIl surface. Cementum formation then proceeds by the continued and altemating deposition of cernenioid following subsequent mineralization. As the developing tooth erupts, cementum is laid dom slow1y and the cementoblasts retreat into the periodontal ligament leaving a thin acellular layer of cementurn immediatel y adjacent to the mot dentine surface. This cementurn is known as acellular cementurn and consists of a variable number of mineralized matris layeers separated bg resting Iines. Concomitantly, col lagen fibers produced by fi broblasts of the periodontal ligament are incorporateci wi thi n the developing cemennim. These fiben are known as entrinsic ligament fi bers and consti tute the majority of the collagen fibers found in acellular cementurn. They are lu1 1y mineralized and are indistinguishable from the intrinsic fibers, which are fibers that are produced by the cernentoblast proper. As the tooth cornes into occlusion more cementum begins to form at the apical portion of the mot. Some cementoblasts are trapped within the extracellular matrix during cementogenesis, in much the same manner as çome osteoblasts (osteocytes) are surrounded by bone matrix during osteogenesis. This type of cementum, which overlies acellular cementurn, is known as cellular œmenturn and those cementoblasts which have been trapped in lacunae wi thin their own matri x are known as cementmgtes. Characteristically, cellular cementurn is found to have a greater content of mineralized intrinsic fiben. Extrinsic ligament fibers, which do pass into cellular cementum, mineralize ody at the periphery and as such retain an unmineralized are. Towards the root apex, the cementum layer becomes progressively thicker and irregular where layers of acellular and celluiarcementum alternate in an apparenfiy random manner with an increasing incorporation of cementocytes. Any resulting fibers that are a continuation of the principal fibers of the periodontal ligament and are associated with cementum are dled Sharpey's fibers. Cementogenesis is a slow intermittent process whereby the tissue continues to thicken wi th increasing age.

MINERALIZZNG CARTILAGE The calcification of cartilage mahx is a prerequisite step in the process of endochondrai bone formation (Poole et al., 1989). 1t is in1 tiated in the growth plate, principally in the hyper~ophiczone of cartilage, although signifimt amounts have ken detected proximal to the hypertrophie region (Rey and Glimcher, 199-). Mineralization occurs in an entmcellular matrïx cornposed principally of fibnls of type II collagen which are closely associated with type X collagen, two of the stnicturai proteins produced by cells called chondroblasts (Schmid et al., 1984). Also present in the matris are the large chondroi~insulfate containing proteoglycans uhich are associated with hyaluronic acid and are also produced by chondroblasts (Poole et al., 1982). 1t is thought that calcification of cartilage involves three distinct phases: preparation for nucleation, nucleation and multiplication/mineral growth (Rey and Glimcher, 1992). Prior to calcification, the proteoglycans and associated hyaiuronic acid becorne focally concentrated in sites where mineral will be depsited (Arsenault and Ottensmeyer, 1983 ; Shepard and Mitchell, 1985). It is believed that this 'collapsed proteoglycan aggregate' triggers the release of inorganic PO$- w hich is mediated by the action of rnatrix vesicle associated alkaline phosphatase (de Bernard et al., 1986). This then leads to the progressive displacement of proteoglycan bound Ca?+and its precipi tation (Hunter et al., 1988). After crystais are nucleated, multiplication acua in spatially distinct foci which eventually coalesce to Ton larger calcifïc nodules in the tissue (Rey and Glimcher, 19%). Unlike bone, cartilage does not undergo intemal remodelling ( resorption and replacement wi th new calci fied tissue) so that, as more crystais are fomed and the Ussw becomes more minedized, the additional mineral crystals themselves cm act as nucleation centen (Rey and Glimcher, 1992). This process rapidly produces more crystals so that the rate of mineralization increases in the calcifieci regions. The &-propeptide of type II collagen becomes concentrated in the mineralizing site, prior to whch it is rnainly associated with type II collagen fibrils (Hinek et al., 1981). However, at the the of dcification, type X collagen remains associated with type II collagen fibrils. It is thought that this may play a role in preventing the initiai calcification of these fibrils, focusing mineral formation in interfibrillar sites (Poole et al., 1989). Subsequent to its formation, most of the heavily mineralized cartilage is resorbed and de rwvo bone formation proceeds.

2) EXT'RACELLULAR MATWX OF MINERALIZED TISSUES As describeci, the mineralized connective tissues of the body are formed by specialized cells which synthesize and secrete an organic matrix capable of incorporating mineral in a systematic and highlp controlled fashion. Although the formation of these mineralized tissues involves complex events, many of which are not properly undentood, some common themes are evident. To undentand the steps involved in the formation of the mineraiid matnx of these tissues, a derailed knowledge of the composition of the matm. and the role of each of the matnx components is a prerequisite.

A) MZNERAL PHASE As reviewed by Christoffersen and Landis ( l991), the inorganic cornponent of mineralized tissue consists of crystailites of biologic apatite. Biologic apatite is essentially a calciurnJphosphate (CaZ+m3- ) sali approxirnatmg the composition of calcium hydronyapatite (Calo(POJJa[OHj2). The unit cell of biologic apati te has the shape of a snort rhombic prism. These, when stacked together, fom the lattice of a csstal. The number of repetitions of this arrangement produces crystallies of various sires. The proper structurai unit is the crystai. whch can vary in size and weight. The hydroxyapatite (HA) of the mesenchyrnal hard tissues is in the fom of crystallites of approsmate dimensions in the range of 10-50 nm (length) s 10-40 nm (width) x 3-5 nm (thickness) (Weiner and Rice, 1986). Direct visualization of the apatite crystalli te can be descn bed as plate-like ( Weiner et ai., 1992). Aroundeachcrystallite isa layer of watercalled the hydration shell. Thereare three interactive surfaces to the apatite crystallite: the crystai interior, surface and hydration shell. Al1 are available for exchange of ions. The Ca?+. P043- and OH- positions are therefore available for substitution- Bone and dentine contain considerable amounts of CO$- . Na+, Mgz+ and citrate with smaller amounts of K+,Cl- and F- (Driessens, 1983). Since the biologic apacite of vertebrate hard tissues is e~chedin carbonate, it is referred to as dahlli te (Kemp, 1984). However, mineral from calci fied cartilage is di fferent to that of bone in chat the carboate content is low (Rey and Glimcher, lm-). Although it is often assumed that HA is a suitable prototype for biologid minerais, it is currently unciear whether or not other forms of CaZ+/P043- such as amorphous calcium phosphate (Cas(P043- )6;ACP), dicalcium phosphate dihydrate (CaHPO$- 3H20;DCPD) and octacalcium phosphate (Ca4H6(FO$- )3 ;OCP) participate in the formation of biological hard tissues (Nancoilas et al.. 1989). Thus it bas been postufared that the formation of an apatitic phase is preceded by the precipitation of a non-crystalline precursor phase of ACP (Nancollas et al., 1989). The simplicity of ACP is useful in explaining some of the features associated wirh the initiation of rnineralization and to explain the fact that with age bne becornes more crystalline. However. studies have shown that neither ACP nor qstalline DCPD murin significant quantities in bone minerai. regardless of age or stage of maturation, and that neither functions as a precursor phase in the formation of bone mineral (Bonar et ai., 1983). Thus, while newly-developed bone has even smaller cqstaili tes, and generaii y has a Caz+IPOJ3- ratio considerably lower than that of HA, with an apparent solubili~greater than HA, minerai that is initially formed in connective tissues can be best described as king a poorly crystalline HA (with very small crystallire size). having a high degree of disorder and number of impurities such as Hm4-.In addition, when first deposited apati te crysrais do not maintain a constant structure (Rey and Glimcher. 1997). Dunng maturation. the cqstal lini ty improves: cryswl size increases. disorder decreases, and most impurity levels decrease (Bonar et al., 1983; Hohling et al., 198 1; Palamara et al., 198 1; Landis and Navarro, 1983; Kemp, 1984). The main changes noted in the Ca2+IW43- ntio of maturing apatite crystals have ken related to an increase in their cryslallinity. an increase in the carbonate content and a decrease in the Hm4-content (Rey and Glimcher, 1992). Further increases in the calcification of the collagen fi brils occur principally by multiplication: a term used to denote an increase in the number of crystals rather than by crystal growth (Glimcher. 1981). However, the continuous turnover of bone tissue, complicates the study of the mineral phases (Rey and Glimcher, 1992). Thus biologic apati te is buil t up on a definite Iartice paneni that pemits considerable variation in its composition through substitution, exchange and adsorption of ions in a manner that allows the apatite qstallites to retain their structural configuration while accommodating these substitutions. The mineral component of mature bone is. therefore. considered to be a poorly-crysialline apatitic matenal mat closely resembiing HA, but w i th very small crystallite size and extensive incorporation of substinite ions (Bonar et al.. 1983).

1. COLLAGENOUS PROTEINS: Collagen is the major organic constituent found in mineralized connective tissues. The major cdlagen found is type 1 collagen wiih small amounts (< 5%)of type V collagen (Bronckers et al., 1986). Type 1 collagen is formed as a heterotrimer of a l(1) and aS(1) pdypeptide chains in a 1:1 ratio. Some type 1 collagen may also exist in a homouimeric form of al(I) collagen chahin embryonic tissues. This collagen is known as al(I) trimer. In rat incisor dentine al(1) trimer represents 3Wc of the salt extractable and 10- 15% of the insoluble fraction of intenubular dentine (Goldberg et al., 1995). The high proportion of d(I), in incisor dentine probably reflects its embryonic characteristics which are linked to its continuous formation throughout life, as the collagen al(I), is not normally detectable in mature mineralized tissues (Bronckers et al., 1989). Although three discrete a-chains [al(V), a3(V), and d(V)] have been described for type V collagen, in bone the type V collagen seems to be formed as a mixed collagen comprising of a I(V), a3(V),and aI(X1) chains in a 1: 1: 1 ratio (Niyibizi and Eyre, 1989). In addition, a copolyrner of type 1 and V collagens can also forni (Birk et al., 1990) in whch 15% of the type V is covalendy cross-linked to type 1 (Niyi bizi and Eyre, 1994). The type V collagen is believed to affect the diarneter and architecture of the fibriilar network in these tissues (Butler and Ritchie, 1995). Although there are conflicting data with respect to the presence of type III wllagen (Wang et al., 1980; Sodek and Mandell, 1982; Steinfort, 1990) in normal bone and dentine, in cementum type III collagen constitutes approximately 5-74 of the collagenous proteins. Type III collagen, which is a homotrimer of a 1(III) collagen chains, is believed to be associatecl with extrinsic fibres in cementum. The presence of type III is thought to prevent minerai deposition in the fibres, although there is no direct proof of ths (Buder and Ritchie, 1995). Under pathological conditions such as hereditar). opalescent dentine, type III collagen can be detected in dentine (Sauk et al., 1980; Magloire ei al., 1988; Bronckers et al., 1989). It is interesting to note that there are differences in the chernical properties and fine stnicture of the type 1 collagen in soft and hard connective tissues. Thus, the type 1 collagens in mineralized comective tissues are more highly hydroxylated (< 7,-fold) than in soft tissues (Volpin and Veis, 1973). Al1 16C telopeptide lysines in bone and dentine are hydroxylated, as are the cross-link aldehyde receptors at helix psition 87 (Lys). This is not the case in soft tissues (Yamauchi et al., 1992). Amineterminal telopeptides of type 1 collagens in bone do not seem to be invdved in cross-linking reactions. The collagen fïbrils in bone and dentine cm undergo reversi ble molecular packing rearrangements on rnineralization. Thus, as hard connective tissues mineralize, the nature of the cross-link changes (Otsubo et al.. 1992). Pyridinoline cross-links are fomed in osteoid and soft tissues, while virtually none are deteetable in the mineral ized cornpartment of bone. This cross-link appears to impede the entry of the ions in between the molecules of mllagen to form mineral (Miller, 1984b). There i s, however, an increase in hydrox y1 ysine-deri ved crossli nks in bone collagen over time. This may be due to a decrease in minerakation associated with ageing (Syftestad and Urist, 1W-). Overail, there are fewer crosslinks found in mineraiized connective tissues compared to sofi tissues. AIthough the signifiouice of differences in giycosylation of hydroxyl ysines found in collapens from di fferent tissues is still uncertain, the sugars are believed to participate in cross-linking (Eyre, lm). Thus, i t has been proposed that sugars may widen the lateral spacing in bone collagen, allowing for ihe passage of ion precursors for the formation of mineral deposits. In addition to the different collagens, the a I pN-propeptide of type I collagen. which is released during the proteoiytic processing of procollagen, is also present in signifiant amounts in the bone matrin (Fisher et al., 198%; Goldberg et al., 1988a) and the propeptide associated witb the mineral appears to be phosphorylated (Fisher et al.. 1987b). In addition, a second. possibly non-phosphorylated form of the propeptide associated with the organic matrix has also been identified (Goldbeq et al., 1988a). Notably, these propeptide forms have the potential to regulate fibril-associated HA formation and, if released during bone resorption, can regulate osteoblast activity by inhibiting collagn synthesis. Two foms (28 and 25 kDa) of a smail collagenous protein. termed "small collagenous apatite-binding" (SCAB) proteins. have also been identified in demineralizing extracts of porcine bone (Kuuata et al., 1987). Since these proteins. li ke the a 1(I) pN-propeptide, bind strongly to HA and are partial 1y depded by bacterial collagenase, it is conceivable that these proteins might represent pN-propeptides, of type V collagen.

II. NON-COLLAGENOUS PROTEINS:

Proteoglycans The major proteoglycans (PGs) that have ken isolated from bone contain chondroitin sulfate (CS) as the principal aüached gl ycosaminogl ycan (GAG) instead of dennatan sulfate (DS) which is large1y found in soft tissue. A large 1,000 kDa CS-% is presen t in the non-mineraiized bone matrix wi th at least three smailer CS-- associated wi th the mineral phase (Fisher et al., l983a; Franzen and Heinegard, 1984a; 1984b; Fisher, 1985; Sato et al., 198%; Fisher et al., 1987a; Goldberg et al., 1988b). One of the small proteogl ycans isolated from human and bovine bone, known as CS-PG 1 or bigl ycan, has an M, of -3M kDa and a protein core of 46 kDa, and two CS chains attached to the core protein. The amount of CS-PG 1 appears to be generally low and variable in the di fferen t species exami ned and was not observed in porcine bone (Goldberg et al.. l988b). A more prominent small proteoglycan (M,- 130 kDa - 200 Ba),CS-PG II (decorin analogue), has a single CS chah and a core protein thaî migrates as a doublet on SDS-PAGEat 45 and 47 kDa The CS-PG 1 and CS-PG II proteogl ycans have been shown to be homologous to the DS-PG 1 and DS-PG II proteoglycans of cartilage (Goldberg et ai., l988b). Using monoclonal antibody probes, Goldberg et al.. ( l988b) have show that CS-PG II core protein is homologous to the smail soft connective tissue proteodematan sulfate proteogl ycan. 1t appears, there fore, that osteoblasts sy nthesize the same proteogi ycan core as found in other tissues but lack the epimerase activity that converts glucuronic acid to iduronic acid in the formation of dermatan sulfate gl ycosarninogl ycan chai ns. As a consequence, the CS-form of proteogl ycan is de psited into the mineralizïng bone matnx. In rat dentine. 5 small proteogiycans (described as PG1-5) with core proteins of 35, 40, 1 I5,70and 4û-M kDa, respective1y, have been characterized (Steinfon et al., 1994). It is believed that: PG1 ma): be similar to the CS-PG III idated from the porcine calvarial bone (Goldberg et al., 1988b); PG4 appears to be BSP with an attached CS chain(s); and the PG5 is probably decorin andfor bigl ycan. The proteoglycam of cementum have ken less well characterized. In human cementum. hyaluronic acid ( 16%), chondroitin sulfate (5369%) and dermatan sulfate (3 1%) GAGShave been identified and are believed to form a population of three distinct proteoglycans (Bartold et al., 1988).

Sidoproteinr: a) Osteopontin (OPN Osteopontin was initially discovered as a phosphoprotein secret& by transfomed cells (Senger et ai., 1979; Chac kalaparampil et al., 1985; Senger et al.. 198%) and was inde pendent1y isolated from bone and characterized (Franzen and Hei negard, 198%; Pn nce et al., 1981; Fisher et al., 1987a). Rat bone osteopontin is a44 kDa phosphoprotein, shown to contain 13 phosphoserines and one phosphothreonine in addition to 5 O-linked oli gosaccharides and an N-linked oli gosaccharide (Franzen and Heinegard, 1 98%; Rince et al., 1981; Fisher et ai.. 1987a). The cornplete pnmary sequence of rat OPN as determined from the cDNA has been show to contain a polyasparthrombin susceptibility has been identi fied (Senger et al., l989a; 1989b) at a centrally located Arg-Ser together with sites of "tryptic" cleavage which generate 23 and 20 kDa fragments that have been identifïed in EDTA extracts (Zhang et al., 1990). That OPN is involved in bone formation is indicated through its expression by pre- osteoblasts and osteoblasts, and by i ts stimulated synthesis in the presence of hormones and cytokines, such as glucoconicosteroids and TGF-p (Kasugai et al., 199 1 ;Wrana et al., 1991), which prornote bone matrix formation. Although bone OPN has many of the characteristics of a nucleator of HA, biosynthetic studies have failed to detect its presence in the collagenous matna prior to mineralization (Kasugai et al., 1991; Nagata et al., 199 1b). However, once mineralization has ken initiated, OPN associates rapidly with the prefonned minerai where it may regulate the groath of the HA crystals. Whether the selective proteolytic fragmentation of the protein (Zhang et al., 1990) is important for this function remains to be determined. It has also ken postulated that OPN functions in the attachent of osteoblasts to osteoid during bone formation (Butler, 1989). The preference of OPN for the vitronectin receptor (Oldberg et al., 1988a). which is ennched on the surface of osteoclasts, together with the strong stimulation of OPN synthesis by vi tamin D3 (Prince and Butler, 1987) and retinoic acid (Kasugai et al., 1991) which promote bone resorption, indicates a role for OPN in osteoclastic bone resorption (Reinholt et al., 1990). The apparent concentration of OPN and the vitronectin receptor in the clear zone of osteoclasts (Reinholt et al., 1990) provide strong support for this proposed role. OPN has also been isolated from porcine dentine matris (Fujisawa et al., lm), and expression of OPN mRNA obsened in rat incisor odontoblasts (Chen et al., 1993). The detection of OPN mRNA in 2 day-old molar tmth germs indicates the expression of OPN in early tooth development (Ritchie et al., 1994). lmmunohistochemical studies have shown that OPN is found in both acellular and cellular cementum (Bronckers et al., 1994).

Bone Sialoprotein (BSP) BSP is a highly glycosylated and sulfated phosphoprotein that has ken isolated (Fisher et al., 19û3a; Franzen and Heinegard, 1985a) from bones of several species including human (Fisher et al., 1987a), cow (Fisher et al., 1983b;Franzen and Heinegard, 1985a). rabbit (Kinne and Fisher, 1987) and pig (Zhang et al., 1990). It has high levels of sialic acid and glutamic acid and is heavily sulfated, the sulfate king primarily linked to tyrosine, as tyrosine sulfate (Ecarot-Charrier et al., 1989; Nagata et al., 1989). Notably, rabbit BSP has a keratan sulfate side chah (Kinne and Fisher, 1981). Although BSP is also phosphorylated. the degree of phosphorylation is lower than for other mineralized connective tissue phosphoproteins (Kasugai et al., 199 1; Nagata et al., 199 1b) . A molecular weight of 57 kDa has ben determined for bovine BSP by sedimentation anal ysis while the size of the nascent protein in several species is close to 34.5 kDa (Oldberg et al., 1988b). This difference reflects the high degree of glycosylation of the protein which has been calculateci to represent 50% of the protein mass (Fisher et ai., 1983b). The protein is characterized by its ability to bind strongly to HA (Oldbeg et al., 1988b) and to mediate ce11 attachment through an RGD site, (Oldberg et al., 1988a; Someman et al., 1988) that mgnizes the vitronectin ad3receptor. A high degree of sequence conservation is observed when the primary sequences of human (Fisher et al., 1990), pig (Shapiro et al., 1993) and rat (Oldberg et ai., 1988b) are cornpareci. The terminal segments are enriched in aromatic amino acids, especially tyrosines which, when anaiyzed according to consensus sequences, are believed to be suifatexi in the C-terminal region and surround the RGD ceIl attachment motif. The cenual region of the moiecule is e~chedin polar arnino acids which are almost entirel y negatively charged and include two to three suetches of polyglutarnic acid, which are believed to be involved in the binding to HA (Oldberg et ai., 19ûûb). Several si tes of serine phosphorylation and N-linlied gl ycosylation are also consenredin the centrai region. The low amounts of hydrophobie residues indicates that BSP is largeiy udolded and flexible, as also reveaied by rotary shadowing (Franzen and Heinegard, 1985b). Expression of BSP is essentially restrkted to mineralized connective tissues (Oldberg et al., 1988b;Fisher et al., 1990) and immunolocalization studies have show that the protein is confined to the bone matnx and directl y associated cells (Chen et al.. 1991b). Studies on the expression of BSP in rat bone by in situ hybridization have shown a strong, specific expression by osteoblasts at si tes of neu bone formation (Chen et al., 1991a). Studies on the biosynthesis of BSP have demonstrated that the expression of BSP is directl y Iinked to bone formation (Kasugai et al., 1991) and that, while most of the protein associates with pre-eaisting mineral, some protein is present in the collagenous matrix pnor to mineralization (Kasugai et al., 199 1; Nagata et al., 199 1b), and appears to be masked by mineral formation (Kasugai et ai., 1%). In dentine, synthesis of BSP and its mRNA by odontoblasts nas demonstrated by immunohistochemistr). and in situ hybridization (Chen et al., 1992a). The BSP was found to be localized to odontoblasts, odontoblastic processes and associated intratubuiar dentine. Although BSP appean to be hi ghl y resmcted to mineralized tissue, being synthesized in bone, dentine and cementum, i t has also been demonstrated in hypertrophie chondrocytes and placenta trophoblasts (Young et al.. l99?).

Dentine Ma& Protein 1 (Dmgl) A unique, acidic, ptentially highl y phosphorylated extracellular rnatrix protein calleci Dentine Maaix Protein 1 (Dmp1 ) has ben characterized (George et ai., 1993 ; 1994; 1995). This protein as predicted from cDNA analysis is to be nch in Asp, G!u and Ser and is secreted into the matrix of dentine. It has a mdecular weight of 53 kDa with a net charge of -81 (pnor to any phosphorylation) (Butler and Ritchie, 1995). The predicted phosphorylated form of Dmp1 is much more acidic than those of bone mauix acidic proteins (George et al.. 1993). 1t has consensus sequences for a potential N- and several O-glycosylation sites, together with an RGD sequence (George et al., 1993). Although the NH1-terminus is identical to BAG-75, a bone derived phosphoprotein, and to the chondroitin sulfate proteoglycan CS-PG III in porcine bone (Goldberg et al., 1988b) it is believed that Dmpl is a distinct entity since the amino acid compositions of these proteins are different (Butler and Ritchie, 1995). The rat hp1gene is localized near the other mineralized tissue related genes of OPN,BMP3 and TGF-p (George et al., 1994). Drnp 1 expression is developmentall y reguiated and is restncted to secretory odontoblasts (George et al., 1993 ; 1995). Dmp t protein has been shown to be present in dentine extracts (George et al., 1993) at relatively low levels, implying a possible regdatory role (George et al., 1994).

Dentine Sialoprotein (DSP) Dentine sialoprotein (DSP)is a dentine-specific, sialic acid-rich glycoprotein. 1t has neither PQ3- nor cysteine but contains large amounts of Asp, Glu, Ser and Gly and has a high carbohydrate content (30%). 10% of which is sialic acid. There are a nurnber of N- and O-glycosides present in its structure. Imrnunohistochemical siudies have shown the presence of DSP in early predentine prior to the onset of minenlization with a more restricted localization to dontoblastic processes at later mature stages (Butler et al., 1992: D'Souza et al., 1992; Bronckers et ai., 1994). The cDNA has ken isolated and the nucleotide sequence predicts 6 potential N-linked glycosylation and 13 potential phosphorylation sites. It is distinct from OPN, BSP and BAG-75 and does not have an RGD sequence (Ritchie et ai., 1994). Recent studies strongly suggest that there are two discrete but homologous DSP genes which exist in rat (Ritchie et al., 1995), and Western BIoi studies reveal two proteins that react with anti DSP antibodies (Butler and Eùtchie, 1995).

Bone Acidic Glycoprutein (BAG 75) BAG-75 is a bne-denved phosphorylated acidic glycoprotein (contaming N- and O- linked sugars; including sialic acids) having a M, of 75 kDa that was originally isolated from rat (Gorski and Shimizu, 1988). Because of its tight association with small bone proteoglycans it is found to co-elute with these molecules during purification. This protein has also been shown to cross react with specific rat antibodies to osteopontin (but not to BSP) suggesting that these molecules are related in some manner. This molecule, however has been shown to be distinct from osteopontin (and BSP) in that there are differences between their N-terminal sequences. Al though the first ten arnino acids in the BAG-75 protein are essentially identicai to Dmpl and CS-PG III no equivalent protein has yet been characterized in other species.

Dentine Phosphproteins (phosphophorym) Some of the Tint and highly characterized molecules isoiated from dentine were called the phosphophoryns. These are the most abundant, non-collagenous proteins extractable from this tissue. They seem to be unique to dentine and represent a heterogeneous group of proteins w hich degrade as dentine ages (Dimuzio and Veis, 197%; Butler et al., 1983; Lee et al., 1983; Linde, 1Wb;Masters, 1985; Linde, 1989; Veis, 1989). Characteristically these rnoIecules have a high content of Ser (P)(354%) and Asp (4550%) with a pI of 1.1. As such, they represent the most acidic class of proteins known (Jonsson et al., 1978). A compiete amino acid sequence for a phosphophoqn has yet to be determined. At present, the phosphophoryns represent tuPodistinct proteins that differ in their degree of phosphorylation (Butler et aï., 1583). During dentine formation. phosphophoryn is thought to be deposited directly at the mineralization front (MacDougall et al., 1985; Nakamura et ai., 1985; Gorter de Vries et al., 1986; Rahima et al., 1988; Gorter de Vnes and Wisse, 1989; Veis. 1989). These proteins strongl y interact wi th Ca?+. binding large amounts with high affinity (Zanetti et al., 1981; Marsh, 1989). A small portion of the phosphophoqn is also associated wi th collagen but it is not known w hether or not ths interaction is covalent (Maler et al., 1983; Kuboki et al., 1984; Stetler-Stevenson and Veis, 1986; Linde, 1989; Veis, 1989). Al though li ttle is known of the orientation of phosphophoqn with respect to the collagen fibril axis (Veis, MG), in vitro studies have shown that it can bind type 1 collagen (Stetler-Stevensonand Veis, 1986) in the gap region of fibrils (Traub et al., 1992) and when the cornplex is incubated under physiological concentrations of CS+ and POj3- can induce the formation of HA (Linde, 1989). Furthemore, in the presence of Ca?+,phosphophoryn forms a p-sheet, a structure which can interact with Ca2+ ions in growing apatite crystals (Addadi et ai., 1992).

Osteocalcin (BGP) Also known as bone Y-carboxygiutamicacid (gla) protein (Hauschka et al., 1975; Hauschka, 1985), ostdcin(Pnce et al.. 1976; Pnce, 1983) is the mat completely characterized of the non-collagenous bone proteins (Rice. 1983 ; Hauschka, 1985). Osteocalcin is a smal15.8 Haprotein which contains 2-3 Gla raidues and one disuIfide bond (Hauschka et al., 1989; Pnce, 199-). AIthough it represents 1% to 2% of total bone protein in rat (Hauschka et al., 1975). the amounts of osteocalcin vary wnsiderably in bone of di fferent species, with human bone contaming approximately one-tenth of the arnount found in rat (hce, 1983). The primaq sequence of osteocaicin has been obtamed frorn a number of species and high sequence conservation has ken mted. 1t is characterized bu the presence of Y-carboxyglutamic (gla) residues that are formed by a vitamin K-dependent post-translational carboxylation of glutamic acid. The gla groups bind Ca?+ions in solution and also tightly to HA, inhibiting HA formation in virro. Inhibition of y-carboxylation by the vitamin K antagonist warfarin nsults in a dramatic reduction of ostdcin in the bone (Pnce and Williamson, 1981). Although hypemineralization has ken observed under these conditions, bone fornation is not irnpaired. The hypemineralization rnay relate to the ability of ostdcinto inhi bit HA formation from supersaturated solutions of Ca?+and F043-. Using specific antisera to determine tissue distn bution (Mark et al., 1988a). it has ken revealed that ostedcin is essentially specific to mineralized connective tissues. Notably. the expression of osteocaicin in rat bone is initiated after bone formation has ocfurred (Yoon et al., 1981). Although the presence of osteocalcin in serum has been used as a masure of bone rernodelling, its function appears to be related more closely with the resorption of bone. Thus, synthesis of the protein is increased significantl y when osteoblasts are stimulated with 1.75 dihydroxyvi tamin D3 (Price and Baukol, 1980). whch is known to stimulate bone resorption. Also, it has ken shown that the presence of osteocalcin is necessaq for osteoclastic resorption of implanted bone panicles to occur (Glowach et al., 1989). Although the exact function of the protein in the resorption process is not known, a proteol ytic fragment released from the C- terminal end of the molecule is chernotactic for the monocyte precursors of the osteoclasts (Mundy and Poser, 1983). indicating a potential role for osteocalcin in the ldized recruitment of resorptive cells. Osteocalcin is also found in dentine (Linde et al., 1981) and is synthesized and secreted by odontoblasts in vivo (Bronckers et al., 1985; lm;Gorter de Vries et al., 1987) and in vitro (Dirnuzio et al., 1983 ; Finkelrnan and Butler, 1985). In human and bovine teeth, however, only mande dentine is immunoreactive to anti-osteocalcin antibodies (Goldberg et al., 1995). More recentl y, the existence of three ost-ocalcin genes have been reponed in the mouse genome (Desbois et al., 1994). Two of the genes (OGI and OGS)are expressed in bone while the third is ody expressed in kidney (Desbois et al.. 1994). Notably, the temporal expression of osteocalan in dentine differs from bone in that in dentine formation it is expressed prior to the production of collagen (Heersche et al, 1992). Osteocalcin in cementurn has ben shown to be compartmentalized, bei ng presen t in ceIIuIarcementum but absent in acellularcementurn (Bronckers et al., 1994). The significance of this distribution is unclear but may be related to the expression of osteocalcin by mature. matnx-forming cells such as lining cells and osteocytes in bone and cementocytes in cementum.

Osteonectin (SMC) Osteonectin was fint isolated from subperiosteal bovine bone and named to reflect its ability to bind collagen and promote HA formation in virro (Termine et al., 1% la; 198 1b). Although the protein represents up to 15% of the non-collagenous proteins in the bones of larger mammals, it represents onl y a small percentage of the nontollagenous proteins in rodent bone despite the fact that the protein is highly expressed by osteoblasts (Wrana et al., 1988; Kubota et al., 1989). Although originallp thought to be specific to bone. osteonectin was shown to be synthesized by fibroblasts (Wasi et al., 1984; Tung et al., 1985) and subsequently shown to be expressed by a variety of ce11 types including panetal endoderrn (Mason et al.. l986a).endothelid cells (Sage eet al., lm),and epithelial cells (Mann et al., 1987). The bone protein has been descri bed as a phosphoprotein (Termine et al., 1981a; Engel et al., 1987). However, several studies of bone formation (Kasugai et ai., 199 1; Nagata et al., 199 la; 199 1b) have failed to observe any incorporation of (3PO$-) into ej ther rat or pig osteonectin (Dornenicucci et ai.. 1988). The amino acid sequence for osteonectin has been deduced from human (bnkat- Buttgerei t et al., 1988). bovine (Bolander et al., 1988) and mouse (Mason et al.. 1986a) cDNA sequences. The sequence is hi eh1 y conserved and comprises four distinct domai ns. An amino terminal domain comprising two glutamate-rich segments that can bind >8 Ca?+ ions. On binding Gaz+ the protein chain in rat osteonectin has been suggested to undergo a coil-ta-helix transition (Engel et al., 1987). The amino acid sequence in this region is the Ieast well conserved and is believed to bind to HA. Thus, differences in the sequence in ths region are thought to relate to differences in the ability of the osteonectin to bind to HA and, consequentîy, to the differences in the arnount of osteonectin in the bones of animals of different species (Domenicucci et al., 1988). The second domain shows some homology to ovomucoid and is characteristically rich in disulfide bridges that stabilize the protein stnicture. This is followed by a segment which is susceptible to proteolysis and is predicted to rom an a-helical structure. The fourth domain contains a single high-affinity EF-hand Gaz+ binding si te with the characteristic helix-loophelix structure (Engel et al., 1987). This si te is expected to be full y occupied at physiologicai concentrations of Cal+ and is, therefore, unli keI y to be involved in Ca?+regulation. Osteonectin is vimiall y absent in rat dentine. but present in porcine dentine, where immunohistochemically intense dentine staining was seen in unerupted teeth (Tung et al., 1985). In bovine dentine, osteonectin consti tutes 46%of the total protein extractable from the tissue. In empted porcine teeth, anti-osteonectin staining in dentine was concentrated around dentinal tubules, with weaker staining in the cells of the pulp, and more intense immunoreaction in odontoblasts (Tung et al., 1985). In human teeth, the predentine odontoblasts and their ceIl processes show a strong immunohistochemical staining reaction (Reichen et al., In situ hybridization studies with mRNA show an intense reaction over the ociontoblasts (Goldberg et al., 1995).

3) MINERALIZATION MECHANISMS

Although the earliest fossil vertebrates, the ostracoderms (more than 500 million years ap), had skeletons which included the hard tissues of calcified cartilage, bone, dentine and enamel, it is believed that the modes of mineralization in these primi ti te tissues have persisted throughout the vertebrate lineage without major change (Kemp, 1984). In addition, it is believed that minerdization of comective tissues share similar features. In general, these fearures include the provision of calcium (Ca?+)and phosphate (PO$-) through the vascular system and tissue fluids (systemic factors) in conjunction with the elaboration of a unique rnatrix by specialized ceil types (local factors). The mosaic of components, under temporal restrictions, generates apatitic calcium/phosphate crysrallites in close association with mavix proteins in a highly organized and controlled manner. To undentand the complexity of this process, however, a distinction between the initial deposition of minerai (initiation phase) from that of mineral which foms on prefomed mineral (pro~iferation/accretion/secondarynucleation/growth phase) must be appreciated. Thus, as new crystals are formed, the tissue becornes more mineral ized and the addi tional mi neral crystals themselves act as nucleation centers. This produces addi tional crystals rapidl y so that the potential rate of mi neralization increases enponentiall y in the more calcified regions, albeit, in a controlled, progressive manner (Rey and Glimcher, 1992). As an additional complexig in bone, there is a continuai growth and remodelling so that in the sarne tissue there are two processes that are superimposed in tirne. Models of mineralization have to futher account for the fact that Ca2+ and FQ3- exist in a supersaturated state in the body fluids. The concentrations of these ions king regulated by hormonal influences on the kidne y, intestine and bone. 1nterestingl y, aithough the CS+and m3- in this supersaturated state will deposi t on preexisting mineral crystals. they are unable to precipitate spontaneousl y. Therefore. the concentration of Gaz+ and POJ3- in body fluids, and presumably in the bone milieu, is described as king metastable (Neuman and Neuman, 1953). Since spontaneous precipi tation of Ca?+and W43- from the supe~turatedbody fluids does not mur, an energy barrier is considered to exist. In theory, this energy bamer can be lowered &ya number of rnechanisms, and once lowered the formation of HA crystals cm proceed. Theories whch try to explain how a lowering of the energy barner may occur in mineralizinp systems faIl into three groups: homogeneous nucleation, heterogeneous nucleation or nucleaiion i nhi bi tion. A brief description of some of these theories follows.

I. THEONES OF MINERAL FORMATION

Homgeneous Nucl eation The theones propose that there is a localized increase in Ca2+ andior PO$- allowing for the formation of a criticai number of ionic dusters which then leads to spontaneous crystaI precipitation. a) The booster mechanism suggests that local PO$- could be increased by enzymes that release PQ3- frorn various inorganic and organic substrates, thereby exceeding the solubility product and initiating Ca2+1W43-precipi tation (Robison, 1923). Interestingiy, the addition of f3-glycerophosphate in osteogenic explants (Tenenbaum and Heenche, 1982) and cell cultures (Bellows et al., 1986) is often a requisite for the initiation of minerai in vivo. b) The secretion theop states thar there is active transport of Ca?+or PQ3- from iniracellular stores to the site of mineralization. Ca?+is thought be actively sequestered, concentmted and packaged by the mitochondrïa (Lehninpr, 1970; 1977) and then dischargeci extracellulady (Azzi and Chance, 1%9). C) The matrix vesicle theory states that vesicles, believed to be derived [rom local cells, provide a microenvironment in which the energy Merfor the formation of the first Cat+lP043- crystallites can .be overcome (Bonucci, 1%7; Anderson, 1969; Bonucci, 1970; Bonucci, 1971; Anderson, 1973). Thus, the increase in Gaz+ and PQ3- inside the vesides leads to HA crystal formation (Wuthier, 1981). These intravesicular crystals rapidly grow and rupture through the membrane bound structure. An exûacellular sphenilitic deposit of HA crystallites then develops and eventually joins with others to fonn even larger mineralized masses (Christoffersen and Landis, 199 1) . Notabl y, al kaline phosphatase and pyrophosphatase acîivities have been associated with these vesicles (Marsuzawa and Anderson. 1971; Majeska and Wuthier. 1975).

Heterogeneous Nucleation Theories involving a Iocalized nucleator, which is thought to lower the energy bamier and bring about crystallization without the necessi ty for an increased concentration of Cal+ and PO$- are general 1y based on the epi iauy theory w hich States that the formation of crystallites occurs on a surface other than a Ca2+/Pû43-crystai (Neuman and Neurnan. 1953; Glimcher, lm;Glimcher and Krane, 1968; Gebhardt, 1973). Hence, a specific suface (ie. a nucleation site) provides an appropriaie structure onto which Ca?+/P043-ions can be comctly oriented for the even tual Ca2+/P043- preci pi tationlcrystal Ii te formation. i) Thatcoll~encanactasaheterogeneousnucleatorisindicatedbythe observation that a site in the microfibril collagen structure known as the gapregion is the location of the fint mineral qstals in mineraiizing systems (White et al., 197). ii) That phosphophoryn cm act as a heterogeneous nucleator in dentine is large1 y based on the protein's rich [-AspSer(m4)-] repeating sequences which have theoreticdly been thought to provide. in a maris extended fonn, t~odistinct rows of negative charges (Stetler-Stevenson and Veis, 1986). One row is formed by the Asp residues which through a Ca2+ bridge interacts wi th collagen. The opposi te row which is composed of Ser-FQ, can simulate a laîtice distance found in the HA unit qstal, a structure that could, in theory, provide a nucleation surface (Veis, 1989; Veis, 1993).

Nucleation inhibition Theory A theory involving minerai cq~talformation by the rernoval, or inactivation of inhibitors is known as the nucleation inhibition theory (Howell, 1976). This theory is predicated upna major event in whch Ca?+/W43- precipitaîion is permitted by the rernoval or inactivation of inhibiton existing locally that prevent minerai fornation (Howell et aI., 1%9; Termine and Corn, 1976; Fleisch, 1983). Once the first crystallite has formed, a supcrsanimted tissue fluid will dlow continued deposi tion of Ca2+ and W43- on that crystallite. The local concentration of available PQ3- is thought to be increased by the degradation of pyrophosphate by pyrophusphatases. Similariy, degradation of pmteoglycans and glycosaminoglycans by enzymes cui result in a local increase in available CS+(Kuettner et al., 1974). Cleariy, the process of rnineralization is comples and may invdve several mechanisms at different levels, in different tissues and is unlikely to be described by a single unifying ooncept. Integral playen in the initiation of mineralization however are believed to involve col lagen and/or associated non-col lagenous bone rnatrix proteins.

II a. COiLAGEN AS AN IMTIATOR OF MINERAL DEPOSITS

Coilagen's rote as a possible initiator of rnineralization was first posnilated by White et al., (197) who proposed that the gap region in the fibrils was the initial site of minerai deposition in connective tissues. This theory was supportcd by the demonstration that collagen fi brils could promote the fomation of crystals under metastable conditions (Glimcher et al., 1957). In addition, modification of the collagen stnicture by blocking the carboxylic acid groups of aspartate and gi utarnate prevented this NI vitro apati te deposition (Davis and Walker. 1972). Furthermore, al tering the col lagen by dehydration also destroys the ability of collagen to promote KA formation (Glimcher and Krane, 1968). Electron microscopie examination of the cystals formed in vitro revealed that the minerai was not randomly packed within the collagen but was located at regular intemais, coincident with the axial repeat correspnding to the gap regions (Glimcher, 1984). In addition, the mineral cqstals were depited in a manner related t the periodicity of the collagen fibrils with the c-ais of the HA mineral usually aiigned parailel to the collagen fibnls (Heeley and Irving, 1973). as those found in vivo (Weiner and Traub. 1989). In vivo studies have also demonstrated that type 1 collagen fi brils isolated from both mineralized and non-mi neral ized sources have an i nherent abil i ty to prornote apati te cqsral formation in the gap regions if they are reconstituted in the native form (Glimcher, 1981). In addition, electmn probe microandysis studies (Hohling et al., 1970) indicated that early in the mineralization of bones and teeth, CS+is bound to collagen while PO$- is only looseiy associated (Hohling et al., 1970). This is of special interest since the telopeptides of collagen contain acidic residw ciusten which could maintain the CS+ion clusters long enough to permit growth into stable nucleation centres (Davis and Walker, 1973; Davis et al., lY75). This could explain how modification of acidic residues can comptetely destroy the ability of collagen to promote HA crystal fomation (Davis and Walker, 1972; Davis et al., 1975). Thus, there is a high degree of specificity required for the nucleation event (Glimcher, 1989). The three-dimensional packing of collagen molecules in native collagen fibrils impa specific threedimensionai geometry and electrical charge distribution of the fibril side chah groups, as well as a volume of space within which the cqstals cm be deposited without disruption of the fibrils (Glimcher, 1989). Although the chernical differences between the hard and soft tissue type I collagens are quite subtle, the following small differences may be significant with respect to initiation of mi neral i zation. Fiat, the collagens in bone and dentine are more hydroxylated, yet they are les crosslinked compared to skin collagen (Miller, 1984b). In addition, while pyridinoline crosslinks are present in non-mineralizing bone tissue they are virtually absent in the minemlized cornpariment (Mechanic et al., 1%). Thus, the pyridinoline cross link appears to impede the entry of the ions in between the molecules of col1agen to form mineral. Second, the intemolecular distance in soft tissue collagens, when reconsti tuted in vitro, is ody 0.3 nm whereas in the mineralized connective tissues it is 0.6 nm (Katz and Li, 1973). Since the interfibnllar volume in collagens of non-mineraiizing tissues is smaller than that in mineralizing tissues, bone collagn is less tightl y packed than soft tissue collagen. Thus, the space amilable in bone and dentine collagen its almost twice that present in slun and tendon collagen (Miller, 1984b). Notably, 56 4 of the mineral in lamellar bone exists within the collagen fibrils. Third, r-gl~~ylphosphate is found only in bone collagen (al CB 3-5 peptide) (Landais et ai., 1989) and is absent in unmineralized tissue collagens (Cohen-Solal et al., 1979a; 1979b). The presence of y-glutamyl phosphate in a structura1 protein such as collagen is unusual, since ths modified glutamate side chain has ken identified on1 y in certain enzymes (Glimcher, 1989). Since the y-glutarnyl phosphate in the collagen molecule ccAocalizes to the gap region it could have the potential to bind Ca?+and initiate apatite formation there. Native collagen is, howeuer, oniy one of a number of macromolecules whose presence is known to facilitate HA formation in vitro (ie. phosphoproteins, proteolipids, etc.) (Boskey, 1981). Thus, the ability to promote apati te formation is not exclusive to col lagen. Moreover, since the nucleation of apati te by collagen may take up to several weeks, this activity may not be of physiological importance (Glimcher, 1984). Indeed, studies of comparative rates of diffusion of Cg+and PO$- in collagenous and agar gels suggest that the presence of collagen neither enhances nor retards the nucleation and growth of HA (Pokric and Pucar, 1979). In addition, studies on telopeptide structure, which exists in the gap region of the collagen microfibrils, suggest that it is not directly involved in nucleation (Miller, 1984a). Thus, it appears that the role of collagen is that of a passive template which provides a structural framework on and in which the oriented deposi tion of minerai can ocnir in an ordered fashion (Glimcher. 1984); mineral deposited at the gap region grows and subsequentl y proceeds between the intefi tmllar spaces delineated by the collagen molecule (Katz and Li, 1973; Weiner. 1984). Interestingly, collagen is absent in intratubular dentine, sirnilar to cernent lines and laminae limitans in bone (Goldberg et al., 1995). This suggests that mineralization mechanisms may be different in these areas. Therefore, while native collagen fibrils are neces- for calcification in collagenous tissues, cdlagen alone is not biologically sufficient (Glimcher, 1984). Thus it is believed that the interaction with one or more of the noncoIlagenous matrix proteins that form mineralued tissues must faciliiate the initiation of mineralization within collagen fibrils.

II b. THE ROLE OF NON-COLLAGENOUS PROTEINS IN THE MINERALIZATION OF HARD CONNECîIVE TISSUES

As eariy as the 199's. type 1 collagen was shown to facilitate minerai formation both in vitro (Koutsoukos and Nancollas, L98T) and in vivo (Mergnhagen et al., lm). As more highly purified preparations of type I collagen became available. the experimental facilitation of mineralization of collagen was demonstrated to be appreciably lower than those in earlier experiments (Koutsoukos and Nancollas, 1987). I t was thererore hypothesized that the "non-collagenous" proteins present in partially purified collagen preparations were the responsi ble factors that facil itated the mi neralization of collagen in these earlier experiments (Termine et al., 198 la: Endo. 1987). A corollary aas that, in vivo, one or a Iimited group of these non-collagenous proteins associated with collapn could have a more direct role in the initiation of minerd crystalli tes [rom a solution of Car+Pû43-(Fisher and Termine, 1985). Consequently, some of these matris proteins could have a roIe in the mineralization of hard connective tissues. A good exarnple of this would be dentine phosphophoqn nhich is a molecule that binds to the collagen (Sretler- Stevenson and Veis, 1986) in the gap region (Traub et al.. 1%) and retains its ability to bind many CS+ions (Stetler-Stevenson and Veis. 1986; 1987) after doing so. Because of these feaaires, it is believed that phosphophoryn could serve as an agent for concentrating Car+ ions in the vicinity of the gap region of the collagen fibrils and specifically initiate the nucleation of the apatite crystals (Veis, 1993). It follows, therefore, that a stereospecific protein-rnediated mineralization mechanimi pro@ for dentine could dso eist in bone. 1II. FEATURES OF A NUCLEATOR

Some of the anticipated requirements of a nucleator, that would facilitate the controlled phase ûansfmation of metastable solutions of CaZ+/PO$- to a crystailine fom of hydroxyapatite in precise locations are as follows: Fiat, a nucleator would be expected to be specifically expressed, synthesized and secreted by osteoblasts in a tissue-specific manner (Triffit and Owen, 1973; Ashton et ai., 1976; Triffit et al., 1976; 1978) at sites of new bone formation. The molecule should also maintain a temporal sequence of synthesis and secretion relative to other proteins in the matrix (Stein et al., 1990a; 1990b) and would localize to the site of mineralization shortly after synthesis and secretion. The molecule should bind in the gap region of collagen at a site correspondhg to the precise location of initial crystal formation, in the unmineralized fibrillar collagen (White et ai., 1977). A logical extension of this aould be thai the molecule should not cause premature or random minedization in the osteoid. Since there is a hi@ degm of specificity required for the nucleation event (Glimcher, 1976; White et al., 1977; Berthet-Colominas et al., 1979; Glimcher, 1985), it is believed that interaction between collagen mdecules and the nucleator transforms the gap regon to a nucleation si te for the formation of HA crystals (Glimcher, 1989). In support of this premise is the demonstration that the irnmobilization of some bone proteins is a pre-condi tion for HA nuckation to occur in vitro (Linde and Lussi, l989a; Linde et al ., l9û9b). 1t has been postulateci that organically bound m43-in the form of Ser-PO4 are likely to play a role in nucleation of Cat+P043-(Glimcher, 1%0; Glimcher and Krane, 1968; Lian et al., 19=; 1982b). Moreover, from a structurai, chernical and functional point of view. the Ser-IQ cornponent in chicken bone phosphoproteins exist as reactive monoesters which are availabIe for interaction wi th Ca?+(Lee et al., 1983). In addition, dentine phosphophoryn has benpostulated to employ Ser-Pû4 binding sites in a similar manner (Lee et al., 1977; Li and Katz, 1977; Zanetti et al., 1981; Lee et al., 1983). It has also ken shown that protein phosphorylation can increase the binding alfinity for Ca?+/Q3- by acting coordinakly with neighbouring acidic groups (Holt and van Kemenade, 1989). Since strong binding of Ca?+ may inhibit any or dl of the physicochemical steps involved in the deposition of a solid phase of Ca7+/P043-(ie. nucleation, crystal multiplication, etc.), the CS+binding of a nucleator should be such that it promotes the interaction with inorganic FQ3- to form temq complexes in a manner that binds ions so that they remain reactive (Lee et al ., lm). More recently, other chernical features pstulated for a nucleator-protein include sulfate groups in the fom of Tyr-S04 which codd interact with conserved polyacidic amino acid stretches (Nagata et al., 1991a). Thus mollusc shell proteins, and synthetic pol yrners that rnimic these characteristics have been shown to act cooperati vel y in HA crystal nucleation (Addadi et al., 1981). Notably, polyacidic stretches of 8 or pater are found predorninatel y in metal binding proteins (Gorski, lm). Ai present, however, there is no fundamentai difference which can distinguish between a protein that acts as a nucleator compared to one that staùxlizes a metastable supersaturated solution or that inhibits CaZ+TPOJ3- crystal growth (Holt and van Kemenade, 1989). The ability of some molecules to bind to the collagenous matnx of bone (Sodek et al.. 1991; Kasugai et al.. lm; Sodek et al., 19E.a; Sodek et al., 1Rb)or dentine (Stetler-Stevenson and Veis, 1986; Traub et al., lW) rnay be the key difference with respect to nucleation. However, even this feature may not necessari1y be essential (Boskey el al., 1990; Hunter and Goldberg, 1993). STATEMENT OF THE PROBLEM / HY POTHESIS

The formation of hydroxyapatite in a collagnous rnatrix is

To isolate, punfy and charactenze osteonectin and further evaluate iü role in minerakation.

To punfy and charactenze the major proteins released in the demineralizing extracts (E-extract)of bone, to determine the potential desof these proteins in minerali zation.

To isolate and charactenze two major, and potentiall y novel, proteins present in dissociative estracts of demineralized bone and den the.

To extend the extraction protocol for the preliminary identification of proteins tightl y bound to the demineralized collagenous matrix of bone. CHAPTER II

THE DEVELûPMENT OF A PROCEDURE TO EXTRACï PROTEINS FROM MINERALEED CONNECIIVE TISSUES.

INTRODUCTION

Isolation and purihiion of macromolecules tha t form connective tissue matrices is hampered by the highl y associative/aggregative properties of the cuntributing components. This has lead to the development of dissociative extraction procedures, such as the 4 M GuHCI extraction prduredeveloped by Sajdera and Haseal1 ( 1%9) for the isolation of cartilage macromolecules. In mineralized tissues the problem is further complicated by the presence of the mineral phase, which necessitates the utilization of demineralizing agents. To facilitate the isolation of proteins [rom mineralired connective tissues a number of extraction procedures have been developed. These tissues have been treated with solutions of weak acids in conjunction with NaCI, or chelators such as tetrasodium ethylenediaminetetraacetic acid (EDTA) (Veis et ai., 1979) to release moIecules during the process of demineralization of the tissue. Other extraction procedures have isolated proteins after dernineraiization but on1 y after collapn degradation, by employing sol utions that contain either alkaline periodate (Schlueter and Veis, 1964; Carmichad et al.. 1971; Smith and Leaver, 1978; Jontell et ai., 1980),cyanogen bromide (Volpin and Veis, 1973; Dimuzio and Veis, 1978a; Curley-Joseph and Veis, 1979; Lee and Veis, 1980; Fujisawa et ai., 1981 ; Linde et al ., 1981) or bacterial collagenase (Jones and ber,1974; Leaver et ai., 19'7%; Butler et al., 1977; Hemng, 1977; Thomas and Leaver, 1977; Linde et al., 1981; Linde et al., 1983). Because degradation of bone proteins is thought to occur coincidentally with the increased mineralization of maturing hard connective tissues, and since large numben d fragmented macromolecules are obtained dwing extraction as a result of enzymatic digestion (Spector and Glimcher, 1977; lm),proteol pic-enzyme inhibitors are routinely added to extracting solutions (Linde et ai., 1980; Termine et ai., 1980). Hence, dissociative extraction procedures (Linde et al., 1980; Termine et ai., 1980) are generall y ernployed to isolate non-covalent1y associate. proteins from mineralized tissues in an intact form. A strong dissociative agent, 4 M guanidine hydrochloride (GuHCl), is used Tint to extract proteins from the soft tissues (and ostmid) and then used in combination wi th 0.5 M EDTA to extract minerai-associated proteins (Linde et al., 1980; Termine et al., 1980). In al1 the extraction solutions an inhibitor cocktail, comprising 1 mM phenylmethanesulphonyl fluoride, 5 mM benzamidine, 5 mM N-ethylmaieimide and 100 mM E-aminocaproic acid ( proteinase inhi bi tors: PI),is included to prevent proteolysis during the isolation procedures. To more cl wly define the macromolecular components that form the mi neralized connective tissue matrices of bne, fetal porcine calvariae were extracted first with 4 M GuHCl (Gl-extract), then with 0.5 M EDTA (E-extract) and then again with 4 M GuHCI (G2-extmct). This extraction procedure is believed to separate the minerai-associateci proteins (E-extract) of bne from proteins associated w i th the m ineralizjng collagenous maaix (G3extract). Using this procedure i t was detemined that most of the previously describeci proteins could be released wi th EDTA alone. 1n addition. the presence of a number of apparentl y nove1 proteins in the demineralized bone matrix (Glextract) were revealed.

MATERIALS AND METHODS

Preparution of Calvariae, Long Bone. Dentine and Cementum For comparative analytical studies of various mineralized porcine tissues, one gram wet weight of each tissue was prepared as follows: a) For calvariae and long bones, fetal pigs were obtained [rom a local abattoir and within two hours of slaughter the caivariae and the midshafts of femurs were isolated. The tissues were kept separate and placed in an ice-cold phosphate buffered saline solution (pH 7.4), deficient in calcium and magnesium (PBS), and containing protease inhibitors (PI): E-aminocaproicacid (100 mM), benzamidine hydrochloride (5mM), Nethylmaleimide (5 mM),phenyImethanesulfony1 fluoride (1 mM). Soft tissue adhering to the bne was removed with tissue paper and the remaining hard tissue was freeze-thawed three times in PBS + PI.The bone was then frozen in liquid nitrogen and crushed using a mortar and pestle in order to ensure the exposure of the i~erbld-containing cavities in bone to the extracting solutions. A total of 10 g of calvariae and long bones were collected and then frozen. b) Dentine and cementum were prepared by obtaining 300 +mthick slices of the apical third of adul t porcine mandi bular incisor root tips by employing a micro-circula saw . The mot slices were immediately placed in ice-dd PBS fontaining PI.Utilizing the fact that there are different refractive properties between dentine and cementum. it was possible to obtain pure sarnples of each of these tissues by dissecting away the cementum and assaiiated regions of periodontal ligament and pulp under a dissection microscope. One gram each of dentine, cementum and whole root slices (ie. root slices with associated regions of periodontal ligament and pulp removed) were obtained and stored at -20°C. EmacRon of Fetai Porcine Long Bones by Various Extraction Procedures For a cornparison between various extraction protocols, long bones were used as the test material. Separate tissue sarnples were then extracted according to one of the fdlowing protocols: 1) deminemlizing extraction procedures under non-dissociative conditions, 2) dissociative extraction procedures of Termine et al., ( 198 la) or 3) a rnodified extraction procedure. Demineralizing extraction procedures under nondissociative conditions involved the treatment of bone chips with PBS + PI. This was then followed by deminerakation of the tissue by four, 24 hr extractions with 0.5 M EDTA, M mM-TnslHCl, pH 7.4 (E- buffer) + PI, providing an E-extract. Similarl y, the dissociative extraction procedures of Termine et al.. ( 198 1a) also involved the treatment of bone chips first with PBS + PI.The tissue was then extracted with 4 M GuHCI, M mM-TridHC1, pH 7.4 (G-buffer) + PI, providing a G-extract. Followed by a PBS + Pl wash, as descnbed above, the tissue was then extracted with 4 M GuHClIO.5 M EDTA, 50 mM-TridHC1, pH 7.4 (GIE-buffer) + Pl; genera~nga G/E-ext=t. The rnodified extraction procedure involved an exhaustive wash of the tissue fragments in PBS + fi, More extraction in 4 M GuHCl + Pi to provide a G1-extract. This was followed by extractions with 0.5 M EDTA + PIto obtain an E-extract, and finally the demineralized tissue was extracteci with 4 M GuHCl + PIto generate a Gl-extract. Between each extraction step, an exhaustive PBS + PIwash was included. For dl the above procedures, an extractanthissue ratio of 200 ml: 1 g was used. All solutions used cuntained Pl and were buffered with a pH of 7.4. Al1 extractions were carried out at 4'C. Each extraction was performed for a penod of 34 hr, with constant gentie stimng. Four consecutive 24 hr extractions with each solution type was employed. A11 exûacts were pooled and filrered, concentrated by ultraiïltration (Y M 10 or UM 10 membranes; at 40 psi), exhaustively dialyzed (Spectrapor I acetylated dialysis tubing) against 50 mM ammonium bicarbonate and freeze-dried for subsequent anal ysis by SDS-PAGEas descnbed below.

Eitmctiun of Mnerdked Connecrive Tissues Employing a Modified Extraction Procedure. For the comparative analysis of sequentially extracted proteins from the rnineralized connective tissues - calvariae, long bone, dentine, cementum and whole root slices - one gram (wet wei ght) of each tissue was placed in 50 ml pol ypropylene screw top centrifuge tubes and, using 50 ml of extractant per gram of tissue, sequential extractions were carrieci out with constant shaking, employing the rnodified extraction procedure. The G1-. E-, and Gkxtracts were then concentrated, dia1 ysed, and freezedried for subsequent anal ysis by SDS-PAGE.

Analysis of Proteins by Various Eitraction Methodr To identify those proteins that cm be released from bne by a demineralizing extractant under non-disxriative conditions. fetal porcine long bones were treated with 0.5 M EDTA (E-buffer). For cornparison, some of the long bones were processed employing the dissociative extraction procedures of Termine ( 198 1a) ; the tissue fint king extracted with 4 M GuHCl (G-buffer) followed by extraction with 4 M GuHCl/O.S M EDTA (GtE- buffer). The proteins extracted by demineralization under non-dissociative and dissociative conditions were analyzed by SDS-PAGE,as shown in Figure 1. Demineralization in the absence of denaturant released essentially the same proteins in a similar proportion as extracted with 4 M GuHCIIO.5 M EDTA. Since the major difference was the virtuai absence of collagen and fibronectin in the Eextract it uas evident that the denanirant GuHCl was not required for the solubilization of most of the non- cokgenous proteins of bone. The orignal dissociative extraction procedure of Termine (1981a) was, therefore, modified so that matris proteins of the mineralized tissues could be separated according to their relative affinities for hydroxyapatite and the collagenous matrix. Comparative analysis of proteins sequentially extracted from fetd porcine calvariae using the modified extraction procedure, which involves treatment of bone first with 4 M GuHCl (G1-extract), then with 0.5 M EDTA (E-extract) followed by 4 M GuHCl (G2- extract) was achieved using SDS-PAGE(Figure 7). The Eextract comprised a limited nurnber of clearly-resolved proteins; the major proteins rnigating with a mas of 67-55 ma,40 kDa and 15.5 kDa (Figure 2, lane 3). When compared with a 4 M GuHCI/O.S M EMA(GE)-exûact (Figure 1, lane 4), the E-extract protein profile aas very similar with the most notable difference king a lower content of collagen a-chains, ( 110 Da) in the E-extract (Figure 2, lane 2). G2-extracts on the other hand revealed strong bands for collagen a 1 and a2 chains (110 Da). Some 60-55 Damaterial was aiso apparent in GZextracts as were some minor bands having a rnass of 55 Da, 40 ma, 33 Da. 28 kDa 26 kDa, 24 kDa and 16 kDa (Figure 2, lane 3). To determine the extraction efficiency of 0.5 M EDTA on bone sarnples. 100 g calvariae was successively extracted with fresh E-buffer every 74 hr at 4' C. It was found that 600 mg, 125 mg, 42 mg, 28 mg and 6 mg of material could be recovered from a total of [ive successive extractions. Most of the EDTA-duble materiai (m) could, therefore, be obtained after two successive 24 hr extractions. Exhaustive extractions on isolateci mineralized tissues were, nevertheless, dways performed to rninimize contamination of the subsequent extractions with 4 M GuHCl.

Comparative Amzysis Beîween Various Mineralized Connective Tissues: Based on the above findings i t was of interest to compare protein profiles of different mineralized conneclive tissues, sequentially treated wi th 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract) and again with 4 M GuHCl (GZ-extract). SDS-PAGEanaiysis with CBB R-250 stained gels of the various extracts are shown in Figures 3 A, B, C. The G1-extracts from long bone, calvariae and cementum were found to be very similar in that these extracts were primari1y made up of type 1, a 1 and a2 collagen chains (molecular weights of - 1 10 k), and low molecular weight proteins of 15 k, 17 k, 18 k and 19 k (Figure, 3 A). Although undetectable in the cementum extract, long bone and calvarial extracts aiso enhibited a prominent protein with a molecular weight of 43 k. A 35 kDa protein was also evident in the long bone extract. In cornpanson, only minor arnounts of type 1 collagen a-chains were extracted from dentine and other proteins were bareiy detectable. Analysis of proteins wi th either Stains-al1 or silver nitrate (not shown). did not provide any additional information on the G 1-extracted proteins. Anal ysis of proteins present in the E-extracts from the wious mineralized tissues revealed qualitatively similar profiles when SDS-PAGEgels were stained with CBB R-950 (Figure 3 B). Long bone and calvarial extracts were vîrtually identicai; each exûact contained abundant amounts oi67 kDa proteins and proteins with molecular weights of 60 k, 40 k and 15.5 k. Notably, long bone preparations also contained a 36 kDa protein. Roteins from dentine and cementum Eextracts were also quditativeIy sirnilar; these dental tissues shared somewhat similar amounb of the 67 kDa and 60 kDa proteins, while demonstrating substantiall y laser amounts of the 67 kDa proteins relative to calvarial and long bone tissues. The most notable difference between bone and dental tissues was the lower arnounts of 67 kDa and 40 kDa protein in the E-ex~tand the presence of small amounts of 34 kDa and 78 kDa proteins in the dentine and the cementum. In addition, a 19 kDa protein and a large amount of a 15.5 kDa protein were entracteci from cementum. When the E-exfract proteins were stained wi th Stains-dl. long bone and cal varial extracts revealed a broad bluelturquoise staining band with a molecular weight of 80 k, blue staining bands with molecular weights of 75 k and 70 k, a broadlarge pink staining band with a molecular weight of 67 k, and blue staining bands with molecular weights of 60 k, 40 k, 28 k, 22/20 k and 15.5 k. A broad blue/turquoise staining band with the rnolecular weight of 80 k was prominent in the dentine and cementum together with blue staining proteins of 75 ma,70 kDa, 38 kDa and 15.5 kDa. Examination of the proteins present in the G-extract of long bone (Figure 3 C, lane 1) and calvariae (Figure 2, lane 3) showed an abundance of type 1 collagen a-chains (-1 10 kDa) and 67 kDa protein. Other proteins present in this extract migrated at 45 kDa, 38 ma,32 Da,78 kDa, 36 kDa, 24 kDa and 16 kDa Although not apparent at the loading concentrations used in Figure 3 C, cementum and dentine reveaied a simpler protein profi le that included collagen a-chahs, and proteins at 67 kDa, 32 kDa and 34 kDa Notably, cementum exhibited substantially lower amounts of the 31 kDa protein. The 37 kDa and the 24 Daproteins, comprised rnost of the proteins in the dentine ex tract, stained well wi th silver nitrate and stained pink with Stains-ail. These proteins mipted at 26 kDa and 72 kDa, respectively, under non-reducing conditions. indicating the presence of intrachain disul phide bonds. Since whole root slices are disproportionate to the relative amounts of dentine to cementum present (dentine > cementum), the proteins released from whole mot slices were more similar to the dentine protein profiles.

DISCUSSION

This study revealed that extraction with 0.5 M EDTA under non-dissociative conditions, (i.e. demineralization in the absence of denaturant) is sufficient to release the previously descri bed major non-dlagenous proteins quanti tativel y irom bone (Figure 1 lanes 2 and 3). This finding subsequently led to the development of a novel extraction method whereby bone proteins muld potentially be separated into a mineral-associated fraction and a collagen matrix-associated fraction (Figure 3). The demonsiration of distinct protein profiles for each extract suggests proteins are compartmentalized within comective tissues (Figure 3). However, i t cannot be concluded that ail proteins found in the E-extract are direct1y associated with the minerai phase, since demineralization could have released diffusion- blocked sites of some proteins which are readily soluble in EDTA but that lack an affinity for the remaining insoluble collagenous rnahin. In addition, it is psible that divalent ion- mediated collagen binding proteins would also be present in the E-extmct. Conversel y, in the G2extract some proteins may acnially represent authentic mineral-associated proteins which are not cumpletely sduble in EDTA-buffer alone and may therefore repent protein 'spi11 over' hmthe E-e,xtract (i.e. the partial insolubility of some of the Eextract derived proteins in EDTA-buffer alone may be due to the removal of divalent ions which may be associated with these proteins but which can then be subsequently solubiiized by a dissociative buffer). Nevertheless. as judged by SDS-PAGEanalysis it appears that the extraction pnnedure has the ability to efficiently entract non-covalendy associated proteins from different tissue compartments in different rnineralized tissues. The efficiency of the procedure is evident from the discrete protein patterns obtained for each extraction step with minimal apparent "spi11 over". The G 1-extract is anticipated to extract proteins from the mineralized osteoid (bone). cementoid (cementum) and predentine (dentine) as well as from any contaminating sort tissue. The comparative analysis between the different rnineralized tissues when assayed for proteins released by the sequential treatment with GuHCl, EDTA and again with GuHCl reveaied a number of interesting findings (Figure 3 A, B. C). The G 1-extracts of long bne, calvariae and cernenturn revealed predominantl y collagens ( - 1 LO kDa) and a group of low molecular weight proteins with molecular weights of 15 k. 17 k. 18 k and 19 k. Notably, small proteins with cell-attachment properties (Wong et al.. 1985) are present in bone and cementum, but are absent in dentine. Except for the presence of ahat is thought to be the 35 kDa protein c hondrocalcin (Poole etal., 1984; van der Rest et ai., 1986; Hineket al., 1987). the E-extracts from long bone and caivarial tissues displayed no remarkable differences and revealed a number of proteins. psibl y proteogl ycans ( 100 ma). sialoproteins (60-70 ma),os teonectin (40 Da)and ostdcin (16 Da) (Termine. 1980). The presence of chondrocalcin in the long bone E-extract would not be surprising since chondrocalcin is a cartilage-derived protein and long bone is a tissue which forms by endochondral calcification. Although the SDS-PAGEresults of long bone and caivarial E-extracts exhibi ted large amounts of osteonectin, this protein was not prominent in cementum and dentine E-extracü. The identity of the prominent 16 kDa protein in the cementum E-extract is not known. Roteins wi th the propenies of phosphophoryn (70 Da)and osteocalcin ( 16 Da), were also detected in the dentine Eextract, as judged by their behaviour on SDS-PAGEand their gel- staining properties. The 70 kDa protein was found not to stain wi th CBB R-250 or silver nitrate but did stain with Stains-dl. The 16 kDa protein stained well with CBB R-250 and silver nitrate and stained blue with Stains-dl. In addition, the E-extracts from ail the rnineralized connective tissues showed simiiar amounts of a molecule with a molecular weight of 60 k; a protein which could be similar to a 60 Dasialoprotein described by Sato et al., (198Sb). The protein profiles from the G2-extracts also showed that long bone and calvariae were qualitatively similar to each other. as were cementurn and dentine. G2-estracàon revealed the presence of a number of discrete proteins in the demineralized bone mauix (Figure 2, lane 3), some of which were thought to be novel. Note that this extract is e~chedwith collagen (- 110 kDa) and, potentially, collagen-associated proteins. The common proteins detected among the G?-extracts of mineralized connective tissues were type 1 collagen and proteins with molecular weights of 32 k and 34 k (for long bone and dentine: Figure 3 C, arrows; for calvariae Figwe 2, lane 3; for cementum detection of these proteins was much less apparent because the low arnounts loaded). The simplici ty of the protein profile and the relative abundance of the 32 kDa and 14 kDa proteins making up the GZextract of dentine made this an interesting finding, especially since proteins with these molecular weights had not been described previously for this tissue. It is probable that in bone the G3-extract also includes matrix Gla protei n (Price et al., 1985). bone morphogenetic protein (Urist et al., 1984; Muthuliumaran et ai.. 1985) and cartilage inducing factors of which, CIF-A and CIF-B,appears to k identical to two forms of TGF-6 (Seyedin ei al., 1987). In addition, it might be anticipated that proteins that can simultaneously interact with collagen (because of the order of extraction) and CaWhydroxyapatite (blue staining with Stains-dl) would also be expected to be in the G2-extracts. Based on these prelirninary findings it appears that the modified extraction procedure separates proteins according to their relative affinities for either the mineral phase or the collagenous phase of mineralized connective tissues. Since most of the previously described proteins of bone can be released without the need of denaturants, it aas anticipated that these proteins may be mineral-associated proteins involved in stabilization/regulation/inhbitionof crystal groulth. rather than being nucleaton of hydroxyapati te mineral. However, since EDTA chelates Ca?+, it is conceivable that some proteins released in the demineralizing extract are associated with the organic matrix through calcium bndging. Therefore, it will be important to study, the binding propenies of the purified proteins to the organic and inorganic components of the bone matrix before conclusions can be made regarding the compartmentalization of individual proteins. Similady, it is also conceivable that in the presence of 4 M GuHCl some proteins may dissociate from the organic matrix and bind to the minerai phase. However, the similarity of the protein profiles in the demineralizing extract performed with and without prior extraction with 4 M GuHCl indicates that this is not a problem for the major proteins. Figure 1: Analysis of Proteins Releascd by Demineralization with 0.5 M EDTA from Feîal Porcine Long Bones.

SDS-PAGE (5% - 20% crossiinked gradient gel) was perfomed under redudconditions and proteins stained with CBB R-250. Tanelow molecdar weight markers; 1.proteins extracted with 4 hl GuHCiIO.5 M EûTA with prior 4 M GuHCi treatment; maneproteins extracted solely with 0.5 M EDTA with no prior 4 M GuHCl treatment. Figure 2: Annlysis of Proteios Released from Fetal Porcine Calvariae using a Modified Extraction Procdurc.

SDS-PAGE ( 12.5 % aassed-linked gel) was periormed under reduced conditions to separate proteins w hich were then stained witb CBB R-250.Tane, proteins extracted with 4 M GuHCl (Gl-extract). This extract qnsents approximatel y 5 % of total extractable proteins; W.proteins extracted subsequentiy with 0.5 M EDTA (E-extract). This extraci represents approsimatel y 90 % of total extractable proteins; Land, proteins extracted with 4 M GuHCl a second time (G2-extract); this extract represents approximately 5 % of toial extractable pmteins; Tane,proteins extracted with 4 M GuHCi!O.S M EDTA (GIE-extract) with prior 4 M WC1 treatment using the dissociative extraction procedure of Tennine et al ., ( 198 1a). Cl-Extract WlZ345

Figure 3 (.A. B, C): Comparative Analysis of froteins Sequeatiaily Extrmcted From Various Mineralized Tissues.

SDS-PAGE (12.5 55 CBB R-250stained gels) analysis of proteins sequentially estracteci from fetal porcine bones, and from dentine and cemen- of duit porcine teeth nui under reduced conditions. showing the proteinprofilespresent ineachof theentracts A, G1-Exuacts;B, E-Extracts;C, G2-Extracts XI,: low molecular weight markers in kDa's; rlong boue extract ;I d2ad extract; M incisor dentine extracts; inasor cemennim enuact; whole mtslico extract. Note: loading for calvaid G2-extract represeiits 1 20 compared to other extracts. 1130th the volume of totd extract was ldedfor beand l! 1ûth the volume of tolal extract was Ided for dentine, cemennun and whole mot slices. Note, anows depict the position of the 32 kDa and 24 kDa pmteins. CHAPTER III A

ISOLATION,PURIFICATION AND CHARACTERIZATION OF OSTEONECT'IN EXTRACTED FROM FETAL PORCINE CALVARIAIE: SOME COMPARISONS WITH PURiFIED PORCINE DENTINE OSTEONECTIN.

INTRODUCTION The isolation of bone proteins has ken facilitated by the introduction of dissociative solvents to extract the proteins and the inclusion of proteolytic-enzyme inhibiton to prevent degradation during their extraction and subsequent purification (Linde et al., 1980; Temine et al., 1980). Several of the major bone proteins have been prepared in this way, inciuding asmall proteoglycan (Fisher et al., 1983a),sialoprotein (Fisher et al., 1983b). osteocalcin and osteonectin (Termine et al., 1981a; Termine et al., 198 1b). More refined purification procedures have subsequen tl y identi fied several fonns of the pro teogl ycans (Franzen and Heinegard, 1984a; Franzen and Heinegard, 1984b;Sato et al., 1985a) and sialoproteins (Franzen and Heinegard, 198%; Sato et al., 1%Sb). Osteonectin, which was originally characterized as a phosphorylated glycoprotein that binds Cd+,represents approximately 25% of the non-collagenous proteins of fetal bovine bone (Termine et al., 1981a). Particular interest in this protein stems from early investigations indicating that osteonectin, as implied by its name, binds to the inorganic (hydroxyapatite) and organic (collagen) phases of bone and may also initiate the precipitation of Ca?+/P043-on collagen fibrils (Tennine et al., 198 1b). Osteonectin purified from aduit bovine bone under non-dissociative conditions also bund to a partially purified type 1 collagen preparation (Romberg et al., 1985). However, preliminary studies in Our laboratoly have questioned the proposed role of osteonectin in hydroxyapatite nucleation (Wasi et al., 1984) and native osteonectin from bovine bne aas shown to strongly inhi bi t hydroxyapati te crystal formation (Romberg et al., 1985). Also, proteins sirnilar to osteonectin had ben characterized as a "culture shock" protein produced by bovine endothelial cells (Sage et al., 1W), and as a s ecreted protein w hich is acidic and rich in cysteine (SPARC)in differentiating mouse parietal endoderm cells (Mason et al., 1986a). To further characterize osteonectin and to determine whether or not the properties of this protein are consistent wi th a nucleator of h ydroxyapati te, the modified extraction procedure (Chapter II) was used to isolate osteonectin from fetal porcine bone and dentine. MATERIALS AMI METHODS

Preparation of Calvan'ae, Long Bone and Dervine A total of 100 g of calvariae and long bones were collected and then frozen as previously described (Chapter II, Materials and Methods). For the quantitative isolation of proteins from root dentine, adult pig jaws were obtained from a local abanoir and within hours of slaughter the lower incison were extracted and placed in ice-cold phosphate buffered saline (PBS) containing PIas described in Chapter II. Tissue adhering to the root dentine including cementurn was removed using a conventional high speed drill with a straight fissure, friction grip 357 carbide bur, maintaining a constant gentle spray of cold water on the tissue at al1 times. With the use of stainless steel plien. the crown and pulp, including the pulp within the root mals, were separated as a unit from the rmt dentine by gentl y appl ying controlled compressive and tensi le forces at the cemento-enamel junction thereby separating the crown portion of the tmth from the roots. To ensure the removal of any residual pulpal tissue within the root canals, the roots were split open with stainless steel pliers to expose any soft tissue which was then wiped away with tissue paper. The rmt dentine was then frozen in liquid N? and crushed with a mortar and pestle. A total of 90 g of root dentine was collected and then frozen.

Extraction of Mineralized Connective Tissues Emp loy ing a Modtj7ed Extracrion Procedure For the quantitative extraction of bones, frozen calvarial and long bone chips ( 100 g each) were thawed, washed with ice cold PBS + PI and al1 subsequent extraction sleps camied out at 4'C with constant stimng. Extraction with PBS +PI (three times for a total of 24 hr) was performed to remove any soluble components including ce11 debris and bld proteins which are weakly associated with bone. The tissue aas then extracted four times with 4 M GuHCl (Sigma Chernical Co., St. Louis. Mo.), 50 mM Tns/HCl, pH 7.4 (G-buffer) containing PI(50 mllg tissue) for a total of % hr to rernove non-covalently associated components in the remaining soft tissue, including osteoid (GI-extract). The residue was then washed three times in PBS + q for 24 hr before extraction with 0.5 M EDTA (Sigma), in 50 mMTrislHC1, pH 7.4 (E-buffer) with PI(four times for a total of % hr at 50 ml/g tissue) to release mineral-associated proteins (Eextract). The bone residue was then washed three times wi th PBS + PIand was then extracted again with G-buffer (four times over % hr) to release proteins which remained associated with the exposed collagenous matrix (GZ-extract). The G 1, E and G3 extracts were then concentrated by ultrafiltration using a YM-10membrane (Amicon Corp. Lexington, Ca., U.S.A.). The G1-, E-and GZconcentrates were exhaustively dialyred against 10 mM ammonium bicarbonate at 4'C with constant stimng using Spectrapor 1 acetylated dialysis tubing. The dialyzed extracts were freezedried in preparation for gel filtration. With some modifications in the procedure, extraction solutions employed for the large scale extraction of dentine were identical to those described previously for fetal porcine caivariae . Al1 procedures were perfomed at 4'C, using an extraction buffer volume (ml) to wet tissue weigh t (g) ratio of 200: 1. The root dentine chips collected from the large sale preparation were fint washed in PBS + PIto remove any soluble components associated with the dentine and then extracted under constant stirring in a solution containing. 4 M GuHCl Tris-HCI buffer + PI,pH 7.4 (G-buffer). to remove non-covalently asçociated components in the remaining soft tissue (G 1-extract). The residue was then washed with PBS + PIand extracted under constant stimng with 0.5 M EDTA Tris-HCl buffer + Pi, pH 7.4 (E-buffer),to release mineral associated proteins (E-estract). The G 1 extract and E-extract were then concentrated, dialyzed and freezedried using the same procedures diat were used for the calvarial G-extract and E-extract above. The residue was again washed with PBS + Pl and then evenly disüibuted into rive 50 ml polypropylene screw-top centrifuge tubes and the contents of each tube extracted with constant shaking with 40 ml G-buffer for one week to release proteins which remained loosely associated with the demineralized collagenous matnx (G?-extract); this procedure was repeated four consecutive times. The extracts were then concentrated and further processed for FPLC chromatography.

Gel Filtration with Sepharose CL-6B & Sephacry i-200 Resirts Chromatography on Sepharose CL-6B was performed either in 4 M GuHCl as described by Termine et al.. ( 1980) or in 7 M urea. For each fractionation, 0.17 g of freezedried material from the E-extract was dissdved in either G-buffer or 7 M urea in 50 mM TrisMCI, pH 8.0 and chromatographed ui th the sarne buffer at 20aCusing two columns (each 2.6 cm x 95 cm) in tandem, packed with Sepharose CL-6B. A flow rate of 20 mllhr was used, with 6 ml fractions collected. For the 4 M GuHC1-fractionated sample, the cdurnn fractions containing osteonectin were pooled. concentrated by ultrafiiltration on Y M- 10 Amicon filters and were further fractionaieci under the same mndi tions on tandem Sephacryl-200 columns (each 1.6 cm x 95 cm). A flow rate of 12 mllhr uas used, with 2.0 mllfraction king collected. Fractions enriched in osteonectin were then pooied. didyzed, freeze-dried and were subjected to anion-exchange chromatography. For proteins separated in 7 M urea, fractions containing osteonectin were directly applied ont0 a FPLC "plyanion" anion exchange resin as descri bed below . Al1 column chromatopphy runs were monitored at Aao - and by analqzing aliquots of the mllected fractions on SDS-PAGE.Preparations chromatographed in 4 M GuHCl were used for primary sequence analysis and for experiments involving the binding of osteonectin to collagen- and gelatin-Sepharose resins. In dl other experiments, osteonectin prepared in 7 M urea was used. For the E-extract obtained from adult porcine mot dentine, a total of 0.04 g of freeze- dned material was dissolved in G-buffer and was subjected to Sepharose CL-6B chromatography under identical conditions as described above for the calvarial derived E- extract. Column chromatography runs were monitored at AzO nm and by analyzing aliquots of the collected fractions by SDS-PAGE.Fractions that were ennched in dentine osteonectin protein were identified by ELISA using rabbit anti-osteonectin antibodies to porcine calvarial osteonectin (see below). Briefly, 3 aliquots of fractions from CL-6B runs were either spotted onto nitrocellulose membranes or directly coated onto ELISA plates and assayed using standard protocols (Rennard et al., 1980). Positive fractions were pooled, concentrated, dialyzed and freeze-dried for subsequent anion eschange chrornatography using FPLC.

Ion Exchge Chromatography Ion-exchange chromatography was periormed at 20°C in 7 M urea dissolved in 50 mM Tris/HCI, pH 8.0 buffer using a linear O to 1.O M NaCl gradient on an automated FPLC system (Pharmacia, Uppsala, Sweden) using either a preparatory grade "polyanion SI" column (1 x 10 cm), or an anaiytical grade "polyanion SI" column (0.5 x 5.0 cm). Fractions enriched in osteonectin from the preparatory mns were pooled, concentrated, freed from NaCl on Sephades G-55,in start buffer, and chromatographed on the analyrical column. Column runs were monitored by absorption at AZ0 nm and the fractions anallzed by SDS-PAGE.Protein fractions conraining osteonectin were pled,diaiyzed against 10 mM ammonium bicarbonate and freeze-dried. The "HR515 Mono Qu(Pharmacia) anion exchange column could aiso be used instead of the polyion cohmn for the purification steps, with best results king obtained using a ?O mM piperazine pH 6.0 buffer. linear 0- 1.O M NaCl gradient, and a flow rate of 1.5 mllmin. Dentine osteonectin protein was purified to homogeneity by applying freeze-dried enriched fractions from the CL-6Bchromatography ont0 a preparatory grade "polyanion SIncolumn run on an FPLC system under identical conditions as described above. Osteonectin enriched fractions were pooled, exchanged into start buffer by ul trafil tration and run on an FPLC anal pcal "polyanion SI" column. Rirified dentine osteonectin was then dialyzed, freeze-dned and prepared for furrher analysis. SDS-PAGEwas performed as descrikd in Chapter II. Sodium Dodecyi Sulf4re-Polyucrylamide Gel Electrophoresis Electrophoresis in the presence of SDS was canied out with 13.5 8 and ?O % linear, or 5-20 % gradient cross-linked polyacrylamide gels, using the discontinuous Trislglycine buffer system of Laemmli (1970) on a Bio-Rad "Rotean Apparatus". Samples were dissolved in 40 of sample buffer containing 1 8 wlv SDS, 2 M urea, and 0.0032 5% wlv bromophenol blue dye. For reduction, 10 of a 75 rnglml solution of dithiothreitol was added pnor to heating the sarnples at 56'C for 30 min. Gels were stained with 0.25 Q wlv Coomassie brilliant blue R-250 (CBB R-250, Sigma Chemical Co., St. Louis. Mo.) using the procedure of Fairbanks et al. (1971), or 0.01 9% wlv Stains-al1 (Eastman Organic Chemicals) using the pro ce du^ of Campbell et ai., (1983). CNBr fragments were analyzed by SDS-PAGEby staining first with silver nitrate (Meml et al., 1983). completely de-stained with Farrner's Reducer (Kodak,Canada Inc. CAT 169 ME),and then re-staining with 0.010 8 wlv Stains-al1 using solutions at 200 mllgel. To completely remove the silver prior to Stains-al1 staining, the gel was treated with three, Emin washes of Farmer's Reducer. This was then followed by three, 30-min washes in hypo-clearing agent (Kodak)to remove any residual Farmer's reagent in the gel. The gel was then washed three times for 40 min with 25% vfv isopropyl alcohol and re-stained with 0.01 8 wlv Stains-ail. Low molecular weight protein standards (LMW: phosphoqlase b, 94 kDa; albumin, 67 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; trypsin inhibi tor, 3 1 kDa; and lactaibumin, 14.4 ma; purchased from Pharmacia) rvere used to calculate relative masses.

Amina Acid Anulysis Purified porcine calvarial and dentine osteonectin (- 5 pg) were individudly h ydrol yed for 24 hr at 110°Caith 1.0 ml of 5.7 N HCl containing 0.4 Q vlv phenol in pyrex hydrolysis tubes which were flushed with nitrogen, and sealed pnor to hydrolysis. Solutions were dried in vacuo in the presence of NaOH pellets, the residue redissolved in 0.39- N sodium citrate, pH 2.2, and anaiyzed on a Beckman ElMautomated amino acid anal yzer. To quantitate recovenes, 5 nMoles of nodeucine was added to the protei n sam ple prior to hydrolysis. Cysteine was measured as cysteic acid in hydrolyses carried out in the presence of 2.5 % vlv dimethylsulfoxide. Tryptophan was determined by hydrolysis of 40 pg of osteonectin with rner~a~toethanesulfonic acid (Pierce Chemical Company) accord ng to the procedure of Penke et al., ( 1974). Aminu Acid Sequence Analysis The amino-ierminal sequence of osteonectin from porcine and bovine bone were determined by appl ying 200 pg of pure osteonectin to a gas-phase 470 "A" Rotein Sequencer (Applied Biosystems Inc.). Porcine osteonectin for these analyses was purified in the presence of 4 M GuHCl and exposed to 7 M urea oniy dunng ion-exchang chromatopphy. Amino acid residues were identified and quantitated using high pressure liquid chrornatography (HPLC)to separate the phenylthiohydantoin amino acid derivatives. Bovine osteonectin used in this study was generously supplied by Dr. John Termine (National Insti tute of Dental Research, NIH, Bethesda, MD.).

Detemination of Extinction Coefficient

The extinction coefficient of porcine osteonectin at 380 nm (Eo 1 Icm) was determined from u. v. absorption measurements on a Cary 719 spectrophotometer and from two independent methods of determining the protein concentration. One method employed chfierenliai refractometry on a Hilger and Watts Static Raleigh interference instrument, with data treatrnent as desaibed by Babul and Stellwagen (1969). Altemativelu, protein concentration was determined by amino acid analysis using norleucine as an intemal standard for protein recovery. The absorbance value at 280 nm was corrected for contri bution of Rayleigh scatter by subtracting 1.7 times the absorbance ïaiue obtaïned at 320 nm. In addition, usine the molar extinction values ai 280 nm for tryptophan and tyrosine of 5,600 and 1,250 respectively. an extinction coeifïcient for osteonectin was calculated from the amounts of these amino acids determined from the arnino acid analysis. An estimated M, value of 29 k as detemined by ultracentrifugation for bovine osteonectin (Rornberg et ai., 1985) was used in this calculation.

Secondary Structural Amlysis Far and near U.V.- circular dichroism spectra were obtained using a Jasco Mode1 J-41A spectropolarimeter at 20'C for a 0.148 mg/rnl solution of porcine osteonectin containing 10 mM phosphate. pH 7.4, or a 0.094 mglrnl solution containing 5 mM TnslHCl at pH 7.4 in the presence and absence of 2 mM Gaz+ ions. The samples were scanned between 190 and 260 nm (far U.V.) using a 0.10 cm cuvette and the signai a\-eraged4 times. Similarly. a U.V.- circular dichroism spectrum of a 0.148 mglml solution of protein in 7 M urea, 5 mM TrisMCI, pH 8.0, with and without Ca2+ (2 mM CS+)nas also determined. The contribution of apparent a-helix and 6-sheet to the secondary structure of osteonectin nas calculated from the far U.V.- circular dichroism spectra using the curve fitting procedure of Rovencher & Glockner ( 198 1). With a 0.148 mghl solution of protein, a near U.V.- circula dichroism spectrum was obtained by scanning the sample between 2% and 320 nm in a 1.0 cm cuvette and the signal averaged 16 times. For al1 samples, the mean residue elipticity [eli was calculated using a mean residue weight of 1 10. which was determined from the amino acid composition. The concentration of protein was calculated by using as Eo- 1 icm the extinction coefficient value of 1-25at 180 nm, as determined by refractometry .

Hydroxyapatire Binding Mies Micro test ~bes(Bio-Rad) containing 0.5 mg of synthetic hydroxyapati te (Bio Gel HTP, Bio-Rad) were pre-washed with three. 1 ml aliquots of G-buffer and then incubated, with pntle agitation, on a rotaton shaker at 10'C for 1 hr wirh 1.O ml of a 5 rg/ml solution of osteonectin in G-buffer or a sample of total E-eutract proteins containing the quivalent of 10 pg of osteonectin. The sarnples were centrifuged at 10.000 xg on a microfuge (Brinkrnann) for 30 sec and were then washed 3 times with G-buffer to remove non-bound proteins. The hydroxyapatite was then resuspended in 200 of E-buffer and gently agitated on a rotatory shaker for a further 20 min at 70°C dter uhich the hydroxyapatite was pelleted by centrifugation on a microfuge at 10,000 sg for 30 sec; ths was repeated until the hydroxyapatite was completelÿ dissolved. The €-extracts and G-supematants were then dialyzed against 10 mM ammonium bicarbonate, freeze-dried and analyzed by SDS-PAGEon 17.5 Ck gels.

Cohgen Binding Type I collagen- and gelatin-Sepharose affinity resins were prepared as follo~~s:5 g of CNBr-activated Sepharose 4B resin (Pharmacia) was prepared according to the manufactures instructions. A solution of porcine gingival type 1 collagen (50 ml of a I mgml solution in 0.5 M acetic acid) which was dialyzed at 4'C against a 0.5 M NaCI. O. L M sodium carbonate buffer (pH 8.5) was immediatel y added to M ml of si milady buffered resin. For the gelatin affinity resin preparation. the collagen was heated at 56'C for 30 min and cooled to 20aCpnor to mixing with the activated Sepharose beads. The suspension was shaken gendy for 1 days at 4'C on a rotary shaker. The resin was isolated by centrîfugation at 100xg for 30 sec, resuspended, and shaken for 2 hr in 100 ml of 1 M ethanolamine, 0.5 M NaCI, in 0.1 M carbonate buffer, pH 8.5. This was followed by washing with PBS, 0.01% wlv sodium azide buffer in which the resin was stored at 4'C. Prior to thexr use in the batch adsorption experiments, the resin beads were repeatedly washed in PBS. Osteonectin (40 of a 125 &ml solution of PBS) and human fibronectin (5 pi of a 323 pg/ml solution of PBS ;used as a psitive control) were incubated with 500 of either collagenlgelatin CL4B affinity resins or Sepharose CL-4B (used as a control for nonspecific adsorption) in the presence or absence of 5 mM CaCL. for 1 hr at 20°C and shaken gently on a rotatory shaker. The samples were then centnfuged on a microfuge at 10,000 xg for 30 sec and the supernatants dialyzed, freeze dried and processeci for SDS-PAGEwhile the pellet was washed with five, 1 ml aliquots of PBS to remove any non-bound materiai. Electrophoresis sample buffer ( 100 pf) was then added to the resin, the mixture vortexed, and incubated at 56°C for 30 min. The resin was then allowed to cool to ?OeC,centrifuged at 10,000 ug for 30 sec and the supernatants anal yzed by SDS-PAGEfor any alfinie-bound material.

Cyamgen Bromide (CNBr} Cleavage Ekified osteonectin (10 rg) was dissolved in M pl of 70 % vlv formic acid (fiushed wi th N2) to which was added 25 or a 400 mglml solution of CNBr in 70 Ck formic acid which aas briefly flushed with N-. The reaction was c-edout at 30°C for 4 hr and samples were agi tated occasional1y during the incubation. The contents were di1 uted 5-fold with water and freeze-dned. The residue was diluted in 5 ml H,O and freeze-dned once again before analysis by SDS-PAGEon 15 9C or ?O 8 gels. In addition, CNBr clea\.age of osteonectin that was reduced and alkylated according to Stevens et al., ( 1976) was also performed. The only modification of this rnethod involved the micredialysis (Overall, 1987) of the reduced and aliqlated samples (100 pi) awnst 10 mM ammonium bicarbonate, instead of using a desalting column.

Prepcuution and Charucterim'ion of Rabbir Anîi-porcine Osteonectin Antibodies Polyclonal antibodies to letal porcine osteonectin were obtained by injecûng female New Zealand White Rabbits subcutanecusly in the Iower dorsal region of the back with 80 rg/!200 of osteonectin in complete Freund's adjuvant once every two weeks for a total of four weeks followed by one 80 @00 injection of osteonectin in incompIete Freund's adjuvant. Injections of 100 pg/ml osteonectin in PBS and subsequent blood collections followed every 10 days. These antibcxiies were then used in the biosynthetic, immunocytochernical and immunohistochemical studies. Although the antiserum appeared to react on1 y wi th a single protein wi th the characteristifs of osteonectin on immunoblots, and in immunoprecipitations, and specifically recognized osteonectin in radiolabelled cultures of conneetive tissue cells, including rat cells, when used initially for immunocytochemical staining, nonspecific reaction was observed in ce11 nuclei. Consequently, the antisemm in 200 pi portions was affinity-purified usi ng pure porcine osteonectin ( 100 rg) immobilized to CNBr-acti vated Sepharose 4B ( 100 d. Pharmacia) as descri bed by Tung et al. ( 1985). This antiserum showed strong cross-reactivity to both human and rat osteonectin (Otsuka et ai.. 1988). The affinity-puri fied antibodies prepared were used at dilutions of between 1:4û and 1:200 with respect to the original antiserum and were judged to be specific by using immunoprecipitations and immunobiots of tissue extracts and from the lack of nuclear staining in immunohistochemical studies. For the aibumin and collagen binding studies belon, mouse anti-porcine osteonectin polyclonal antibodies were used. Mouse anti-porcine osteonectin pdyc:clonal antibaiies were prepared by injecting BalbIC (Charles River) mice every 2 weeks, first aith 25 pg of osteonectin in Freund's complete adjuvant, followed by 25 pg of osteonectin in Freund's incomplete adjuvant and then boosted with 30-50 pg of osteonectin in PBS.

AIbumh and Colkgen Binding A modification of the Enzyme-Linked Immunosorbent Assay (ELISA) system of Rennard et al. ( 1980) was used to test for osteonectin binding to various proteins. Bovine semm alburnin (Pentex grade, Miles Laboratories, Inc.) and type 1 collagen (porcine and rat) at concentrations of 1.ûû, 0.35,O.1 0 and 0.025 pg well ; were dissolved in 10 mM carbonate buffer, pH 9.5, containing 0.E Q wlv sodium azide (Voiler's Buffer) and were alloaed to adsorb to the microtiter wefls (Linbrflitertek PVC Plates) for 48 hr at 4'C. PIates were then washed three times at 3 min intends wi th PBS-Tween 20 (0.05 % Tween ?O in PBS) and 50 ng of osteonectin in 150 of PBS-Tween 20 aas allowed to incubate for 1 hr with the proteins adsorbed onto the microtiter wells. The plates were washed as above and the procedure described by Kuwata et al. ( 1987) was folloned. using 150 of mouse anti-osteonectin antiserum ( 1:Wû diluted in PBS-Tween 20 ) as first antibody and 150 pi rabbit anti-mouse horseradish peroxidase-linked IgG as second antibody (Cappel; 1: 1000 diluted in PBS-Tween 20) for 1 hr ai 20°C. As a positive control, binding of fibronectin (1.6 pg in 150 PBS-Tween)) to the collagedgelatin coated wells was also tes ted.

Cell Cultures and Radiolabellittg Erperiments Bone cells from fetal porcine calvariae were prepared using methods previousl y described (Brunette et al., 1976; Rao et al., 1977). Briefly. cells obtained from collagen digests were grown to confluence and were then useci. either for pulse-chas expenrnents with %-methionine (Otsuka et al., 1984) or continuous 32POS- labelline as desci bed by Rince et al. ( 1981). The medium from these experiments was prepared for speci fic immunopreci pi tation as descri bed previousl y (Wasi et al ., 1984). The speci fic irnmunoprecipitates were then anal yzed by SDS-PAGE and fl uorograph y or were electrophoretically transferred to nitrocellulose for 45Ca2+ binding anal ysis or immunoblotting anal ysis.

Electrophoretic Tronsfer, lmmbiotting and 45Ca2+ Binding For the electrophoretic transfer of proteins onto nitrocellulose membranes (0.45 m) proteins were first run on linear 15% gels (0.75 mm thick, 5 cm long) using a Minislab gel system (Hoefer Scientific) and then transferred ont0 the nitrocellulose sheets usi ng an LKB 7 117-750 Nova Blot electrophoretic transfer system (Pharmacia). Irnmunoblotti ng (Western blotting) was perfomed using a modification of the original methcd by Towbin et al. (1979) as described by Kuwata et al. (1987) using rabbit anti-osteonectin antisera (1150 dilution) as first antibody and peroxidase-conjugated sheep anti-rabbit IgG (Cappel; 111000 dilution) as second antibody. The ability of reduced and non-reduced forms of purified osteonectin and reduced CNBr cleavage fragments of osteonectin to sequester Ca*+ions was determined by incubating nitrocellulose membranes containing the eiectrophoreticall y transferred protei ns wi th 'Kaz+ ( 1 mCilL) as descri bed by Mani yama et ai., ( 1984). The sequestered Wa2+ was visualized by autoradiography.

Proteins in the E-extract were separated by gel filtration on Sepharose CL-6B in the presence of 4 M GuHCI. A composite elution profile characteristic of the chromatographic runs is shown in Figure 1 A for fetal porcine calvariae and adult porcine dentine E-extracts. The fractions containing osteonectin were identified from the mobility of the protein on SDS-PAGE (an apparent mass of 40.3 Daunder reduced conditions and 37.6 kDa in the absence of reductant), and were pooled as shown in Figure 1 A, bar 3. Separatel y pooled material from the CL-6B runs was then applied ont0 a Pharmacia "preparatoq plyanion" column run under dissociative conditions for either calvarial or dentine proteins (Figure 1 B). Fractions highly enriched in calvarial and dentine osteonectin (shown by bar in Figure 1 B) were separatel y re-chromatographed, using a Pharmacia "analpcal pol yanion" column. As illustrateci in the composite chromatogram in Figure 1 C, a symmetrical peak was obtained for the major components which eluted between 0.150.18 M NaCl for the dentine protein and 0.17-0.20M NaCl for the calvarial protein. The purity of the protein in the peaks was confimed by SDS-PAGE using CBB-WM (Figure 2) and Stains-dl, both of which stained the single protein band blue; silver staining also showed a single protein band. The behaviour of the protein on SDS-PAGEunder reduced and non-reduced conditions (Figure 1).the amino acid composition (Table 1) and the Stains-al1 staining properties were used to identi fy the pun fied proteins as osteonectin. From 100 g of calvariae (wet weight) approximately 6 mg of osteonectin was obtained in pure form. From 90 g of dentine (wet weight) approximately 1.4 mg of osteonectin was obtained in pure form. From other pools made from the fetal porcine cal varial CL-6B sepration. two small proteoglycans (see Chapter III C), 3 or psibl y 3 sialoproteins (see Chapter III C), small wllagenous apati te-binding (SCAB) proteins (Kuwata et al.. lgû'i), a 1(1)pN- propeptide (see Chapter II 1 D) and osteocalcin have ken isolated. Fractions enriched in these proteins are illustrated in Figure 1 A. The amino acid composition of the porcine calvarial osteonectin are compared (Table 1) with the compositions of fetai (Termine et al., 1981a) and adult (Rombeq et al., 1985) bovine long bne osteonectin and those of bovine endothel ial "culture shock" protein (Sage et al., 1984) and murine SPARC protein (Mason et al., 1986a). In each case the compositions were similar, and charactenstically rich in acidic amino acids andlor their amides and ha1 f-cystines. The hal f-cystine value for feta! bovine osteonectin, however, was much lower than for the other proteins, wherûas proline, serine and aspanic acid were low in the munne SPARC protein composition. An extinction coefficient, E o-l%/280,Icm, of 0.83 was found for a sol ution of porcine osteonectin whose concentration was determined by amino acid analysis, ahereas an extinction value of 1.01 was calculated from the tryptophan and tyrosine content of the molecule. Both of these values are lower than the value of 1 25 obtained by refractometc, but are much higher than the values of 0.36 and 0.46, respectively as determined by refractometry and amino acid anaiysis reported for bovine osteonectin obtained from long bone (Romberg et al., 1985). The amino acid sequence of the fint 35 residues of porcine osteonectin and the first 38 residues of bovine osteonectin, was determined by automated sequencing with a repetiti ve yield of % 4c and 89 Ck obtained respective1y, [or each of the two proreins. As shown in Figure 3, the sequence of porcine osteonectin was dmost identical to that of bovine osteonectin; the only difference king the absence of a "Val-Ala-Gluntripeptide alter residue 20 in the porcine protein. Although there are notable differences with the short sequence reported for adult bovine osteonectin (Rornberg et al., 1985), both the fetal porcine and fetal bovine osteonectin sequences (residues 1- 19) are consistent wi th the bovine cDNA sequence (Young et al.. 1986). The porcine sequence, representing approximately one-tenth of the molecule, is enriched in small hydrophobic (approx. 50%) and acidic ( approx. 25%) amino acids and is notabl y deficient in aromatic amino acids. The amino-terminal sequences of the rnurine SPARC protein and BM4,and the corresponding DNA sequences of mouse SPARC (Mason et al., 1986a) and bovine osteonectin (Young et ai., 1986) are shown in Figure 3; the sequences king aiigned to maximize identity with the published DNA nucleotide sequences of bovine mteonectin and murine SPARC. An apparently blocked N-terminus for adult porcine dentine osteonectin precluded direct sequenœ analysis of this protein. Tu detenine the presence of secondary structure in the dissociativelyextracted osteoneciin, the circular dichroism spectra of the protein were studied in the presence and absence of Ca?+. The far U.V.- circular dichroism spectnim of osteonectin in the presence of 10 mM phosphate, pH 7.4 was similar to that in 5 mM TrisIHCI, pH 7.4 shown in Figure 5. When analyzed by the circular dichroism cunie-fiiting procedure of Rovencher and Glockner (1981) the following best fit values were obtained: 27 8 a-helical content. 39 R p-sheet structure, and the remaining 34 8 consisting of both unordered structure and g-tums. Although these values represent the chosen solution of the alprithrn, visually acceptable fits of the data were aiso obtained in the following ranges: a-helix, 14-19 8; 8-sheet, 3545 9%; unordered structure and $-tums, 26-41 8. Quali tativel y, the far U.V.- circular dichroism spectm was changed slightly in the presence of 2 mM CS+(Figure 4), whereas no appreciable change could be detecred in the near U.V.- circular dichroism spectrurn (not shown). In addition, when osteonectin was re-exposed to 7 M urea, the far U.V.- circular dichroism spectrurn revealed that ail a-helical structure in the molecule was abolished (Figure 4) and was not restored upon the addition of Ca?+ions. In al1 cases the concentration of Car+ used in these experirnents (i.e. 2 mM CaCI2)was well above the binding constant for Ca2+ determined for bovine osteonectin (Romberg et al., 1985). Osteonecti n cleaved wi th CNBr generated a characteristic pattern of fi ve si1 ver staining fragments wi th masses of 33.1 kDa, $7.9 kDa, 27.8 Da, 17.6 kDa and 12.4 kDa (Figure 5, Iane 2) as determined by SDS-PAGErun under reduced conditions. When the gel was stained subsequently wi th Stains-dl, al1 bands except the 22.8 Da and 17.6 Da fragments, both of which were stained pi&, were found to be stained blue (figure 5, lane 4). A 10.3 kDa fragment was also observed in cleavage products transferred to nitrocellulose and stained with arnido black (Figure 6, lane 3). Analysis of CNBr fragments mn under non-reducing conditions on SDS-PAGEshowed that the majority of Stains-al1 staining bands, al1 of which were blue, occurred between 40 kDa and 29 kDa (Figure 5, lane 5). Notabl y, CNBr cleavage of reduced and alkylated osteoneciin generated the same fragments as above, in addition to a 7.3 kDa fragment, al1 of which were visualized with silver staining (not shown). Calcium binding snsdies, usi ng protei n electrophoreti cal 1y iraosferred onto nitrocellulose sheets, showed that both reduced and non-reduced forms of puri fied osteonectin were able to bind Wa2+(Figure 6, lanes 8 & 9, respectively). Of the CNBr cleavage fragments, the 12.4 kDa band was the only fragment to selectively sequester Ca2+ ions (Figure 6). When incubated in the presence of 4 M GuHCl. purified osteonectin bound quantitatively to hydroxyapatite crystds (Figure 7). However. under sirnilar conditions, the osteonectin present in the whole E-extract did not bind significantiy to the hydroxyapatite. Notably, not al1 proteins that bound to the hydroxyapatite in the presence of 4 M GuHCl stained blue with Stains-dl; a number of pink-staining proteins were also bund (Figure 7). To study the binding of osteonectin to collagen. the purified protein was incubated with both native and denatured (gelatin) collagen- Sepharose CL4B resins in the presence and absence of added Ca?+ions. From analyses on SDS-PAGE(Figure 8),on1 y a small amount of osteonectin was bound to the collagen and gelatin aflinity resins in the absence of Ca2+ (6 and 7% respective1y) and in the presence of Cal+ ( 14 and 1 1 % respective1y), as measured by densitometric scanning. However. in each case, the proportion bound was substantiall y less than was bound to the CL-4Bresin aione ( 13 Q in the absence and 33 % in the presence of Ca?+)which was used as a control. Consequently . there is no apparent affinity of osteonectin for collagen, or gelatin, both of which appear to prevent osteonectin binding to the CL-4Bsupport resin. Control experiments wi th fi bronectin, however, showed that 1.5 pg (290 5%) of fibronectin could bind to bath the collagen and gelatin affinity resins. In agreement with these observations are the resulü of the ELISA binding assays. which showed that osteonectin bound to native or denatured collagen could not be detected over control wells. Using this method, however, it could be shown that fibronectin binding to gelatin was four-fold greater than to native collagen, as was previously descnbed by ELISA assays (Karp et ai., 1986). In addition, M ng of osteonectin was quantiiativeiy bound by 1.0 pg of highly purified aibumin (Pentex) coated ont0 the ELISA plate wells. To determine whether osteonectin is phosphorylated, porcine and rat calvarial osteoblast-like cells were cultured in the presence of 32FQ3- . Al though a num ber of phosphorylated proteins could be resolved by SDS-PAGEno radiolabelled proteins in the pition of osteonectin could be detected in autoradiographs expsed for two weeks, nor was any phosphorylated protein immunoprecipitated with osteonectin antisenim (not shown). To confirrn that the osteonectin had been immunoprecipitated, duplicate samples were first electrophoretically transferred to nitrocellulose and then the transferred protein was detected by irnmunoreactivi ty wi th an ti bodies. As expected, the immunoprecipitated proteins were found to behave identically to the EDTA-extracted protein nin on SDS-PAGE under reduced and non-reduced conditions (resul ts not shown).

DISCUSSION

In this study, 1 have shown that osteonectln together with the other major non-collagenous proteins of mineralized bone matrix. incl uding proteogl ycans (Gold berg et al., 1988b),sidoproteins (Zhang et al., 1990) and osteocalcin could be extracteci quantitatively with 0.5 M EDTA under nondissociative conditions (Figure 5). These proteins, therefore, appear to be bound to the surface of hydroxyapatite crystals where they may influence crystal growth or dissolution. More than 95 8 of the osteonectin in bone couId be extracted with 0.5 M EDTA, whereas only small quantities (less than 5 9)were recovered in the subsequent 4 M GuHCl extract ((32-extract) from the bone fragments. More exhaustive extractions with EDTA were found to remove most of the osteonectin. Consequently, osteonectin would not appear to have a marked affinity for any components in the demineralized collagenous mauix of bone. Using the sarne procedure it u-as possible to quantitatively extract and purify osteonectin from porcine incisor dentine. clearly demonstrating that this protein is not specific to bone. Although there were some minor differenca in the amino acid compositions of the proteins (slight incrûases in senne. glycine and isdeucine; slight decreases in histidine and lysine in the dentine osteonectin) and chromatographie properties, these psibl y reflecting post translational modifications. The calvarial and dentine proteins however are likely to be the sarne. This conclusion is based on the behaviour of the dentine protein on SDS-PAGEunder reduced and non-reduced conditions, similar staining behaviour (CBB R-2%. Stains-al1 and silver nitrate) and its immunoreactiui ty to an ti-osteonec tin antibody. The lower recovery of osteonectin from dentine probabl y reflects a lower arnount present in this tissue as indicated previously (Chapter 11). Since bovine osteonectin was reported to be tissue specific, bind collagen and promote hydroxyapatite crystal formation (Termine et al.. 1981a; Termine et al.. 1981b), the properties of the porcine osteonectin were corn pared to the bovine protein. The puri fied proteins were found to behave similarly on SDS-PAGE,to have similar arnino acid compositions and amino terminal sequences. The chernical similarities between bovine and porcine osteonectin were aiso apparent in the CNBr cleavage patterns generated under non-reduced conditions. Although not show for fetal bovine long bone osteonectin. CNBr cleavage of porcine calvarial osteonectin (Figure 6) generated relative1y large mo1ecula.r weight fragments indicative of i ncomplete cleavage of the mol ecule under these conditions. In addition, there appeared to be preferen tial cleavage of the molecule into 27.9 kDa and 12.4 kDa fragments as shown by rhe relative staining intensity of fragments with Stains-dl. Since CNBr cleavage of methionine residues in proteins is usuaily highly efficient. the resistance of osteonectin cleavage to CNBr could reflect a compact tertiary structure stabilized by extensive dxsui phide bridging. Interestin@y, the major CNBr cleavage fragments (the 12.4 kDa and 27.9 kDa fragments) retain the original blue staining properties of uncleaved osteonectin wi th Stains-all. Stains-dl has ken reported to interact with different groups present in proteins produchg a distinct blue/turquoise colour (Campbel1 et al., 1983). For exarnple, it has been shown that Stains-dl will stain sialic-acid nch proteins (King and Momson, W6),phosphate-contaming proteins (Cutting, 1984) andor Ca2+ binding proteins (Campbell et al., 1983) blue, whereas other proteins stam pink The Mue-staining 12.4 kDa fragment selectively sequestered aXa2+ ions, indicating that i t contains the Cs+binding site in porcine osteonectin. Of note, an 'EF hand' Ca2+ binding site has been predicted from Chou-Fasrnan analysis of the cDNA-derived arnino acid sequence to be near the C-terminus of the SPARC protein (Mason et ai., 1986a); indicating that the 12.4 kDa CNBr fragment is likely to be part of the C-terminal ponion of the porcine osteonectin moIecu1e. In the case of the 77.9 kDa fragment, the blue staininp aith Stains-al1 li kely reflects the acidic character of this part of the osteonectin sequence, since the N-terminus is known to be acidic and osteonectin synthesized by porcine and rat osteoblast-like cells (see Chapter III B) is not phosphorylated, as was also observed for "culture shock" protein (Sage et ai., 1984). Analysis of the far U.V.- c.d. spectra of porcine osteonectin revealed appreciable amounts of a-helix (77%) and 8-sheet (39 %) structure. Since p-sheet structure has been shown to be a stereochemical requirement for the adsorption of a number of acidic proteins to hydroxyapatite (Addadi and Weiner, 1985) this may be involved in the binding of osteonectin to hydroxyapati te. Furthemore, the refoldi ng of osteonectin structure after anion exchange chromatography under dissociative conditions is also indicated by ths hi gh content of secondary structure, w hich is abolished upon re-exposure to 7 M urea (Figure 5). The remvery of secondary structure is li kel y to be facilitated by the extensive disulphide bridging which is apparent from the high haif-cystine content (Table 1) and the behaviour of CNBr (Figure 6. lane 5) and proteolytic (Kuwata et ai., 1985) fragments on SDS-PAGEin the presence and absence of dithiothreitol. In contrast to these findings, adult bovine osteonectin, isolated without dissociative agents, has been reported to Iack a-helical structure and to have oniy 30 % p-sheet structure (Romberg et al., 1985). Although the spectra were andyzed by different procedures, perhaps olmore sigmficance is the lower amount of negative elipticity generated by a solution of bovine osteonectin (ie. approx. 3.5 times less than porcine osteonectin at 202 nm). This difference is reflected in the extinction coefficients of these molecules; 1.25 for porcine osteonectin and 0.36 for bovine osteonectin, as rneasured by refractometry. Since the tryptophan content appears to be the same for these proteins, these differences may be due to the variation in tyrosine and phenylalanine (Table 1; colurnn L vs 3) and perhaps to differences in the local environment of the tryptophan. The lack of a-helical structure in bovine osteonectin (Romberg et al., 1985) is surprisinp, in view of its homology to the SPARC protein (Mason et al., 1986a) in which the Gaz+ binding domain appears to have an 'EF hand' structure, a structure with appreciable amounts of a-helis. In addition. Chou-Fasman analyses of amino acid sequences based on the cDNA of the SPARC protein predicts a helical stmcture in the arnino-terminal segment of this protein (Mason et al ., 198th) while 30 1 a-helix has been indicated in BM-40 protein (Mann et al., 1987). Although not readily apparent in the far u.v.c.d. spectrurn of adulr bovine osteonectin (Romberg et al., 1985). fetal porcine osteonectin showed a slight change in the secondary structure in the 7 17 nm cd. region upon exposure to saturating amounts of Ca?+. The lack of any spectral changes in the aromatic region (250-330 nm) of the near U.V.-cd.spectra (not shown) upon the addition of saturating amounts of Ca?+ suggests that a Ca?+binding site is not close to, nor does its binding affect, aromatic groups. Thus the Cal+-mediatedeffeci on the far U.V.-cd.spectrum is more libly to represent a small change in secondary structure than a perturbation of short wavelength aromatic c.d. bands. Notably, a more prominent spectral shift in the presence of Ca?+ has ken observed in B M-40 (Mann et al ., 1987). In contrat to bovine osteonectin (Termine et ai., 198 1a), porcine osteonectin was not selectivel y bound to hydroxyapati te (in the presence of 4 M GuHCI) nhen cornpared to other proteins in the E-extract nor did osteonectin show any particular affinity for collagen or gelatin by either the EUSA binding assay or by affinity to collagenigelatin-Sepharose resins. Since the lack of affinity for collagen has also been shown for cell-synthesized porcine osteonectin (Otsuka et al., 1%). the resul ts obtained wi th the tissue-entracted osteonectin do not appear to be a consequence of expure of the protein to denanirants, and are consistent with the quantitative extraction of osteonectin from bone with EDTA alone. Despite apparent differences between porcine and bovine osteonectins in binding properties, spectrai properties and some chemical properties, these proteins generally show closel y sirnilar chemical characteristics, many of which are shared by bovine endothelid ceIl "culture shock" pmtein (Sage et al., 19û4) mouse endodermal SPARC protein (Mason et al., 1986a; Mason et al., 1986b) and BM-40 protein (Mann et al., 1987). Some structural and immunological similaxities between "culture shock" and SPARC protein have also been documented (Mason et al., 1%). Al1 these proteins are acidic glycoproteins, rich in cysteine with similar biosynthetic forms. They al1 show characteristic migration on SDS-PAGEunder reducing and non-reducing conditions. Cornparison of nucleoiide sequences of SPARC and bovine osteunectin have revealed a high degree of identity (Holland et al ., 1987). However, the murine SPARC and porcinelbovine osteonecti ns display major differences in the highly acidic amino-terminal region whch may also be involved in apatite binding. It is conceivable, therefore, that the low arnounts of osteonectin in mineralized tissues of rodents (Chapter III B) may reflect a lower binding affinity to hydroxyapatite of the SPA RC-li ke osteonectin. Although osteonectin was thought to be specific to the osteoblastic phenotype (Whitson et al., lm),an immunologicaily timilar protein has been show to be synthesized by various mesenchyrnal cells (Wasi et al., 1984), and can be extracted [rom platelets (Stenner et al., 1986), and periodontal ligament ( Wasi et al., 1984). Moreover, bovine "culture shock" protein does not bind to collagen or geiatin, but does bind to BSA and a 67 kDa semm component Consistent with these obsenrationsare the results of porcine osteonectin tissue localization (Chapter 111 B), which has shoam the protein to be prominent in cells of soft as well as hard tissues and results in this study showing the affinity of porcine osteonectin for serum albumin but not for collagen. Furthemore, like "culture shocknprotein, porcine osteonectin synthesized by cells in vitro does not appear to be phosphorylated whereas fetal bovine osteonectin has been reported to be a phosphoprotein (Termine et al., 1981a). In view of the homology of osteonectin to SPARC,BM-40 and "culture shock" protein, the apparent lack of specificity to bone, the absence of seleetive binding of porcine osteonectin to collagen and the unknown function of these proteins, it would, in accordance with the recently proposed nomenclature for bone proteins (Hauschka et al., 1986) be more appropriate to replace the functional term 'osteonectin' with a more descriptive term, 'bone SPARC protein'. tluüon Vduao (ml) EIuUon Volume (ml)

Figure 1 A, B. C: Composite Cbromntographic Profila For Fetal Porcine Calvarial and Adult Porcine Dentine Ecxtracts: Gel Filtration and FPLC.

Figure 1 A. sohd line represenb cbromatographic profile of Ealvarial Ecsiract proteins (0.12 g) on CLdB Sepharose. Bars throughout ihe profile reptesent ftauioos dched in vhous pro": harl: fractions emiched in proteoglycans. W;fractions enriched in sialoprottins. h;n-i; fractions emiched in osteonectin. k4;fractions enriched in SCAB proteins and the related 53 kDa peptide fragment of OPN. hari; fractions enricheci in ostdàn. Hatcbed iine represents chromatographie profile of dentine E-extract proteins (0.12 g) on CLdB Scpbe. Bar 3 is also represmtati ve of fractions enrichcd in dentine ostconectin. as umî~rmedby positive raults from ELISA and imrnuwblot analyses employing spxific ami-sera raised against fetal porcine calvarid osteonectin.

Figure I B. FPLC chromaiography of orteouectin poded from CL- gel filtratim (Figure 1 A) using a Phwnaaa HRlOi IO mlunm packcd with Pharmaaa preparatory grade 'poiyanion' exchange min nui unda dissociative conctitions of 7 hi urea, in 5û mM TridHCl, at a pH of 8.0 employing a Linear gradient of Naa. The bar rcpesmts fractions paolal for re-chomatowhy;sdid line: calvariac; hatched line: dentine

Figure 1 C, FPLC dution pmfie of ostoonectin on 'dyticai pol yanion' resin. rot^ [For dv&ac: 117th of pmled fracfions fmm ihe pmpmîory nm 1 B); for dentine ail of the cnriched fractions from plcparatory m]was nm on a HR515 mlumn under dissociative conditions of 7 M urea, in 50 mG1 TnslHCi. ai a pH of 8.0 ernploying a lin- NaCl gradient; solid line: calvariae; hatched line: dentine. The bars represent podd fractions analyzed by SDS-PAGE; ba 1: dentine osteonccrin and bar 2: calvariai osteonecfin. Figure 2: Comparative Analysis of Purified Osteonectin from Fetal Porcine Calvarial and from Adult Porcine Dentine,

SDS-PAGE (12.5 % mss-linked gel) nui under reduced conditions for proteins stained with CBB R-250. M,, LWmarken ; Lana, 5 pg purified porcine ostwnectin fmm calvariae and dentine, run under reducing conditions W.respective1 y) and non-reducingconditions -, respective1y).

190 21 0 230 250 Wavelength (nm)

Figure 4: Circular Dichroism Studies with Ostconectin.

The far U.V.- cd. spectlum of osteonectin in the presence and absence of 2 mb1 Cd+ions for samples dissolveci in 5 rnM TrisiHC1 at pH 7.3.Superimposeci is the measurabIe part of the specvum of osteonecbn in the presence of 7 M urea, 5 mhf TrisiHCl at pH 8.0. Figure 5: SDS-PAGE Analyses of CNBr Cleavage Fragments of Osteonectin.

Fragments were separateci on a 20 % SDS-PAGE cross-linked gel. Linci, 2 pg of reduad osisoaechn staincd wiih silver; W.reduced CNBr cleavage fragments fmm IO pg of osteonectin stained with silver; W.pm

Purifiexi porcine osteonectin (2 pg) and C3Br cleavage fragments (10 pg) generated under non-redzLced amditions were electmphoresed on 15 % minislab SDS-PAGEgels, aud transferred onto nitrocellulose membranes. Transferred proteins were tested for reactivity against poiydonai antibdes to fetai porcine osteunectin and for the ability of osteonectin and its CNBr fragments to sequester WS+.Lanes 14: Amido black staining of proteins transferred onto uitdluiose: fane 1.- markers; W.wiuced osteonectin; W.fragments generated by CSBr cleavage obtained under non-reduœd conditions but m mder reduced amditiaas; r;ine3,non-reduced osteunectia. Lmes 5-7: Immunoblot of proteins transferred ont0 nitroce11ulose: W.non-reduced osteoriectin; I,reduced os teoneçtin; W.fragments geaerated by CM3r cleavage obrained under non-reduced co~lditioasbut nm under reddconditions. hes 8- 10: 45Ca2+ binding to proteins trausîerred onto nitrocellulose: manereduced osteonectin; lane. fragments genaated by CM3r cleavage obtained under non-reducdconditions but nm under reduced conditions ; non-reduced osteonectin. Figure 7: SDS-PACE Analysis of Ostconectin and E-extract Proteins Bound to Hydroxyapatite in the Pnsmœ of 4 M GuHCI.

Electrophoresis was performed on a 12.5% cross-linked gel nui under reduced anditions. land. L!!W markers; lane.5 pg osteonectin (amount employed in the ùinding expriment); Land3,Eexiract proteins employai in the binding experiment (amtaining - 10 pg ostmnectin);1. axlai y sis for non-bound osteonectin; I-.analysis for any osieonectin released aftef sucwssive washes in G-buffer; IAIICZ, non-bomd E-extract proteins; I.non-noo-bound E-extract proteins released after successive washes in G-bufTu-,LimlQ, bound osteonectin released after treatment with 0.5 ,Li EDTA;hg 11 U,analysis for bound os teonectin reieased after successive washes in E-buffer ;I, Siains-ail shed oounterpart of Lane 10 from a duplicate experimeot, LmeM bound E-extract proteins released after ueatment with 0.5 M EDTA ; I;nie,E-exeract proteins released after successive washes in E-buffer. M. qmseats the Stains-al1 stained counterpart of Lane 14 from a duplicate experiment. Note that al1 lanes excep Ianes 13 and 17 represent proth stained with CBB R-250.The colours of the Stains-ail stained bands in Laae 17 are indicateci. On: osteonectin. Figure 8: Analyses for Osteonectin Binding to Callagen and Gtlatin.

SDS-PAGE (12.5 8 crosslinked gels) analysis under reduced amditions for proteins stained w ith CBB R- 250. I,LMW markers; W.osteonectin not bound to collagen in the presence of 2 mM a+; W,osteonectin not bound io collagen in the absam of CS+; M.osteonectin not bound to gelatin in the presence of C$+;Land, osteoncctin not bodto gelatin in the absence of CS*;m. fontrol; osteonectin not bound io Sepharose CL4B in the vaof CS+; W, mntrol; osteonectin not bowid to Sepharose U4Bin the absena of G$+;W. "cdlagen bound" osteonectin in the presena of W+; M."wllageo bound" osteondn in the absence d CS+; W."gelatin bound" osteonectin in the presence of Cas+; Liinell. "gelatin bound" ost~in absence of CS*; W,control; osteonectin bound to Sepharose CL4B in the presence of Cs+;lane. control; "Sepharose CL4B bound" ostmnectin in the absence of CS+ions. Collagen ùdsobserveci represent protein non-covalent1y associated with the Sepbarose CL4B-bound collagea. On: ostfonectin. -. Table 1: Arnino Acid Composition of Calvarial Osteonectin Compared With Proteins From Other Sources

Hydroxy proline Aspartate Threonhe Serine Glutamate Proline Giycine Alanine ~atf-Cystlne+ Valine Met hionine lsoleuclne Leucine Ty roslne Ph8flyhlanifle Histidine Lysine Arglnlne Try ptophan CHAPTER III B BIOSY NTHESIS AND IMMUNOLOCALIZATION OF OSI"EONECTIN/SPARC

INTRODU~ION The occurrence of macromolecules that are unique to a particular connective tissue matrix provide important dues to understanding the basis of the distinctive biophpsical properties of that tissue. If such macromolecules are synthesized by cells of the same tissue, they can be extremely useful as phenotypic markers which can then be used to identify specialized cells. In rnineraiized mnnective tissues, the presence of unique cornponents may also suggest their psible involvement in the formation or stabilization of hydroxyapati te crystals. Thus, as nas discussed previousl y, tissue specificit): is a prime characteristic of a potential nucleator of hydrosyapatite crystal formation. In the studies described in rhis chapter, one of the prime objectives was to detemine the tissue distribution of osteonectidSPARC in the various hard and soft tissue that comprise teeth and their associated structures. In addition, to funher investigate potential functions of osteonectinfSPARC, die biosynthesis of the protein (in vifro and in vivo ) and was studied together with its developmental expression.

MATERIALS AND MI3HODS

Extraction of OsteonectinlSPARCfiom Rot Calvariae. Long Bone. and Denrine ut Vurious Developmenral/Growth Stages Calvariae, long bone and dentine were dissected from CBL Wistar rats at raious developmentai and groath stages: beginning with 18 dayald fetuses (a time at which bone formation had just begun), 3- and 1 1 &y-old pua (representative of young bone, dentine formation), and 3-, 4.6-and 9 week-old young adults (represeniative of mature bone, dentine formation). For the long bone sarnples, the midshafts of femurs were isolated. For dentine sarnples, enamel and soft tissues from the mandibula incisoa were removed under a dissecting microscope. However, dentine sarnples from fetai and 3 day-old rats could not be obtained in sufficient quantities. Tissues were weighed, frozen in liquid nitrogen, crushed into small fragments, and exmted according to the methods described by Termine et al. (198la). Extracts were diaiyzed against three changes of 0.5 M sodium acetate, followed by three changes of distilled water and then freeze-dried. Equal aiiquots from the entracts of each tissue were analyzed by SDS-PAGEunder reduced conditions (Laernmli, 1970) on 12.5% cross-linked acrylarnide slab gels and the proteins were stained with CBB R-73or Stains-al1 (Campbell et al., 1983). Roteins were quantitated by scanning positive photographie transparencies at 550 nm on a Gilford Spectrophotometer fitted with a film indexing aifachment and digital intepatm which was used for quantitating the density of fiuorographic bands. Separated proteins were also trahcferred elecuophoretically onto nitrocellulose papa for immunoblot analysis (Towbin et al.. 1979) using rabbit anti-porcine osteonectin antibody diluted 1:2ûû and used as desctibed previously (Wasi et al., 1984).

Oszeonectin/SPARC Biosynthesis In Vivo Seven, adult male CBL Wistar rats ( 180 g) were each pena singe inuapentoneal injection of 0.5 Ki PsSlrnethionine (7800Cilmmol, New England Nuclear Corp. ; 1 Ci = 37 Gbq) and one rat sacrificed after 0.5. 1,2,4,6, 19 and 24 hr. Calvariae, long bones. and incisor dentine were fragmented and sequentiaily extracted with PBS, G-buffer and GE-buffer, in the presence of Pi as describeci aboce. Aliquots of the dialped extracts were freeze-dned and anal yzed by SDS-PAGE on 13.5 Q cross-linked gels or 5-20 9 gradient gels run under reduced conditions and the radiolabel led protei ns were visualized by fluorography . Fluorographic tracks were scanned at 5% nm on a Gilford spectrophotometer.

Osreonectin/SPARC Biosynthesis In Vitro TO study the ce11 free synthesis of pre-osteonectiniSPARC, total mRNA was extracted from fetal porcine calvariae using guanidinium thiocyanate, purified by acid- ethanol precipi tation as previousl y descri bed by Kuuata et al. ( 1985). Osteonectin/SPARC RNA was translated using a rabbit reticulocyte lysate translation system that was purchased from BRL. Each reaction was dedout in a 30 J volume containing 10 lysate. 3 reaction mix, and 50 pCi pJS]methionine. The optimai translation conditions with respect to K+, Mg?+ and RNA concentrations were determineci as 100 rnM K+,1 .O mM Mg2+ and 1.0 pg RNA. Total translation products and proteins precipitated with ad-osteonectin anti bodies were electophoresed on SDS-PAGEand subsequently processed for fiuorography (Kuwata et al., 1985). To study the biosynthesis of osteonectidSPARC from cells of various tissues, the fint to third subcul tures of rat calvarial bone -11s (Aubin et al., 1982) and rat pingival and periodontal Iigarnen t fibroblasts, obtained as descri bed previousl y (Cornor et al., lm), were grown to confluence in a-MEM - 15% FBS in 35 mm tissue culture dishes. Confluent cells were washed in serum- and methionine-free DMEM and labeIIed for 30 min with 75 rCi (W]methionine in 1.0 mL of the sarne medium containing M &mL of vitamin C. Sorne cultures were subsequently chased for 4 hr in complete medium containing 1- % fetal bovine serum. Radiolabelled osteonectinISPARC in the pulsed cells and in the chase media was immunoprecipitated as described previously (Wasi et al., 1984; Kuwata et al., 1985) using protein A - Sepharose to isolate the antigen-antibody cornples. The specific immunopreçipitate was analyzed by SDS-PAGEand fluorography (Kuwata et ai., 1985).

Rat OsteonectinlSPARC Immunos)tochemistry/Comparisons wirh Types I di 111 Collagens Rat bone -11s and rat periodonial ligament and gingival fibroblasts were plated ont0 glas oover slips and analyzed for osteonectinlSPARC and collagen synthesis using the same immunocytochemical procedures described previously for porcine cells (Otsuka et al., 1984; Wasi et al., 1984). Bnefly, cells were plated sparsely ont0 glass cover slips and, after 2. days, the cells were fixed with periodate-lysine-paraformaldehydeand then immersed in methanol at -20'C. The permeabilized cells were first incubated with affinitu- puri fied anti bodies to osteonectin/SPARC, type 1 collagen, or type III collagen (Otsuka et al., 1984; Wasi et ai., 1m), followed by fluorescein-conjugated F(A b)? fragments of the second antibody to rabbit (osteonectinlSPARC)and sheep antibodies (collagens).

Immu~hisfochemicaiDistribution of OszeonecrinlSPARC in Rat and Porcine Dental TissueslCornparisons with Types I & Ul Collageru Lower jaws [rom -50 g adult Wistar rats were fised in 10 9 neutral formalin or 3% glutaraidehyde for 24 hr, then dernineraiized for 3 weeks in a 10 5% solution of sodium citrate in 22.5 5% formic acid. The tissues were then dehydrated and embedded in paraffin. Before staining, 6 sections attached to microscope slides were de-paraffinized in toluene, and then hydrated in graded alcohol and PBS. The tissue sections were incubated ovemight at 4'C with pnmqantibody diluted with PBS containing 2 4c wfv BSA. After again washing with PBS,the sections were incubated for 1 hr at 22'C with peroxidase-wnjugated antibody directed at the primary antibody. For rabbit anti-osteonectidSPARC antibodies, pero?ridase-conjugated sheep anti-rabbit IgG ( 12.0 mglm1 protein) was used. Second antibodies were obtained frorn Cappel (Organon Teknika N.V.,Belgiurn) and were used at a dilution of 1:M in PBS containing 3 8 BSA. The peroxidase activity was revealed with a solution of 35 mg 3,3diaminobenzidine tetrahydrochloride in 50 ml of 50 mM tris-HCl buffer, pH 7.6, containing 100 4 of 5 8 hydrogen peroxide. The stained sections were dehydrated and mounted in Rot-Texx mounting medium (Lerner Laboratones, New Haven, CT,U.S.A.) and examined and photographed under a Leitz Orthoplan microscope. The specifici ty of the immunoreactions was controlled by substituting primary antibody with the equivalent fractions from the respective affinity columns upon which normal or pre-immune sera were fractionated, and in other cases by ornitting the second antibody in the reaction sequence. Because of the putative association between osteonectidSPARC and collagen (Termine et ai., 1981a; 1% 1b), an immunohistochernical mmmson of type I collagen ( representative of hard and soft tissue collagen) and type III collagen ( represen tative of soft tissue collagen) was also performed, as described above. Affini ty-purified antibodies to pig type 1 and III collagens were prepared and used as descri bed by Rao et al. ( 1979) and Wang et al. ( 1980). Similarl y, the study of osteonectinf SPARC distri bution was perfomed on tissues associated with unerupted teeth and erupted teeth from fetal and adult pigs. Tissue blocks were sliced into 2 mm sections fixed in penodate-lysine-Wormaldehyde (PLP)and demineralized pnor to embedding. Sections (6 m) were stai ned using affini ty-puri fied osteonectidSPARC antibodm and the pero?udase-mti-peroxidasetechnique similar to that described by Tung et al. ( 1985). For both these studies affinity- puri fied polyclonal antisera to puri fied porcine osteonectin/SPARC were used at dilutions of between 1:40 and 1:?00 with respect to the original antisenim.

RESULTS

Earaction of Osteonectin/SPARCfrom Rar Calvariae. Long Bone. and Dentine ar Variour DmeloprnentallGro wth Stages To detemine developmental changes in ostmnectin/SPARC content in calvariae. long bone and dentine, the analysis by SDS-PAGEof hydrosyapatite-binding proteins of calvariae, long bones and incisor dentine extracted (GIE-estract) from rats at different stages of growth and development are show in Figures 1-3. Within each tissue, the panerns of the CBB R-250stained proteins were similar. Most of the protein migrated with a mass in the 60-70kDa region, with a number of relatively minor protein bands at various higher and lower molecular weights. Generally, on1 y slight quantitative differences were observed for the individual protein bands. Notably, a 25 kDa protein was prominent in fetal tissue samples of calvariae and long bone, but the quantity of this protein appeared to decrease substantially following parturition. Almost ail the proteins, including a protein in bone that CO-migrated with porcine osteunectin/SPARC, stained blue with Stains-dl. The Stains-al1 reagent also revealed some proteins that did not stain with CBB R-250, whereas collagen chahs and a 35 kDa protein in the bone samples stained pink with Stains-dl. The Stains-al1 positive, CBB R-250 negative proteins were most evident in the 60 kDa region of dentine proteins and indicâte the presence of sialoproteinsf phosphoprotei ns (Fisher et al., 1983 b) . To psitivel y identi fy osteonectinlSPARC, proteins were separated on SDS-PAGE. then electrophoreticaily transferred to nitrocellulose, the effectiveness of the transfer king rnonitored by amido black staining of duplicate transfen. OsteonectinfSPARC was identified by immunoreactivity with anti-osteonectidSPARC antibodies. As shown in Figure 1, osteonectin1SPARC could be demonstrated in long bones and in most calvarial sampla, but was barely detectable in dentine extracts. The irnmunoblot results were consistent with the relative amounts of CBB R-2%) staining protein in the osteonectin/SPARC position for the various tissue sam pies. The rat osteonectin/SPARC in each tissue cornprised a single band on SDS-PAGE,migrating with a masof - 39 kDa under reduced conditions. From dupliûite densitometric sans of CBB R-250 stained proteins from each developmental stage, the osteonectidSPARC was calculated to

represent approximately 2.5 + 1.5 Q of the GE-extract proteins in long bone, 0.9 t 0.5 R in calvariae, and 4.1 4c of incisor dentine proteins. In contrast. osteonectinlSPARC could not be detected in the G-extracts of these tissues (results not shown). In long bone, the relative amount of osteonectin/SPARC in the GJE-extracts appeared to increase wi th ag from 1.2 to 3.8 &, whereas in calvariae and dentine no consistent changes were apparent.

Rat OSt eonectinlSPARC Biosynrhesis In Vivo (in Long Bones) The biosynthesis of osteonectin1SPARC was studied in Young adult rats over a 14 hr chase period following a single intraperitoneal injection of 0.5 mCi pjslmethionine. Figure 4 illustrates the densitometric profiles of long bone proteins racholabelled mvivD and separated by SDS-PAGE. From nuorographsc analyses of PBS entracts, G-extracts, and GE-extracts, a radiolabelled band correspondi ng in electrophoretic mobil i ty to osteonectin1SPARC was only evident in some of the GIE-eutracts of long bones and to a lesser extent in calvariae in long exposures of fluorographs. The osteonectin/SPARC band in long bones was detectable at 4 hr and increased in amount through the 6 to 24 hr time points. However, the maximal radioactivity in the band represented on1y 1 9 of the toral radiolabelled proteins in the extract Notably, collagen bands were prominent ar ail time points in dl three extracts analyzed. In addition, at 24 hr a strong but diffuse protein band similar in behaviour on SDS-PAGEto sialoprotein (Fisher et al., 1983b) nas evident in al1 tissues examined.

Rat OsteonectinlSPARC Biovnîhesis In Vitro (in Calv~n'ae) To study the biosynthesis of osteonectin/SPARC, [3sS1-methonine-labelled proteins synthesized from total porcine calvarial mRNA under cell-free conditions. and by porcine calvarial œlls in vitro w ere subjected to i mmunopreci pi tation using osteonectinlSPARC specific antibodies (Figure 5). Although not readily apparent in Figure 5, lane 3, wi th each system, a radio1 abelled protein band could be identified by SDS-PAGE having the electrophoretic mobility comparable to [ljC]-methylated osteonectin/SPARC prepared from the dissociative extracts of porcine calvariae. The cell-free synthesized protein migrated with a mass of 45 kDa (Figure 5, lane 3), but in the presence of microsornes containhg signalase the immunoprecipitated protein (although not apparent in Figure 5, lane 3) c-umipted with the dl-sqnthesized (Figure 5, lane 4), cell-secreted (Figure 5, lane 5) osteonectidSPARC and the tissue extracted protein (Figure 5, lane 1); al1 migrating with a mass of 39 kDa Similarly, a 39 kDa component was immunoprecipitated from radiolabelled proteins synthesized by fetai rat calvarial cells (lane 8) and rai pngivai (lane 6) and periodonral ligament (Figure 5, lane 7) fi broblasts. However, the proportion of radiolabelled materid immunoprecipitaied from the rat calvarial cells appeared to be lower than that obtained the fibroblast cultures, as is reflected in the weaker band in lane 8 of Figure 5. To compare relative amounts of osteonectin/SPARC synthesized by different cells in vitro cultures were pulsed with [3sS]methionine and c hased for 3.5 hr (Figure 6). The osteonectin/SPARC imrnunoprecipitated from rat calvarial cells was extremely low cornpareci wi th pmtein imm unopreci pitated from porcine calvariai cells. Radiolabelled protein corresponding to osteonecrin/SPARC was also immunoprecipitated from culture media of rat gingival and periodontal ligament fi broblasts and again appeared to be synthesized in significantly greater amounts than that produced by rat calvarial cells.

Rat osteonectin/SPARC Imunocytochernistry 1mrnunocytochernic.lstaining of fixed and permeabilized cells (Figure 7) provided results consistent wi th the immunoprecipitation anal ysis (Figure 6). OsteonectirûSPARC staining, which is observed as an in~acellularvesicularlpunctuate pattern of fluorescence typical of secreted proteins, showed variable staining in different cells. Thus, while imrnunocytochemical staining of rat fibroblasts and calvarial bone cells showed strong staining for collagen types 1 and III, a more pronounced staining for osteonectin/SPARC was observeci in fibroblast populations [rom periodontai ligament and gingivae compared to calvarial cells which showed weak or no staining (Figure 6).

Immunohist~~hem~caiDistribution of OsteonectidSPAR C in Rat and Porcine Dental Tissu es Using ostconectidSPARC antibodies, affinity-purifid on porcine osieonectinlSPARC-Sepharose,the distribution of osteonectin1SPARC was studied in porcine dental tissues associated with unerupted (fetai) and enipted (adult) teeth. As shown in Figure 8, strong staining for osteonectinlSPARC was found in fetai bone and dentine and weak to good staining in soft tissues such as pulp. stratum inkrmedium. stellate reticulum and endosteal tissue. Bone and dentine staining appeared weaker in the adult tissues, and dentine staining was largely resuicted to intertubular dentine. Cementum showed littie reaction with osteonectidSPARC antibodies but ligament and reticular tissue in the endosteal spaces were strongly stained. Notably. cells in both soft and hard tissues were stained using the osteonectidSPARC anti bodies. Imrnunosraining for oskonectidSPARC protein wi th affinity-pun fied antibodies was dso performed in rat dental tissues (Figures 9.1 - 9.11). A relatively strong reaction was obtained in a number of soft tissues including periodontal ligament, pulp, endosteal tissues, marrow spaces and muscle, as shown in the sagittal section through a rat mandible. More moderate staining was apparent in the gingival connective tissues with weak staining in the deminedized bne tissues. No immunoreaction above background was evident in either the dentine, cemennim or the enamel matrix at this resolution. The strong staining in the molar periodontai ligament and dentai pulp shown at higher magnification in Figure 9.2 contrasts with the unstained cementum and also with the weak staining in the alveolar bone. A slight, diffuse immunoreaction appeared to be present in the demineralized dentine main'; at this magnification. However, this may be the result of reagents king trapped in the dentinal tubules. At even higher magnification, the fibrous pattern of siaining that was evident for type 1 collagen was similar to the osteonectinlSPARC staining in the molar ligament. However. osteonectidSPARC stained more strongly and was more uni foml y distri buted in the soft tissues of the endosteai spaces. The diffuse and patchy staining of the dentine and alveolar bone with osteonectinlSPARC antibodies contrasted with the more uniform staining for type 1 collagen in these tissues. However, it was clear that type 1 staining in these tissues was compromised by the demineralization of these tissues. as has ken found before (Raoet al., 1979). The distribution of osteonectin/SPARC in muscle was evaluated at higher magnification with the aid of successive tissue sections stained with haematoxylin and eosin. The osteonectidSPARC protein was revealed both on muscle fibre surfaces and between fibres. where i t produced a floccular appearance. No particular association of the osteonectinlSPARC with muscle cells was apparent. The similarity in the distribution of osteonectiniSPARC and type III collagen was evident in the incisor ligament, which is shown in a series of successive sagittal sections that included an adjacent molar tooth root. The histological features of the tissues at medium and hi& magnification are shown in sections stained with haematoxylin and eosin. Type III collagen was revded to be distributd in both rnolar and incisor ligaments with circumferential staining in the dental pulp and somewhat stronger staining of the incisor ligament adjacent to the tmth in some regions. The osteonectin/SPARC showed a similar distribution to type III collagen in the ligaments but was stained more intense1y and uniformly in the molar pulp. An intempted band of staining for osteonectin/SPARC was dso apparent benveen the dentine and cemennim. The mineralized tissues of bone and dentine were diffusely stained, whereas the soft tissue in the endosteal spaces staned strongly. At high rnagnification of the incisor ligament, osteonectinlSPARC protein had a fibrous appearance. In the region shown, panicularly strong staining was observeci in material adjacent to the tooth. which appeared to run paralle1 to its length, whereas in the middle region of the ligament the staining materid appeared to foilow a more oblique distri bution.

DISCUSSION

To further evaluate osteonectin/SPARC as a bone- or mineralized tissue-specific marker and as a potential regulator of biologcd mineralization. studies invoiving the biosynthesis and the tissue distribution (Otsuka et al., 1984; Kuwata et al.. 1985; Tung et al., 1985; Wasi et ai., 1983 ; 1984) of this protein were performed. Results [rom these and additional studies, which are surnmarized here, do not support a specific role for osteonectidSPARC in mineralization, nor was the tissue specificity of the protein apparent

Bioqnthetic Studies and Propenies of OsteonectidSPARC Studies on the biosynthesis of ostmnectidSPARC showed that the nascent protein chan has a M, of 45 k on SDS-PAGEwhich may inciude a signal sequence (Kuwata et al., 1985). In bovine osteonectin the signal sequence is 17 arnino acids long (Findlay et al., 1988). From cornparisons of the size of the processed pre-osteonectinISPARC, the cell-synthesized osteonectin/SPARC and the osteonectin/SPARC extracted from bone, no further processing of the osteonectdSPARC molecule wsapparent These results also demonstrated that the osteonectin/SPARC puri fied from tissues usi ng dissociative extraction is recove~din an intact form. Furthemore this study has shown that a protein wi th the ph ysical and chernical propemes of osteonectidSPARC is preseni, albeit in low amounts, in the mineralized matrix of rat calvariae and long bones. The tissue-extracted osteonectinISPARC, and the osteonectin/SPARC synthesized and secreted by rat calvarial cells and fibroblasts in vitro, were found to CO-mipteon SDS-PAGEunder reduced conditions wi th porcine osteonectidSPARC at an M, - 39 k. The radiolabelled osteonectidSPARC frorn rat ce11 cultures was also found to hnd to hydroxyapatite in the presence of 4 M GuHCl (results not shown), consistent with the properties of porcine osteonectinlSPARC (Otsuka et ai.. 1984; Wasi et d., 1984) and bovine osteonectinJSPARC (Termine et al., 198 1a; 1981 b; Romberg et al., 1985). Although the amount of osteonectinfSPARC appeared to change in long bones during the development and growth of rats, it remained at levels reiatively low (-2.5 4c) compared with that found in fefai porcine (-15 Q) (Kuwataet al.. 1985) and bovine bones (20-359) (Termine et al., 198 1a; Conn and Termine, 1985). The low levels of osteonectin/SPARC in rat do not apprto be the result of a rapid turnover since immunoreactive degradation products were not evident on irnmunoblots, and both in vivo and in vitro labelling experiments indicate low levels of osteonectin synthesis in bones and by bone cells. The apparent increase in osteonectin1SPARC in long bones of growing rats rnay result from an age-dependent decrease in the amount of chondroid tissue that becomes incorporated into the larnellar bone of the long-bone shaft. The demonstration that osteonectinlSPARC is a relativei y minor non-collagnous protein cornponent in rat bones and that i t is virtually absent in rat dentine and porcine cementum (Tung et al.. 1985) suggests that the amount of ostmnec tin/SPARC in a particular mineralized rissue ma. reflect detaled diffe~ncesin the formation of specialized types of mineralized tissue. Notably, osteonectin/SPARC appears to be more abundant in mineralized tissues of larger marnmals where the bones are proportionately larger and stronger. performing a correspondingiy greater load-beanng function. The prominence of osteonectin/SPARC in porcine and bovine fetal bones and in adul t bovine bones (Conn and Termine, 1985), therefore, ma. be indicative of the earl y formation and subsequent maintenance of a strongr larnellar bone. The biosynthetic studies funher suggest that phenotypic differences exist between osteoblasts of different species, while the difference observed in the osteonectidSPARC contents of long bne and calvariae in rat may reflect more subtle variations in the composition of bone matrices within individual animals. The virtual absence of osteonectin/SPARC in rat dentine is consistent with the results of more detailed analyses of non-collagenous proteins in this tissue by Butler et al., ( 198 1) and by Linde et ai., ( 198%). These results, however. conmt analyses of bovine (Termine et ai., 1981b) and porcine dentine (Chapter III A), in which appreciable quantities of osteonectin has been dernonstrated.

Distribution of OsteonectidSPARC Although it was dready known from biochemicd studies that osteonectidSPARC is abundant in porcine bone and dentine, these studies revealed that it was also present in a nurnber of soft connective tissues, with a psuticularly strong reaction in periodontal ligament. These observations support biochemical studies in w hich osteonectin/SPA RC protein has ban extracted from various soft tissues including gingiva and periodontal ligament (Wasi et al., 19&0, and in vitro studies, in which osteonectinfSPARC was shown to be synthesized by fibroblasts at levels similar to those of bone cells (Otsuka et al., 1984; Wasi et al., 1984). The irnrnunostaining of adui t rat dental tissues has now shown that osteonecti niSPARC is a prominent protein in several soft tissues in periodon tal ligament, endosteal spaces, dental pulp and muscle in rat. As illustrated in Figure 8, the sraining of ligament and endosteal tissue is consistent with the findings in porcine tissues (Tung et al., 1985). However, pulp was more strongly and unifomiy stained in the rat, whereas the weak staining of rat alveolar bone and the even weaker staining of rat dentine contrasts with the observations in porcine tissues. 1n porcine dentine, the osteonecti n/SPARC has been shown immunohistochemically to be associated predominantly with the hypermineralized pentubular dentine (Tung et al., 1985). which is known to be more extensively developed in teeth of animals that grind their food and which are mechanically stronger ((Bradford. 1x7). Conversel y, peri tubular dentine is poorl y developed or absent in teeth of animais such as rats and mice that do not pnd their food appreciably. The staining for osteonectin/SPARC in porcine bone and dentine appears to be decreased in adul t tissues (Tung et al., 1985), but i t was also less than anticipated from biochemical anal ysis. Because osteonectidSPARC is bound to the hydroxyapatite cqstals (Domenicucci et al., 1988), it is possible that some of the osteonectin/SPARC was lost frm the mineralized tissues dunng demineralization, even thou* dernineralization was carried out in the presence of fixative. However, in rat bone, osteonectin/SPARC is present in substantially lower arnounts than in the larger marnmals such as man, pig and cow. It has been suggested that the low content of osteonectidSPARC in rat bone could be attri buted to rat osteonectin/SPARC having a lower affinity for hydroxyapatite because of differences in the structure of the putative hydroxyapaîi te-binding region (Domenicucci et al., 1988). Osteonectin/SPARC has subsequendy been found in platelets (Stenner et al.. 1986) and basement membranes (Mann et al., 1981) and is produced by parietal endoderm (Mason et al., l986a), endothelid (Sage et al., 1984) and epithelial cells (Dziadek et al., 1986). Northem blot analysis (Mason et al., 1986b) and in situ hybridization studies (Holland et al., 1987; Nomura et ai., 1988) have indicated that the osteonectint SPARC mRNA is expressed in a variety of tissues, wiih a particular abundance in placenta. In the fetd mouse, tissues associated wi th developing teeth showed relati vel y strong hybridization, i ndicating hgher Ievels of osteonectin/SPA RC expression in these tissues (Nomura et al., 1988). The preponderance of osteonectin/SPARC in fetal tissues (Tung et al., 1985; Mason et al., 1986b;Nomura et al., 1988) and its stimulation in response to trauma (Sage et ai., 1984; Sage et ai., 1986) and wound-healing hormones such as transforming growth factor-p (Wrana et al., 1988; Sodek et al., 1988) indicate a function for this protein in developmentally related processes. The relatively high content of osteonectifiJSPARCin periodontal ligament is consistent with the embryonic features of this tissue, which include rapid remodelling, higher content of type III collagen and the type of collagen crosslinks (reviewed by Shuttleworth and Smalley, 1986). Although the immunostaining for osteonectin1SPARC protein in the periodontai ligament has a fibrous appearance similar to that of collagen, a direct association between these proteins appears unlikely, as osteonectidSPARC protein is quantitatively extracted from bone by 0.5 M EDTA in the absence of denaturants and porcine osteonectinlSPARC does not bind to collagen in either the presence or absence of calcium ions (Domenicucci et al., 1988). It is interesting, however, that the majority of the osteonectinlSPARC protein in porcine periodontal ligament can only be extracted in the presence of 0.5 M EDTA (Wasi et al., 1984). A sirnilar observation was made for basement membrane osteonectidSPARC protein (Mann et al., 1987). Changes in secondary structure occur on calcium binding (Engel et al., 1987; Domenicucci et al., 1988) and i t has ken suggested that calcium-induced conformationai changes may be responsible for osteonectin1SPARC binding to tissue matrices (Mann et al., 1987'). Despite considerable knowledge of the structure, biosynthesis and distribution of osteonectinlSPARC that has ben accumulated in recent years, its function has nor been determined unequivocally. The orignai suggestion (Termine et al.. 198 1a) that osteonectinlSPARC may bind to collapn fibrils and act as a nucleator of hydroxyapatite crystal formation has not been supported bg these results or subsequent experimentation. Al though Termine et al. (1981 b) and Romberg et al. ( 1985) have indicated that bovine osteonectidSPARC binds to collagen, these studies on porcine osteonectin/SPARC have failed to demonstrate appreciable binding to either native or denatured collagen (Kuwata et al., 1985). Further, osteonectidSPARC was quantitative1y entracted from porcine bone and dentine tissues by EDTA under non-dissociative mndi tions. These obsemations suggest that osteonectiniSPARC, with its high affinity for hydroxyapatite (Romberg et al., 1585). may bind to prefomed hydroxyapatite rather than king an integral component of the tissue matrix. The eariy appearance of radiolabelled osteonectin in the G/E-extracts of long bone puise-labelleci in vivo would support this possibility. Consequently, osteonectin1SPARC is more likely to affect crystal growth or stability, rather than initiate cry stal formation. Di fferences in surface properties of hydroxyapatite cry stals may affect the arnount of osteonectin1SPARC bound, and ihis could also explain the variable levels of osteonectin/SPARC found in different types of bone (e.g. lamellar versus woven (Conn and Termine, 1985)),in different species and in diseased bone (Termine et al., 1984)). Regardless of the discrepancies round between the bone-derived osteonectins and the disputed functions that it may have in bone mineralization, more recent studies have suggested that osteonectin1SPARC may play a pivotal role in the proliferation pathway of angiogenesis and wound healing (Lane and Sage, 1994; Sage and Vemon, 1994). In this respect the soft tissue-derived molecule has ken shown to inhbit ceIl spreading, diminish focal contacts (Sage et al., 1989; Murphy-Ullrich et al., 1991) and alter endothelid barner functions (Goldblurn et ai., 1994). OsteonectidSPARC also has demonstrabie effects on the production of extracel1ular matrix components (Iniela-Arispe et al., 199 1). For example it can regulate the pne expression of fibronectin, thrombospondin- 1 and plasminogen activator- 1 (Lane et al., 1992). Furthenore it can regulate the activity of platelet-derived growth factor (Raines et ai., 1%) and basic fibroblast growth factor (Hasselaar and Sage, 1992). It is therefore believed that osteonectinlSPARC exhibits these growthlrn~dulato~properties as a result of its effect on ce11 shape (Sage and Vernon, 1994). Peptides generated from osteonectinlSPARC have also been shown to have an inhibitory effect on cell-cycle progression and stimulate angiogenesis in vivo and in vitro (Funkand Sage, 1991; Lane et al., 1991: Funkand Sage, 1993). In fact osteonectinfSPARC peptide derived from dornain II of the molecule contains the sequence GHK which is the sarne as the plasma tri-peptide which stimulates the proliferation of cul tured cells and augments wound healing and angiogenesis (Lane and Sage. 1994). Furthemore prote01 ysis of osteonecrin/SPARC in the extracellular matris is quired for the expression of angiogenic andlor proliferative activig (Lane et al.. 199 1 ). Conversely, intact osteonectin/SPARC acts as an inhi bi tor of' endothel ial ce11 pro1 iferation that might be functional during the latter stages of angiogenesis, when the growth of new endothehl vessels ceases and rernodelling continues (Sage and Vemon, 1994). Thus by studying the high levels of expression, synthesis and elaborarion of osteonectinISPARC by cells in tissues undergoing remodelling as a consequence of injury, disease andor development (Lam and Sage, 1994) a clearer understanding of its function not on1 y in soft tissues but also in bone will be revealed. Long Bone a b c

*11.**, 1114s.r 1?S.*.?1i

Figures f -3: Extraction of Ostconectin/SPARC from Rat Calvariae, Long Bone and Dentine at Varioas Developmental/Growth Stages.

GIEuiract proteizu from rat bone and dentine dy.rid by SDS-PAGE and immuno~ansfer.Proteins in quai aliquots of GIE-extracts of rat long bone figure 1). calvaiae (Figure 2) and inusor dentine figure 3) wexe qmated by SDS-P.4GE on 12% cross-liuked gds under rcduced woditions. Gcls were either siained with CBB R-550(a) or with Stains-al1 (b). Reteins in otha gels were dectrophoretically transfemd onto nitrmllulose pepcr and eithcr s tained with amido bladr (c) or incubateci seguentially wi th alti- osteonectioiSPARC aotibodia. peroxidaseconjugd second antibodies. and 4-chIoro- 1-naphth01 substrate to identify rntematiniSP.4RC (Oh? (d). The Iane nrmibas 1-7 of tbe bone samples mrrespond to sampla obiaiiicd from 18 day-dd fetuses, 3- and 1 1 day-old pups. and 3-. 4.6-and 9 week-oid adult rats. mpe&vdy. The lane numbers 1-5 for dentine correspond to sampla obtained from 11 day-dd and 3-. 4. 6- and 9 wd-old rats. M,,UIW protein standards. O 0.2 0.4 0.6 0.8 1

Relative Mobility

Figure 4: Rat Osteonectin/SPARC Biosynthesis In Vivo.

Demitometric profiles of long-bone proteins radiolabelled in vivo and separateci by SDS-PAGE. Seven duit (200 g) rats were given 0.5 Ki p%]methionine by intraperitoneal injection and individuai animals were sacrificed over the subsequent 24 h tirne period. Dissected fernuis were fragmenteci and the proteins in E-extracts were separaied b y SDS-P.4GE and visualized by fluorography. To quanti tate the radiolabelled proteins. individual tracks were scanned and the density ar 550 nm was measured using a digitai integrator. Daisitornetric profiles for the 24- (a), 12- (b), 6- (c)and 2 h (d) time @ods are shown. The open arrow indicates a prominent broad peak wi th electrophoreuc mobility simiIar co siaioprotein, and the asterisk indicates the position of coiiagen a £hainse Figure 5: Rat OsteonectidSPARC Biosya t hesis In Vitro.

Composite fluomgraph showing SDS-PAGE analysis of 35s-metbionine labelled osteonectia SP.4RC protein synthesized from mRNA and by various cells in vitro. I,1-4 C-methylated tissue-exmted fetal porcine calvarial osteonectinlSPARC; I,ceil-free s ynthesized irnmunoprecipitated osteonecanlSPARC in the absence of microsornes ; T,cell-free synthesized immunoprecipi tated osteoneWSPARC in the presence of rniaosomes (containing signalase). although not apparent here a band could be detected wi th a longer exposure time; Lm& œlI-synthesized immunoprecipitated osteonectin/SPARC; cd-secreted immunoprecipi tated osteonech'SPARC; rat gin@ val fibroblasts W.rat periodontal ligament fibroblasts Iane,rat calvarid cells. RC PC RGF RLF I '-8 \ 1 u nsuns ~,oss

Figure 6: Immunoprecipitation of radiolabelled osteooectin/SPARC synthtsized by rat bone cells and fibroblasts.

Rat calvarial bone œlls (RC).gingival (RGF) and periodontd ligament fibroblasts (RU'), and porcine calvariai œlls (PC) were pulse Iabelled with P5S] methionine for 30 min. Following a 3.5 h cbage period. the radiolabeiled osteonectidSPARC in media samples (containhg approximately 300,000 dpm) was immimoprecipitatd using spacific antibody bound to protein A - Sepharose. Samples of unbound (u), ncmsmcaily bmd(n). and s~cally bound (s) proteins were separated by SDS-PAGEon 12 1aoss- Linked gels undn redllced condi tiais and visualized by fluorography. o. [14Chethylaied porcine calvarid oste~~lcctin.The arrowhead shows the psihou of the weak band irnmunoprecipitated from the rat calvarial ceil media. Figure 7: Rat Osteoaectin/SPARC Immunocytochcmistry. immuaocytochemical anal y sis of osteoneçtint SP.UC synthesis by rat calvariai bone cells and fibroblasrs. Sparse culnues of rat calvarial bone œlls (.4-C) and gingival fibroblasts (D-F) were fised. permeabilid and stained by indirect fluorescence for osteonectin: SPARC.(A and D). type 1 collagen (B and E) and type III collagen (C and F). Magnification, x 113. Figure 8: ImmunohistochemicaI Locdization of Ostconcctin/SPARC in Porcine Dental Tissues.

Rcpfesentati ve longinidinal sections of unerupied (a) and empted (b) teeth showing osteonectin staining in bone (B),endosteai spaces (ES),dentine (D),enamel (E), stellate reticulum (SR), pulp (P),cementun (C), periodontal ligament (PL) and stratum intermedium (1). Figutes 9.1-6: Immunohistocbemical Localization of OsteoaectlnlSPARC and Type 1 Collagen in Adult Rat Dental Tissues.

Tissues fixed in glutaraldehyde and stained with specific antibodies to osteonectin;SPMZC protein and collagen type 1. Panel 1: Sagittal section of lower jaw from a 250 g aduli rat displaying immunoreactivity for osmnedinlSPARC protein in tissues associated with molar teeth. Panet 2: Cross-section through the fint molar and associated periodontal tissues stained with osteonectin/SPARCantibodies. Panel 3: Enlargeai section tbrough the periodontal Ligament showing immunostaining for collagen type 1. Pauel 4: Secbon as shown in Figure 3 stained for osteonectidSPARC protein distribution. Panel 5: Section thmugh a bundle of muscle fibres stained with hamatoxyiin and eosin. Panel 6: Section as shown in figure 5, but stained for osteonectidSPARC protein.

Ml, M2 and M3, molar teeth 1.2 aud 3. respectively; AB, dvedar boue; D, dentine; C. cementun; ES, endosteal space; PL, pexiodontal ligament; P. dental pulp Figure 9.7-12 Immuaohistochernical Locaiization of Osteonectin/SPARC and Type III Collagen in Rit Dental Tissues.

Panels 7-12. Sagittal sections through the mandibulx incisor, including the discal mot of the second molar. Panels 7.9, I 1, and enlarged for the incisor ligament, Panels 8. 10. 12. Tissues were fixed in giutarddehyde and sections stained with haematoxyiin and asin (Panels 7 and 8). wi th ancibodies 10 eoiiagen type III (Panels 9 and 10) and osteonectin,SP.4.RC protein (Panels 1 1 and 12)

AB, alveolar bone; D, moIar dentine; ES, endosteal space; iD. incisor dentine; IL, incisor periodontal Ligament; P. moIar dental pulp; PL, molar periodontal Ligament. CHAPTER III C

PURIFICATION OF PROTEOGLYCANS: CS-FG II (DECORIN) AND CS-PG III AND SIALOPROTEINS: OfN AND BSP ROM E-EXTRACTS

INTRODUCTION

As discussed previousl y in Chapter III sections A and B. it appeared unli kely that osteonectin/SPA RC is a nucleator of h ydroxyapati te in bone but rather a mineral-associated protein aith unclear functions. To determine whether any of the other proteins could be better candidates for the nucleation of hydroxyapatite. it was of interest to determine whether the proteogl ycans and sialoproteins, which are prominent components of fetal bone could be classified as mineral-associated or collagenous-associated molecules. The presence of proteoglycans and sialoproteins in bone was first established by Hemng ( 1968, 1973). More recentl y, these molecules have since been isolated in an intact form and partially characterized. For the proteoglycans (Fisher et al., 1983a; Sato et al., 1985a), studies with fetai bovine bone have shown that there are two Iow rnolecular weight foms (called CS-PG 1 and CS-PGII) associated with the mineralized collagenous matrix. Further characterization has demonstrated that these proteoglycans differ in their amino acid content and in the number of chondroitin sulfate (CS) side chains (CS-PG 1: two-chain form; CS-PG II: one-chain fom) (Fisher, 1985). In addition, CS-PG 1 and II have been shown to be homologous to the dermatan sulfate-containine proteogiycans of cartilage (Hassel1 et al., 1986). However, the existence of additional proteoglycans in bone has been demonstrated (Franzen and Heinegard. 1985b). Two sialic acid rich, aridic (high contents of aspartic and dutarnic acid) proteins have ben isolated (Termine et al., 1% la). One of the molecules is now referred to as osteopontin (OPN) and the other as bone sialoprotein (BSP).Both migrate on SDS-PAGEgradient gels with M, values of 60-80 k. and stain blue with the cationic dye Stains-dl. Both proteins are phosphorylated (Pnnce et al., 1986; Butler, 1989; Nagata et al., 1991b; Kasugai et ai., 1993) and sulfated (Ecarot- Charrier et al., 1989; Nagata et al., 1991a; Kasugai et al., 1992). Complete amino acid and nucleotide sequences have been obtained for the rat bone sialoproteins from the corresponding cDNAs (Oldbeq et al., 1986; Oldberg et al., 1988b). Both proteins have (G)RGD(S) sequences through which they have been shown to mediate ce11 attachment (Oldberg et al., 1986; Oldberg et al., 1988a; Somerman et al., 1989). BSP is more acidic than OPN and contains greater amounts of glutamic acid and siaiic acid. Interesring1y, the BSP in rabbi t is reported to have a keraÿui sulfate gl ycosaminogl ycan side chan (Kinne and Fisher, lm. The objective of the studies described in this section was to determine wether the previously described proteoglycans and sialoproteins of bone could be predorninately separated into a mineral-associated or a collagenous matrix-associated group. This oecessitated an efficient purification procedure to obtain sufficient quantities of prote@ ycans and sialoproteins from fetal calvanal bone sa a detailed assessrnent of these proteins with respect to their potential role in the mineralization of bone wuld be made.

MATERIALS AND mHODS isolation and preparatiort of proteins from fetal porcine calvarin1 bone The protocml for the extraction of proteins is descri bed in detail in Chapter III A.

Sephorose CL-6B Gel Filtration and FPLC Chromatography (pulyunion. hydroxyapat ire and Mono Q) The proteoglycans were separated and purified as follows. Approxirnately 20(! mg dry weight of EDTA extract was dissolved in 10 ml of chromatography buffer (50 mM Tris-HCI. pH 8.0, containing 7.0 M urea, Buffer A). The mixture was clarified by centrifugation at 30,000 x g, for 30 min at 4'C and applied to two Sepharose CL-6B colurnns (2.6 x 95 cm each) mn in tandem. The columns were monitored at A20 nm and aliquots (5% pl) of the fractions (6.0 ml) anal yzed for @ycosaminoglycanslpmteog1yc;uis using the protocol of Farndale et al., ( lm). The fractions containing the bone proteoglycans (retarded on Sepharose CL-6B)were pooled. concentrated by ultrafiltration, treated with 18(vk) 8-mercaptoethanol, and applied to a FPLC-polyanion SI- 17 (preparative grade, Pharmacia) column (1 a 10 cm) at 3 rnlimin. The column was eluted with a linear NaCI gradient (0-1.0M) in Buffer A at a flou rate of 3.0 mlfmin. The fractions containing the proteogl ycans were pooled, diluted 1:3 with Buffer A, rapplied to the polyanion column, and eluted with a linear NaCl gradient (0-2.0 M NaCI) in Buffer A. The fractions containing the proteoglycans were poled and buffer exchanged into 10 mM Tris-HC1, pH 7.4, containing IO mM sodium phosphate and 7 M urea (hydroxyapatite chromatography buffer. Buffer B), using Pharmacia PD- IO columns (packed with Sephadex G-73.The sample was then applied to a hydroxyapatite column ( 1 x 10 cm) that was also operated on the FPLC system, at a flow rate of 0.15 mllmin. The proteogl ycans that were fractionated, were pooled separatel y as CS-PG II and CS-PGIII. Mono Q (Pharmacia) chrornatography was performed on the hydroxyapatite-purified proteoglycans in an attempt to purify them further. Aliquots were diluted in chromatography buffer (50mM Tris-HCI, pH 7.4. containing 7 M urea), applied to a Mono Q column (0.5 x 5 cm)washed with start buffer and eluted with a linear gradient of 0.51.5 M NaCl in chromatography buffer. Removal of the salt and denaturants was performed by desalting on Sephadea G-25 or dialysis in Spectrapor 4 (Spectrum Medical Industries Inc., Los Angeles, CA) dialysis membranes. Best recovenes of small amounts of proieoglycans were achieved using the micro-diaiysis technique descnbed by Overail ( 1987) -

Enzyme Digestions: Chondroitinuse AC and ABC Enzyme Digestions - Chondroi tinase AC and ABC digestions were perfomd as descri bed by Oi ke wi th the following modifications. Chondroi tinase AC (Sigma or Miles) or chondroi tinase ABC (Miles) was added (0.W5units) to 10 pg (protein weight) of proteogl ycan in O. 1 M Tris-HCl, pH 7.3, containing O. 1 M sodium acetate in 1.5 ml microcentnfuge tubes, and reaction solutions were incubated at 37°C for 2 hr. Reactions were teminated by the addition of SDS-PAGE sarnple buffer containing reductant (65 mM dithiothreitol) and heated ta 56°C for 30 min. Papain di gestions were performed to study the attached gl ycosami nogl ycans. Papain (0.5 units) was added to 10 +~g(protein weight) of proteogl ycans in 100 4 of O. 1 M sodium acetate buffer, pH 6.0, containing 5 mM EDTA,6 mM cysteine-HCI in 1.5 mi microcentrifuge tubes. Sol utions were incubated for 24 hr at 65'C and samples frozen and freezedned.

Polyacry lamide Gel Electrophoresis and Western Bioning A variety of techniques were utilized for the detection of the bone proteoglpns. SDS-PAGEwas canied out in either 5 4c linear polyacrylamide gels by the method of Weber and ûsbom (1975) or 5-20 % gradient cross-linked polyaqlarnide gels, using the discontinuous Tris-glycine buffer system of Laemmli (1970). Samples were dissoived in 10-50 of sarnple buffer containing 1% SDS, ?.O M urea, and bromphenol blue rnarker. Dithiothreitol, 15 mglml (wlv),or 1 9% (vlv) $-mercaptmthanol (final concentration), was added and the samples heated at 56'C for 30 min, or at 95'C for 5 min. Sixteen cm Rotean (Bio-Rad) or 5 cm Minislab (Hœfer) polyacrylamide gels with a thickness of 1.5 or 0.75 mm were us&. The minislab gradient and 15 % linear gels were electrophoresed for 16 hr at 7.5 mA, whereas the 16 cm, 5 8 polyacrylamide gels were electrophoresed for 30 min at 50 V, 36 mA,and then at 100 V, 74 rnA for 6 hr. Gels were stained with either 0.25 8 (wlv) CBB R-250, 1 % (wlv) Alcian blue in 9- % acetic acid, O.? 5% (wlv) silver nitrate or 0.ûX 46 (wlv) Stains-al1 as described previously by Domenicucci et al., ( 1988). Immunotransfer analysis (Western blots) was performed using a modification of the original method dearibed by Towbin ( 1979) or by the technique described by Pnngle (1985). Both nitrocellulose (BieRad) and charge-modified nylon membranes (Zeta-Rep. BieRad) were utilized.

Cellulose Acefnte Elecirophoresis Cellulose acetate electrophoresis for the separauon and identification of g1ycosaminoglyca.n~was performed using the method of Hamh and Krk ( l%7), as modifieci by Prout ( 1969). Approximatel y 100-200 ng of gl ycosarninoglycans were appiied to Beckman cellulose acetate electrophoresis membranes (Microzone membranes) and electrophoresed for 70 min at 6 mA in a solution of 0.7 M zinc sulfate. Strips were stained with 1 Q Alcian blue in 2 8 acetic acid for 10 min and de-stained under mming water.

Chernical A mlysis Purified preparations of proteoglycans were hydrolyzed in 5.7 N HCI contaming 0.4 96 phenol, for 21-24 hr at 1 1O0Cin nitrogen-nushed, screu-cap (Teflon-coated), acid- washed Pyrex test tubes. Aliquots of these hydrolysates nere analyzed for amino acid content using a Beckman 131M amino acid analyer. The level of hexosarnine was determined after hydrol ysis of samples for 16 hr at LûûaCin 4.0 N HCl (Pearson and Gibson, lm). Hydrolysates were applied to the amino acid analyzer on an extended short column and were eluted in 0.35 M sodium citrate buffer, pH 5.75, whch allowed for the separation and quanti tation of glucosamine and galactosamine. Sarnple content of uronic acid was determined using the protocol of Bitter and Muir ( 1963) scaled down 1 in 4, with glucuronolactone (Sigma) used as a standard. The presence of proteoglycans and/or glycosaminoglycans wsdetermined using the technique of Farndaie et ai., (1982). Briefiy, the sarnple (550 pi made up to 50 pi with H20) was dispenseci into a quartz cuvette ( 1.5 ml) of 1 cm path length. Dimethylmethylene blue solution (1.0 ml) was added and the contents rnixed once wi th a plastic msfer pipette. A bsorbance at 535 nrn was measured immediakl y dermixing. Solutions contai ning urea or guanidine HCI did not adversely affect the reaction. With chondroitin sulfate (whale cartilage, Sigma) as a standard, 1 pg gave an As5 nm di fference of 0.040. The values shown on the chromatographie elution profiles represent the actual Aus ,based on a 50 pl sample aliquot Men less than 50 sarnples were tested, the colour absorbance was adjusted for the dilution as follows: (Aus nm (sample) - As5 ,, (background)) n dilution factor. In later snidies, the modified version of this protocol was used (Farndale et al., 1986), with absorbance measured at 525 nm. Purification of Sialoproteim Fractions containing the sialoproteins in the 60-80 kDa region of the CL-6B Sepharose chromatography were pied and concentrated and equilibrated by dialysis against 7 M urea in 10 mM Tris-HCI, 10 mM sodium phosphate buffer. pH 7.4, and applied to a 1 x 10 cm oolumn of hydroxyapati te (BiuGel HTP. BioRad Laboratones) operated on a FPLC system at a flow rate of 0.35 ml/min. The proteins that bound io the hydroxyapatite were eluted at 0.5 mhin with a lin- gradient of 10-MO mM sodium phosphate. The fractions coniaining sialoproteins were pooled, diluted 1:2 with start buffer (7 M urea, 50 mMTridHCI, pH 7.4) and applied ont0 a Mono Q coiumn. The proteins were eluted with a linear salt gradient (0.0 - 1.0 M NaCl) at a flow rate of 1.O mllmin. Aliquots from the fractions were anal jzed by SDS-PAGE using 12.5 Lk or 15 9c cross- linked mini-gels and stained with Stains-al1 and CBB R-750to monitor sialoprotein purification (Domenicucci et al.. 1988).

Amino Acid Sequence Anabsis The arnino-terminal sequences of purified proteins were determined by applying lm-200 pg of protein to a gas-phase 470 "A" Rotein Sequencer (Applied Biosystems Inc.). Residues were identified and quantitated using high pressure liquid chromatography (HPLC), as descn bed in Chapter II 1 A

RESULTS

Bone-proteoglycam :CS-PG II and CS-PG III The purification of the mineral-associated proteoglycans and sialoproteins was achieved using gel filtration (Sepharose CL-6B) and FPLC (polyanion, hydrouyapatite and Mono Q). For the low molecular weight minerai-associated proteogiycans of bone. proteins were first enriched by chrornatography on Sepharose CL-6B and polyanion-FPLC (Figure 1). The addition of 5 mM N-ethylmaieimide to the extractant solutions resul ted in a 2-fold reduction in the amount of protein eluting in the V, of the Sepharose CL-6% chromatography (results not shoan). The inclusion of 1 % b-mercaptoethanol was also necessaiy for removing contaminating proteins of lower molecular weight (67 kDa and 40 kDa) that eluted in the fractions contai ning the proteogly cans. A fter repeared ion-exchange chromatography on polyanion resin, the proteoglycans were fractionated by chromatography on hydroxyapatite (Figure 2). As shown in Figure 2 the proteoglycans were fractionated into three pools. Pool 1 was found to contain CS-PG II, while pools 2 and 3, respectively, containeci PG III one fonn eiuting prior to (designated CS-PG IIIa) the second (designated CS-PG III b). From 100 g of fetal porcine calvariae approximately 1.1 g of E-extract (dry weight) was obtained and from this approximately 1 mg of CS-PGII and 0.8 mg of CS-PG III couid be recovered. The recoveries of CS-PG III however, varied depending on the techniques used to prepare these proteoglycans for analysis. Best recoveries were achieved by the dialysis of concentrated samples in the presence of a surfactant such as Brij 35. For srnall vohmes, the use of the microdiai ysis technique (Overall, 1987) in conjunction with dialysis against 10 mM ammonium bicarbonate contaming Brij 35 improved recoveries (ie 0.05% vlv in the first and 0.0005% v/v Brij 35 in the last didysis step). The proteoglycans present in the G 1-extract as well as that in the G3-extract, were also examined. Both extracts were processed as described for the E-estract: Sepharose CL- 6B gel filtration, FPLC with polyanion or Fast Q resin and hydroxyapatite. From the elution profiles on hydroxyapatite chromatography as well as SDS-PAGE,there was no indication of any proteoglycan with the properties of CS-PG III. However, some proteoglycan with properties similar to CS-PG II was observed (Chapter IV). Further experimentation identified the proteoglycan to be deconn based on similarities in chromatography on hydroxyapati te. chondroi tinase ABC generating a 45 kDa pink staining doublet and immunoreactivity with a monoclonal antibody to proteodematan sulfate proteoglycan (not shown). Notably, total proteoglycan in the G3-extract was only 5 8 of that isolated in the E-extract. Aliquots of the bone proteoglycans, some of which were pretreated with chondroirinase AC, were electrophoresed on gradient minislab gels and subsequently stained with a variety of reagents. The CBB R-250 and Stains-al1 srains are shown in Figure 3. Uniike CS-PG II and its protein core, neither intact CS-PG IIIa, CS-PG IIIb nor their core proteins, were stained with CBB R-250 (Figure 3 A). With silver staining, w hich is reported to be 10- lm-fold more sensitive than CBB R-29 (Meml et al.. 1982), "negative staining" of CS-PGIIIa and CS-PG IIIb was observed initially (i.e. the bands were clear mmpared to the background), but with increasing time this staining pattern merged into the background stain with no evidence of positive1 y staining the proteogl ycans (results not shown). Alcian blue stained ai1 the undigested proteoglycans but did not stain any of the protein cores (results not shown). With Stains-dl, ail the intact proteoglycans and the protein cores were stained. The protein core of CS-PG II çtained pink, contrasting the characteristic turquoise blue observeci for the protein cores of CS-PG IIIa and CS-PG IIIb (Figure 3B). As well, the high and low molecular weight markers used for the SDS- PAGE also stained pink with Stains-dl. The average M, of the proteoglycans estimated from the gradient 5-20 5% SDS- PAGE was approximately 110 k - 120 k for CS-PG II and 100 k - 110 k for both CS-PG IIIa and CS-PG IIIb The protein core of CS-PG II rnigrated aith M, of 45 k, whereas the protein core of CS-PG lIIa and CS-PGIIIb migrated with M, of 37 k - 38 k. On linear gels the M, of CS-PG IIIa and CS-PG III b were 30 k (results not shown), w hile that of CS-PG II was 45 k. In addition the core proteins of CS-PG IIIa and CS-PGIIIb on linear gels revealed a closel y spaced doublet when sufficient material was examined (3-3vg of protein weight; results not shown). Minor protwgl ycan species of larger M, were evident in the fractions of CS-PGII and CS-PG IIIb The minor proteoglycan associated with CS-PG IIIb gave rise to a protein core of 60 kDa - 65 kDa (see Figure 3 B, lane 8). whereas the one associated with CS-PG II, although not visible on Figure 3 B. was approximately 90 kDa These components are possibly dimers of the respective proteoglycans. However, if these are dimeric forms they are unlikely to be covalently linked through interchain disulfide bridges, since dithiothrei toi or 6-rnercaptoethaol were used to reduce the proteins prior to electrophoresis. Treatment of the three porcine cal varial proteog l ycans wi th chondroiti nase AC indicated that the attached gl ycosaminogl ycan on each type of proteogl ycan was chondroi tin sulfate (see Figure 3). Additional evidence for this was obtained by electrophoresis of the papiun-digested proteogl ycans on ceIl ulose acetate membranes where the free glycusarninoglycan chains in each case showed electrophoretic mobili ties similar to whale cartilage chondroi tin sulfate (results not shown). The relative levels of uronic acid, glucosamine and galactosarnine in CS-PG IIIa and CS-PG lIIb are shown in Table 1. The only difference between these proteoglycans was in the relative amount of glucosamine. However. there were distinct differences in the amino acid compositions between the two main types of proteoglycans. Whereas CS-PG IIIa and CS-PG III b had almost identical composiiions, CS-PG II differed rnarkedl y in the amount of charged amino acids present: CS-PG IlIa and CS-ffi IIlb contain 50% more Asx and Glx than CS-PG II. As well, CS-PG IIIa and CS-PGIIIb contained lower amounts of hydrophobie and aromatic amino acids. These and y ses were repeated on three different preparati ons of purifieci proteogl ycans with essentidl y identical resul ü obtai ned. Amino-terminal amino acid analysis was perfomed on CS-PG IIIa with the following sequence obiained: Asn-Pro-Val-Ala-Arg-Tyr-Gln- (average amino acid repetitive yield aas 78%). Attempts to sequence CS-FGII were not successful due to a blocked NH2-terminal amino acid. Bone-sialoproteins: OPN and BSP Purification for the sialoproteins also involved partial resolution initial1y by gel filtration. When analyzed by SDS-PAGE,a characteristic feature that was used for purification was the prominent Stains-ail blue staining, but poor CBB R-750 staming in the 60-80 kDa molecular weight range. A Sepharose CLdB chromatographic run demonstrating which fractions are enriched in sialoproteins is illustrated in Figure 1 A, Chapter 1II A. Figure 4 in this chapter demonstrates the chromatographic profiles of fractions from the gel filtration run that were subjected to chromatography first on hydroxyapatite (A) and then Mono Q (B). The fractions wntaining siaioproteins are represented by the bars. As shown in Figure 4 B the 'ragged' profile dernonstrates charged side-chain rnicroheterogeneity as resolved by die Mono Q resin (pH 7.5) for siaioproteins. The proteins were eluted in a linear sait gradient between 0.4 - 0.5 M NaCI. As illustrated by the inset of SDS-PAGE gels the staining with Stains-ail could be used to detect the blue-staining sialoproteins. Re-chrornatography on Mono Q for fractions represented in Figure 4 B (first and second bars) generated purified proteins (Figure 4 D, lane 1 representing OPN and Iane ? representing BSP). If, however, the original materiai pooled from in Figure 4 A aas chrornatographed under conditions of 7 M urea, 70 mM N-methyl piperazine (pH 4.5). three 'less-ragged' peaks could be resolved where the 1st of the 3 peaks was identified as OPN, the last peak as BSP and the middle peak as a 75 kDa unknown protein (results not shown). The arnino acid compositions of purified OPN and BSP are shown in Table II. In general the amino acid compositions were similar for both purified proteins in that they exhibited a hi@ content of acidic arnino acids. OPN was charactenstically rich in aspartic and senne whereas BSP showed higher levels of glutamic acid cornpared to aspartic acid. From 100 g bone it was found that 5.5 mg OPN, 7.1 mg BSP and 2.8 mg of the 75 kDa protein could be purified. Amino-terminal acid sequence analysis of the purified OPN and BSP proteins confirmed their relaiionship to the corresponding hurnan (Fisher et al., 1987a; Kiefer et al., 1989) and rat (Oldberg et al.. 1986; Oldberg et al., 1988b) proteins and also to the mouse (Fet et al., 1989) protein (Figure 5). For the porcine OPN, identities between species in the first 30amino acids were 75.55 and 60 9 for human, rat and mouse, respectively, and for porcine BSP, 85 '7c when cornpared with both human and rat. As with the proteoglycans isolated from G3-extracts, the total sialoprotein in the G2-extract represented -5 B of that isolated in the E-extract. Based on chromatographic behaviour (FPLC:hydroxyapati te and Mono Q) , SDS-PAGE and protein staini ng behaviour (silver nitrate, Stains-ai1 and CBB R-2X) under reducing and non-reducing conditions) and immunoreactivity on1 y BSP was presen t (results not shown). DISCUSSION

In this s~dytwo small chondroitin sulfate proteoglycans, CS-PG II and CS-PG III, and two sialoproteins, OPN and BSP, were purified from fetal porcine calvariae E-extracts. The pun fïed proteins were used for more detailed characterization (Goldberg et ai., 1988; Zhang et al.. 1990) including the preparation of antibodies to the sialoproteins to study their biosynthesis (Nagata et al., 199 la) and tissue distribution by light (Chen et al., 1991b) and electronmicroscopy (Chen et al., 1994). When compared to core protein of the proteogl ycan isdated by Fisher et al. ( 1-a) from bovine bone, which is homologous with the core protein of the iduronic-rich proteodematan sulfate (PDS)proteogl ycan present in bovine skin, periodonial ligament and gingiva (Pearson and Pringle, 1986; Pearson et al., 1983),CS-PG II was show by immunoblotting to have a similar core protein. The dermatan sulfate proteogl ycans (DS-PG)are aiso related to the DS-PG II isolated from cartilage (Hassel1 et al., 1986) that are dso recognized by the trivial narne "decorin"(Ruoslahti, 1989),reflecting the discrete association of these proteogl ycans to cullagen fi bnls (Pringle and Dodd, 1990). The core protein of decorin has an M, of -38 k, characterized by eleven leucine-rich repeats, with a singie gl ycosaminoglycan chain attached near to the amino terminus (Neame et al ., 1989). Thus, based on the size of the proteoglycan and the composite protein and glycosaminoglycan chain, together with its immunoreactivity, CS-PG II of bone appean to be the mineralized tissue equiulent of decorin, perhaps differing only in the absence of iduronic acid in the carbohydrate moiety. Notably, from bioqnthetic studies the CS-PG II has been show to associate with the collagenous matrix prior to mineralization (Nagata et al., 1991a). The second small proteoglycan, which was recovered in two fons, appears to be novel and has been named CS-PG III (Goldberg et al., 1988b) in keeping with the current nomenclature for small proteoglycans CS-PG 1 biglycan and CS-PG II (decorin) found in cartilage (Rosenberg et al., 1985) and other tissues (Vogel and Fisher, 1986; Heinegard et al., 1985). Characteristical1y the core protein of CS-PGIII does not stain with CBB R- 7K), pmbabl y reflecting the acidic nature of the protein, but li ke the bone sialoproteins stains blue aith Stains-dl. Although the amino acid composition is simiiar to the sialoproteins (Zhang et al., 1990), the lack of any sequence sirnilarity or cross-reactivity of specific antibodies would appear to mle out the possibility that CS-PG III is a proteogl ycan fom of either OPN or BSP. Although CS-PGIII has not been reported in bone of other species its unusual staining characteristics and lack of binding to nitrocellulose membranes for immunobloaing may explain why this proteoglycan may have ken overlooked in previous studies. However, based on chromatographie behaviour, it may be related to a proteogl ycan HTP 1V identified in rat bone (Franzen and Heinegard, 19&la). 1nterestingl y, recent cornpansons of the amino terminal amino acid sequence of CS-PG III have shown a high level of identity wi th the coiresponding sequences in BAG-75 (Gorski and Shimizu, 1988) and dentin matrix protein (George et al., 1993). Although the size of these proteins are markedl y different they muid share a cornmon evolutionary on gin. 1t is notable that dermatan sulfate protmgl ycans are not presen t in mineralized tissues of bone whereas chondroitin sulfate counterparts to both biglycan and decorin have been identified (Fisher et al., 1983a; Franzen and Heinegard, 1984a; Vogel and Fisher, 1986; Goldberg et al., 1988b). This may be explained by the lack of the epimerase needed to convert the giucuronic acid into the iduronic acid found in dermatan sulfate (Silbert et ai.. 1986) in differentiated osteoblasts. Since the dermatan sulfate proteogl ycans are expressed by differentiating bone ce11 cultures (Beresford et al., 1987), it has been suggested that the lack of this epimerase may be a marker of differentiated osteoblasts (Nagata et al., 1991a). The sidoproteins OPN and BSP were found to have similar physicochemical propenies to their counterparts isolated from bovine bone (Fisher et al., 1983b; Franzen and Heinegard, 1985a). The characteristic behaviour of these sidoproteins on gel electrophoresis, including their slow migration, absence of staining with CBB R-250 and blue staining with Stains-ail, reflects the acidic nature of these proteins and the presence of phosphate groups (Heinegard et al., 1986; 1988a; Fisher et al., 1983a; 1987a; Rince et al., 1987). Biosynthetic studies have also reveaied the presence of signifiant arnounts of sulfate on both proteins (Nagata et ai., 1989; 199 la; 1991b; Kasugai et al., 19P-), which has ken located to tyrosine residues in mouse BSP (Ecarot-Chder et al., 1989). Despite the presence of keratan sulfate in rabbit BSP (Kinne and Fisher, lm),no glycosarninoglycan component was associated with the BSP isolated from porcine bone (Zhang et al., 1990) al though the presence of BSP with an aitached chondroi tin sulfate has been indicated in demineraking extracts of rat dentine (Steinfort et al., 1994). The amino acid sequence of the arnino terminal region of BSP shows high identity with other species (Oldberg et al., 1988; Fisher et al., 1987a; lm)and reflects the high conservation of the arnino acid sequence throughout the molecule. as deduced fmm the subsequent cloning of the porcine cDNA (Shapiro et al., 1993). Conserved regions include several series of polyglutarnic acids sequences, whch are believed to mediate the attachment of the protein to hydroxyapati te crystals, and sites of tyrosine sulphation around an RGD motif (Shapiro et al ., 1993) that can mediate ceIl attachent (Oldberg et al., lgûûa). In cornpanson, the amino terminal sequence of the porcine OPN shows a lesser degree of sequence conservation when compared to other species (Zhang et al.. 1990). This also reflects a lower sequence conservation in the rest of the molecule (Wrana et al., 1989) when compared to OPN frorn other mammalian species (Oldberg et al., 1986; Craig et ai., 1W;Kiefer et al., 1989) and chick OPN (Moore et al., 199L). Nevertheless, the polyaspartic acid sequence, through which OPN binds hydroayapatite (Oldberg et al., 19%). the thrombin susceptibility site (Senger et ai., 1988) and the RGD motif, involved in ce11 attachent (Oldkrg et al., 1986; Somerman et al., 1988).are ail consend together wi th several potential si tes of serine phosphorylation. The development of polyclonal antibodies that specifically recopze the porcine bone OPN and BSP (Zhang et ai., 1990) have been used to demonstrate the tissue specificity of these proteins (Chen et al., 1991a) as well as aid in the identification of the proteins in biosynthesis studies (Zhang et al., 199û; Nagata et al., 1991a). The tissue distribution of porcine BSP was found to be essentidly restncted to rnineralized connective tissues of bone, cartilage and teeth (Chen et ai., 1991a; 1991b) consistent with the anaiysis of BSP mRNA expression (Oldberg et al., 1988a; Bianco et al., 1993; Shapiro et al., 1993). hmuno-electronrnicroscopic studies have further show BSP localized to the eari y mineral crystailites in the osteoid of feial porcine bone (Chen et al., 1994). In contrast, studies of porcine OPN revealed a more widespread distribution, as obsened in other species (Butler, 1989; Denhardt and Guo, 1993), but was consistent with sites of mRNA expression (Nomura et al., 1988). At the elecvon microscope level the OPN is found concentrated at the mineraiizing front and in cernent and reversal lines (McKee et ai., 1995; Chen et al., 1994). Frorn the chamcterîzation of the two sidoproteins it is evident that the. share many similarities (Sodek et ai., 19m; 199nb). Both are glycoproteins of similar size that are su1 fated and phosphorylated. They bind Gaz+ and hydros yapatite and mediate ce11 attachment. However, they differ markedly in their tissue distribution and their temporal expression during bne formation (Chen et al., 19=a; 1993) which has impomt implications with respect to their functions in the process of mineralization, as will be discussed in Chapter VI. : ri W 'CC ';t

Figure 1: Chromatography of the EDTA Extract and Isolation of ProteogIycans.

A. 200 mg of EDTA estract was dissolved in 10 ml of Buffer .A (50ml1 Tns-HC1. pH 8.0, containing 7.0 M um).sample clarifieciby cmcrifugation. and applied to two Sepharose CL-6Bwlumns in tandem (2.5 x 95 cm. each). The columns were run at 20 ml%at 22°C. and 6.0 mi fractions were collecteci. Fractions mntaining glymsaminoglycansiproteog1ycans wae pooled (bar) and concentrateci by ultrafiltration. B. approximaiely 7.0 ml of wnanmted proteoglycan solution was nduad witb ~mergptoethanol.then applied to a Sephamse CL4B colurnn (2.6 x 100 cm)and elutcd at 22'C in Buffèr 4 at 20 ml hr. Fractions of 4.0 ml were collected and those fractions mntaining the proteoglycans, as determined by Ans,, (a-) were pooled. V,. fraction 13: V,. fraction 120. C .the proteogl ycans from the Sepharose CL-6B re- cbromatograph y (B) above, were du& with ~mercaptwthanol.and applied to a Pol yanion oolumn ( 1 S 10 cm).The colurnn was washed with Buffer A, and sarnples were eluted with a SaCi gradient (0-2.0M) in Buffer A. Fractions of 1.4 ml were collected and those conüüning the proteoglycans (Ans nm . *.) were pooled. D. the pooled proteoglycan fractions from C above were diluted 3-fold with Buffer A. and reapplied to the Polyanion column. ïhe proteoglycans were eiuied from the column and collected as described- Figure 2: Further Puri ficatfon of Fetai Porcine Calvarial Protcoglycans by Hydroxyapatite Chromatography .

The piedproteogl ycan preparations obtained lrom pot y anion chomatography (Figure 1 D) were desal ted on PD- IO colurnns and applied to a hydroxyapatite column ( 1 s 10 cm),at a flow rate of 0.25 ml min. The proteoglycans were eluted with a Iinear mentof sodium phosphate ( 10-500 di).at a flow rate of 0.25 m1,'rnin. The proteoglycans were eIuted with a Iinear gradient of sodium phosphate (10-500 IL!^), at a fîow rate of 0.5 mi,min. .LUiquots (10-50pi) of the fractions ( 1 .O ml) wwe anaiyzeci for proteoglycansi glycosaminoglycans(.A535 am .L--) p001ed as shosn, dial>zedagauist water and freeze-dried. Figure 3: SDS-polyacrylamide Gel electrophoresis of Bone Proteoglycans.

Aliquots of the purifieci proteoglycans from the chromatography on hydroxyapatite were veated with chondroitinase .4C (Sigma) and approximately 1.5 pg (protein equivalents) were applied to @en t gels (5- 20 %) on the rninislab system. The lanes are as follows: 1, high moledar weight standards; 2, low motecular weight standards; 3 and 4, CS-PG 11; 5 and 6,CS-PG IIIa; 7 and 8, CS-PG III b; lanes S. 6,and 8 were the sped"iedproteoglycans treated with chonciroi tinase AC, which contains dbumin (67 kDa). A, SDS pol yacrylamide gel stained with CBB R-250. B. SDS-polyacrylamide gel stained with S tains-dl . Note that the -70 kDa band seen in figure 3 B, lane 8 is ûelieved to represent a dimer of the origuial35 kDa are materiai. See text for details. These analyses were perfomed by Dr. H. Goldberg. Fmctlon No.

Figure 4: Purification of Fetal Porcine Boue Sialoproteins by FPLC.

A. hydroxyapatite chromatographg ( 1 x 10 an)of an enricheci sample contairing siaiopmteins (Chapier III A, Figure 1 A, pool 2 fmm CL-6B run) on a column of hybxyapari te. Proteins were eluted [rom the column witb a linear giadient of 0.01-0.5 bl sodium phosphate and the absorbance monitored at 230 rn with 1.0 ml fractions collected. Bar represents region analyzed by SDS-P.4GE for sialoproteins and fractions pooled for subsequeflt chromatography on Xlono Q. Note that the sialopteins eIuted (rom the hydroxyapatite in a hoad peak and produceci a broad Stains-dl positive blue band - 67 kDa as shown by the corresponding SDS-PAGE andysis of Figure 4 C flanes 14 represesr fractions analyted through the chromatogram represented by the bar). B. Mono Q chromatography of saxnples enriched in sidoprotein as represented in the bar in Figure 4 A. Proteins were eluted from the column with a iinear gradient of 0- 1 .O M M. Aliq~tsfrom the fractions were analyzed on 15 8 SDS-PAGE gels and Stains-aiI used to detect the blue-s*g sialoproteins as above. Note that the BSP was separateci from the OPN (SPP-1) on Mono Q as shown in the amespondhg SDS-P.4GE analysis of sampIes taken from each end of the pooled pcak lanes 1 (bar rqrresented as "SPP-I" on the Mono Q chromatograrn) and 2 (bar represented as "BSP*on the Mono Q chrornato~of Figure 4 D. Xote that the eariier eluting OPN (SPP-1) was reveaied as a sharp band and the BSP was revealed as a broad band close to the 67 kDa marker. LM,, LW markers. 1 ~umui ED E EOC Y RE ED P 71ELUC; BSP

OPN

Figure 5: Amino-terminal Amino Acid Sequence Aaalysis of OPN and BSP.

The amino acids are represented by the single Ietter code. The human BSP sequence was derived from the corresponding cDNA nucleotide sequences. .hnoads that were identical in tbe corresponding psitions for ail species have been piaced in thick-Iined blocks, and conservaiive replacements in tbin-lined blocks. Alignments made to maumize homologies required the assumption that a single amino acid has been lost in position 16 at the amino temiinus of moue OPN. Table 1: Amino Acid Analysis of Porcine Boue Protcoglycans

CS-PG II

Asx 154 Thr 55 Ser 131 Glx 215 Pro 55 G~Y 134 Ala 63 CYS N.D. NmDm Val 24 Met N.D. N.D. N.D. Ile 6 6 Leu 50 49 Tyr 12 16 Phe 9 9 His 24 22 LYS 30 28 =g 39 35

Pro teinb I 1 I Uronic Acidc 1. 7 949 .882 Gaiactosamined NeDe ,876 ,904 Glucusamined NeDe .O73 .O54

8N .D., not determined. bAmount of protein determined by amino acid analysis. cReIative amount of the respective constinients to protein by weight. W. D., CS-PG II was contarninated with an indeterminant amount of degraded proteoglycan or glywsaminoglycan. Amino acids are in residues per 1000. Table II: Amino Acid Anmlysis of Porcine Boue Sialoproteins: OPN and BSP Cornpirisons 4th the Compositions for the Humaa Prottins

porcine

protein

Asx 160 207 Thr 71 57 Ser 109 80 Glx 133 226 Pro 57 57 G~Y 66 132 Ala 67 59 =Y= N-D-C N.D,C Val 60 18 Met N.D.C N-D-c Ile 19 9 Leu 72 29 Tl?= 4 42 Phe 30 Il ais 42 14 LYS 65 33 -g 43 24 lamino acid corn psition from Fisher et ai., 1990. barnino acid composition derived from OPN cDNA from Zhang et al., 1990. N .D .c not detennined. Amino acids are in residues per 1000. CHAPTER III D

PN-PROPEPTIDES ASSOCIATED WITH THE DIFFERENT COMPARTMENTS OF BONE: COMPARISONS WITH SERUM-DERIVED PN-PROPEFTIDE

INTRODUCTION

The interstitial collagens of connective matrices (collagen types 1, II, III) are synthesized in precursor foms that are characterized by globular extensions which function in the formation of the procoilagen moleniles and facilitaie their transport through the ceIl (for review see Rockop et al., 1979). The procollagen peptides also appear to be involved in mllagen fibril formation extracellularly, but in most cases are subsequenily cleaved by specific proteinases (Leung et al., 1979). The occurrence of propeptides that remain associated with type I and III mllagen fibrils has been observed in embryonic and adult connective tissues (Mandell and Sodek, lm; Sodek and Mandell, 1982) where they appear to be located aithin, or on the surface of thin fibrils (Fleischmajer et al., 1981 ; Fleischmajer et al., 1985). Moreover, both ami no and carboxyl extensions of type 1 collagen have ken shown to be associated with extracellular fibrils in embryonic bone and are beiieved to regulate fibril formation and size (Fleischmajer et al., 1987). Procollagen peptides released from procollagen molecules by proteolytic processing are resistant to degradation as indicated by their abundance in fetal blood (Rohde et al.. 1976). Potentiai functions for the procollagen peptides have been indicated in a number of studies. For example, the propeptides of type 1 collagen. the amino propepude in particular, can inh bi t the transcription and subsequent translation of the collagen gene and may, therefore, be important in regulating collagen synthesis by a feedback inhibition rnechanism (Paglia et al., 1979; Weistner et al., 1979; Aycock et al., 1986; Wu et al., 1986). In addition. the carboxyl-terminal propeptide of type II collagen has been observed to be e~chedin mineralizing cartilage (Poole et al., 1984); where, under the control of vitarnin D metabolites, it is thought to ifluence calcification (Hinek et al., lm. In bone and dentine the collagen in the mineralizi ng matrix is almost entirely type 1 collagen, the fibrils of which appear to form a iemplate for the formation of the hydroxyapatite crystals (Glirncher, 198 1). However, despi te the spatial relationship that exists between collagen fibrils and hydroxyapatite crystals, as discussed earlier collagen itself is not a good nucleator of hydroxyapatite crystal formation. Consequenrly, tiere has been considerable interest in the characterization of other mineralid tissue proteins that may act as nucleators of hydroxyapatite crystals in association with the collagen fibres. During the course of characterizhg some of the prominent molecules constituting fetai porcine bone extracts, it was obsewed that there were proteins ( wi th molecular weights - 2û k) in both the E- and G2extracts of bone that stained poorl y with CBB R- 250, stamed blue Stains-al1 and were susceptible to bacterial collagenase. Because of these properties some of these molecules were funher characterizai to detemine wether they could be candi date nucleators. Isolation. purification and comparative characterization of similar molecules present in the E- and G2-extracts was pursued with a preliminary identification of these molecules as pN-propeptides of type 1 collagen. As was described previously (Chapter 1) there are subde differences between hard and solt tissue-derived collagens, however it is not known if there are an y di fferences between hard and soft tissue-derived pKpropeptides: differences whch could ultimately help explain why bone collapn mineralizes. Because of the presence of mu1 tiple forms of pN-propeptides in bone, it was important to determine which of these forms represent endogenously bone synthesized pN-propeptides or serum- derived propeptides. This was considemi because of the high circulating quantities of pN- propeptides in serum which could be incorporateci into bone prior to andor during the mineralization process. Thus a cornparison of the properties of semm-derived pN- propeptide with those of the pKpropepdes isolated frorn bone was perf'mned so chat the origin of the isolated bone pN-propeptides could be determined. Moreover, charactemation of these molecules codd help el ucideate their potential involvement in the nucleation of hydroxyapati te of bone collagen.

MATERIALS AND DHODS

Extraction Procedures For Bone Proteins The procedures followed for extmcting bone proteins from fetai porcine calvariae are similar to those descri bed in chapter II (Dornenicucci et al., 1988). Briefly, calvariae were carefully cleaned of soft tissue. fmen in liquid nitropn, and fragmented in a mortar and pestle. The fragments were washed ovemight with PBS in the presence of protease inhibitors (Pr),and exmted sequentially with G-buffer, E-buffer and then again with G- buffer. A tissue to extractant volume of t :2ûû was used with PBS washes inçluded between the extractions.

Punpcation Procedwes for Bone pN-Pmpopti& Proteins in each extract were concentrateci 200-foid by ulMltmtion on a Y M- IO Diaflo membrane (Amicon Corp., Lexington, MA) and dialyzed against water and freeze- dried. Approximately 200 mg freeze-dned maienal of the E-extract and 60 mg of G2 (equivalent to 20 g and 100 g, respective1y, wet weight of bone) was dissolved in 4 M GuHCI, 50 mM TrisIHCl buffer, pH 7.4, (5-8 ml), clarified by centrifugation (30,000ug; 45 min) and fraaionated on tandem columns (1.6 X 95 cm) of Sepharose CL-6B. Proteins eluting in the M, 20-35 k region from die E-extract and from the G2-extract were pled separately, concentrated by uluafiltmtion and the buffer eirchanged by dialysis against hydroxyapatite start buffer ( 10 mM TridHCl, pH 7.4, containing 7.0 M urea and either 1 mM or 10 mM PO$-; prepared as a buffer consisting of 19 4c NaH2HP04 and 81 & Na?HPOJ). Aliquots of 5-15 ml (equivalent to 75 9 of the pooled region from CL-6B) were applied to a KTP-hydroxyapati te (BioRad) column ( 1 X 10 cm) that was pre- equilibrated in start buffer. After applying the sarnple at a flow rate of 0.25 mllmin, the column was washed with 15 ml of start buffer at a flow rate of 0.5 mlhin and the proteins eluted with a linear gradient (1 or 10 mM - MO rnM) cf FQ3- at 0.5 mllmin using the FPLC system (Pharmacia). Fractions containing proteins that showed susceptibility to bacterial collagenase were further purified by chrornatography on Mono Q resin. The collagenous peptides in hydroxyapatite buffer, were diluted 12with 50 mM TrislHCl. pH 7.4, containing 7.0 M urea, and applied to an analytical column (0.5 X 5 cm) of Mono Q resin (Pharmacia). The proteins were eluted wi th a linear gradient of NaCl (O- 1.O M) at a flow rate of 1.0 rnlhin. The purity of the protein preparations was assessed by SDS-PAGEin Tris/glpcine buf fers on 15 Lk linear crossli nked mini slab-gels (Hoder). with 5 Q stacking gels, using CBB R-750,Stains-al1 and silver nitrate staining to visualize the proteins. Fractions containhg the proteins of interest were desalted, either by chromatogaphy on Pharmacia PD- 10 columns, equilibrated and eluted in 0.5 M acetic acid, or by dial ysis (Spectrapor 3 ;3.5 kDa cusoff) against 7 changes of 100 mM ammonium bicarbonate containing 0.058 (vlv)Brij 35, and then a single dialysis against one-tenth strength buffer.

Preparation of Senun-derived pN- Propeptide Fetal pigs were obtained from a local abattoir and within 7 hr of slaughter 40 ml of blwd was collected. The blood was allowed to dot and the serum separated by centrifugation at 4,000 rpm at 4'C. A total of 27 ml of semm was recovered. A ? M urea, M mM Tris HCI (pH 7.4), serum suspension (41.5 ml) was obtained by adding appropriate volumes of stock solutions to the isolated semm (Tris Stock 50 mM Tris HCI, pH 7.4; urea Stock: 8 M freshl y de-ionized urea). The suspension was dari fied by centrifugation at 1,600 rprn for 5 min and was further processed by chromatogaphy ( first by CL-6B gel filtration then by hydroxyapatite, Mono Q and HPLC ). Pwification Procedures of Serum-derived pN-Propeptide Using a QAE filter cartridge (BioRad) that was washed with 200 ml of lûading buffer (2 M urea, M mM Tris HCI, pH 7.4), preparatory ion-exchange chromatography was performed on the 41 -5 ml of clarifieci 9 M urea serum suspension. The serum was Ioaded onto the filter at a fiow rate of 10 rnlhin and non-band proteins eluted with a funher 100 ml of loading buffer. Using a step gradient, and 10ml each of the loading buffer containing 0.15 M, 0.20 M, 0.25 M, 0.30 M and 1.0 M NaCI. bound proteins were eluted. Four, 35 ml fractions were colIected per step. Fractions were rnonitored by using the strong silver staining behaviour of pN-propeptides when run on SDS-PAGE under reduced vs non-reduced conditions (see Figure 4). A fraction enriched with pN-propeptides (3ml) was concentrated by ultrafiltration (YM 10 membrane) to a volume of 5 ml and subjected to gel filtration using CLdB Sepharose under denaturing conditions of 4 M GuHCl. Enriched fractions, as determined by SDS-PAGEaere pooled, concentrated and exchanged into 10 mM phosphate buffer and run on FPLC hydrovyapatite chromatography. Non-bound fractions were pooled, concentrated and exchanged into 1 mM phosphate buffer by repeated ul tmfiltration and subjected to FPLC hydroxyapati te. Bound fractions were directly subjected to anion-exchange FPLC cchromatopphy using a Mono Q column under denaturing conditions of 7 M urea The pN-propeptide was punfied to homogeneity on HPLC using a C3 reversed phase column (trifluoroacetic acid/acetonitrile gradient). Fractions containing purified protein were then freeze-dned before lurther analysis.

Amino Acid Amiysis This was performed on a Beckman 121 MB anal-r using a program to ailotr. for separation of hydroxyproline from aspartic acid, followi ng h ydrol ?sis of the samples under nitrogen in 5.7 M HCI, containing 0.4 4F phenol, at 1 10°C for 20-22 hr.

Amino Acid Sequencing Fifty pg aliquots of SCAB 3b protein, one of which was treated wi th pyrogiutamate arninopeptidase (Boennger Mannheim, West Germany). in an enzyme:substrate ratio of 120, were subjected to sequence analysis on an Applied Biosystems gas-phase Sequencer. lmmunononrfer AM1ysi.s The basic rnethod of Towbin et al. (1979) was used with some modifications. Proteins were first separated by SDS-PAGEon 15 % cross-Iinked polyacrylamide gels and then electrophoreticaily transfened over a 1- 1.5 hr period to nitrocehiose or charge- mochfied nylon (Zeta-prep, BioRad) in 39 mM glycine. 48 mM Tris, and 20 % methanol, at 0.8 mA/cd of gel, using a LKB Multiphor II Nova Blot apparatus. The membranes were incubated for 90 min with 3 % BSA (for nitrocellulose) or overnight in 10 % "BLOTTO" (for nylon). The proteins on the membranes were probed with specific rabbit antisenim raised against sheep type 1 amino-propeptide (generously provided for these studies by Dr. Rupert Timpl, Max-Planck-1 nstitut fur Biochemie, Martinsried bei Mtinchen, West Germany). Protein-bound antibod y was detected using peroxidase-linked sheep anti-rabbit IgG antiserum (BioRad) and rlchloro 1-naphthol (BioRad) as substrate. Fol lowing electrophoretic transfer some mem branw were incubated with 45Ca2+, to stud y calcium binding (Mamyama et al.. lm),using porcine osteonectidSPARC as a control.

Bacterial Collogenare Digestion Digestions with highly-punfied bacterial collagenase were carried out as describeci previousl y (Kuaata et al., lm),using an enzyme to substrate ratio of 1: 10, and incubation times up to 30 min., at 37'C.

Cyanogen Bromide Cleavage

Freeze-dned protein samples were dissolved in 700 pl of 70 Q (ulv) formic acid. flushed with nitrogen for 1 min, and approsimately I mg of cyanogen bromide cqstals added to 50 pg of protein. The contents were flushed briefly with nitrogen, and then incubated at 2530aCfor 4 hr with occasional shaking. The solutions were then diluted 20- fold wi th water and freezedried. Samples were redissolved in water and freezedried again to remove residual cyanogen bromide.

Bone pN- Propeptides Bone proteins in the initial 4 M GuHCl (Gl-extract), in the subsequent 0.5 M EDTA (E-extract) and in the second 4 M GuHCl (GZextract) estracts were fractionated on Sepharose CL-6B under denaturing conditions. Aliquots frorn each fraclionation were analyzed by SDS-PAGEbefore and after digestion with bacterial collagenase to identify collagenous proteins. mer than interstitial type 1 cotlagen a chains, no other collagenase- susceptible proteins were readil y apparent in the G 1-extract (resul ts not show). In contrast, in both the E-extract and in the G3-extract a prominent protein, M, of 19 k, generated by collagenase digestion, could be identified (Figure 1). Of note, SCAB 1 and 2 proteins in the E-extract are not readil y visualized by ei ther CBB R-250or silver staining. The collagenase-susceptible 28 kDa proteins were purified to apparent homogeneîty by chromatography, first on hydroxyapatite then on Mono Q resin. The E-extract protein on hydroxyapatite was separated into a retarded (pool 1, SCAB 3a) and a bound (pool II, SCAB 3b) form, using 10 mM PO$- in the start buffer (Figure 1 A). These elution profiles were unchanged when SCAB 3a and SCAB 3b were re-chromatographed on hydroxyapatite. InteresUngly, SCAB 3a (Iike G2-28 kDa protein and serum-derived pN- propeptide) can bind COhydroxyapite under low phosphate concentrations and denaturing conditions of 7 M urea, but require higher phosphate concentrations in order to el ute adsorbed molecules. Following bacterial collagenase digestion of the bound protein (SCAB 3b), the 19 kDa collagenase-resistant fragment generated was found to bind hydroxyapatite and eluted at approximately 100 mM Pû$-, indicating that the hydroxyapatite-binding properties of this protein reside in a non-collagenous region of the molecule (result not shown). The G2-estract 28 kDa protein (G?-28)behaved like SCAB 3a on hydroxyapatite when start buffer contained 10 mM PO$-. However, neither SCAB

3a nor G2-28 bound to hydroxyapatite when start buffer contained only 1 mM POj3-, and were both eluted at approximately 100 mM Q3-(Figure 28). These proteins could be extensively purified by this two-step procedure on hydroxyapatite. However, final purification was achieved on Mono Q as shown in Figure 3C. Notably, the proteins were eluted at increasing concentrations of NaCl in the order: GZWSCAB 3a and then SCAB 3b. The purified proteins, SCAB 3b and G3-28Da, gave a single band on SDS-PAGE, migrating with an M, of 23 k under reduced conditions and 27-18 k under non-reduced conditions, and al1 stained strongly with silver (Figure 3). Each proiein aras degraded to a 19 kDa fragment with bactenal collagenase, as shown aith the material from the CL-6B chromatography (Figure 1). indicating the presence of a collagenous region of approximately 9 kDa. Also, each protein, including the bacteriai collagenase-derived 19 kDa peptide. stained blue with Stains-all, indicating that these proteins are either phosphorylated, highly acidic or both. Treatment with neurarninidase to remove sialic acid failed to cause a shift in molecular weighf or affect Stains-al1 staining. For cornparison the amino acid compositions of porcine bone-denved and bovine skin-derived a 1 pN-propeptides are shown in Table 1. When compared to the porcine protein, the bovine protein demonstrated significant differences in Asp, Ser, Ala, Cys, Tyr, Phe, His and are believed to reflect species differences. The 38 kDa and SCAB 3 proteins however were similar to bovine a 1 pN-propeptide, in that they al1 characteristically showed a high content of acidic arnino acids. The contents of glycine, hydroxyproline and proline were consistent wi th approxirnatel y one- hrd of the molecules having a collagen-li ke structure. Although the amino acid cornposi tions of the porcine bone-derived pN- propeptides were sirnilar, there were differences in the Ala and Pro values for SCAB 3b when compareci to other pN-propeptides. Also, cornparison of the G2-18 kDa and SCAB 3 protein compositions demonstrated differences in Hyp and Tyr. The absence of methionine was confirmed by the raistance of the proteins to CNBr cleavage. Attempts to sequence the bone-proteins were unsuccessful indicating a blockeù amino- terminal amino acid, aithough a low yield consisting rnostiy of acidic amino acids were obmned through the fim 10 cydes. In addition, the treatment of the proteins with pyrogiutamate arninopeptidase before sequencing did not i mprove the yields si gnificantly. For funher chamcterization. the proteins were separated on SDS-PAGE and electrophoretically tiansferred onto either nitrocellulose or modified nylon membranes. The transferred proteins were then tested for irnmunoreactivity against a specific antiserum to the sheep a 1 (1) pN-propeptide. Transfer to the modified nylon was found to be superior. Non-reduced 78 kDa proteins s howed strong cross-reactivi ty to the propeptide anti body w hereas the reduced proteins were less reactive (Figure 3); a resul t consistent wi th the propenies of the antibodies in this antiserum (Rohde and Timpl, 1979). Of note, the sarne antibody was able c specifically immunoprecipitate [W]-glycine radiolabelled procollagens and pKpropeptide synthesized by porcine calvarial bone cells in vitro (results not shown); the latter peptide king generated by treatment of the culture medium with CNBr. Although electrophoretically transferred osteonectidSPARC in reduced and non-reduced foms bound 45Ca2-, none of the 28 kDa collagenous proteins showed affinit~for Ca--, under these conditions. Based on amino acid anal ysis at least 0.1-0.2 mg of SCAB 3 b protein, and 0.5- 1.O mg each of SCAB 3a and G2-18 proteins could be purified from 100 g wet weight of bne. These recoveries were comparable to recoveries of the major nonsoIlagenous proteins extracted from bone under the same conditions. When fetal bone was extracted with 0.5 M EDTA, in the absence of denaturants, and the proteins fractionated on Sephacyl-200 under non-denaturing conditions, a collagenase-sensitive protein with the same characteristics on SDS-PAGE as the 78 kDa protein was identified in fractions that eluted with an M, of 70-80 k. On subsequent fractionaiion of this material on Sepharose CL-6B under denaturing conditions, the protein CO-eluted with the 28 kDa protein extracted under denaturing conditions, indicating that, pior to denaturation, this protein exists in a tnmeric form, as would be expected for procollagen peptides. Sem-derived pN-Propepride SDS-PAGE analyses of effluents from the preparatory QAE chrornatographic run reveaied that al1 of the semm pN-propeptide was located in the second 25 ml collection of the 0.3 M NaCl step gradient (Figure 4, lane 4). Demonstration of the intense sensitivity of the pN-propeptide to silver staining under reducing conditions and pwr staining under non-reducing conditions is also shown (Figure 4, lane 2 vs lane 4). This peculiar staining behaviour with silver however, facilitated the and ysis of the protein's chrornatographic behaviour. The semm pN-propeptide was also show to be the only proteidpeptide in the fraction to stain blue with Stains-ai1 (results not shown). Sepharose CL43 separation of protein from pooled and concentrated fractions from the QAE preparatory chromatographic run 1s shown in Figure 5. SDS-PAGE analyses of collected fractions through the chromatographic run when stained with silver under reducing vs non-reducing conditions are also shown (Figure 6 A and B. respective1y). The bar show in Figure 5 and the arrow illustrated in Figure 6 indicates fractions that were concentrated and exchanged into 10 mM hydroxyapatite start buffer. Hydroxyapatite chromatographp under Ioading conditions of 10 mM POJ3- revealed that pN-propeptide muid not bind to the column (Figure 7 A, bar). When non- bound fractions were concentrated and exchanged into I mMhydroxyapati te nart buffer and re-chromatographed (Figure 7 B) it was found that bound proteins were ennched with pN propeptide (Figure 7 B. bar). When pooled fractions were direcdy applied ont0 a Mono Q column and chromatographed by FPLC (Figure 7 C) a sharp peak which eluted between 0.17-0.19 M NaCl was found to contain fractions ennched with pN-propeptide. Pmled fractions from the Mono Q mn (Figure 7 C, bar), when directiy applied onto a C3 revened phase column and chromatographed by HPLC (Figure 7 D). demonstraied a sharp symmetrical peak eluting at 34 ck acetonitrîle. SDG-PAGE analysis with silver staining under reducing vs non-reducing conditions dernonstrated pure protein (Figure 8). A composite chromatogram of bone pN-propeptides and serumderived pN- propeptide on Mono Q resin îs shown in Figure 9. SCAB 3b when compared to SCAB 3% GI-28 and senun-derived pN-propeptide demonsmted a greater retention elution time when chromatographed on Mono Q resin. Interestingl y, the on1y discemable difference between SCAB 3a, G2-Z3 and sem-denved pN-propeptide îs that SCAB 3a and G?-28 exhi bi t doublets upon chrornatography w hereas the serurn-derived pN- propeptide is resolved as a single sharp peak. Further cornparisons between SCAB 3%G2-33 and senirn-denved pN-propeptide are shown in Figures 10. 11, 12 and 13. From these studies similarities in SDS-PAGE behaviour, staining properties (especially wi th silver nitrate under reducing and non-reduci ng condi bons), bacterial collagenase suscepti bility , raistance to CNBr cleavage and immunoreacti vi ty to pN-propeptide an tibodies are evident. In addition amino acid compositions were comparable suggating thar each of these molecules represents forms of pN- propep tide.

DISCUSSION

Using a dissociative extraction procedure Fisher et al. ( 1981b) reportecl that a phasphorylated form the cx 1-pN propeptide of type 1 procollapn comprises 5 % of the 'non-collagenous" protein of developing bovine and human bone. Results of these studies have demonstrated that small collagenous proteins in fetai porcine bone are present in substantial amounts and have the physical, chernical and immunological properties of a 1 (1) pN-propeptide, thereby confirming and extending the obsenations of Fisher et al. ( lm). By using a seqwntial extraction procedure to separate the mineral-binding proteins [rom those proteins associated ui th the underlying collagenous rnamx, evidence for the existence of several forms of this protein in bone has dso ken provided. Two forms appear to be assmiated with the hydroxyapatite cqsstals, since they are extracted by EM'A. One form (SCAB 3b), like a number of the other proteins in the E-extract, binds to hydroxyapatite in the presence of protein denaturants and 10 mM P0.13-; whereas the second fonn (SCAB 3a) is not bund under these conditions. A third form (G2-28)is associated with the collagnous matrix that is expûsed foilowinp demineralization, but othenvise its behaviour on hydroxyapatite chromatography is similar to SCAB 3a. The difference in behaviour of the three forms of the pN-peptlde on hydroxyapatite and on Mono Q chromatography could reflect either truncated foms or differences in post-translational modification, such as phosphorylation. Evidence for differences in phosphorylation is indicated by the variable amounts of phosphoserine found in the bovine and human proteins (Fisher et al.. 1987b). However, since 3?Pû43- incorporation into collagenase digestible protein synthesized by rat ostmblastic cells could not be documented (Kubota et al., 1989) the degree and significance of pN-peptide phosphorylation is unclear. Based on distribution, immunoreactivity, chromatographie propemes and the lack of susceptibiliiy to CNBr. the a 1 (1) pN-peptide foms do not appear to be related to the 25 LDa and 28 kDa SCAB proteins (SCABs 1 and 2) described previously (Kuwara et al., 1987). In addition, since the minerd binding proteins, including osteonectin/SPARC and the other small cdlagenous proteins (SCABs 1 and 2) are quantitatively extracted with EDTA in the procedure used, the presence of the propeptide associated with the demineralized coiiagen rnatnx (ie- G2-28)dœs not appear to be due to inadequate extraction of the rnineraiized bone with EDTA. Consequendy, this would imply that the different foms of this protein have vwng affinities for both hydroxyapatite and the underl ying minerali zi ng collagenous matrix. The presence of appreciable amounts of the amino-propeptide of ype 1 procollagen in porcine and bovine (Fisher et ai., lm)bone, is interesting in view of the potential activity of this molecule in regulating calcification and collagen synrhesis in mineralized connective tissues. Although the mechanism of hydroxyapatite crystai nucleation and growth in minedized comective tissues is not known, it is known that the hydroxyapatite crystals are siniated predominantly in close proximitp to the gap region between successive collagen molecules in fibril structures (White et al., 1977). As found in embryonic bone (Fieischmajer et al., 1987) and dentine (Sodek and MandeIl. 1982) procollapn peptides, would lie in the gap region ahere they could influence the calcification process, either negatively or positively. The propeptides may be cleaved subsequently, but some (SCAB 3b) could remain associated with the mineral crystal through an affinity that is derived from pst-translationai modifications, while other foms (SCAB 3a and G3-3)may bridge between the mineral and the collagenous matrin. Because the aminopropeptide of type 1 collagen cmact as a feedback inhibitor of collagen synthesis by connective tissue cells (Paglia et ai., 1979; Weistner et al., 1979; Wu et al., 1986), a role for the propeptide in bone remodelling is also indicated. It is conceivable thar propeptide released from mineralized bone tissue during osteoclastic resorption could inhibit the synthesis of collagen by neighbouring osteobiastic cells, ternporarily blochng the synthesis of new bone in that location. The similarities between the SCAB 3a, G2-28 kDa and serum pN-propeptides isolated are remarkable. Thus it is possible that at least sorne of the SCAB 3a and G?-3-8 kDa could be derived from sem. The G2-28 kDa pN-propeptide could have been absorbed during the pre-minerdized stage in bone formation and the SCAB 3a pN- propeptide dunng the rnineralizing phase of bone formation. Alternativeiy, SCAB 3a could represent an adsorbed form of the sem-derived pN-propeptide that was released in the E-extract whereas the G2-'S kDa protein represents spillover from inefficient extraction with E-buffer. However, it is also possible that SCAB 3a and G2-78 kDa pN-propeptides are produced endogenously during bone formation as altemate forms of SCAB 3b. This possibility is supported by the knowledge that the =,-type I collagen chan in type 1 coilagen can be phosphorylated in bone collagens (Glimcher, 1981) but not in soft tissue associated collagens. As such, assays selectively detecting SCAB 3b in serum could be ernployed to selectively measure nomai and pathological bone turnover, and to rnoni torhest for bone diseases Iike osteoprosis. Interestingly, like CS-PG III and OPN, there was no detectable SCAB 3b in the G?-extnct, indicating that it is a minerai-associated fom of pN-propeptide and that i t is extmcted efficient1 y wsth the extmction procedure. Based on these preliminary findings it is believed that the pN-propeptides isolated from bone are unlikely candidates for the nucleation of bone collagen. Figure 1: SDS-PAGE Analysis of Srnall Collagenous Proteins Extracted from Bone.

Mineral-associated proteins extracied hmfetal porcine bone with 0.5 EDTA (Esxuact) and proteins from the underiying demineralid maaix exaa~tedwith 4 $1 GuHCl (G2-extniçt) were fractionated on Sepharose CL-6B. SDS-PAGE anaiysis (15 % cross-Linked gel) of the small hl, proteins stained with silver are shown from the E-extract (E) and G2cx-t (G). The collagenous prottins were idenliried by susceptiùility to bafferial cdlagenase (Ec) and (Ge). .4ltbou& the mntmst is somewhat strong to clearly see proteins in some of the lanes. the amws indicate the position of a 23 kDa protein which was degraded to 19 kDa fragment by ~Uagenase.hlr, moldar weight &er pmtàns. hmc~L6.sampla from every third fraction (fiactions 125- 140). 6 d~fracrion.from the CL-oB cohmn. O 20 40 60 Volume (ml)

Volume (ml) Figure 2: Purification of the Small Collagenous Proteins from E- and G2-extracts of Boae on Hydroxyapatitc.

The fractions from Sepharose CL-6Benriched in the 28 kDa coIIagenous protein were pooled, çoncentrated to 10-20 ml and dial yzed against hydroxyapatite start buffer. A. E-extract (representing 25 % of the protein extracted from 100 g bone) applied with a start buffer containhg 10 mM PO&. Fractions enriched in SCAB 3a were then pooled, exchanged inio 1 mM PO$- and chromatographed on hydroxyapatite. A profile similar to ihai illustrated in 28 was obrained. where the previousl y non-bound protein was adsorbed onto the column under a lower phosphate concentration (results not shown). Bound fractions were then chromatographed on Mono Q see Figure 9. B. G2-extract (representiag 10 % of the protein extraccd from 100 g bone) applied wiih a start buffer containing 1 mM PO$-. Bound fractions were then chromatographed on Mono Q see Figure 9. See Figure 9 for composite elution profiles of the different pools of SCAB 3a.bIG2-28 proteins on .Mono Q chomatography. NOTE: Bars represent regions çontaining the various pools of pN-propeptides proteins. Figure 3: Characterizatfon of the Purifid Small Collrigenous Proteins

SDS-PAGE (15% camposi te gel) analpis of small collagenous protMs. SCAB 3b protein (Lane 1) digested wilh bacterial dagemse(km&); bactaid cdlagcoase aione. Lin&. SCAB 3b stained blue with Stains-al1; note that although the background is hi&, a broâd 28 kDa band can be seen. Irmes.SCSCAB 3b non-reduced and reduced. respectiveiy, transferred ro a modif~ednyion membrane and incubated with rabbit initibodies to sheep a 1 0)propeptide. hus&axi8, immunoreaction of oon- rediiced and &ced G2-28 procein. W.19 kDa collagenase resistant fragment from G2-28.M. 02-28 kDa pmtein after hydroxyapatite chromatography. ~1.2.stainedwiih silver. Bi,. Figure 4 Prepratory Fractionation of Fetal Porcine Serum by Ion Exchange Chromatography on a QAE Filter Cartridgc

Profiles are from fractions enriched in pN-propeptide using a ; 03 51 NaCl Step gradient. SDS-PAGE (12.5 % cross-iinked mini-gei) nin under reducing and non-reditcing conditions for proteins stained with silver. W.LW markers; I;me~&, 10 10 aiiquots respectively from the 2nd and 3rd fractions (25ml each) coilected through the 0.3 Xi SaCl step gradient nui under non-reducing conditions; Lane 4 10 pi aiiquots from the 2nd and 3rd fractions (25 mi each) collixted hughthe 0.3 hl NaCl step gradient nrn under reducing conditions. Note the prominent staining of a 28 kDa band in Iane 4 when nin under reducing conditions and the apparent lack of any detection of this mareriai when run under non-reducing conditions (Lane2). The 2nd fraction (35mi) collected througb the 0.3 hcli SaCl step gradient represents materiai that was used for subsequent fractionation on gel filtration. .kow muking position of p'i-propeptide. Note. mow shows position of s- p~-mpep~de.

40 60 80 100 120 140 160 Fractlon no. (6 ml) Figure 5: Chromatogrsm of Partially Fractionated Fetil Porcine Serum on CL68 Sepharost. hfdeof pN-propeptiQ-enriched fractions from the QAE ion exchange cartridge (Figure 4, Lad)run on Sepharose CL- under the denaturing conditions of 8 M GuHCT. Flow rate of 20 mlih was used with 6 ml fractions collected. Bar represents fractions containhg protein eruiched in pN-propeptide. Figure 6: SDS-PAGE Analysis for Serum pN-propeptides Separated by Cbromatography on Sepharose CL-6B under the Denaturing Conditions of 4 M GuHCI.

SDS-PAGE (12.5 % aoss-linked gel) nin under reducing A) and non-reducing B) conditions for proteins stained with silver. 1,MVmarkers; Limes, are 10 pl aliquots from 6 ml fractions collected through the CL-6B profde. Note the prominent staining of a 2û kDa band in lanes indicated by dotted bar (Figure 6 A) when run under reducing conditions compared with the poorer staining in this region when run under non-reduang conditions (ianes indicated by dotted bar, Figure 6 B). Fractions represented by lanes indicated by dotted bar were pooled, concentrated and exchanged inio I O mh4 hydrox y apati te start buffèr in preparation for FPLC. Note: arruws depict position of senun pN-propeptides. O 20 40 a] 80 Fractlon na (1.5 ml)

f O 20 30 40 voîumo (ml) Figure 7: FPLC and HPLC Chromstograpby of Serum pN-propeptide Pooled After CL-6B Gel Filtration.

Figure 7 A, Hydroxyapatite chromatography of fractions pledas shown in Figures 5 and 6 and KKI under denaturing conditions using 10 mM phosphate in the starting buffer and 500 mM phosphate in the eluting buffer. Non-bound fractions were found to be emiched in pN-propeptide and were pooled (as shown b y the bar). concentrateci and exchanged into 1 raM hydroxyapatite start buffer for re-chromatography. Dotted lines represait linear phosphate gradient

Figure 7 B. Hydroxyapatite chromatography of fractions as shown in Figure 7 A and nia under denaturing amditions using 1 rnM phosphate in the starting buffer and 500 mM phosphate in the eluting buffer. Bound fractions represenled by the bar were found to be enriched in pN propeptide. Fractions were pooled and directly loaded onto a Mono Q anion exchange column. Dotted lines represent phosphate linear gradient

Figure 7 C. Mono Q anion exchange chromatography of fractions pledas show in Figure 7 B and nm under denaturing conditions using 0.0 M NaCl in the starting buffer and 1.0 M NaCI in the eluting buffer. 'Ihe sharp peak eluting between 0.17-0.19 M NaCi was found to be enriched in the pN-propeptide. The bar represents f'ompooled and directly loaded onto a C3 reversa! phse HPLC column. Dotted lines represeat linear sa1 t gradient. Figure 8: SDS-PAGE Analyses Demonstrating Purfty of pN-propeptide.

SDS-PAGE(12.5 % cross-linked gel) run under reducing and non-reducing conditions for proteins stained with silver. TaneLW%'markers; lane 2 pg of fetal porcine senim pS-propeptide purified by C3 reverseci phase HPLC and nin under reducing Oane 2) and non-reducing (lane 3) condi rions.

1.2 0.6 1 serurn pN propeptlde

volume (ml)

Fîgure 9: Composite elution profile of SCAB 3a, SCAB 3b, G2-2û kDa protein and serurn pN-propeptide ran on FPLC Mono Q chromcitqraphy.

Note single peak for serum pN-propeptide (. ...) wiuting wi& the major peaks for G2-23kDa protein (hatchd) and SCAB 3a (solid line), both of which have a minor second peak. Figure 10: SDS-PAGE Analysis of pN-propeytides.

Cornparison of purified SC.G 3a, G2-28kDa protein, and serumderived pS-propeptide using SDS-P.AGE under reducing and non-reducing conditions. Lane 1. LLW markers; Lanes 24,the G2-28kDa protein. SC.- 3a and serum pN-propeptide,respectiveiy ,nui under reduang condi tiom and srained with silver. Lam 5-7. same proteins ntn under non-reducing conditions. Sote the different apparent molecular weight and the differential staining intensity betweea reduced and non-reduced sampleç.

Figure Il: Collagenase Sensitivity of pN-propeptides.

SDS-PAGE ( 15 % Tris-giyciw, stained with silver) anal pis undw reducing conditions demonstrating the sensitivity to bactena1 collagenase at various time intervals. Lane 1, LMW markers; Lana 2.7 and 12: Vehicle (Collagenase Assay Buffer CAB);Lanes 3.8 and 13: original G2-28kDa protein. SCAB 3a and senun pN-propeptide. respectively. @or to dagenase digestion; Lanes 4.9 and 14: O min treaûnent with mlagenase; Lanes 5. 10 and 15: 5 min treatment with çollagenase; and Lanes 6, 1 1 and 16: 30 min treatrnent with collagenase, respective1y. CoIlagenase digestion is very rapid as is evident in laaes 4.9 and 14. h. h. ---- - ZCc Figure 12: Susceptibility of pN-propeptides to CNBr.

SDS-PAGE (15 % Tris-glycine. stained with silver under reducing conditions) analysis demonstrating the resis~ceto the tnament with CI\;Br. LLIU'markers; Lanes2.4and6: original G2-28kDaprotein, SCAB 3a and serum pS-propeptide respective1y prior to ueatment with C';Br. Lanes 3.5 and 7: origtnal G2-28kDa protein. SCAB 3a and serum pK'-propeptide. respectively after treatment with ChBr.

Figure 13: Immunorenctivity of pN- propcptides.

SDS-PAGE(15 % Tris-glycine. stained wi th silver under reducing conditions) dysis demonstrating the immunoreactivity of G2-28kDa protein, SCAB 3a and senim pN-propeptide to specific antibodies against ph'-propeptides Note the greater immunoreacti vi ty against these proteins under reducing çondi tions w hen cwipared to non-reduQng conditions. LWmarker Lane; Lanes 1 -3: G2-28 kDa protein, SC.G 3a and serum pN-propeptide respective1y tested under reducing conditions; Lanes 4-6: G2-28 kDa protein, SC.- 3a and senun pN-pfopeptide,respective1 y, tested under non-reducing condi tions. Table 1: Cornparison of Amino Acid Compositions Between Bone and Skin pN-propeptides.

(A) porcine tmne SC.4.B 3a; (B)pane bone SCAB 3b (C)porcine bone G2 îû kDa (D) calf-skin pK-jxopeptide (from Horiein et al., 1979)

WT' ASS Thr ser GIS Pro G~Y -4ia Cys Val Met ile Leu TF Phe His Lys -kg

ND not determined. amino acids are in residues per 1000. CHAPTER IV

IDENTIFICATION, PURIFKATION AND CHARACTERIZATION OF THE MAJOR PROTEINS FROM THE G2 EXTRACTS OF MINERALIZED CONNECTIVE TISSUES

Since hydroxyapatite is associated with cdlagen fibrils it rnighr be anticipated that proteins involved in nucleation in vivo, including collagen iwl f, would be associated wi th the collagenous matrin, wherûas those proteins that modulate crystal growth would be associated with the mineral crystals. However, the proteinaceous molecules that associate with collagen have not been clearly identi fied. Thus, an extraction procedure was developed to isolate non-collagenous proteins from different tissue compartments comprising boneldentine matnx (see Chapter II). Briefl y, in this procedure 4 M GuHCl is first used to extract non-cowdentiy bound proteins from osteoid/predentine (G 1-extract) and 0.5 M EDTA is then used ro release proteins bound to the hydroxyapatite crystals (E- extract). Following the demineralization step, proteins previously masked by the mineral that are non-covalentiy associated with the collagnous matris are then içolated wi th a second series of extractions wi th 4 M GuHCl (G1-entract). As descri bed in Chapter 1II. this procedure aas shown to release the major proteins associated wi th the mineral phase in fetai porcine bone whch have been subsequentiy characterized (Domenicucci et al.. 1988; Goldberg et al., 1988b;Zhang et al.. 1990). However, based on the premise that hydrogapati te nucleation would i n\.olve a col lapn-bundfi nieracting/associated protei n, studies were focused on the identification of proteins in the G3-extracts of bone and dentine. SDS-PAGEanalysis of the G?-extract revealed two proteins wi th M,s of 32 k and 24 k, which appeared to be absent in the E-extracts, that were common to the rnineraiized connective tissues and comprised the major G2-proteins in dentine (Chapter II. Figure 3 C). In view of these findings, attempts were made to purify and characterize these proteins to determine whether they could be candidates for HA nucleation.

MATERIALS AND METHODS

Isohrion and Preparation of ProteimJrom Fetai Porcine Bone The procedures for preparing fetai porcine cal variae and for isolati ng bone-associ ated proteins were described in detail in Chapter II with some modifications. Subsequent to demineralization of the tissue with 0.5 M EDTA, the bone chips were exhaustively washed with PBS and then re-extracted with 4 M GuHCI, 50 mM Tns-HCI, pH 7.4 (G71-extract). This was followed by exhaustive washes with PBS, and the chips re-extracted wi th 4 one-week extractions with 4 M GuHCl buffer (labelled G2 II, 111, IV, V, VI-extracts, respective1y) and tissues prepared for analyses as discussed in Chapter II1 A. For the quantitative isolation of GZproteins [rom root dentine, lower i ncisors were demi neralized as described in Chapter III A and the residue exhaustively washed with PBS. The demineralized tissue was distributed equally into five, 50 ml polypropylene screw top centrifuge tuband then extracted again with 40 ml 4 M GuHCl in Tris buffer (G3-extract) by shaking gently on a platforrn shaker at 1 revhec. Following centrifugation, four more G2 extractions were pedormed and pledseparately. Pooled extracts were then concentrated ?O-fold (UM- IO membrane) and prepared for FPLC chromatography.

Gel Filrration Chromatography with Sephorose CL-6B and Sephacyl S-200 Resins Gel filtration chromatography nas performed in 4 M GuHCl as described previousl y (Chapter III A). Al1 column chrornaiography runs were monitored for protein by rneasuring As0 nm, and Amnm, and by analyzing aliquots of the collected fractions on SDS-PAGE.Roteoglycan wcs determineci by Farndale anal ysis (Ass nm)as descn bed by Goldberg et al.( l988b).

FPLC Chrornatography A11 hydrosyapati te runs were as described in Chapter III D and were performed at 21' C over 3.5 hr using a loading flow rate of 0.25 mllmin and an eluting flow rate of 0.5 mlhin and the absorbante king monitored at 230 nrn. Both bound and non-bound fractions from the hydroxyapatite chromatography mns were subjected to anion-exchange chromatography using an HR 515 Mono Q (Pharmacia) column as described previously (Chapter III). Highly purified proteins from Mono Q ruswere pooled and subjected to HPLC reversed phase chrornatography. For anal yticd purposes (involving ei ther collagenase-sensi ti ve proteins, proteogi ycans, sialoprotein-like protei ns) , pools from the CL-6B gel filtration mns were also subjected to anion exchange chrornatopphy employing Fast Q Sepharose (Phannacia) on the FPLC system. Briefly, pooled fractions were exchanged in start buffer (M mM Tris-HCI buffer, pH 7.4, containing 7 M urea) and eluted with a Iinear gradient of 0-2 M NaCl in start buffer. The column was developed at a flow rate of 3 mllmin and 1.4 ml fractions were collected. Proteins were monitored at 230 nm and samples were anaiyzed by SDS-PAGE. To determine the pI of partially purified 32 kDa proteins from the G2 I-estract. samples were subjected to FPLC chromatofocusing (pH intemai 4-5) employing an HR 5/20 Mono P (Pharmaaa) column. Briefly. freeze dried samples ( 100 pg) were dissolved in 0.5 ml 25 mM N-methylpiperazine (pH 5.7) start buffer, filtered (0.23 w),and applied onto a pre-equilibrated column for a pregradient volume of - 10 ml ( flow rate: 1 mllmin). The sample was then eluted at the same flow rate with 10% Polybuffer 74, pH 4.0, HCI for a total eluate volume of -25 ml. Runs were moni tored at Am nm and by anal yzi ng aliquots of the collected fractions on SDS-PAGE.The determination of pH through chrornatographic runs were detennined using a pH meter (Radiometer AI S. Copenhagen, Denmar k). For the dentine preparation, the first G?-extract was exchanged into 7 M urea, 10 mM Tris-HCII 10 mM PO&, pH 7.4, buffer by repeated ultrafiltration through a YM- IO membrane (Amicon Corp., ) and then filtered through a protein-blocked, 7 M urea washed 0.2 prn Nalgend disposable filter. The filtrate containing - 1 mg protein in 10 ml buffer was subjected to chromatopphy on hydrosyapatite as described in detail previously (Goldberg et al., 1988a; Goldberg et al., 19ûûb) using either 1 mM or 10 mM P043- in the starting buffer and 500 mM PO$- in the eluting buffer. Al1 separations were performed at room temperature (31°C) with a flow rate of 0.25 mllmin for loading and 0.5 mllmin for elution. Absorbnce was monitored at 9-30 nm. Both the bound and unbound fractions were then fractionated by anion-exchange chromatography on a HF2 515 Mono Q (Pharmacia) column as detailed previously (Chapter III). Briefly, a linear gradient of O - 1.0 M NaCl in 7 M urea, M mM Tns-HCLbuffer, pH 7.4, at a f2oa rate of 1.0 ml/rnin was used to elute the proteins, which were monitored at A,, ,,.

HPLC Reversed Phase Chromrography (Beckman Sysrem Gold ) Pooled samples from Mono Q fractions were loaded directly ont0 a Beckman UltraporeN C3 reversed phase column. Purification of proteins was achieved by employing a linear gradient of @ 100% (vlv) solvent B (containinp O. 1 C/c trifluoroacetic acid in acetonitrile) in solvent A (containing O. 1 Q tnfiuorœcetic acid in H20)over M min at 2 1'C. A constant flow rate of 0.5 mlimin was used and the absorbance was monitored at both 230 and 180 nm. Fractions (0.5 ml) were collected using a Gilson FC 3-03 fraction collecter. Peaks of interest were pooled, freeze-dried and subsequently resuspended in appropriate solutions for further anal y sis.

Reduction Md AUyIation Fractions enriched with 32 kDa protein were reduced and alkjlated as descriùed by Wu et al., (1971) with some modifications. Briefl y, 100 pg of protein was dissolved in 200 pl of 6 M GuHCl - 0.2 M Tris-HCl buffer, pH 8.0 containing 100 molar excess of dithiothreitol per mole of disulfide (assuming 6 disulfides per molecule; 290 pg dithiothreitol per 200 pi incubation buffer was added). The sample was purged for 20 sec wi th nitrogen and left at 31'C for 3 hr. At the end of 3 hr a I :1 molar ratio to total sulfhydryls of Cvinyl pyridine was added (assuming II sulfhydryls per molecule; 4.3 rl of a 95 % pure solution of Cvinyl pyridine (Aldrich] was added and left at 71 'C for a funher 3 hr. At the end of the incubation, removal of the reduction and alkylation buffer from proteins was achieved by re-chrornatography on a Beckman Ulmporem C3 reversed phase column as demibed above. Appropriate fractions were pooled and freeze-dned for preparative gel electrophoresis.

Prepnrotive Gel Electrophoresis Preparative gel electrophoresis was used to separate the 33 kDa protein from contaminants. A BRL 1100 PG preparative gel electrophoresis system was used, as descri bed by Zhang et ai., ( 1990). wi th some modifications . A pproximatel y 200 rg of freeze-dried sample (from either bone or dentine) was dissolved in 100 pi 4 x SDS-PAGE sarnple buffer and Ioaded onto a 6 cm 10 4c cross-linked polyacqlamide tube gel and electrophoresis performed in the presence of O. 1 9 sodium dodecyl sulfate (SDS)at 200 V for 3 hr. using the discontinuous Tris-glycine buffer system of Laemmli ( 1970). Electrophoresis was stopped at the end of 3 hr to allow for the attachment of the collection apparatus and then electrophoresis was then resumed for a further 3 hr. Using the sarne Tris-glycine buffer contaming SDS,an elution/flow rate was set at 43 mllhr and 7M) lil fractions were collecteci. The separateci proteins were detected by SDS-PAGEand appropriate fractions were pled and proteins freed from SDS as described below.

Remval of Sodium Dodecyl Sulfae from Pro teins by Ion- Pair Extracrion Prior to ion-pair extraction, pooled samples from the preparatory gel electrophoresis runs were dialyzed against two changes of 0.01 9c SDS,0.01 M ammonium bicarbonate solution at 31'C over 16 hr. After noting the approximate volume of the diaiyzed samples. the samples were then freeze-dried and subjected to ion-pair extraction by the method described by Henderson et al. ( 1979). Briefly, a minimum of 100 pi of freshly-made extraction solvent mntaining reagent grade acetone: trieth ylamine: acetic acid: water (86: 5: 5: 4, vlvlvlv) was added per ml of diaiyzed sample (thereby keeping the final SDS concentration below 10 mghl). The mixture was kept on ice for 1 hr and briefly centrifuged at 10,000 rpm in a microfug for one minute. The supernatant was decanted from the protein pellet which was then washed twice with reagent grade acetone and the removal of any residuai acetone was then achieved by vacuum desiccation. 100 pg of purified 32 kDa protein was subsequently cleaved with CNBr as described in Chapter III A and prepared for sequene analysis.

Anulytical Gel Electrophoresis (SDS-PAGE) Analyiical polyacrylarnide gel electrophoresis was performed in the presence of O. 1 4c SDS using 2 M urea 5-20 ';E gradient rnini-gels and 7.5 %; 15 %; 10 4c T,3 ck C; and 16.5 8 T,6 % C continuous gradient mini-gels (on a Hoefer Scientific 6 cm mini-gel Instrument). Sarnples were prepared for anal y sis under reducing and non-reducing condi bons using the discontinuous Tris-gl ycine buffer system of Laemmli ( 1970) for the 2 M urea 5-20 B gradient gels and 7.5 9c and 15 5% continuous gradient gels as previously descri bed in Chapter II 1. Addi tionall y the discontinuous Trisftricine buffer system of Schagger and von kgow ( 1987) was used for the 10 % T,3 8 C and 16.5 % T. 5 Q C gels. The mini-gels were mn at 150 volts for 1.5 hr. Gels were then stained and de-stained either individuall y or consecutively wi th silver nitrate first, then Stains-dl, and final1y wi th CBB R-250 as described previously (Chapter III). The only modification in staining procedures involved gels mn under the discontinuous Trisitncine buffer systern. In this case the 100 ml. 3 u 10 min 75 9isopropy l alcohol washes preceding staini ng with Stains-al1 were replaced with 3 x 10 min buffer exchanges containing a solution of 15 Q isopropyl alcohol, 7.5 C/c formamide and 30 mM Tris-HCI; pH 8.8. The M, of proteins were dculated by using M, standards (Pharmacia)or pre-stained M, marker proteins (Bio-Rad Laboratones).

Western Bloning and Amino Acid Sequence Amlysis On Immobilon P* Polyvinylidene DiJluoride (PVDF)Membranes Preliminary amino-terminal sequence analysis of the 32 kDa and 34 kDa proteins were unsuccessful, apparently due to N-terminal blockage. Consequentl y, reduced and dkylated proteins were trated with CNBr to pnerate peptide fragments that cuuld be sequenced. Briefly, 40 pg of CNBr-generated peptides from the 33 kDa or 24 kDa proteins were mn under reducing conditions usinp 16.5 % T, 6 8 C Tridtricine gels as described above. After electrophoresis the gel was placed in 300 ml of transfer buffer (39 mM glycine/ 48 mM Tris-HCI; pH 8.9) for 20 min. Meanwhile, an Immobilon PVDF membrane was prepared as described by the manufacturer and equilibrated in the transfer buffer for 15 min. Peptides were electrophoreticaily transfemed for 2 hr at 0.8 mA/cmz onto the Imrnobilon membrane using an LKB 2.1 17-93Nova %lot Systern. The PVDF membrane was rinsed in distilled water and stained in a 0.2 % CBB R-qS/ 45 % methanol/ 10 4c acetic acid solution. The sheet was de-stained first for 2-5 min with 100 ml of 50 % methanoIl 10 % acetic acid and then for a further 1-3 min with 100 ml of 90 9% methanoIl7 CPt acetic acid. The membrane was washed 3 .u with 400 ml of H20 for 5-10 min, blotted dry with Whatman Elter paper and then ailowed to air dry for 1-3 hr. Selected bands were cut out with clean scissors and stored at -20°C.Direct sequencing from the PVDF membrane was achieved by employing a gas-phase 470 A Rotein Sequencer (Applied Biosystems). The amino acid phenylthiohydantoin derivatives were identified and quantified by HPLC. To determine the identity of the 24 kDa protein and its isofoms, sarnples of the individual proteins were first separated by SDS-PAGE on 10 % T, 3 8 C,Tris-tricine gels and transferred ont0 an Irnmobilon-P membrane using a modification of the onginal method by Towbin et al. ( 1979). After transfer of the proteins the membranes were incubated with 3 % bovine semm albumin to block non-specific sites, washed and then incubated in a 1: 1000 dilution of rabbit anti-porcine TRAMP anti-senun (generously provided by Dr. David S.J. Hulmes, University of Edinburgh, Scotland). Immunoreactive protein was identified using goat anti-rabbir IgG horse-radish peroxidase conjugated second antibodies and the substrak 4-chloro- 1-naphthol (Bio-Rad).

Amino Acid Amlysis Using a minimum of 5 pg per sample, proteins for amino acid anal ysis were prepared as previously described (Chapter III A). Briefi-, sarnples were hydrolysed for 24 hr at 110' C with 5.7 M HCl contaîning 0.4 47c (vk) phenol. Solutions were dried in vacuo and the hydrolysates were analyed on a Beckrnan 17 1 M automated mino acid analyzer. To quanti- recovenes, 5 nanomoles norleucine was added to the protein samples before hydrol ysis.

Enzymgraphy Proteolytic activity in the G1, E, G2 1, II, III, IV, and V-estracts was assayed by the previousl y descri bed method of Ovedl and Limeback ( 1988). SDS-PAGEgels ( 10 9c wlv) were cast with heatdenatured (60°C, ?O min) type 1 collagen (platin) and casein (Sigma), at protein concentrations of 40 pg/ml. From each of the G 1, E, G2 1. II, III, IV, and Vextracts 1% equivalents were dialyzed, freeze-dned, and prepared for elecîmphoresis under non-reducing conditions without prior sarnple heating ( 19 of G? 1-extract represents -15 pg of freeze-dried material). After electrophoresis at 150 V for 40 min. the gels were nnsed twice in 2.5 % (vlv) Triton X-100for 10 min each to remove SDS,then incubated in 50 mM Tris/HCl assay buffer, pH 7.4, or in 50 mM sodium acetate assay buffer, pH 5.5, at 37'C for 24 hr. The gels were then fixed for 5 min in 15 % vlv acetic acid, and the gels stained with CBB G-350( 1.O mg of CBB G-250 /ml in 0.2 M H,W,I(~),SO, (50 mg/ml) (Neuhoff et al., 1985). Proteolytic activity was detected as cleared bands against the blue background stain of the undigested substrate in the gel. Roteinase activity was assayed in the presence of 10 mM EDTA, 5 mM PMSF or 5 mM NEM (Sigma) addd to the assay buffers. Human gingival fibroblast gelatinase, present in 0.25 pi of serum-free 24 hr conditioncd cell culture medium ( 1-35 ml1 106 cells). was used as a positive control. Tissue inhi bi tor of metalloendoproteinases (TIMP-1), paniall y punfied from ROS 1713.8 rat osteosarcorna cells (Overall and Sodek, lm,was also testeci at a concentration of 10,000 unitslml (in this assay 1 unit of TIMP- 1 inhibits 2 units of collagenase (EC3.4.24.7) by 50 %; 1 unit of collagnase degrades 1 pg ol soluble collagenlhr at 37'Cl. To activate any latent neutral metalloendoproteinases, samples were incubated with 1 mM paminophenylmercuric acetate (APMA) for 60 min at 31'C before electrophoresiS.

In ihe G? 1-extract, 44 mg (dry weight) of material was obtained from 1M) g wet weight bone. In the GS 1-extract from dentine, 1 mg of matenal (dry weight) was isolated from 90 g wet weight tissue. When compared to the CL-6B chromatograrn of the bone E- extract (Chapter III A, Figure 1 A), the bone G2-extract a1asfound to be different. as was the SDS-PAGEanalysis for protein (Figures 1 A and B, respectively ). To detennine sorne basic properties of the GZextract proteins, SDS-PAGEgels were run under reduced and non-reduced conditions and stained with various protein staining dyes. From these analyses, relative molecular weights together with the sensitivity to reducing agents and differences in their staining characteristics were used as a means to identify and differentiate some of these molecules. NotaMy, the G3-extraci compnsed high and low molecular weight (HMW and LMW) proteoglycans, type 1 collagen, HMW collagenase-sensitive proteins (> 110 kDa), a 60 kDa collagenase sensitive protein, and proteins with rno1ecula.r weights of 60,55,38,32,28, 24 and 16 k. Employing the techniques from previously developed isolation procedures for other bone proteins (Domenicucci et al., 1988; Goldberg et al., lm;Goldberg et al., 1988b;Zhang et al., 1990), a purification scheme for some of the GZextract proteins was developed (see Appendices A, B 1, B2). Similarities of some of the G2-extract proteins with E-extract proteins identified the LMW proteogl ycan as decorin and the 60 kDa 60-55 kDa, 38 kDa and 28 Daproteins, as BSP, fetuin/a2-HSglycoprotein-like proteio, osteonectinlSPARC and pN-propeptide. respectively. In general, these findings were based on properties of which included the behavior of proteins on SDS-PAGEgels run under reducing and non-reducing conditions; staining characteristics using silver nitrate, Stains-dl, CBB R-250; chromatographie properties on CL-6B, Sepharose S-200, hydroxyapatite, Fast Q and Mono Q resins and HPLC revened phase resins; and other anaiysis including Farndale analysis, chondroi tinase AUABC digestion and immunoreactivity to specific antibodies. Notabl y, the G2-extract was devoid of OPN, the related 23 kDa gl ycoprotein and osteocalcin/BGP (proteins found in the E-extract; Chapter III A, Figure 1 A). Although some of the properties of the proteins in the G2-extract revealed some differences when compared to their E-extract counterparts, the high degree of similarity between molecules discouraged any further characterization. Similarly, no further characterization of the HMW collagenase-sensitive proteins (M, of >110 kDa) was attempted since these were anticipated to represent and forrns of crosslinked collagen =-chahs. The low amounts of purified 16 kDa protein also precluded any further anal ysis. When compared to the calvarial G1-extract (Chapter II, Figure 7, lane 3) the G3- extract protein [rom dentine revealed prominent bands at 33 kDa and 34 Da(Figure 2: also Chapter II, Figure 3C,lane 3) in addition to proieins in positions expected for collagen a chains. In dentine the 32 Daand 24 Dawere cleari y the major proteins in the extract and compnsed ~805% of the total protein in this extract (ie. 800 pg from the original 1 mg of dry ufeight material obkuned from 90 g wet weight tissue). Since the 32 and 24 kDa proteins were cornmon to the G3-estracts of both mineralized tissues, these proteins were selected for funher characterization. Purification of the 33 kDa and 24 kDa proteins from bone and dentine was achieved using hydroxyapatite chrornatography. In the presence of 10 mM KI$- both the 31 and 24 kDa proteins eluted in a major unbound protein peak, whereas a small peak of proteins, with electrophoretic and staining characteristics of proieoglycans, was eluted around 100 rnM PQ3- (Figure 3 A). Re-chromatography of the unbund protein (pl1, Figure 3 A) starting at 1 rnM PO$- separated the 32 kDa protein in the unbound fraction (pool 1. Figure 3 B) from the 24 kDa protein which was bound with a retention time of 46 min (pool 2, Figure 3 B). The 32 kDa protein was further purified on Mono Q anion-eschange resin from which it was eluted in a NaCl gradient as a ragged peak at 0.12-0.23 M NaCl (Figure 3 C). However, the 32 kDa protein pooled from the Mono Q run eluted as a single, homogeneous peak at 3 1 8 solvent B when chromatographed by an HPLC reversed phase C3 column (Figure 4 A). Sirnilarl y, the various foms of 34 kDa proteins that were separated on Mono Q (Figure 3 D)also displayed a single homogeneous peak when indi viduall y chromatographed by HPLC reversed phase chromatography. An example of a composite HPLC reversed phase chromatogram for bone and dentine 24 kDa proteins is shown in Figure 4 B. From bone ( 100 g) the amounts of puri fiable 32 kDa and 24 kDa proteins were found to be approximately 600 pg and 170 rg of each protein. respectively. From dentine (90 g) a total of 600 pg and 227 pg of the 33 and 74 kDa proteins, respectively could be isolated. Both proteins migrated more rapidly on SDS-PAGEunder reducing conditions suggesting the presence of intrachain disulphide bonds (Figure 3), stained well with silver nitrate and stained pink with Stains-dl, while neither protein stained with Alcian Blue or Toluidine Blue. Collectively, these data suggest that they are unlikel y to be phosphorylated, sulfated or highl y acidic proteins. When analyzed by SDS-PAGEunder reducing conditions the protein bands at 24 kDa and at 6 kDa, together representing approximately 5 % of the total protein (as judged by CBB R-250 staining), were found with the 31 Daprotein, whereas a broad band at 26 Dawas obtained under non-reducing conditions (Figure 5 A, note 6 kDa band not LSisible under these loading conditions). Although a number of procedures (gel filtration, ion exchange, chromatofocusing 4.7) and revened phase chromatography) failed to separate these proteins, they were successfully içolated, after reduction and alkylation, bj preparatory gel electrophoresis (Figure 5 8). Demonstration of the separation of the 32 kDa protein from the contaminant 24 kDa protein after preparatory SDS-PAGEis show in Figure 5 C. Since protein sequencing of the intact 32 kDa protein aas unsuccessful for both bone and dentine proteins, CNBr fragmentation was carried out generating peptides of 20 Da, 15 Da, 13 kDa and 6 kDa Follo~vingthe transfer of these peptides (Figure 6: shown only for the bone proteins) to an Immobilon-h membrane the amino terminal amino acid sequences were determined for the bone-derived 6 kDa and 13 kDa peptides and the dentine-derived 13 Dapeptide. These sequences were found to match arnino acid sequences in the C-terminal half of rat, mouse, human, chicken and porcine Iysyl oxidase (Hamalainen et al., 1991; Kenyon et ai., 1991; Trackman et al., 1991; Mariani et al., 1992; Wu et al., 1992; Cronshaw et al., 1995);human lysyl oxidase-like protein (Youngho et al., 1995);and the nzs recision gene (Mariani et al., 1999-) as shown in Figure 7. The dentine-derived 74 kDa protein pled frorn the second hydroxyapati te colurnn run (1 mM HA start buffer) was separated into al least five peaks (pools I-V, Figure 3 D) when run on Mono Q resin, with each pool pnerating essentially identical elution profiles on reversed phase HPLC (Figure 4 B), and with the major peak eluting at 35 % solvent B. Based on SDS-PAGEanalysis the proteins appeared to be >99 % pure, with a trace amount of an 18 kDa protein king evident in pools II-V when protein was overloaded and stained with silver nitrate (results not shown). Similarly, the bone-denved 24 Daaas also found to separate into multiple peaks on Mono Q resin (4 peaks, Figures 3 D and 8 A). Three of the four peaks from bone had identical el uhon characteristics to dentine proteins on Mono Q, while one did not have a dentine analog (Figure 3 D: Bar 2, Figure 8 A: Lanes 4 and 5). Similarly, these three bone proteins were also found to have identical elution profiles on reverseci phase chromatography to their munterparts frorn dentine. To determine ahether the 24 kDa proteins were related, CNBr-cleaved proteins were analyzed by SDS-PAGE. Each 24 kDa protein generated a prominent, broad -5 Daband on SDS-PAGE for both bne- and dentine-derived proteins (resul ts not shown). A ttempts to obtain pnmq sequence data from ei ther the intact protein or from the CNBr fragments were, however. unsuccessful . Since the physicai and chernical characteristics of the 24 kDa protein were remarkably similar to a recently described tyrosine rich afidic rnatrix protein (TRAMP) that m-purifies with lysyl oxidase (Cronshaw et al., 1993). the Mono Q-dentine purified 24 kDa proteins were run on SDS-PAGE and transferred to Immobilon-P membrane and reacted wi th specific antibodies to porcine TRAMP. A strong positive reaction was obtaned for each of the 54 Daproteins, but no immunoreactivity was observed with an); other protein in the E-extracts (Figure 9). However, the most acidic of the proteins mîpted slightly faster than the others on SDS-PAGE. A cornpanson of the amino acid compositions between the 24 kDa proteins purifid from bone and dentine with that of TRAMP isolated from various tissues is shoun in TabIe 1. The corn psitions of al1 but the most acidic 24 Daprotein were similar to TRAMP. the most acidic protein (34.V kDa) reveaiing a composition close to a 74 kDa placentai protein with higher amounts of glutamic acid/glutarnine and glycine and lower levels of tyrosine. With successive GuHCl extractions the quantities of the proteins originally present in G? lextract decreased as expected, so that by the fourth extract minor quantities of proteins could be isolated (Figure 10, Lanes 1-4). In the fifth extract however, an unexpected increase in collagen a, 8 and chains (greater than present in the 1st extract), and the absence of an): lower molecular weight proteins (ie < 100 k) was obsemed. Total quantity of freeze-dried material recovered was f8.45 mg per 100 g bone from the G? V-entract and 4 mg per 100 g bone for the G2 VI-exuacts, demonstrating thai the de@ ymerizationldi sassociation phenomenon was 1imited to the fif th extraction s tep. When analyzed for protekase acûvity, no collagenase or caseinolytic activity was detected. However, gelatinase activity was detected in the fourth G?-extract. with none king detected in either the G1, E or G2 1, II, III, V-extracts (Figure 11). The gelatinase activity was detected on enzymography in two bands at 59 kDa and 41 kDa; the 59 kDa band corresponding to the activated form of matrix metalloproteinase gelatinase (MMP-1). DISCUSSION

Sequential extraction wi th guanidine-HCI and EMA can effective1y i dateproteins from the pre-mineralized matrix from those that are bound to the mineral crystals (Chapter III). By introducing a second extraction with guanidine (G1-extract) after demineralization it was anticipated that proteins associated with the collagenous matrix and masked by the mineral, could be obtained. This is of importance since it is believed that a nucleator must first localize and possibly interact with collagen in specific regions pnor to the subsequent nucleation of Cd+and PO,$- ions into crystailites (as discussed in Chapter 1). The G2-extract, therefore. is of particular interest since proteins that function in hydroxyapatite nucleation could conceivably be emiched in this extract. Notably, in pulsechase studies Ni virro this procedure has been used to demonstrate that BSP, a protein implicated in the initial nucleation of hydroxppatite crystal formation (Sodek et al., 19%; Sodek et ai., 1%b) appears sequentially in each of the guanidine and EDTA extracts, indicating that this protein initially binds to the organic matrix but becomes masked by the formation of mineral (Kasugai et ai., 1991). Here also, BSP comprises a portion of the G3-extractable proteins (Appendix, Figure 1). Whether the BSP in this extract represents a collagn-associated form of the protein or whether it is the result of inefficient estractions with EDTA is unhown. However, the absence of OPN and the reIated 23 kDa fragmentation peptide in the G2-estract are in accordance with the in vitro work by Kasugai et ai., (1992), and would argue against an inefficient estraction. In dentine, phosphophoryn is believed to be the nucleator of hydroxyapatite crystal formation (Veis, 1989; Veis, 1992). Like BSP (Kasugai et ai., 1992; Fujisawa et al., 1993). a small percentage of phosphophoryn has been shown to bind to collagen (Maier et al., 1983; Glimcher et al., 1986). HowelPer,in the G?-estracts of porcine incisor dentine no protein resem bling phosphophoryn was detected, oniy minor amoun ts of proteins resembling the G2-proteoglycans from bone (Goldberg et al., 1988a) and the pN- propeptide (G2-28 kDa protein) from bone (Goldberg et al., 1988b) were evident. In this study the focus was to identify two prominent proteins in the G3-extracts of mineralized tissues that migrate on SDS-PAGE with M,s of 34 k and 24 k. In porcine calvaria and incisor dentine, these proteins have been identified as lysyI oxidase and TRAMP,respectively; proteins that are present in dense wnnective tissues and which are known to bind to collagen (Siegel, 1974; MacBeatb et al., 1993). The identity of lysyl oxidase was deduced from cornpanson of primary sequence data obtained from CNBr peptides and published sequences for the rat, mouse, human, chicken, and porcine enzymes. Lysyl oxidase (EC 1.4.3.13). which was Tint purified by Narayanan et al., ( 1974), catal yzes the oxidation of speci fic lysine &-aminogroups to a -arninoadipic+ semiaidehyde in collagen a chains and tropoelastin as a prerequisite to the formation of covalent crosslinks that stabilize the collagen and eiastin fibre stnictures (Siegel. 1979). However, the recent demonstration that lysyl oxidase is synonymous with the ras recision gene (vg) has lead to suggestions that i t rnay be involved in tumour suppression (Mariani et al., 1992) and in the retinoic acid modulation of adipoqte differentiation (Dimaculangan et al., 1994). The accumulation of the enzyme in the extracellular matnx of co~ectivetissues further suggests that Iysyl oxidase may serve additional functions in matrix architecture. As observed in this study the existence of mu1tiple forms of 1ysyl oxidase has ken observed in preparations obtained [rom chick cartilage (Stassen, 1976) bovine aorta (Sullivan and Kagan, lW) and human placenta (Kuivaniemi et al., 1984). Since lysyl oxidase is coded by a single gene (Trachan et al., 199 1; Mariani et al., 199-) i t is probable that these differences reflect pst-translational modifications. In addition, man' preparations of lysyl orüdase have been reported to be contaminated by smaller proteins which have ken suggested to be degradation products (Sullivan and Kagan, 1982; Kuivaniemi et al., 1984; Burbelo et ai., 1986; Shackleton and Hulmes, 1990) or unrelated proteins (Kuivaniemi et al., 1984). Co-purification of 26 Daand 6 Dadong with other fragments (Figure 5 B) Iysyl oxidase in bone and dentine extracts indicates the presence of degradation products of lysyl oxidase in this study. TRAMP is a recentiy characterized rnauiï protein thai co-purifies with lysyl oadase (Cronshaw et al., 1993) and dermatan sulfate proteogl ycans (Neame et al., 1989). The bovine protein, isolated as a 12 Damolecule, has been sequenced and show to have tpsines enriched in the amino tenninal region and three repeat regions in which the consensus sequence AspArg-Glu-TrpAsnIGlnlLys-Phe/Tyris contained within disulphide bridged domains believed to interact with other matrk components (Nearne et al., 1989). The porcine protein has a broad distribution, king present in skin, skeletai muscle, heart, lung cartilage and bone osteoid and is characterized by the presence of sulfated tyrosines (Forbes et al., 1994). Notabl y, the human form of TRAMP has ken named "dermatopontin" and has been sequenced at the nucleotide level (Superti-Furga et al., lm).Although the precise function of TRAMP is unknown the bovine protein has ben shown to have ce11 adhesion properties that are abrogated by dermatan sulfate proteogl ycans (Lewandowska et al., 1991) and the porcine protein has ken shown to accelerate collagen fibril formation (MacBeath et al., 1993). Similar to 1ysyl oxidase, multiple (5) forms of TRAMP have been reported (Cronshaw et al., 1993). These also appear to be pst-translational modifications which rnay result from carbamylation of lysines that can occur in the presence of urea (Cronshaw et al ., 1993). In these studies, i t was observed that approximately five possible isofoms of the 74 kDa protein from dentine could be identified by speci fic antibodies to TRAMP. Bonederived 24 kDa proteins were found to exist in 4 forms, 3 of which appeared analogous to dentine forms. Similarities in chromatographie behaviour, amino acid composition and imrnunoreactivity indicate it likely that the 24 kDa proteins of bne and dentine are related to TRAMP. A lthough the ure-a used in Our studies was freshly de-ionized, we motmle out the psibili ty of carbarnylation and hence the presence of multiple foms for the proteins purified in our studies. While this could also have blocked sequencing of these proteins, it is notable thar a pyroglutamic acxd has been reporied at the amino terminus of bovine TRAMP (Neame et al.. 1989). Notabl y, the most acidic 24 kDa component which migrates slightly [aster on SDS-PAGEhas an amino acid composition that is closer to a 74 kDa plaental protein which shares sirnilarit). to TRAMP (Kuivaniemi et aI., 1984). The sipificance of the unexpected. but reproducible finding of a marked increase in collagen a-chains in the fifth GuHCl extracts of demineralized bone chips is at present unclear. Although there are a nurnber of enzymes which are knonn to be active in the presence of denaturants, including GuHCl it is possible that this phenomenon is caused by reversibly crosslinked collagens and not mature crosslinked collagns (Otsubo et al.. 199-). However, gelatinase activities were also present in this extract indicating that a depol ymerase activity may be involved. Notabl y, several tissue proteinases, including cathepsin B (Burleigh et al., 1974), cathepsin N (Etherington, 1972). cathepsin D (Scott and Pearson, 1978),leucocyte elastase and cathepsin G (Starkey et al., 1977), are al1 capable of solubilizing coilagen monomen from fibrillar collagen. In addition, proteolqtic activi ty released into the medium from porcine gingival explant culture can degrade native fibril l ar collagen to soluble fragments (Pettigrew et al., 1978) by a neutrd proteinase/telopeptidase which acts on the extra-helical, carboxy-terminal cross-linhng region of the a 1-chin of fibrillar type 1 collagen (Goldberg and Scott, 1986). Thus. the presence of gelatinase in the extract just pnor to the collagen depolynerization is of interest with respect to depolymerase activity, especially since this enzyme is capable of extensive degradation of the major comective tissue glycoproteins, including denatured collagen and proteoglycans (Pettigrew et al., 1981). However, further studies are required to determine the significance of this phenomenon. In summary, these studies have identified lysyl oxidase and TRAMP as major non-collagenous, collagen-associated proteins in porcine bone and dentine. Although it is conceivable that bone and dentine 1y syl oxidase and TRAMP could have ancillary functions in hard tissues, based on the known properties of these proteins in soft tissues, i t would appear that they are likel y to be more important in the organization of collagenous rnatrix of bone and dentine than to have any profound influence on matrix mineralization. 40 60 80 100 120 140 160

Fraction no. (6ml)

Figure 1 A: Gel Filtration Profile of Fetai Porcine Calvarial G2 1-extract Protein Rua on Scpharose CL-dB Rein.

Gd filtration profile of G2 1-extract protein (44mg) run on Sepharose CL-6B under the denanuing conditions of 4 SI MC1in 50 &t Tris-HCI at pH 7.4. Xbsorbance at 535 nm represents proteogl y can mntaining fractions as detennined by Famdale anal ysis. V, and V, for Lhe column bave kenindicated. Bars throughout the profde represent fractions enriched in various proteins: Bar 1. HX.W proteogi y cans (fractions 50-57); Bar 2. LMR' protmglycans (fractions 60-90).type 1 coIlagen (fractions 53-92), H5fW collagenase-sensitiveproteins (53-92) and 60 kDa collagenase-sensitive protein (fractions 78-87); Ba.3.60 kDa protein (BSP-üke protein; CBB R-250-vefSA blw staining; fractions 87-99);Bar 4.60-55 kDa recîuctim-semitive protein (fetuinla2HS-likeprotein, fractions 93- 108); Bar 538 kDa protein osteon~~tin.~SP.4RC-iikeprotein (fractions 105- 11 1); Bar 6,32 kDa iysyi oxidase (fractions 1 1.I 117); Bar 7.28 kDa pN propeptide (fractions 1 14123); Bar 8.24 kDa TRLW (fractions 120- 129);Bar 9, 16 kDa protein (fractions 1M- 135). Fractions çontaining glycosaminoglycans!proteo~ycansas &tennined by AS5 mn (+ ) employing the pmtcxx>ls of Famdaie et al.. ( 1982; 1986). figure 1 B: SDS-PAGE Analysis of Fetal Porcine Calvarial G2-extract Proteins in Fractions From Gel Fiitratioa with Sepbarose CL6B Resin.

SDS-PAGE (0.5 3f urea 5-20 % gradient gels) analysis for proteins from fractions on the Sepharose CL4B gel filtration run . Samples were run under reducing and non-reducing conditions and then stained for proteins with CBB R-250and Stains-aii (silver stained gels not shown). LadiineLLLCW markers (Pharmacia); limes, aliquots. 5 $ and 1 +drespective1 y, from the original G2-extlact sample (6 ml) *or to CL4B chromatography. T,represttptesent 75 pi aliquots from each corresponding fractions (6 ml) wilected for every third tube starting with tube number 38. A, represents fractions nui under reducing conditions and stained for CBB R-250;8, régresen& fractions nm under reducing conditions and stained for Stains-dl; C ,represents fractions nui mder non-reduung conditions and stained for CBB R-250; D, represents fractions run under non-reducingamditions and stained for Stains-ail. Figure 2: SDS-PAGE of the Major Proteins Comprising the G2-extract of Dentine

Samples were sepal-ated on 15 % gels md scained with CBB R-ZY). Lanel) low rnolcfular wQght mark= in kDa's, -2) deutine G2-extract nm under reducing conditions (- 5 pg pth),-3) dainne G2utmrn unda non-reducing conditions (- 5 pg protek). Sote the simplicity of the dentine protein profile when compared to that of boue, Figure 1 B. O 20 JO 60 80 !üû EToUon Volume (ml) Elation Volume (ml)

Figure 3 FPLC Chromtograpbic Profila for the Partial Purification of 32 kl)a and 24 kDa Proteins.

Figure 3 A. Hydroxyapatite chromatographic profile of G2-extract proteins run under dissociative conditions of 7 Si urea in 10 mM phosphate, 10 mM Tris-HCi buffer, pH 7.4, ( 10 ntC1 EL4 buffer) employing a linear gradrent of sodium phosphate ( 10-500 mbi). Bar 1 represents fractioas pooled and exchanged by ultAïltration in 1 mM KA buffer and subsequently subjected to hydroxyapatite chromatography (Figure 3 B). Bar 2 represents fractions enriched in protmgiycan-like materiai.

Figure 3 B. Hydroxyapatite chromatographic profile of pool 1 proteins from Figure 3 .A nin under the dissocïative conditions of 7 M urea in 1 m!1 phosphate, 10 mbl Tris-HCl buffer. pH 7 4 (1 raM KA buffer). employing a linear gradient of sodium phosphate ( 1-500 di).Bar 1 represents fractions hi $11 y enriched in a 32 kDa protein while bar 2 represents fractions hîghiy enriched in a 21 kDa protcin.

Solid lines represent ctiromatographic profiles for dentine pro tein w Mehatched fines represent chrornatographic profiles for hneprotein (bars 6 and 8 from Figure la). O 40 ra 60 eo O 20 40 6a 80 Elmtlon Volume (al) Elution Volume (ml)

Figure 3 C. Anion exchange chromatographic profile of hdf of the pool 1 proteins from Figure 3 B directly loaded onto an HR 55 Mono Q column m under dissociative conditions of 7 hi urea in 50 m-Lf Tris-HCI buffer (pH7.4) while employing a linear grachent of 0- 1 .Li NaCI. Bar represents fractions pooled and su bsequentl y subjected to HPLC revmed phase chromatography. For furiher purification and characterization of the 32 kDa protein see Figures -17.

Figure 3 D. Xfono Q anion exchange chromatographic profile of pool 2 proteins from Figure 3 B nui under similar conditions as in 3C. Bars represent fractions pooled and subsequentl> subjected to HPLC reversed phase chromatograpby. .a1 pools were enriched in 2-î kDa proteins and were thereby designated 24 (1-i-) kDa for the dentine proteins, and 24 1.4. B,C. D] kDa for the calvarial proteins. * represents fractions enriched in pSpropeptide. Note that when comparing peaks from the bone chrornatogram to those of the dentine chromatogram, it is found that: B=I. C=III, D=V. Yote also that peak '-4' does not bave a dentine analogue. For further purification and characterization of the 24 kDa proteins see Figures 4.8 and 9.

Soiid lines represent chromatographic profiIes for dentine protein w hile hatched lines represenc chromatographic profiles for bone protein (bars 6 and 8 from Figure la). O 10 ?O 50 40 50 Tlmc (min)

Figure 4: HPLC Reversed Phase Chromatography for the Purification of 32 kDa and 24 kDa Proteins.

Figure 4 A. HPLC C3 reversed phase chromatography of fracrions pooled from Figure 3 C. Sote that SDS-PAGEanaiysis demonstrated that the single major eluted peak compriseci the 32 Daprotein.

Figure 4 B. HPLC C3 reversed phase chromatography of fractions piedfrom Figure 3 D. The dentine profile of fractions designated by bar I in figure 3 D (rqresenring the 24.1 kDa protein) is illustrated. For cornpanson. the bone profile generated from fractions designated by the letter "B" in Figure 3 D (representing the 23.B kDa protein) is shown. Xote. when other designateci pools comprising 24 kDa proteins 3 D) were subjected to HPLC C3 reversed phase chromatograph y. similar chromatographic profiles were generated. In addition. when subjected to SDS-PAGE anaiysis. a single major eluted peak compnsing 24 kDa protein was demonstrated.

Soiid lines represent chromatographic profiles for dentine protein whle hatched lines represent chomatographic profiles for bone protein (bars 6 and 8 from Figure 1 A). Figure 5 A, B and C: Purification of the 32 kDa Protein from the Contaminant 24 kDa Protein in a Mixture of 32/24 kDa Proteins

Figure 5 A: Properties of the 32/24 kDa Proteins.

SDS-PAGE analysis of the 32/24kDa protein when stained by silver nitrate under reducing and non- reducing conditions. T,low molecular weight markers; I.reduced G2 32/25 kDa protein; Lane 3, non-reduced G2 32124 kDa protein. Note the reduction sensi tivity of both the 32 and the 24 kDa compents. Note that this particular preparation of protein has been subjected to gel filtration (CL-6B, S-200and Superose 6B);FPLC chromatography with hydroxyapaci te @th 1 mV and 10 mM start buffer), anion exchange (plyanion and Mono Q resins at vasious pH's: 7.4.7.0.6.5.and 6.0) and cbromatof'ing (Mono P at pH gradient of 64;+=4.7); and HPLC reversed phase chromaiography (Beckman Ulvapore U and BioRad C 18). These chromatographie similari ties suggest that these molecules have similar chernical properties. Figure 5 B : Preparativt SDS-PAGE of Mixture of Proteins for the Purification of 32 kDa Protein.

Purification to homogeneity of the 32 kDa protein from the 32#'24kDa mixture was achieved by reduction and alkylation of start material followed by preparative SDS-P.4GE (10 oC Trisglycine). SDS-PAGE(15 % Tris-glycine) analysis of 10 pi aiiqouts for each 700 pi collected rhrough the preparative ruis shown. Note that this parùcular preparation of start materid had other lower molecular weight contaminants (possibly degradation products) present pnor to chromatography. Bar A represents the 24 kDa contaminant, Bar B represents 32 kDa protein that was purifieci, pooled and subjected to further andysis.

Figure 5 C: Dtmonstratfon of the Separation of the 32 kDa and Relatai 24 kDa proteins from a mixture of 32/24 kDa Protcin fiom Bo& Porcine Boae and Dentine by SDS-PAGE

SDS-PAGE (15 % Tris-glycine. staining with silver) analysis to demonstrate the separation of ihe 32 kDa protein from the contaminant 24 &.Daprotein Ws7 & 3. respective1y) after preparatory SDS-PAGE as in Figure 5 B. For cornparison purification of the 32 kDa and 24 kDa protein from porcine adul t incisor dentine is also shown respectiveIy). hd.low moIecular weight markers. Figure 6: CNBr Cleavage Sensitivity of Bone-deri ved 32 kDa Protein and Electrophoretic Transfer Properties of Peptides onto PVDF Sequencing Membrane.

Figure 6 A, SDS-PAGE (16.5 %Ti6 '3 C Tris-tricine, CBB R-250stained gels) analysis of ChBr fragments from the 32 kDa protein. Major peptides generated were 20 kDa, 13 kDa and 6 kDa T. LW marlrers; Land, ext LWmarkers; hL3.peptides frm ChBr-treated 32 kDa protein nin under reducing conditions. lime, peptides from m'Br-treated 32 kDa protein nui under non-reducing conditions.

Figure 6 B, .Mysis of a duplicate geI as in Figure 6 A, demonstrating CSBr-peptides remaining in the gel after electrophoretic transfer onto ImmohIon P\DF sequeacing membrane. Lanes are as above in Figure 6.4.

Figure 6 C. ChBr peptides stained with CBB R-250after electmphoretic transfer onto Immobilon PCBF sequencing membrane. Lanes are as above in Figure 6 A.

'a' repments low moIecular weight markers in kDa; 'b' qxesents smaller low molecular weight markers in kDa than those represented in 'a'

Note that dentinederived 32 kDa protein demonstrated a similar CXBr cleavage profile with a similar eiecaophoretic transfer behaviour of peptides (not shown). Comparisons of Pcptidoderived Sequeoces from Bone and Dentine 32 kDa Proteins:

Porcine 13 kDa peptide: 6 kDa peptide:

BONE : DtPS-YDLLDASTQ- -NL--AAEGN-LAS--

DENTINE : -t?SLYDLLDPBTQR ---II------SKIN : DEFSHYDLLDABTQR YNLRCAAEENCLASTA

Comparisons with published lysyl oxidase cDNA-derived aequences:

Buian DEFSBYDLLDANTQR YNLRCAACENCLASTA or rrg

Rat DEFSEYDLLDABTQR YNLRCAACENCLASBA

XOUS~ DC~SBYDLLDANTQR YNLRCAAEENCLASSA

Chick DEFSHYDLLDABPER YNLRCAAEENCLASBA

Human DEPSBYDLLDAATQK YNLRCAAEEICLASTA LOL

Figwe 7: Identification of the 32 kDa Protein of Porcine Bone and Dentine as Lysyl Oxidase by Sequence Analysis.

Sequence match of the rtspecti ve 6 kDa and 13 kDa peptides obtained from m'Br treatmeat of bone- and daitiw- derived 32 kDa protein with the nucleotidekmino acid deduced sequeme of the cDXA endng lysyl oxidase. Comparisons with the C-terminal sequence of huxnan, rat. mouse, chick and human lys y1 oxidase; rrg (m recition gaie); and lysyl oxidase-like protein mrresponding to residues 234-248 for the 6 kDa peptide and 299-3 13 for the 13 kDa peptide. Amino acids designatecl wi th bold Ietters represent

ciifferences in sequencx when related to the human cDNAderived seqwnce. " - " designates regions wirhin ihe CMlr-derived peptide for whicb the amino âcid could not be ideotifiddetenniaed Note- the adut t porcine dentine sequeoœ obtained was not identical to fetal porcine bone or to that of the published sequence from porcine skin in position 1 1 (Cronshaw et al., 1995). Eigure 8: Properties of the 24 kDa Proteins Isalated from Porcine Bone.

Figure 8 -4.SDS-P-4GE ( 15 % Tris-@ycine, stained with silver nitrate) anai y sis of the proteins from the FPLC Mono Q cbromatographic profile shown in Figure 3 D. Tm pi sarnples fmm 1.5 mi fractions wiiected were analpd: fmie LM%' markers; W.fractions illustrateci by Bar * ,are representative of pN-propeptide. Fractions illustrated by Bar A -AS), Bar B (Lan 6-59. Bar C (hues 10-12) and Bar D (hnss 13- 14) correspond to fractions with similady tabelleci peaks. Lanes + 14 show purified 23 kDa proteins.

Figure 8 B. SDS-PAGE (15 % Trisglycine, stained with Stains-ail) dysisdernonstrating the reduction sensitivity of the -ed protein represented by Bar B in Figure 8 A. M,LbIU' markers; 1,24 kDa protein nm uwIer reducing conditions;Tirne î4 kDa protein run under non-reducing conditions. Note the shift in moldar weight under reducing and non-reducing conditions. Protein stains prnk with Stains-al]. Figure 9: Identification of the 21 kDa Proteins Isolated from Adult Porciae Dentine as TRAMP by Immunoblotting.

Figure 9 A, SDS-PAGE (IO % T;3 % C Tris-tricine gels CBB R-250 stained gels) m under reduced conditions for dentine samples from Figure 3D dernonstrating proteins prior to transfer onto Immobilon-P membrane

Figure 9 B, Western blot analysis of immunoreactive proteins after transfer of proteins onto Immobilon-P membrane I,cmloured LbfW markers (Bio-Rad); Lane.2. LMU' markers; T,dentine E-extract (5 ~g);I, dentine G2 Iextract (5 pg); Ts9.respective1 y contairing 2 pg each of the 24 kDa proteins from pools comesponding to peaks 1 to V from Figure 3 D. The band shown in lanes 9 demonstrates a faster migrating protein (arrow illustrates position) when compareci to other proteins (lanes 5-8). Figum 10 and Il: Demonstration of Collagen Depolymerase Activity and Gelatinolytic Activity in Successive G2-extractions.

Figure 10: Depolyrnerase Activity-

SDS-P-AGE (15 % Tris-glycine. staining with CBB R-29)analysis of 5 consecutive exuactions wlth 4 hI GuHCI. Note: 1) rhe quantitative decrease in the lower molecular weight proteins with successive extractions. 2) the increase in collagen alpha chans in the fifth extract (pater tban that present in the in the first extract!)

Figure 11: Gelatinase Activfty of Boue Protein Extracts.

G 1. E. and G2 extracts were electrophoresed on geIatin (JO pgîml) sutstrate. 10 5 (w,v) cross-linked polyaxylamide gels under non-reducing co~ditionsand assayed for gdatinolytic activity by enzymogmphy (24 hr incubation). Gdatinolytic activity at 59 Daand 41 LDa was identifid ody in GZextract four. bfatrix metalloproteinase-gelatinase (MbIP-2)affinity-pdled frorn the conditioned medium of human fibroblasts dtiped either in the presence (+) or absence (-) or 20 pgld concanavalin A, was decûophoresed as standards and are shown in the right panel. Minor gelatinolytic activity in the kBP-2 standards. detectable at 43 kDa (Con A-) and 31 kDa (Con A+), was also eviht. Mr, low molecular wught marker proteîns. Gl. Tmt 4 M GuHa bone extract: E. EDTA bone estract; G2. saoad 4 M GuHCl bone extract, sequentidly extracteci 5 times. Table 1: Amino Acld Compositions of TRAMP and Related Proteins.

Cornparison of Amino acid ampositions for the 24 kDa proteins from bone and dentine with TkJLLfP and related proteins isolated from various tissues. A, TR.L!lP from porcine skin; B. 23 kDa protein from bovine skin; C, 22 kDa protein from human placenta; D,Z4.B kDa protein from porcine fetal calvaria @eak B represented in Figure 3 C); E. F. G,24.1 kDa, 24.111 kDa and 24.V kDa proteins, respectively. from duit porcine incisor dentine. VaIues expresseci as raidues; lûûû amino acids. .A. from Cronshw et al., 1993; B. from Nearne et ai., 1989; C, from Kuivaniemi et al.. 1%.

Asx Glx Ses G~Y His Jw Thr Ala Pro TF Val Met =YS Ile Leu Phe LYS CHAPTER V

PRELIMINARY ANALYSIS OF PROTElNS COVALHWLY BOUND TO THE COLLAGEN MATRIX OF BONE.

INTRODUCTION

Although the analysis of the GZextract denved-proteins identified proteins involmi in the formation of the coIIagen matrix (lysyi oxidase and TRAMP), these proteins which were enriched in this extract did rot reved a nucleator. As early as 1%7 compelling evidence was reponed for the existence of proteins tightly associated with dentine collagen (Veis and Peq, 1%7; Cmichael et al., 197 1 ; Dickson et al.. 1975). These proteins were not extractable by conventionai methods nor with the more recen t protocols incl udi ng solutions of satumted neutrai EDTA (pH 7.4) and GuHCl-EDTA (pH 7.4). One of the molecules was found to be similar to the EDTA-soluble form of phosphophoryn. Subsequent evidence suggsted the existence of a covalent association between phosphophory and collagen in both rat and bovine dentine (Dimuzio and Veis. 1978b; Curley-Joseph and Veis, 1979),and treatment of dernineraiized dentine matrix with cyanogen brornide was shown to release covalent phosphopho~nîollagenconj ugate (Lee and Veis, 1980). Based on these findings, it aas hypothesized that the marris-bound phosphophosn might sen-eas the epiiactic agent in dentine mineralkation (Dimuuo and Veis, 1978b; Lee and Veis, 1980). Sirnilarly, the collaborative studies of bone formation in vitro using fetai porcine cdvariae (Nagata et ai., 199 la) have show that some proteins are not extractable wi th the 4 M GuHCl in the G?-estract and remain tightly bound to the demineralized collagen rnauix. These snidies identified proteoglycan-Iih material, BSP, pl-procollagen peptide and a 38 kDa (on 15 Q Tris-glycine SDS-PAGE)sulfated protein. Similarly. in studies of bone fomed in vitro by fetaI rat calvarial œlls using 35S04 label proteogl ym-like material, BSP and a 38 kDa protein were identified when exhaustive1y extracted bone nas treated with bacterial collagnase (Kasugai et al., 199"-). Based on these observations, preliminary experirnents were canied out to determi ne how these proteins that remained tightl y associated to the exhaustive1y extracteci demineralized bone chips might be isdated. Two possible approaches were considered to solubilize the proteins. One approach would be to use hialy-purified collagenase to selectivel y degrade the collagen. Al tematively chernical fragmentation of collagen residue with CNBr can be used. In view of the large amount of material required to isolate proteins the CNBr cleavage was atiempted. Al though this would likel y fragment some of the proteins it has the additional advantage that an): covalent linkages between the protein and collagen couId be more readil y analyzed.

MATERIALS AND MET'HODS

Tissue PrepratiOn Fifteen grams (wet wr) of demineralized and dissociatively extracted fetal porcine bone chips were rinsed with ten, M ml changes of Caz+/Mg2+deficient PBS. The bone chips were then placed on a Whatman #4 filter paper and rinsed with 3 L of ice-cold distilled nater on a Buchner funnel and vacuum dried The bone chips were frozen in liquid N, and crushed to a fine powder with a mortar and pesrle. Ten grarns of the bone chip powder was recovered and stored frozen for further processing.

CNBr Tremeni of Demineralized Bone Powder In a 50 ml polypropylene screw-top centrifuge tube, 10 g of demineralized bone chip powder was suspended in 30 ml of N, flushed 70 9 formic acid, and 6 g of hydrated CNBr, dissolved in 15 ml N, flushed 709 fonic acid, was then added and the volume brought to M ml with formic acid. The suspension was flushed wi th N, and the centrifuge tube re-capped, sealed wi th paraffin and covered wi th tin foil. The tube was placed on a rotary shaker at mmtemperature (27'C) for 6 hr. It was found that the bone chip powder was cornpletely solubilized within 1 hr. To stop the reaction, the contents of the reaction vesse1 was poured into 400 ml dHrO. The resulting solution was divided into 4 equal volumes that were each poured into 600 ml mers. The solutions were shell-frozen and freeze-dried. The freeze-dned material was re-dissolved in 15 ml dH20and freeze-dned

Acetic Acid Precipitation of Proteins from CNBr -Trpated Demineralized. Bone Powder From the 10 g wet weight of bone chip powder, 1.3 g of freeze-dried CNBr-ueated bone powder was recovered. The CNBr digested material was then suspended in M ml of ice-cold 0.1 N acetic acid. The suspension was agitated on a rotary shaker for 15 min at 4'C and was then centrifuged at 3,000 rpm for 15 min at 4'C. The supernatant was decanted and the pellet re-suspendeci in 50 ml O. 1 N acetic acid. Re-suspension, extraction and precipitation of the pellet at 4'C with O. 1 N acetic acid was then repeated a further 5 times. The pellet was freeze-dried and 36 mg of protein recovered. The precipitated proteins were subsequentl y processed by FPLC. FPLC Chromtography of Acetic Acid Precipitated Proteinî from CNBr-Treated Demiireraiized Bone Po wder The acetic acid-washed pellet was neutraiized with 1 N NaOH (30 pl) and dissolved in 1 mM phosphate hydroxyapatite buffer (16 ml). A 4 ml aliquot of this solution was then subjected to h ydroxyapati te chromatography as descri bed previousl y, wi th a loadi ng flow rate of 0.5 rnllmin. Non-bound protein was reîhromatographed on hydroxyapatite at a loading fIow rate of 0.25 mllrnin. Bound fractions were then subjected to FPLC on Fast Q resin. Al1 chrornatographic runs were moni tored by SDS-PAGEwith subsequent staining wi th silver. Stans-ail and CBB R-250. In these experiments, on1 y proteinslpeptides that stained blue with Stains-al1 were analyzed further. The reason for this is mo fold. First, the well chamcrerized CNBr collagen peptides stain pink with Stains-ail and anaiysis of these proteins can be avoided. Second, the blue staining wi th Stains-al1 could potenual 1y identify proteins which are acid nch (King and Momson, 1976), phosphate-contaning (Cutting, 1984) andlor Cat- binding (Campbell et al., 1983) feanires of whch are believed to be associated wi th a nucleator (Chapter 1).

RESULTS

Demineralized and dissociatively evtracted fetal porcine calvarial bone when treated with CNBr/formic acid could be solubilized within one hour of treatment at room temperature. The freeze-dried material was found to have a weight of 2.35 g. Treatment with 0.1 M acetic acid $vas used to selectively solubilize the collagen fragments while acidic proteins would remain insoluble. Analyses of the acid-soluble fractions demonstrated that the first three treatrnents with acetic acid contained 96.3 5% (2.215 g), 1.4 Ck (0.033 g) and 0.1 9c (0.001 g) of the totai starting material. The acid insoluble fraction made up 2.2 R (0.036g) of the start material. SDS-PAGE and chromatographic anal ysis of a 25 mg sarnple of acid soluble matenal showed that vimially dl of the material was mrnposed of proteins that did not stam blue with Stains-al1 (pi& or no staining at dl) or that did not bind to hydronppatite under starting conditions of 1 mM phosphate (results not shown). Hydroxyapatite chromatographic anaiysis of the acid insoluble material when applied as a 1 mM phosphate solution under loading flow rates of 0.5 ml per min showed three fractions (Figure 1; Bars 1, II and III, respectively). Fraction one compnsed non-bound proteindpeptides which stained *th CBB R-XI, silver nitrate but did not stain blue with Stains-dl. Fraction two demonstrated non-bound proteindpeptides that were slightl y retardeci on hydroxyapati te chromatography . Proteins in this fraction contained some proteins that either did or did not stain with CBB R-250or silver nitrate, and proteins that either stained pink or blw with Stains-dl. Fraction three revealed bound proteinslpeptides which did not stain with CBB R-250 and did not stam blue with Stains-al1 but did stain with silver nitrate. Sarnples of fraction two (Bar II) were pooled and re-chrornatographed on hydroxyapatite, with a lower Ioading flow rate of 0.35 ml per min. SDS-PAGEanalysis of fractions containeci within the non-bound peak dernonstrated proteinslpeptides which stained with CBB R-350and silver nitrate but did not stain blue with Stains-dl. SDS-PAGEanalysis through the bound peak demonstiated two predominate proteinslpeptides which did not stain with CBB R-750,stained with silver nitrate and stained blue with Stains-dl. One of the proteins/peptides had a mass of 120-200 kDa (represented by fractions represented by Bar I) and the other a mass of 43 kDa (represented by fractions represented by Bar II), with the 120-200 kDa protein having a staming pattern reminiscen t of proteogl ycans. Fast Q chromatography on FPLC of the bound fractions dernonstrated separation of proteins/peptides, with the 170-200kDa material eluting between 0.65-0.70 M NaCl when anaijzeci on SDS-PAGE. The purified 170-100 kDa protein could also be positively stained with alcian blue and toluidine blue. Surpnsingly,the 4043 Daprotein was lost and a 10 Daprotein (eluting between 0.3-0.4 M NaCl ) appeared after chromatopphy and which stained blue with Stains-dl. Al1 other material through the chromatographic mn stamed with CBB R-250, and silver nitrate, but did not stam blue with Stains-dl. SDS-PAGEanalysis of the original matenal exhaustively dialyzed against water prior to Fast Q chromatography, illustrated an absence of degradation products and the presence of the -3 kDa proteidpeptide.

DISCUSSION

Since the proteins exnacted with 4 M GuHCl after demineralization lacked a number of the expected characteristics for a nucleator of hydroxyapahte (ie. lack of bone speci ficity ) , these studies were initiated to search for non-collagenous proteins that were more strongiy associated with the collagenous matn?; of the fetai bone. Using the fact that the CNBr derived peptides are well characterized (properties, M,, etc.) fragments, are readiiy soluble in O. 1 N acetic acid (Scott and Veis. 1976a; 1976b) and do not bind to hydroxyapatite under denatunng conditions (Lee and Veis, 1980). it was anticipated that peptidedproteins tightly associated with the collagenous frame work could be isolated in a manner similar to that previousl y shown for phosphophoryn (Lee and Veis, 1980). These preliminary snidies have shown that, following CNBr cleavage of demineralized and dissociatively ex- ted fetal porcine cal varial bone an enric hed fraction of protei nslpeptides with propenies, as judged by their acid-insol ubil i ty ,chromaiographic and SDS-PAGUgel-staining properties (with silver, Stains-dl, alcian blue, toluidine blue and CBB R-2M) that are similar to rnany of the previously described mineral-associated proteins of the Eextract can be isolated. In view of the findings obtained in studies involving the G1, E and G2 1-VI extracts it is reasonable to assume that the bone chips used in this report were more li kel y to be free from the more loosely associated bone proteins (E- and G3-associated proteins) prior to treatment with CNBr. This has lead to a dearer resolution hence successful isolation of a tightly or psibly covalend y assaciated bone protein ennched fraction wi thn the residual demineralized collagen cross linked matrix. It is possible that previous analyses of proteins associated with crosslinked bone collagen were probabl y unsuccessful because of the complexity of the protein profiles. Aiso if proteinase inhibiton were not included in the extraction protocois proteol ysis could have further corn plicated anal y ses. These preliminary studies wi th CNBr-treated demineralized bone collagen suggests there are ad-insoluble peptidesiprotein present in this extract that could be involved in the formation of hydroxyapatite wi thin the collagen fibrils. Consequentiy, these proteins would be of particular interest in future studies. Frittlan no. (1.5 ml)

Friction no. (1 .S ml) Friction no. (1.5 ml)

Figure 1: FPLC Chromatography of Acetic Acid Insoluble Proteins.

Figure 1 A. hydroxyapatite chromatography of O. 1 M acetic acid insolubie proteins nui at a loading flow rate of 0.50 milmin in 1 &I phosphate hydroxyapatite buîfer. Stains-ail blue staining proteins were only found within Bar II.

Figure 1 B. re-chromatogrqhy on hydroxyapatite of fractions represented by Bar II at a loadmg flow rate of 0.25 d/min 1 mM phosphate hydron yapatite buffer. Bar 1 represents fractions mntaining a 40-13 kDa protein plus some 15û-200 kDa protein. Bar II represents fractions oontaining 150-200 kDa protein plus some 40-43kDa protein. Because there was no clear enricbment of either 10 kDa or 150-200kDa peptiddprotein fracfinis represented by Bars 1 and Il were pmled for subsequent FAST Q chromatography.

Figure 1 C. Fast Q chromatography of poded fractions represented by Bars 1 and II from the hydron yapati te run figure 1 B). Bar 1 represents fractions enriched in IO kDa Stains-al1blue staining peptideiprotein. Bar represents fractions enriched in 150-200 kDa Stains-al1 blue staining protein.

Figure 1 D. SDS-PAGEanalyses (10 pl samples per well) of fractions thmugb Bar II and Bar III as shown in Figure 1 A. 10 % Tl3 % C Tris-tricine gel nui unda reducing conditions and stained for proteins nith silver: M,. LMW markm. Lane-, wnsecutive fractions through Bar II. Lam 52.evq fourth fraction througb Bar III. Arrow rqm~ents30-43 kDa protein. CHAPTER VI

DISCUSSION

In an atternpt to segregate the potential nucleator of hydroxyapatite from other bone matrix proteins, a procedure was developed for extractinp noncollagenous proteins according to their relative affinity for collagen and hydroxyapatite cqstals. 1t was postulated that proteins that could nucleate hydroxyapatite crystal fornation would be collagen-associateci whle those which regulate the growth and dissolution of the mineral crystais would be mineral-associakd. It was further hypothesized that a potential nucleator, consistent with the initial appearance of mineral crystals in the gap region of collagen fibrils, would display affinities for both collagen and minerai. However, the isolation, purification and characterization of osteonectinISPARC. and other major proteins released in the E-extract (mineral-associated proteins), did not reveal a molecule with al1 the properties of a nucleator in the gap repon of collagen fibrils (as descnbed in Chapter 1). In addition, the isolation and characterization of the two major, collagen asçociated pro tei ns present in the G3-extracts of bone and dentine (1 .y1 onidase and TRAMP) were also identified as unlikely candidates as nucleaton of hydroxyapatite. Thus an extension of the extraction protocol for the preliminq identification of proteinslpeptides which are more tighdy associated with the demineralized and dissociatively evtracted collagenous mauix of bone was developed. It was concluded that the residual collagen matrix could harbour previously uncharactenzed proteins including the putative nucleator of bne rnarrix.

Distribuion of Proteins in the Born Extram. The dissociative extraction procedure developed in these studies has ken used to isolate the major non-collagenous proteins from newly-forming porcine bone according to their affinity for the hydroxyapati te mineral cqstals and the collagenous bone matrix, prior to (osteoid, G1-extract) and after (G2-entrac:) mineralizaiion. The same basic procedure was also used in biosynthetic studies descnbed in Chapter III B and in subsequent studies of porcine and rat bone formation Ni vitro (Nagata et al., 199 la; 1991b; Kasugai et al., 1992). Based on the collective results of these studies some proteins, including the SPARC/osteonectin, OPN, CS-PG III and osteocaich, were quantitatively extracted from demineralizing extracts indicating that they are associated with the mineral crysds. In support of this, temporal studies on the tissue compartmentalization of SPARC,OPN and CS-PG III (Nagata et al., 199 la; 199 1b; Kasugai et al., 1997) have shown that within the first few hours of synthesis. pulse-radiolabelled proteins move directly from the cellular comparunent to the mineral cornpartment in the extracellular rnaaix, with no significant amounts of radiolabelled protein recovered in either of the 4 M GuHCl extracts. Although it is possible that some of these proteins could be covalentiy bound to collagen soon after biosynthesis, this was not apparent from the anal ysis of collagenase-digested demineralized tissue residues. However, a more detailed analysis of the proteins remaining with the cdlagen after the G2-extract is required to venfy these obsemations. The presence of the SPARC/osteonectin, OPN, CS-PG III in fetal porcine E-extracts is consistent with their known ability to bind Ca2+ and hydroxyapatite. Thus, the acidic character adorlow affinity CS+binding ability of the arnino-terminus could be involved in the binding of SPARCIosteonectin to the surface of hydroxyapatite crystals (Engel et al., 1987). Notabl y, the shorter and less acidic character of the amino-terminal sequence of the rat SPARClosteonectin, is believed to be responsible for the low amounts present in rat bone and dentine (Chapter III B ;Zung et al., 1986). Conversely, the bovine and porcine SPARClosteonectin, which bind quantitatively to hydroxyapatite and are present in hi& amounts in fetal bone, have a longer and more acidic arnino terminus (Chapter III A; Domenicucci et al., 1988; Termine et al., 1981a). Thus, differences between the amino terminus of ras and porcine osteonectin/SPARC may reflect differences in the hydroxyapatite binding properties of these proteins whch is in tum believed to be responsible for the lower levels of protein observed in rai and the higher levels present in porcine bones (Domenicucci et al., 1988). While OPN has a large number of Cat+ binding sites (Butler, 1989),binding to hydroqapatite is believed to occur through the polyaspanic acid region (Oldberg et al., 1986). Although OPN has also ken reponed to bind to collagen (Chen et al., 19n), pulse-chase studies of bone formation in vitro have failed to show OPN in either G 1- or GI-entracts (Nagata et al., 1991a; 199 1b; Kasugai et al., lm). CS-PGIII has also been show to bind hydronyapatite soon after secretion (Nagata et al., 1991a) and it is believed that both the acidic protein core and CS- glycosaminoglycan are associated with the mineral crystals (Goldberg et al., 1988b). In contrast to the mineral-associated proteins, the 32 kDa and 24 kDa proteins, lysyl oxidase and TRAMP,were present as collagen-associated proteins in the Gî-estract, but not in the E-extract. Immunoblot analysis has also shown TRAMP to be present in the G1-extracts where it is ennched in the low-density fractions of fetal porcine bone particles (unpublished obçervations). The location of TRAMP in newly-forming osteoid (Levene et al., 19%; Forbes et al., 1994) or predentine (Hayakawa et al., 1985) would be consistent with its perceived role in collagen fibnl formation (MzcBeath et al., 1993). since this is a precondition to bone formation (Glimcher, 1984). Based on its function in the formation of collagen cross-links, and its association with TRAMP (Cronshaw et al., 1993), it would be anticipa that lysyl onidase would also be present in G1-extracts. aithough analyses were not performed to establish this. The remaining prominent proteins identified in the exaacts of fetai porcine bone; the proteoglycan decorin, SCAB 3 proteins and BSP demonsmted affinities for both collagen and hydroxyaptite. The presence of deconn in G1-and G?-extracü would be anticipated from its specific binding, with a regular periodicity, to the surface of collagen fibrils (Pnngle and Dodd, 1990). However, most of the decorin was recovered in the E-extract, which probably reflects the binding of the CS glycosaminoglyon side chan to the minerai crystals. Similarly, whle it might be anticipated that the putative pN-propeptides of type V collagen (SCABs 1 and 2) and the pN-peptides of type 1 collagen (SCAB 3) would be present in G-eatracts, both were recovered in çigniîïcant arnounts from the E-extracts. This ma. reflecr the anionic characteristics of these molecules, as indicated by Stains-al1 staining, as well as the possibility that some of the pN-propeptide may also be phosphorylated (Fisher et al., 19û7b). The presence of BSP in the G?-extract (Chapter IV), albeit in much smaller amountç (-5%) than in the E-extract, is consistent with the ability of BSP to bind to collagen (Fujisawa et al., 1995), and with the resul ts of pulse-chase studies of bone formation in viro (Nagata et al., 199 1a; 199 1b; Kasugai et al., 1W-) in which radiolabelled BSP was identified in both G1- and G?-extracts However. the vast majonty of BSP molecules recovered in the Eextmct are beiieved to bind hydroxyapatite through the polyglutamic acid stretches (Oldberg et ai., 1988a; Fisher et al., 1990; Shapiro et al., 1993). 1t is not possible from the extraction procedure done to determine w hy the deconn, SCAB proteins and BSP are distri buted between the mineral and collagen cornpartmenB. Although di fferent foms of the protei n rnay exin no apparent differences have ken observed in ei ther the decorin or the BSP that have been isoIated from the E-extracts and GI-entracts (Chapter II 1 C and 1V). However, chrornatographic differences were clearl y evident in the type I collagen pN-propeptide which may distribute according ta the Ser(FQ43-), which has only ken observed in boue (Fisher et ai., 1987b). Thus, the semm pN-propeptide, whch is not known to be phosphorylated, has properûes similar to the bone protein obtained in the GZ-extracf whereas the predominant fom of pN-propeptide in the E-extract shows stronger binding to hydroxyapatite on chromatography (Chapter II1 D). For decorin, the presence of the proteoglpcan in the E-entract may resul t from excess protein after the collagen binding sites have been filled, or the removal of the deconn from the gap region of dlagen fibrils during the mineralization process. Similarly , the sites of collagen binding for BSP may be limi ting. Houtever, i t is also conceivable that the recovery of these proteins from the E-and G3extracts reflects their relative affinities for the collagen and hydroxyapatite. Although a small amount of SPARClcMeonectin was obsemed in G3-extracts in this study, it was not observed in the equivalent extract in pulse-chase studies (Nagata et al., 1991a; Kasugai et al., 1992), which may be due to the facility of achieving exhaustive extractions with srnalier amounts of tissue. Whle this obsewation is consistent with the inability of the porcine SPARClosteonectin to bind collagen or gelatin (Chapter III A; Domenicucci et al., 1988) and the poor affinity of the bovine protein for collagen in the studies of Tyree (1989) and Sage et ai (1984). it contrasts the original results of Termine et al., (1981a). Interestingly, it has ken show that in the Mov 13 mouse (Iruela-Arispe et al., 1996), and in the human and bovine forms of osteogenesis imperfecta (Termine et ai., 1%; Fisher et al., 1986; Vetter et al., 19911, in which the formation of collagen type 1 is abnormal, the presence of SPARClosteonectin is depleted in both soft and hard connective tissues. This apparent discrepancy could, in part, be explained by an indirect association of SPARCIosteoneciin with collagen fibrils. However, the apparent absence of the SPARC/osteonectin from the G-extracts of bone would be difficult to esplain by an indirect association of osteonectin with collagen.

Potential Roles of the Non-Collagenous Proteins in Bone Fomrion. Having punfied and partially characterized a number of the non-collagenous proteins from the different extracts of fetai bone, it is of interest to speculate on the possible roles of these proteins in bone formation and remodelling based on the properties of these proteins determined in studies in this and other laboratones.

SPARC/osteonecrin Although it was believed that a nucleator of hydrosyapatite uouid demonstrate affinities for both collagen and hydroxyapatite, studies in this thesis were initially concentrateci on SPARC/osteonectin. Despi te the quantitati ve recover): of SPARC/osteonectin from the demineralizing extracts, indicatine an association only wi th the crystal phase, previous studies have shown that SPARC/osteonectin was able to nucleate hydroxyapatite following binding to type 1 collagen (Termine et al., 198 la). However, the lack of tissue specificity shown in immunolocalization studies (Chapter III B), isolation from diverse tissues (Wasi et ai., 1984; Sage et al ., 1984; Mason et al., 1986; Dziadek et ai., 1986). and in situ hybridization analyses (HoIland et al., 1987; Nomura et al.. 1988). together with the small amounts of SPARClosteonectin in rat bones (Chapter III B) are inconsistent with a nucleating function in bone. It has been argued that the low amounts of SPARUosteonectin in rodent bones is related to the higher proportion of woven bone in rodents relative to larnellar bone (Gehron Robey, 1989);which is believed to contain hgher amounts of SPARC/osteonectin (Conn and Termine, 1985). However, the ability of SPARClosteonectin to bind to type I collagen is also questionable (Domenicucci et al., 1988; Sage et al., 1984; Tyree, 1989) and the tempord expression of SPARCIosteonectin in bone occurs prior to the mineralization stage both in vivo (Y oon et al., 1987; Chen et al., 19%) and in vitro (Y ao et al., 1994). Thus. the presence of SPARUosteonectin bone is more likel y to influence the growth of hydroxyapatite crystals through its strong binding (Kd-8 .Y 108 M) to apatite which has ken shown to be higher than other calcium binding proteins (Romberg et al., 1985). That SPARUosteonectin has a more fundamental biological role than regulation of mineral formation is indicated by the expression of this protein in the invertebrate C. elegm(Sch warzbauer and Spencer, 1993). Moreover, anti bodies to SPARClosteonectin injected into the blastome1 cwity of tadpoles causes developmental abnormalities (Damjanovski et al., 1W-). Although the precise role of SPARC/osteonectin is unknown the hgh amounts of the protein in rapidly remodelling tissues (Salonen et al., 1990; Lane and Sage, 1994) and its ability to affect cell-substrate and ceIl-ce11 interactions indicate that SPARClosteonectin may facilitate ce11 migration and differentiation in a calcium dependent manner (Lane and Sage, 1994). A comparable function may be performed in bone where ceIl migration and differentiation are important components of the rernodelling process (Delmas and Malaval, 1993).

Osteopontin The lack of tissue specificity for OPN (Butler, 1989; Denhardt and Guo, lW3), its uncenain affinity for collagen and its temporal-spatial expression in relation to rnineralization of osteoid (Chen et al., 1992a; Yao et al., 1994) are inconsistent with ths protein having a nucleating role (Sodek et al.. 19%; 199%). It has also been shown that OPN in solution lacks nucleating activi ty in a steady-state system in vitro (Hunter and Goldberg, 1993) and that in this form OPN is a good inhibitor of crystal nucleation and growth (Hunter et al., 19%). Studies of chemically modified OPN have indicated that the phosphate groups on OPN are particularl y important for these effects (Hunter et al.. 1994). However, the presence of OPN on the ceIl surface of pre-osteoblasts in rat bone rnarrow ceIl cultures has ken show to correspond with the formation of minera1 accretions which cdesce to form a mineralized cernent layer on which bone tissue is subsequently formed (Shen et al., lm).The early expression of OPN mRNA in these cultures was related to the formation of a partially phosphorylated fonn of OPN (Yao et al., 1994) which could conceivably, as an immobilized protein (Linde et al., 1989a; 1989b) act as a cvstal nucleator (Sodek et al., 1995). Notabl y, sirnilar collagen-poor cernent layers are formed as reversal lines in bone remodelling and are characterized by an enrichment in OPN (McKee et al., 1996; 1995; Chen et al., 1994). In addition to the early expression of OPN in bone rnarrow ce11 cultures, a second larger peak of expression occurs following the initial mineralization of the newly-forming bone (Yao et al., 1994). This OPN is thought to be produced by osteoblasts and is highly phosphorylated. This biphasic expression of OPN in the marrow ceIl cultures can be related to the deposi tion of an OPN-ennched lamina limitans layer that is characteristical1y present beneath the lining cells that mark the end of a bone formation phase (McKee et al.. 1995; 1996). Here the OPN is thought to provide an attachment for the lining cells to the mineralized bone through the OPN RGD motif and the polyaspartate sequence ahich can mediate the ce11 attachment and hydroxyaptite binding functions, respectively. During bone resorption the OPN in the lamina limitans is thought to mediate the attachent (Reinholt et al., 1990) and stimulate the activity of osteoclasts (Miyauchi et al., 1991) after the lining cells have been moved away. In other tissues such as kidney (Shiraga et al., 1992), the inner ear (Swanson et al., 1989) and marnmary glands (Senger et al., 1989b) OPN is present in tissue secretions (kidney tubule fluid, cochlear fluid and milk) where ii is believed to regulate the formation and growth of various crystal foms of calcium salts (Denhardt and Guo, 1993). However, it is also possible that its presence in these fluids is related to its anti-infection activity since OPN is also produced by various lining epithelia and is characteristically espressed by activated lymphocytes and macrophages (Patarca et al., 1989). Noiably in mouse, the OPN gene has ken mapped to the rickettsia resistance gene (ricr) locus (Fet et al., 1989). OPN is also enpressed in several pathological conditions including atherosclerotic plaque (Giachelli et al., 1993) and tumours (Senger et al., 1988). While the role in plaque may be to prevent rnineralization, the induction of OPN in transformed cells is less clear. Since OPN expression has been related to the ability of cells to metastasize (Senger et al., 1989b;Craig et al., 1990) i t has ken postulated that OPN could act as an anti-adhesive molecule by blochng ce11 attachment and ailouringcells to mipte. Altematively, it may facilitate ce11 movement through its association with the CD44 receptor (Weber et al., 1996) and avB, integrin (Ross et al., 1993), or induce an invasive phenotype by signalling through these receptors (Denhardt and Guo, 1993).

Bone Sialoprotein While BSP has many of the structural and binding characteristics of OPN it is essentially unique to mineraking tissues. Moreover, its ability to bind to collagen (Fujisawa et al., 1995) as well as hydroxyapatite are consistent with the postulated characteristics of a nucleator. Studies in vivo (Chen et al., 1992a) and in vitro (Yao et al., 1994) have shown that BSP is expressed in association with mineral formation in bone tissues and in steady-state systems the purified protein has been shown to have nucleating activi ty (Hunter and Goldberg, lm).Imrnuno-electron microscopy has shown that BSP is associated with minera1 crysrallites that fonn between the collagen fibrils in osteoid of fetal bone (Bianco et al., 1993; Chen et al., 1994). BSP has also ken detected at ectopic sites of minerai deposition in breast tumours (Bellahcene et al., 19%). Expression of BSP has also ken shown to be induced by gl ucocorticoids (Oldberg et al., 1989; Kasugai et al., 1992) and bone morphogenetic protein (Li et al., 1996). which promote osteoblast differentiation, whereas vitamin D3,which suppresses osteoblastic differentiation. suppresses BSP expression (Kim et al., 19%). Thus, the BSP gene appears to be regulated in accordance wi th mineral tissue formation. Collectively, these observations provide strong evidence for BSP king a nucleator of hydroxyapatite. However, while there are indications that BSP is associated with minerai crysral induction in newly-foned woven bone, and it is associated with the interfibrillar crystallites, its relationshp with mineralization in lamellar bone, where mineraiization is predominandy within the collagen fibrils, is less clear. In ths regard it is interesting to note that BSP is expressed at high ievels at sites of de novo bone formation, but expression is decreased markedly thereafter, even though bone growth and remodelling are still taking place. Thus, it is possible that BSP rnay be involved only in the initial rnineralization of bone tissue and that a different mechanisrn is operative at later stages. That BSP may also regulate crystal growth is indicated by the abundance of the protein in the demineralizing extracts. A similar dual role in mineral nucleation and conuol of crystal growth has also been observed for phosphophoryn which is implicated in the nucleation of hydroxyapatite in dentine (Veis, 1993). BSP also has a conserved RGD sequence that recognizes the avB, integrin (Oldberg et al., 1988b) and addirional integnns (Flores et al., 1996) suggesting that BSP could have additional functions involving ce11 attachent and signailing. Studies in vitro have indicated that BSP rnay influence the differentiation of pre-osteoblasts grown on a substratum of BSP (Zhou et al., 1995). Additionally, several studies have shown that BSP acts as an atmhment protein for osteoclastic resorption (Ross et al., lm;Helfrich et al., 1992; Fiores et ai., lm) and can increase osteoclastic activity (Raynal et al., 19%). Interestingly. phosphorylation of both BSP and OPN appear to be important for osteociast attachent (Ek-Rylander et al., 1994). Proteogiycans Although the association of the decorin with the gap region of cdlagen fibres places ths molecule at the site of crystal formation, the ubiquiious presence of ihis proteoglycan in comective tissues, albeit with a dermatan sulfate glycosarninoglycan ide chain, would not support a nucleator role. However, removal of the deconn from the gap region may be required for mineralization to take place, indicating that this proteogl ycan could prevent mineralization (Nagata et al.. 199la). Thus, its absence from the gap region in type I collagen of bone, contrasting its presence in this region in soft connective tissues, is a particularly intriguing observation. The association of the bone decorin with mineral crystais is likely to involve the chondroitin sulfate side chain, which could regdate crystal growth and dissoiution. However, the principal role of decorin in bone is likely to be similar to its role in other connective tissues; that is to regulate the formation of collagen fibrils and mediate interactions with other matris macromolecules (Vogel et al., 1984). In contrat, CS-PG III does not have ans apparent association u.i th wllagen and i ts acidic core protein suggests that i t could potentially interact with mineral crystals more selectivel y than the CS chain. Its apparent restriction to bone tissues and similarities of the core protein to bone sidoproteins tend to support a specific roie for CS-PG III in mineralization. Its rapid association to the mineral crystais (Nagata et al., 1991a) point to a regulatory role in crystal formation. However, a more complete chamctenzation of this molecule is needed to provide a more meaningful assessrnent of its potential functions.

SCABs The occurrence of procollagen peptides derived from type 1 and type V collagen in the bone matrix is not surprÏsing in vieu of the amounts of these collagens, especially the type 1 collagen, that would undego processing dunng bone formation. While the SCAB 1 and 2 proteins, which are believed to represent the pN-peptides of type V collagen, appear to bind to the mineral, the pN-propeptides of type 1 collagen are praent in both E- and GZ-extracts. The small amounts of SCAB 1 and 2 preclude a signifiant efCect on mineralization and these proteins, like many others found in the E-extmcîs, may remain in the bone simply through their affinity for hydroxyapatite without having a specific role in the mineralization process. However, the type 1 collagen pN-propeptides are present in signi ficantly higher amoun ts, reflecting the relative arnounts of the type I and V collagens in bone. The type 1 collagen propeptides, through their binding affinity for wllagen and hydroxyapatite, couid mediate collagen crystal interactions (Sodek et d., 1989). Similar to deconn, the pN-peptide if not cleaved from the pmllagen molecule would occupy the gap region of collagen fïbnls and could prevent minera1 nucleation. In this regard, the presence of uncleaved pN-propeptides in procollagen molecules has been observed in collagen fi brils in both bone (Reischmajer et al., 1987) and dentine (Sodek and Mandell, 1982). Earlier studi es have shown the type 1 collagen pN-propeptides cmdown regulate collagen synthesis in a feedback mechanism (Paglia et al.. 1981) indicating a third possible role of these peptides in regulating the formation of the bone mairix during bone remodel ling. In addition to the proteins descnbed in this thesis a number of other proteins have been identified as components of the bone matrix. in rnany cases possible functions for these proteins have been suggested as indicated in Table 1. ahich is based on an eariier survey by Triffit (1987). In summary, a modifïed extraction procedure has ken developed to selectively isoiate the major proteins from bone, and the purifid proteins have ben characrerired and evaluated for their candidacy as nucleaiors of hydroxyapatite. Although one of the proteins (BSP)has recently been suggested to be a potential nucleator of intefibrillar cqstals, a nucleator that resides in the gap region of the collagen fibnls has yet to be identified. Based on a preliminaq analysis of proteins tightly bound to the dernineralized collagenous matrix, it is anticipated that such a nucleator ma? be found within ths tissue fraction.

APPENDIX A

Figures 1 A. B, C: FPLC Purification of CS-PG II and BSP from Fetal Porcine G2-extracts.

O 20 40 60 Fraction no. (13 ml) Figure 14: Hydroxyapatite Chromatography of G2-extract Proteins from CL-6B Gel Filtration Eriched in CS-PC II and BSP.

CL-6Bchrornatographic profile of pooled fractims (86-102. Chapter IV,Figure 1 -4) nin on Hydroxyapatite Chromatography under denaturing conditions of 7 hi urea. pH 7.4. Bars represent fractions enriched in the various protans: Bar 1. 120 kDa CS-PGII (fractions 41 43); Bar 2.60 kDa BSP (fractions 60-70).Fractions represented by Bars 1 and 2 were thw separately poaled and were subjected to FPLC anion exchange cbromatography with hiono Q min. Chromatograms of rhese runs are shown belou. in Fi,wes 1 B and 1 C. Chromatography w as performed as described in materials and methods . Chapter IV.

01020304050 01020304050 hactlon no. (1.5 ml) Frectlon no. (1.5 ml) Figure 1 B and C: Purification of CS-PG II and BSP from GZ-extracts.

B,anion exchange chromatography with Mono Q resin for pooled fractions represented in bar 1. Figure 1 A, bar represents purifieci CS-PG LI. C. anion exchange chromatography with Mono Q min for pooled fractions represented in bar 2, Figure I A. bar represents purified BSP. Chromatography was performd mder simila conditions de!scribed for other proteins in materials and methods, Chapter IV. Purification Scherne for Low Molecular Weight Proteins from the G2 1-extract P 'd Aîîer gel filtration on CL-66 Sepharose In 4 M QuHCI, pH 7.4, pisfor Ihe various prdelns illustraled in Chapler IV, Figure 1 A were subfected Io FPLC and WLC as descrlbeâ in 8 the columns below. Representative chromatagrams for both FPLC and t 1PLC runs described here are shown In a similar rowlwlurnn lashion In Appendi~82. z d II: Pool 6 III: Pool 7 IV. Mixture of Pools 7-8 VI: Pool O 5? m # 32i24 kDa Prdein 28 kDa Prdein 24 kDa Prdein 16 LrOa Prdein 1 1 1 1

Hyôroxyepal~e Hydroryapatlte tîydroxyapalite Hydroxyapalile Hydroxyapatite Hydroxyapatite 10 mMPi siart bulier 10 mM Pi &art bufîer 10 mM PI start butter 10 mMPi start bufler 10 mMPi dari bufler 10 mMPi start butfer 500 mM Pi eMhMer 500 mMPI eluüon bufier 500 inM PI elution buîier 500 mMPi ekitlon butfer 500 rnMPi elutlon bufier 500 mM Pi elutkm butler FPLC, 7 M uree FPLC, 7M urea FPLC, 7M urea FPLC, 7M urea FPLC, 7M ma FPLC, 7M ma 20 mM TrietîU, Ph 7,4 2û mMTrlam=l, pH 7.4 20 mMTrtsHCî, pH 7.4 20 mMTris-HCI, pH 7.4 20 mMTris-Ha, pH 7.4 20 mM TbHCI, pH 7.4 Pod Unbound Fredbns PdBwnd Fractions Pool Unbound Fractions Pool Unbound Fractions Pool Unbound Fracîbns Paoi Unbaind Fracth Mereuchange 0uffer exchange Butter exchange Buîier E xchange Bufier Exchange 4 C 1 C C

Hyôroxyapalile Hydronyapallle Hydroxyapatne Hydroxyapatite Hydroxyapalite 1 mMPi dart Mer 1 mM Pi slart bulier 1 rnM Pi statî bufier 1 mMPl slari butler 1 mMPI start bulier 500 mM Pi elutkm butter 500 mM Pi elution butter 500 mM Pl olulion butter 500 rnM Pi elulmn bufter 500 mM Pi elution butter FPLC, 7M uea FPLC, 7M urea FPLC, 7M uree FPLC, 7M urea FPLC, 7M urea 20 mM Tris-Ha, pH 7.4 20 mM Tns- ta, pH 7 4 20 mMTris-KI, pH 7.4 20 mM Tris-Ml, pH 7 4 20 mM TrieHCL, pH 7.4 Pd&und Fraclions Pool Unbound Fraclms Pool Unbound FracIions Pool Unbound Fractions PdUnbound Fractions i 1 1 1 1 mo Mono Q MonoQ MMK)Q Mono Q Morio0 O M NaCl In siart butter O M NaCi In slatî buff er O M NaCl in dari buiier O M NaCl In datl butter O M NaCl In start bufler O M NaCI In starl Mer 1 M NaCl In eMlon Mer 1 M NaCl In elutkm butfer 1 M NaCl in ehition butfer 1 M NaCl in elution butier 1 M NoCl in eMbbutter 1 M NaCl In eMim bufier fPLC 7 hi wea FPLC 7 Murea FP LC 7 M utea FPLC 7 M wea FPLC 7 M urea FPLC 7 M urea 6û mMTrWCl, pH 7.4 50 mM TdsHCI, pH 7.4 50 mMTris-HCI, pH 7.4 50 mM Tris-KI, pH 7.4 50 mMTris-KI, pH 7.4 50 mMTrlaHCI, pH 7.4 PdBoutrd Frecths Pod &und F raclions Pool Bounâ Fractions PdBound Fraclms Pool Bound Fractions PdUnbound Fraclions 1 1 1 1 1

HPLC WLC WLC WLC G3 reverse phase C3 reverse phase C3 reverse phase C3 reverse phase TFAlacetonilrile TFAIacetonitrile TFAlacetonitr ile T FAlacetoniîriie 1

teduclion and alkylation HPLC: C3 reversed phase preperatory SOS-PAGE ion pair extraction of SDS FPLC

32/24 kDa proteln 28 kDa proteln 24 kDa proteln 16 kDa prdeln

The conditians for T;PtC chmmaiography witb 1lA and Mono Q are as describeû iri Chapier IV. inaicrialu and rnethods. Bars illusiraied in [LA runs repmnt pools defor subsequeni chronwiography irs indicaied by arrowu. Bars illusiratd iii Mono Q runs represeni pools maâe for final purification as indicated on the campsite IlPl X3 chromatograrn helow. 'Ihe x-mis for dl çhromaiograms iiidicata fraction numkrs (1.5 ml lfmtion). 'ïhe left y -cuis for al1 chromatograins indicatw ahsorhanw ai 230 nm. Ihc righi y -mis for 10 mM and I mM I 1A chroniaiogra~ns indicaies a phosphate gradient in M and iu depicicd as a hatçhcd line (.-). 'lhc right y-anis for Mono Q nins inûiticaies an NaCl gradieni in M (-.).

Compodta HPLC Chromrtogram tor Plotdm Partkliy PurMed from Mono Q Rurn:

( hiditions for 1 1151 .( ' chroiriatography arc iu dwçrihed in Chapter 1V ,matcriais wd meth&. tlakhd C3 line rcpresents linw acctaniirilc gradient. hified protciiiu itre lahelled as 1= 32124 klla proiein reversed 4 (lysyl oxike); 2= 24 kIh protcin (TRAh.II': A J3.t: ürid 1) fonns); 3= 28 kDa pn~iein(type 1 pN- runs: propcpiide); 4= 38 klla protein (SPAM 'iosicoiicctiii); 5= 5.5 kDa protcin (feiuinln214S g1yu)proiein).

am (iinmwœ r- P-l Addadi, L. and Weiner. A. ( 19û5). "Interactions betwetn acidic proteîns and cxy stals: sterexxhemical . . requirernents in biomineralization." Proc.82: 41 103114.

Addadi. L., Moradian, J., Shay. E., Maraudas. 3i.G. and Weiner, S. (1987). "A chernid mode1 for the cooperation of sulfates and 6xylates in calate aystd nucieation: relevance to biomineralization." . . Proc.84: 2732-2736.

Addadi, L., Moradian-Oldak. J.. Furedi-Mihofer, H.. Weinser, S. and Veis, .4. (1992). Stereochemicai aspects of qsiai regdation in cdcium phosphate-associated miaeralized tissues. -. -. H. Slavkin and P. A. Rice. New York, Elsevier: 153-176.

Anderson, H.C.( 1969). "Vesicles associateci with calcification in the matris of epiphysed cartilage." JXd BiaL 4 1 : 59-72.

.4nderson, H.C. (1973). Calcium-accumdating vesiclw in rhe intercellular matrix of bone. -. -. C.F. S. 11. Ansterdam. Amsterdam Excerpta Media= 213. henauit, .4.L.and Ottensmeyer. F.P. (1983). "Quantitative spatial distributions of calcium. phosphorus and sulfur in calcifying epiphysis by hi& resofution eiectron spectroscopie imaging." LUL LA&. SUSA80 : 1322- 1326.

Ashton. B.A., Hohling, H.J. and Triffit, J.T. (1976). "Plasma proteins present in human cortic. bone: . . enrichment of the qHS-glycoprotein." CahLkdh22: 27-33.

.4ubin. J.E.. Heersche, J.N.M.. hlerrilees, M.J. and Sodek. J. (1982). "Isolation of bone ce11 clones with differeoces in growth, hormone responses and extracelldar marris production." J. ~~92 : 152- 461.

.4ycock, .4.S., Raghow. R., Stricklin. G.P.,Seyer. J.M. and bg,A.H. (1986). "Post-transaiptiod inhibition of collagen and fi bronectin s ynthesis by a synihetic homolog of a portion of the carbonyl- terminai propeptide of hiiman type 1 collagen."J. 26 1: 14355-13360.

Azzi, A. and Chance, B. ( 1%9). 'The "energized state" of mitochondria Iifetime and XTP equivaience." . . 189: 141.

Babd, J. and Stellwagen, E. (1969). "kleasurement of protein concentration with interference optics." Anal. Biochem. 28: 216-221.

Bartold. P.M..Miki, Y., McAllister, B.. Narayanan, A.S. and Page, R.C. (1988). "Glycosaminoglycans of human cementum."- 23: 13-17.

Beilahcene, A.. Antoine, N., Clausse, N., Tagliabue, E., Fisher, L.W., Km.J.M., Jatrs, P. and Castronovo, V. (19%). 'Detection of boue sialoptein in human breast-cancer tissue and ceil-lines at both protein and mesxnger-ribooucleic-acidlevels." 757 203-210.

Bellows, C.G., Aubin. JE.. Heersche, JN.-M. and Antosz MA.(1986). ".Mineraiized bone nodules iormed M vitro from eazymatidly released rat calvariae œil populations." Calat.3 8 38: 143- 1S.

Beresford, J.N.. Fedarko, N.S., Fisher, L.W.. ,Midura, RJ.. Yanagishita. .M.. Tennine, J.D.. Gehron Robey. P. (1987). Anal ysis of the proteogl ycans synthesized by human bone cells in vitro. J.26 2 : 17164-17172. Berthet-Colouinas. C., Miller. A. and Whtte, SU'.(1979). "Strud study of the calafying collagen ln turkey leg tendons."~134: 43145.

Bianco, P.. Riminueci, M.. Siivestrini, G., Bonucci, E.. Tennine, JB.. Fisher. LW.and Robey. P.G. (1993). ''Localization of bone sialoprotein (BSP) to Golgi and pst-Golgi secretory structures in osteoblasts and to discrete sites in dyhe matrix." J. 4 Cvtochcm. : 193-203.

Birk. D.E.. Fitch. J.M., Barbiaz, J.P.. Doane, KJ. and Linsenmayu, T.F. (1990). '%olIagen fibrogenesis in vitro: interaction of types 1 and V collagen regdates fiùril diameter." JL 95: 649-657.

Bit~er,T. and Sluir, HM. ( I%2). AmUkxhm 4: 330-334.

Bolander. .M.. Young, M.F.,Fisher, LW.,Yarnada, Y. and Termine, J.D. (1988). 'ûsteonechn cDNA sequence reveals potenual binding regions for calcium and hydroxyapatite and shows homologm with bofh a basement membrane protein (SP.4RC) and a serine proteinase inhibi tor (ovomucoid)." EkwJatL , . &dLhLU85: 2919-2923.

Bonar, L.C.. Roufosse, AH..Sabine, W.K.. Grynpas, MD. and Glimcher, M.J. (1983). "X-tay diffraction studies of the crystallinity of bone mineral in new 1 y synthesized and densi ty fractionated boue." C;rIcif. Tissue 35: 2û2-209.

Bonuai, E. (1%7). "Fine structure of early cadage calcification."JLV 20: 33-50

Bonucci, E. (1970). "Fine structure and histochemistry of calcifying globdes in epiphyseal cartilage."Z, ZelIforsch 103: 192-217.

Bonucci, E. (19'71). 'The locus of initial dcification in cartilage and bone." Clin.78: 108- 139.

Boskey, AL. (1981). "Current concepts of the physiology and biocbemistq of calcification."Ch C)ahap. 157: 325-357.

Boskey, AL. (1989)."Noncollagenous matrix proteins and their role in mineralization."Borie 6 11 1- 123.

Boskey, -4.L..Maresca, M., Doty. S.. Sabsay . B. and Veis, A. ( 1990). "Concentration-dependenteff~ts of dentin phosphophoqn in the regdation of in vivo hy droxyapati te formation and growth." Bane khd1 1: 55-65.

. . . - Bradford, E.W. (1x7).. . -4. E. W.Miles. Xew York. .Academic Press. n: 3-34.

Bronckers. ALJJ.. Gay, S.. Dimuzio, M.J. and Butler, W.T. (1985). "immimolacalization of y- carbosyglutamic acid-amtaining proteins in developing molar tooth germs of the rat." CdLReh R~L5: 17-22.

Bronckers, A.LJJ.. Gay. S., Lyaruu, D.M., Gay. R.E. and bliller, EJ. (1986). '*Localizationof type V collagen wi th monocional antibodies in developing dental and periodontal tissues of the rat and hamster." Coli.6: 1- 13.

Bronckers. ALJJ.. Gay, S., Finkelman, R.D. and Butler, W.T. (1987). "Developmend appearance of GIa proteins (osteocalch) and alkaline phosphatase in (00th gams and bones of rat." Bose 2 2: 36 1- 373. Brmckers. A.L.J.J.. Lyaniu, DM.and Woltgens, J.H.M. (1989). "Immunohistochemistry of extracetluiar matrix proteins during various stages of dentinogenesis." Conn.2 2 : 65-70.

Broackers, A-L-JJ..Fd-Carson. MC.,Van Waveren. E. and Butler. W.T. ( 1994). "Immunolocalization of osteopontin, osteocalan, and dentin sialoprotein during dental root formation and eariy cementogenesis in the rat." J.9: 9:3-&51.

Brunette, DM..Sielcher, .4.H. and Moe. H.K.( f 976). "Culture and ongin of epitheliurn-like and fibroblast- Iike cells from porcine penodcmtd ligament explants and ceIl suspensions." A&BdBid 2 1 : 393- m.

Bmklo. P.D.,.Monckeberg. -4. and Chichester, C.O. (1986)."'Llonoclonal anfibodies to human Iysyl osidase." 6: 153-162.

Buritigh. M.C., Barrett, .A J. and T,G.S.( 1974). "Cathepsin B 1 : a l y sosomaI -me that degrades native collagen." Biochem. 137: 387-398.

Butler. W.T.. 3likulski. -4. and Crist, M.R. ( 1977). "Soncol~agenousproteins of a rat dentine matnx possessing bone morphogenetic activity." ,Jlka&s 56: 2%.

Butler. W.T., Bhown. M., DimuPo. 51.T.and Linde. A. (1981)."Soncollagenous proteins of dentine. Isolation and partial characterizarion of rat dentin proteins and proteogl y ans using a the-step preparauve method." Col)aPen l(2): lm-199.

Butler, W.T., Bhown. hi., Dimuzio, M.T.. Cothran, U'.C. and Lmde, -4. (1983)."Xfuitiple fomof rat dentin phosphophoqa." 225 : 178-1%.

Butler, W.T. ( 1989). 'The name and significance of osteopontin." ConnÊct. 23: 123-136.

Butler. W.T.. Bhown. Xi.. D'Souza. R.N., Farach-Carson. h1.C.. Happonen. R.P.. Schrohenloher. R.E., Seycr. J.M.. Somermaa. UJ., Foster. R.A., Tomana hi., et al. ( 1992). "Isolation, characterization and immunoIocalization of a 53-kDa dentin sialoprotein." lfahxi 12 : 343-351.

Butler, W.T. and Ritchie. H. ( 1995).'The name and functionai significancr: of dentin extracellular matnx proteins." h.J. Jkv. Ri& 39: 16%179.

CampklI, .\-A.. J3rahimpur, A., Perez L.. Smesko. S..A. and Nancollas. G.H. (1989). 'The dual role of pdyelectrolytes and proteins as mineralîzation promocers and inhibitors of calcium oxalate monohydrate."Calc. 45: 122-128.

Campbell, K.P., .MacLennan, D.H. and Jorgnsen, -4-0.( 1983). "Staining of the Caz-binding proteins, caisequestrin. dmoduiin, troponin C, and S- IOO, nith the catioaic carbocyanine dye "Stains-al1"." L chem. 258: 11267-11273.

Carmichael, DJ., Veis. -4. and Wang, E.T. ( 197 1). "Dentine matris coilagen: evidence for a covalentl y linked . . phosphoprotein anachment." 7: 33 1.

Cawston. TE.,blurphy. G.,Mercer. E. Galloway, W.X., Hazleman, B.L.and Reynolds, J.J. (1983). 'The interaction of purifia! rabbit bone collagenase with purif~edrabbit bone metalloproteinase inhibitor." B~Q&RLL 21 211: 313-318.

Chaddaparampil, 1.. Banejee, D.,Poirier. V. and ,Mukherjee. B.B.(1985). "Ntered processing of a major secret& phosphoprotein correlates with tumorigenici ty in Rous sarcoma virus-transformedmiimmalian œlls." J- Vird 53: 841-850. Chen. J., Shapuo. H.S., Wrana. J.L., Reimers. S.. Heersche, J.N.M. and Sodek. J. (1991a). "Lcdization of bone sialoprotein (BSP)expression to si tes of mineralid tissue formation in fetal rat tissues by in situ hybridizatian." Matria 1 1 : 13343.

Chen. J., Zbang. Q.. iMcCullûch, C.A.G. and Sodek. J. (1991b). "Immunohistochemid localization of boue sialoprotein (BSP)in fetai porcine bone tissues: cornparisons with secreted phosphoprotein I (SPP- lhteopontin) and SP.4RC (osteonectin)." HkimkmL 2 3 3: 28 1-9.

Chen, J., Shqro, H.S.and Sdek. J. (199%). "Developmental expression of bone sialoprotein mR.V.4 rn rat mineralized tissues." 7 : 9W-99'7.

Chen, J.. .McCulloch, C.A.G. and Sodek, J. (1993). "Boue siaioprotein in developing porcine dental tissues: cellular expression and cornparison of tissue Iocaiization with osteopontin and osteonectin." A&Lhi Bd.38: 241-249.

Chen, J.. .McKee, J.D., Nanci, A. and Sodek, J. (19%). Histûchem. 26 : 67-78.

Chen. Y., Bal, %.S. and Gorski. J.P. (1992b). "Calcium and collagen binding properties of osteopontin. bone sialoprotein, and bone acidic giycoptotein-75 frorn bone." J. 26 7 7: 2487 1 -24378.

Christoffersen. J. and Landis. IY J. ( 199 1). "-4 contribution with review to the description of mineralizatioo of bone and other calcifieci tissues in vivo." ,Snat. 2 30 : 435450.

Cohen-Solal, L., Cohen-Solal, M. and Glimcher. M.J. ( 1979a). "Identification of y -glutamyl phosphate in the a2 chains of chicken bone collagen." -ci. 1 321 7 6 : 13274330.

Cohen-Solal. L., Liao. J.B., Kossiva, D. and Ghcher, M.J. (1979b). "Identification of organic phosphonis covalently bund to collagen and non-collagenous proteins of chicken-bone matris: The presence of O- phosphoserine and O-phosphothreoaine in non-collagenous proteins, and their absence from phosphorylated collagen." Biochem 177 : 8 1-98.

COM, K.W. and Termine, J.D. (19û5). "Maûix protein profiles in cdf bone development." Borie 6: 33-36.

Cornor. S.S ., Aubin, JE. and Sodek. J. ( 1983). "independent expression of tpe1 collagen and fibronectin b y normal fibroblast-like ceiis." I_ 63: 233-244.

Cormack, D.H. (1979). Dense comective tissue, cartilage, bone. and joints. -. Phdadel phia. J.8. Lippincott Co.: 157- l98.

Craig, X.,Ll., Nemir. Xi.. Mukherjee. B .B..Chambers. A.F. and Denhardt. D.T.( 1989). "Osteopontin. a transformation-associated ceil adhesion phosphoprotein. is inducd by 12-O-tet~adecanoylphorbol13- acetate in mouse epidennis." J.264: 9682-9689.

Craig, .4.M.,Bowdtn, G.T.. Chambers, AS., Spearman, M.A., Greenberg, AH.,Wright, J..4., hlcLeod. kf. and Desbardt, D.T. ( lm)."Secretcd phosphoprotein mZiYA is induced during mui tistage carcioogenesis in mouse skin and correlaies with the metastatic potential of murine fibroblasts." U GUXX46: 133-137.

Cromhaw, A.D.. MacBeath. J.R.E.. Shackleton, D.R..Collins. J.F., Fothergill Gilmore, LA. and Hulmes. DJ.S. (1993). 'TRAhrP (tyrosine rich acidic matris protein). a protein that co-purifies with Iy sy1 oxidase from porcine skin: Idenification of TRiLCfP as the dennatan suIpbate proteoglycan associateci 22K extracellular maüix protein." Ahûi~13 : 355-266. Ooashaw. A.D., Fothergdl-Gilrnore, L.A. and Hulmes, DJ.S. (1995). 'The protedytic processine si te of the precursor of lysyl oxidase." Biochem, 306: 279-2494.

Ciniey-Joseph, J. and Veis. A. ( 1979). 'The nature of covalent complexes of phosphoproteins with coilagen in the bovine dentin matnx." L5 8: 1625.

Cutting. J.A. (1983). "Gel protein stains: phosphoproteins." 104: 451-355.

Damjanovski, S., Liu, F. and Ringuette, M. (1992). "Molecuiar analysis of Xenopus laevis SPARC: A highly conserved acidiccalcium-bindingprotein." &&emJ 28 1: 513-517.

Davis, N.R. and Wallcer, TE(1972). 'The role of carboxyl groups in collagen calcification." Biochem. 48: 1656-1662.

Davis, N.R., Risen. O.,M. and Pringie, G.A. (1975). "Stable, non-reducible cross-links of mature collagen." 14 : 203 1-2036.

de Bernard, B., Biancu, P., Bonucci, E., Constantini. M.. Ld.G.C., Martinuzzi, P., XIodricky, C., Moro. L., Panfili, E., PouesIo. P.. et al. (1986). "Biochemical and immunohistochernical evidence that in cartilage an dkalinepbosphataseis aCa2+-bindingglycoprotein."J. CelL103: 1615-1623.

Delmas, f.D. and -Maiaval, L. (1993). The proteins of bone. Handzmk of F&x ' ntnl Fhmaa&gw. 10'7 Phpof bofG.R. Mundy and T. J. Martin. Berlin Heidelberg, Spnnger-Vedag .107: 675-723.

Denhardt, D.T.and Guo, S. (1993). "Osteopontin: a protein with diverse functions." 77( 1475-1482).

Denhardt. D.T., hpe~C.A.. Rollo. E.E.. Hvc-ang, S., .4n, S. and Walther, S.E. (1995). Osteopontin-induced modifications of cellular fuuctions. t.D. T. Denhardt, W. T. Butler, -4. F. Chambers and D. R. Senger. New l'ork, The Xew k'ork Academy of Sciences. 760: 127- 142.

Desbois, C., Hogue, D. A. and henty. G. (1994). 'The mouse osteocalcin gene cluster contains three genes with two separate spatial and temporal patterns of expression." J. 2 6 9 : 1 183- 1 190.

. Dickson, I.R., Dimuzio, MT., Volpin. D., .banthanarapan, S. and Veis, A. (1975). 'The extraction of . . phosphoproteins from bovine dentine." CaIcif,_Tissue 19: 5 1 -61.

Dimactilangan, D.D., Chawla, .4., Boak, A., bgan, H.11.and hzar, M.X. ( 1994). "Retinoic acid prevents . . downregulation of rat recision gendlysyl oGdase in adipocyte differentiation." r)lfferentiahon 58: 47- 52.

Dimuzio. M.T. and Veis, A. (1978a)."Pbosphophoryns - major noncollagenous proteins of rat incisor dentin." Calc.25: 169-178.

Dimurio, M.T.and Veis, A. (1978b).'The biosynthesis of phosphophoryns and dentine collagen in the mntinuously erupting rat incisor." J.Biol. 253: 6845-6852.

DimuPo, MT.,Bhown, M. and Butler, W.T.(1983). 'The biosynthesis of dentine y-carboxyglutamicacid- containhg proteins by rat incisor odo~toblastsin organ dture."bdmnJ. 2 16: 249-251.

Domenicucci, C., Goldberg, H..4., Hofmann, T., Isenman. D., Wasi, S. and Sodek, J. (1988). "Characterization of porcine osteotlectin extracteci from foetal calvariae." U 253 : 139- 151. Driessens, F.C.M.(1983). Formation and statility of calcium phosphates in reiiüïon to the phase composition of the mineral in caicified tissues. baaammof -. K. de (hot. .4msterdam. CRC Ras. Inc.: 1-32.

Dtiadek, M.. Paulsson. M., Adlley,M. and Timpl, R. (1986). 'Purification and tissue distribution of a smail protein (BM-U)) extracteci fmm basement membrane tumor." EuL&&mu 16 161 : 455464.

Ecarot-Chanier, B., Boucbard. F. and Delloye, C. (1989). "'Bose sialoprotein II spthesized by culturd osteubIasts antains tyrosine sdfate." J. 2 6264 : 2MM9-2OOS.

Ek-Rylander, B., Fiores. M.. Wendel, D.. Heinegard, D. and Andersson. G. (19%). "Dephosphorylation of osteopontin and boue sialoprotein by osteoclas tic tartrate-mis tant acîd phosphatase. Mdulation of osteoclast adhesion in vitro." J,269: 14853-14856.

Endo, A. (1981). "Potential role of phosphoprotein in collagen mineralization: an experimental study in vitro." 6 1: 563-575.

Engel, 3.. Taylor, W., Paulsson, M.. Sage, H. and Hogan, B. (1987). "Calcium binding domains and calcium- induced conformational transition of SP.~C-B~14~0steonectin,an exiracelIuIar gl ycoprotein expressed in mineralized and non-rnineraiized tissues." 26: 6958-6965-

Etherington. D.J.(1972). 'The nam of the uiiagsnolytic cciikpsin of rat Iiver and its distribution in other rat tissues." Biochem. 127: 685492.

Eyre, D.R. (1980). "Collagen: molecular diversity in the body's protein scaffold." Sarnce 207: 13 15-1322.

Fairbanks, G.. Steck, T.L. and Wallach, D.F.H.(1971). "Electrophoretic dysis of the major polj-peptides of the huma. qthrocyte membrane." l3b&m&~1 0 : 2606-2617.

Farndale. R.W.. Sayers. C.A. and Barrett, A.J. (2932). "A direct spectrophotometric microassay for sulfateci gl ywsaminogl y ansin cartilage cul hues." Connect. Tissue9 9: 217-2158.

Farncide, R.Ur.,Buttie. D.J. and Barrett, A.J. (1986). Biochim. 883: 173-17.

Fet, V., Dichnson, 1I.E. and Hogan, B.L.M. (1989). "Localization of the mouse gene for secreted phosphoprotein 1 (SPPI) (îar. osteopontin, bone sidoprotein 1. UkDa booe phosphoprotein, nunor- secreted phosphoprotein) to chromosome 5. cioseiy linked ta Ric (Rickettsia resistance)." Genomics 5: 375-377.

Fidielman. R.D.and Butler, W.T. (1985). "Appearance of dentin-y-carboxyglutamic acid-containhg proteins in developing rat molars in vitro." J,Dent. 6 4: 1008- 1015.

Fisher, L.W.,Termine, J.D., Dejter Jr, S.W., Whitson, S.W., Yanagishita, M., Kimura, J.H.. Hrrscall. V.C., Kleinman, H.K., Hassel, J.R. and Nisson, B. (1983a). "Proteogiycans of developing bone." JiEd Chem. 25 8: 6588-65%.

Fisher, L.W.,Whitson. W.S., Avioli, L.V. and Termine, J.D.(1983b). "'hlaûix sidoprotein of developing bone." J. 258: 12723-12727.

Fisher. L.W. (1985). The nature of the proteoglycans of bone. Tissueç. W. T.Butler. Birmingtiam, Alabama. Ebsco Media: 18&1%.

Fisher, L.W.and Termine, J.D.(1985). "Noncoliagenous pmteins influencing the idmechanisms of caIcification."ChQrh2p 200: 362-385. Fisher, L.W.. Denhotm. LJ., Conn, K.M. and Termine. J.D. (1986). "Mineralized tissue protein profiles in the Australian form of bovine osteogenesis imperfecta." - -. 38: 16-20.

Fisher, L.W.. Hawkins, G.R.. Tuross. N, and Tennine. J.D.(19û7a). '*Purificationand partid characterization of smdl proteoglycaos I and II. bone sialoprottins 1 and n, and osteoaectin from the mineral cornpartment of developing human bcme." JAdLkm2 6 2 : 902-9708.

Fisher, L.W., Gehron Robey, P.. Tuross. N., Otsuka. X.S., Tep, DA.Esch, F.S., Shimadi, S. and Termine, J.D. (lm).'The M, î4.000 phosphoprotein from developing bone is the '\ï-irtenninal pqxptide of the a 1 chain of type 1 collagen." J. 26 2: 13457- 13466.

Fisher, L-W.. Termine, J.D.and :Young.FM. (1989)."'Muceci protan sequene of bone sdlproteogl ycan 1 (hg1ycan) shows homology with proteogl ycan Il (decorin) and severai non-comective tissue proteins in a variety of species." I. 264: 45714576.

Fisher, L.W.. McBride. O.W.,Tennine, J.D.and Young, M.F. ( 1990). "Human bone sialoprotein. Deduced protein sequence and chromosomal location." I. Biol.5 : 2347-2351.

Fieisch. H. (1983). Biophosphates: Mechanisms of action and clinical application. Annrial. W. A. Peck. .4msterdam. Excerpta hiedica: 3 19-357.

Fleischmajer, R.. Timpl. R.. Tuderman. L., Raisber, L.. U'eistner. M., Perlish, J.S. and Gaves, P.S. (198 1) "*Lltrastructurdidentification of extension amino propeptides of type I and III coiiagens in human skin." W.? 78 : 7360-7364.

Fleischmajer. R., Perlish. J.S. and Timpl. R. (1985). "Collageu fibrillogenesis in hiimmi skin." Ann. . ExkA&3i 460: 256-29.

Fleischmajer, R.. Perlish, J.S. and Olsen. B.R. (198'7). ".hune and carboxyl propeptides in bone collagen fibrils during embr).ogenesis." Cell2Res.747: LOS- 109.

Flores, %LE.,Sorgard. M., Heinegard. D.. Reinholt, F.P. and .Andersson, G. (1992). "RGD-directed attôchment of isolateci rat ostdasts to osteopontin. bone sialoprotein and fibronectin." 20 1 : 526- 530.

Fiores, ME., Heinegard, D., Reinholt, F.P. and Andersson, G. (19%). "Boue sialoprotein coated on glass and plastic surfaces is recognized by different beta (3) integins." EspLdRs227: JO4.

Forbes. G.E., Cmmhaw, .4.D.,biacBeath, J.R.E.and Hulmes. D.J.S. (1994). 'Tyrosine-rich acidic rnatrix protein WfP)is a tyrosine-suiphated and wideiy distributed protein of the extracellular matrix." EF.J3S =nS, 35 1 : 433-436.

Franzen, .4. and Heinegard, D. ( l984a). 'ExExtraction and purification of prote@ ycans from mature bovine bone." Biûchem, 224: 47-58.

Eranzen,A. and Heinegard. D. (1 984b). ''Chamcterization of proteogl ycans from the calcified mahx of bovine bone." &cdemJ 224: 59-66.

Franzen, A. and Heinegard. D. (1%). 'lsdation and characterization of two sidoproteins present ody in bone calcified matrix." Biochem. 232 : 7 15-72.1.

Franzen. A. and Heinegard, D. (198%). Protwglycans and proteins of rat bone. Purification and biosynthesis of niaior noncollagenous ma~omolccuies.a. '. 15.. TT. Butier. Birmingham, Alabama, Ebsco Media: 132-151. Fujisawa, R., Takagi, T.,Kuboki. Y. and Sasaki. S. ( 1% 1). The characterization of collagen-phosphophoqn complex in bovine dentine. mf-1 - PA. A. Veis. New York. ElsevierlNorth-Holland: 483-

Fujisawa. R., Butler. W.T.. Brunn. J-C., Zhou. H.Y. and Kubok, Y. (1993). "Differences in composition of œii-anachment siaIoproteins between dentin and bone." J. 7 2 : 1222-1226.

Fujisawa. R.. Nodasaka, Y. and Kuboki .Y. (1995).-. "'Further characterization of interaction becween bone sialoprotein (BSP) and dagen."Calnf. 56 : 130- 133.

Funk. S.E. and Sage. E.H.(1991). 'The CS+-binding gI ycoprotein SPARC modulates ceil cycle progression in bovine aortic endothelid cells." 7 88: 2648-2652.

Funk, S.E. and Sage, EH.(1993). "Differential effects of SPARC and cationic SP.4RC peptides on DU synthesis by endûthelia1 cells and fibroblasts." J,154: 53-63.

Gebhardt. M. (1973). ""Uberbiokristallisation und epitaue." L C- 20: 6.

George, A., Sabsay, B.. Simonian, P.A.L. and Veis, A. (1993). "Characterization of a novel dentin matris acidicphosphoprotein." J. Riol. 268: 12624-12630.

George, A.. Gui. J.. Jenkins, N..4., Gilbert. DJ., Copeland, S.G. and Veis, A. (1994). "ln siru locaiizationand ' chromosomal mapping of the .4G1 (Dmp 1) gene." J, Hi. .-1 Q&.km. 42 : 1527-153 1.

George, A., SiIberstein. R. and Veis. A. (1995). "In situ bybridization shows Dmpl(.AGl) to be a developmentall y regulated dentin specific protein produced b y mature odontoblasts ." V &S. 33: 67-72.

. * Gerbon Robey, P. (1989). 'The biochemistry of bone." 18 : 859-902.

Giachelli, C.M.. Bae, S., Aimeida. M.. Denhardt. D.. Alpers, C.E. and Schwam~Shi. (1993). "Osteopontin expression is eievated during neointima formation in rat arteries and in human atherosclerotic plaques." J.92: 1686-16%.

Glimcher, M.J., Hodge, A.J. and Schmitt, F.O.( 1957). "biacromolecular aggregation States in relation to . - mineralization: the collagen-hydronyapatite system as studies in vitro." Roc NadAcad Sn I .C -i 53: 860-867-

Glimcher. MJ. (lm). Specificity of the molecular stmcture of organic matrices in mineralization. ... Calnficaiion.B. FR.Sognnaes. Washington. D.C..American Association for the Advancement of Science: 42 1487.

Glimcher. M.J. and he,S.M. (1%8). The organization and structure of bone, and the mechanism of

calfication. -on G. N. Ramachandran and B. S. Gould. New York. Academic Press. IEl: 68-251.

Glirncher. MJ. (1976). Composition. structtm aud organization of bone and other mineralized tissues and the mecbanism of calcification. 7: F-. R. O. Greep and E. B. Astwd. Washington, DC, American Ph ysiological Society. BII: 25- 116.

Glimcher, MJ. ( 19%1 ). On the fonn and fundon of bne: From. molecules. to orgaos. Wolf s Law revisi ted. The..4. Veis. North Holland, Elsevier: 6 17-673. Glimcher, MJ. (1984).'Recent studies of the minerai phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds." miil. 304 : 439-508.

Glimcher, ,M J. ( 1 985). The deof mllagen and phosphoproteins in the calcification of bone and other collagenous tissues. 1.R. P. Rubin, G. Weiss and J. W. Putney Jr. Sew York, Plenum: 607-616.

Glimder, M.J., Lefteriou, B. and Kossiva, D. (1986). 'Dn the pmbiem of covalent lrnkages between phosphoproteins and collagen in bovine dentine and bne." J. 1: 5û9-522.

GIimcher. MJ.( 1989). "Mechani sm of calcification: Role of collagen fibril s and co!lagen-phos phoprotein complexes in vitro and in vivo." .4nat. 224: 139-153.

Glowacki. J., Re)-, C.. Cox. K. and Lian. 1. ( !989). "Effecrs of bone matris components on osteoclast differentiatim." Conn.2 1: 12 1 - 129.

GoIdberg. H.4. and Scott. P.G.(1986). "Isolation from dnired porcine gingival explants of a neutral proteinase with coilagen telopeptidase activity." Connect. 15: 209-2 19.

Goldberg. H.A.,Maeno. Xi., Domenicucci. C.. Zhang. Q. and Sodek. J. (1988a)."Identification of sdl collagenous proteins wi th properties of procollagen a I 0)pK-propeptide in fetal porcine calvarial bone." Coll.8: 187-197.

Goldberg. H. A.. Domenicucci. C., Ringle. G.4. and Sodek, J. ( l988b). "Minerai-bindng proteogl ycans of fetal porcine cdvarial bone.*'I, 263: 12092-12101.

Gddberg, Si., Septier, D., Lecolle, S.. Chardin, H.. Qutntana, S1.A.. Acevedo, AC.. Gafni. G.. Dillouya D.. Vermelin, L., Thonemann, B., et al. (1995). "Dentai mineralization." Lnfc J. kv.Biof. 39 : 93- 1 10.

Goldblum, S.E., Ding. S.. Funli. S.E. and Sage, E.H. (1995). "SP.4RC regdates eudothelial ce11 shape and . . hanier function." 9 1 a. 3443-3452.

Gorski. J.P. and Shimizu. K. (1988)."Isolation of new phosphoqlated glycoprotein from mineraiid phase of bone that e.dubits limi ted homology to adhesive protein osteopontin." J.. Cha~263 : 15938- 159-55.

Gonki. J.P.( 1992). ".Acidic phosphoproteins from bone matris: .4 structurai rationalization of thtir role in . . knomineralization." Calctf.50 : 391 -3%.

Gorter de Vries, 1.. Quartier, E.. Van Steineghem, .4., Boute, P., Coomans, D. and Wisse. E. (19%). "Characterization and immunohistochemical localization of dentine phosphoprotein in rat and bvine teeth." &&Qd&d. 3 1: 57-66.

Gorter de Vries, 1.. Quartier, E.. Boute, P.. Wisse, E. and Coomans, D. (lm."Immunoc~~ochernical localization of ostdcinin developing rat teeth." J-ntBgs. 66: 7&1790.

Merde Vries, 1. and Wisse, E. ( S89)."Ultrastruccural localization of dentine phosphoprotein in rat tooth germs by irnmunogold staining." H&Q&RL 9 1: 69-75.

Hamahineo. ER., Jones, T.A., Sheer, D..Taskinen. K.. Pihlajaniemi, T. and Kivirikko, K.L. (1991). "Moldar cloning of human lysyl oxidase and assignment of the gene to chromosome 5q233-3 1.2." Genonncs 11: 508-516. Hanrki. F. and Kirk, J.E. (1%7). "A method for separation of chondroitin sulfate B from isomers A and C by papet elecvophoresis with zinc sulpbafe or zinc acetate solution as electrol yte medium." Biachim. 136: 391-393.

Hasselaar, P. and Sage. E.H. ( 1992). "SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothehl cells." J. 49 : 272-283.

hseli, J.R., Kimura. J.H.and Hascall, V.C. (1986). "Proteoglycan core protein families. (Review ).*O bu. Bev.55: 539-567.

Hauschka. P.V., Lian. J.B.and Gallop. P.M.(1975). "'Direct identification of the calcium-tnnding amino acid - - t-carboxyglutiimnie, in rnineralized tissue." L SA 7 2: 3925-3929.

Hauschka P.V. ( 1985). Osteocalcin and its functional domains. +and laf mi W. T. Butler. Birmingham, Alabama, Ebsco Media: 149- 158.

Hauschka. P.V.. .Mann, KG.,Price, P..4. and Termine. J.D.(1986). '';omenclahire recomrnendatioas: Boue proteins and growih factors." Cdlagen 6 : 6:53455.

Hauschka, P.V., han. J.B.. Cole, D.E.C. and Gundkg, C.M. (1989). "Osteocalcin and rnatrix gla protein: vitamin K-dependent proteins in bone." Phvsiol. 669 : 990-1037. byakawa, T., Numata. Y. and Hashimoto. Y.(1985). LysyI-oxidase activity in predentine layer isolated from bovine incisor teeth. The.and. of. I3ut.k. Birmingham, AL, Ebsco Media: 423.

Heeley, J.D.and Irving, J.T. (1973). "A cornparison of histological metbods for demonstration calcification." . . Catctf.1212: 169-173.

Heersche, J.Y., Reimers. SM.,Wraaa. J.L.. Waye. 1f .lf. and Gupta. A.K. ( 1992). Thanges in expression of al type 1 collagen and osteocalch rnRV.4 in osteoblasts and dontoblasts at different stages of maturity as shown by in situ hybridization." 8û Suppl. 1 : 173-182.

Heinegard, D., Bjome-Persson, A., Coster, L., Franzw. A.. Gardeil, S., hialnistrom, A.. Padsson. hl., Sandfalk, R. and Vogel . K. ( 1985). 'The core proteins of large and small intersti tial proteoglycans from various comaive tissues fom distinct subgroups." Riochem. 230: 181 - 1%.

Heinegard, D.. Franzen, .4., Hedborn, E., and Sommarin, J'. (1986). "Common stnrcnws of the are proteins of interstitial proteogl ycans." ClbaFourirIation 24 : 69-88.

Heinegard, D.. Antonsson, P., Hesborn, E., Larsson, T., Oldberg. .4.. Sommarin. Y .. and \Vendel. Xi. ( 1%). bLNoncollagenousmairis oonstituents in cartilage." Path. 7 es. 27-3 1.

Helfrich. J.H..Nesbitt, S.A. and Horton. M..4. (1992). J. 7 : 7:3J5-351.

Henderson, L.E.,Orosdan, S. and Konigsberg. W. (1979). "A Micromethoci for complete removal of dodecyl sulfate from proteins by ion-pair extraction." Ad5h&m. 9 3: 153- 157.

Herring. G.M.(196û). "S tucties on the prottin-bound chondroitin suiphate of bovine cortical bone." BmshmL 107: 4149.

Herring, G.M. (1972). . of Rof. G. H. Boume. New York. Xcademic Ress. 1: 127-189. Hcning, GM.(1977). "Methods for the study of the gfycoproteins of bcme using bacterid collagenase." Calcif. Tissue 2 4 : 29-36.

Hinek. A., Reiner. A. and PooIe. A.R. (1987). 'The calcification of cartilage matrix in chondrocyte culture: Studies of the C-propeptide of type II oollagen (chondrdcin)." J. 10 4 : 1435- 1U 1.

H.. R. 'The Hohiing, HJ., Schopfer. Hohling. R.A.. Hall. T..4. and Gieseking, (190).- orgaaic matris of developing tibia and femur. and macromolecuIar deii berations." kW * 57(7): 357.

Hohling. HJ...4ithoff, J., Barkhaus. R.H..kfting, E.R.. Lissner, G. and Quint. P. (1981). Mystages of crystal nucIeation in hard tissues. L - I.G. Schweiger. Sew York. Springa-Veriag. 974-982.

Holland. P.W.H., Harper, S J., McVey , J.H. and Hogan. B.L.Ll. (1987'). "ln vivo expression of mRXA for the Ca2+-bindingprotein SPARC (osteonetin) revealed by in situ hybridization." J.1 05: 473- 482.

Holt, C.and vaKesnenade, M.J.J.>l. (1989).The interaction of phosphoproteins with calcium phosphate. CalafiedTissue. D. W. L. Hukins. hndon. ,Llacmillan: 175-213.

Horitin. D., Fietzek, P.P.. Wachter. E., Lapme, GAI. and Kuhn, K. ( 1979). ".4mino acid sequence of the aminoterminal segment of dermatosparatic calf-skin procollagen type 1." Eur. J. Biochem 99: 3 1-38.

Howell. D.S.. Pita, J.C., Marquez, J.F. and Gatter. RA.(1%9). "Demonstration of macromolecular inhibitor(s) of calcification and nucieational factor(s) in fluid fmm calcifying sites in cartilage."U~R Invest.48: 630-641.

Howell. D.S. (1976). "Caicification Xfechanisms." Israel 12: 91-97.

Hunter, G.K.. IVong. K.S. and Kim, JJ. (1988). "Binding of caidum to glycosaminoglywis: .An equilibrium diaiysis study." Xrch, 260: 161- 167.

Hunter, G.K.and Goidkg, H..4. ( 19%). "Sucleation of hydrosyapatite by bone sialoprotein."hx.Jd,L . * CI. I S.4 90: 8562-8565.

Hunter, G.K.. Kyle, CL. and ûoldberg. H.A. (19%). "l\.lodulation of crystal formation by bone phosphoproteins: stmd specifici ty of the osteopontin-mdated inhibition of h y drox yapati te foxmation." BmjhemJ 300: 723-728.

Hunter, G.K.,Hauschka, P.V., Poole, AR.. Rosenberg, L.C.and Goldberg, H.A. (19%). "Nucleation and inhibition of hydroxyapaü te formation by mineralized tissue proteins." Bioctiem. 3 1 7 : 59-64. hefa- Arispe, ML.. Hasselaar, P. and Sage. H. ( 1991 ). "Differential expression of extracellular proteins is correlaied with angiogenesis in vitro." lab 6 4 : 174- 186. hela-Anspe, ML., Vernon. R.B., Wu. H.. J~EIIMLR. and Sage, E.H.(1996). 'Type 1-collagendeficient mov- 13 miœ do not retain sparc in the extracellular-matnx: implications for fibroblast function." V- V- 207: 171-183.

Johnson. D.R. ( 1986). The cadaginous skeleton. of %Sk&kd l&&gmem. New k'ork, Oxford Vniversity Press: 4û- I 17.

Jones. I.L. and Leaver. A.G. (1974). "Studies on the minor components of the organic matris of human dentine."Arch. 19: 371 -380. Jomson, M., Fredriksson, K.S..Iontell, M. and Linde, A. ( 198). "Isoelectric focusing of the phosphoprotein of rat incisor dentin in ampholine and acid pH gradients."J. 157: 235-242.

JonteU, M., Linde, A. and Lundvik, L. (1980). "Comparative studies of phosphoprotein preparations from rat incisor dentine." Riochem. O : 235-253.

Karp, W., Sodek, J.. Aubin, J.E. and Melcher, A. (1%). "-4 cornparison of fibronectin and laminin binding to undemineralized and demineralid tooth root surfaces." l. 2 1 : 30-38.

Kasugai, S., Todescan. R., Nagata, T., Yao, K.L., Butler, W.T. and Sodek, J. (1991). "Expression of boue matrix proteins associated with mineralized boue tissue formation by adult rat bone marrow cells in vitro: inductive effects of dexamethasone on the osteoblastic phenotype." J. CleUJhpd 147: 11 1- 120. bugai, S.. Nagata, T.and Sodek. J. (1992). 'Temporal studies on the tissue compartmentaiization of bone sialoprotein (BSP),osteopontin (OPN).and SP.4RC protein dunng bone formation in vitro." JLe phpld. 152: 467477.

Katz, E.P. and LI, S.T. (1973). 'The intermolecular space of reconstinited wllagen fibrils." J. 73: 351 -369.

Kemp. N.E. (IS). "Orgaaic marrices and mineral qstailites in vertebrate scales. teth and skeletons." &.EL ZQQL24: 965-9'76.

Kenyon, K., Contente, S., Trackman, P.C., Tang, J., Kagan, HAf. and Friedman, R.31. (1991). "Lysyl osidase and mg messenger RXA." W 2 53 : 802.

Kiefer. MC.,Bauer, D.M. and Bar. P.J. (19û9). Sucletc 1711: 3306.

Kim. R.H.. Li, J.J., Ogata, Y.. i'amauchi. Xi.. Freedman, L.P. and Sodek, J. (1996). "1dentif"icatioa of a vitamin D3-respoase elemmt that overIaps a unique inverteci T.4T.A box in the rat bone sialoprotein gene."Biochem. 3 31 8 : 2 19-226.

King Jr, L.E. and 'Llonison, M. ( 1976). 'The visualization of human qthrocyte membrane proteins and gl ycoproteins in SDS polyacrylamide gels employing a single staining procedure." Anai. 7 'f 1: 223-230.

Enne, R.W. and Fisher, L.W. (1987). "'Keratan sulfate protmglycan in rabbit compact bone is bone siaioprotein II." ,LhUhm26 2 : 10206- 102 1 1. . - Kivirikko. KI. and Raili, M. (1985). '%st-translational prcassing of procollageris." Annals hi. 1 . SGL 460: 187-201.

Koutsoukos, P.G.and Nancollas, G.W.(1987). 'The mineralization of coIIagen in vitro ." Coiloids 28 : 95- 108.

Kuboki, Y ., Fujim R., Tsuzaki, M., Fang Liu, C. and Sasaki. S. (19û-t). "Resence of lysindanine and histidinoalanine in bovine dentin phosphoprotein." CaIc.36 : 126-128.

Kubota, T., Zhang, Q., Wrana, J.L., Ber, R., Augin, J.E.. Butler, W.T.and Sodek, J. (1989). "klultiple forms of SPPl (secreted phosphoptein, osteopontin) synthesited by normal and transfomed rat bone ce11 populations: reguiation by TGF-p." RPF Commun. 16 2 : 1453- 1459. Kuettner, K., Sorgente, N., Croxen, R., Howell, D.S.and Pita, J.C. (1974). "Lysozyme in preosseous cartilage. W. Evidence for a physiological role of lysozyme in no4endochondral calcification." . . 372: 335. Kuîvaniemi. H., Savolainen. ER.and Kivirikko, K.I. (1981). "Hunan placental IysyI oxidase: Purification. parual characterization. and preparation of two specific antisera to the enzyme." J. Rinl C_liem. 2 5 9 : 6996-7002.

Kuwata, F., Y ao, K.L., Sodek, J.. Ives. S. and PuJleyblank, D. ( 1985). "Identification of pre-usteonectin produced by œil-free translation of fetai porcine calvarial &A." f, Blol. 26 0: 6993-4998

Kuwata. F.. .Maeno. M., Yao, K.L., Domenicuui, C.. Goldberg. H.A., Wasi . S., Autnn, J.E. and Sdek, J. (1987). "Characterization of a monoclonal antibody rmgnizing srnaIl collagemous proteins in fetal bone." Cdl.7 : 39-55.

Laemmli, C.K. (1970). "Cleavageof structufal proteins during the assembly of the head of bacteriophage T4." 227: 680-685.

Landais, J.C., Cohen-Solai, L. and Bonaventure, J. (1939). "Locaiization of y-glutamyi-phosphate residues to the a2CB3-5 peptide of Type 1 chicken bone collagen."GmnJhdk 1 9 : 1 -9. hdis,W.J. and Savmo, 'ci. ( 1983). "Correlatai physicochemical and age changes in embryonic bovine enamel." Calc.3 5 : 43-55.

Lane, T.F.. IrueIa-.hspe, L., Johnson, R.S. and Sage, EH. (1991). "SPARC is a source of copper-binding peptides btstimulate angiogenesis." J.125: -9-943.

Lane, T.F.. Irueia-.Gspe. ML. and Sage. E.H. (1992). 'Regdation of gene expression by SP.%RCduring angiogenesis in vitro: changes in fibronectin. thromtxlspondin- 1, and plasminogen activator inhibitor- 1." J. Riol. 267: 16736-16745.

Laue. T.F. and Sage, E.H. (1994). 'The biology of SP.LRC, a protein that modulates ceil-matrix interactions." EASERL 8: 163-173.

Lankat-Buttgereit, B., .LiaM, K.. Deutzmann, R.. Timpl, R. and Krieg, T. (1988). "Cloning and cornpiete amino acid sequaces of human and murine basement membrane protein BXI-UI (SP.UC. osteonectin)." ER332 36 6: 352-356. haver. AG.. Holbrook, I.B., Jones. I.L., Thomas. hl. and Sheil, L. ( 197%). "Components of the organic matrices of bone and dentine isolated oni y after digestion with collagenase." AnLQdhL20 : 2 1 1- 2 16.

LRe, S.L., Veis, .4. and Glonek, T. ( 1977). "Dentine phosphoprotein: .An exmcellular calcium-binding protein." 2 16 : 2971-2979.

Lee, S.L. and C'eis, .4. (1980). "Studies on the structure and chemisüy of dentine miiagen-phosphophoxy . - covalent camplexes." Calclf.31: 123-134.

Lee, S.L., Kossiva D. and Glimcher, kfJ. (1983). Thosphoproteins of bovine dentin: evidene for polydi&ty dunng cooth maturation." 22 : 2596-2601.

Lehninger, AL. (1970). "Mi twhondria and CS+transport." Biochem. 1 19 : 129- 138.

Lehninger, AL. (1977). Mitochondria and biologicai mineraiization processes. An exploration. Hmzmsm u-E. Quaiaiello, F. Palmieri and T. P. Singer. Addison Wesley. 4: 1.

Lang, M.K.K., Fessler, L.I., Greenberg, D.B. and Fessler, J.A. (1979). "Separate amino and carbonyl procollagen peptidases in chick embryo tendon." J.25 4 : 224232. kvene, C.I., Sharman, D.F.and Callingham, B.A. (1992). "Inhibition of chick embqo lysyl oxîdase by various lathyrogens and the antagonistic effect of pyridoxal." IRLLF- 73: 613625.

Lewandowska, K.. Cboi. H.U., Rosenberg, L.C.. Sasse, J., Neame. PJ. and Culp. LA. (1991). "Extracellular matris adhesion-promohng activi ties of a dermatan su1phate proteogl y can-associated protein (22K)from bovine feu skio." JX&ki. 99: 657-668.

Li. I.W.S., Chiefetz, S., McCulIoch. C.A.G., Sampath. K.T.and Sodek, J. (1996). "Effects of osteogenic protein- 1 (OP-1. BMP-7) on bne matris protein expression by fed rat calvarial cells are differentiation stage specific.**- 169: 115-125.

Li. S.T.and Katz, E. (1977). "On the state of anionic groups of demineralized matrices of bue and dentin." -. Calat 22: 275-284.

Lia. J.B.. Cohen-Solal, L., Kossiva, D. and Glimcher, M.J. (19û2a). "Changes in phosphoproteins of chicken bone matrix in vitamin Ddeficient rickets." FEBS 149: 123- 125.

Lian, J.B.,Roufosse, AH.,Reit. B. and Glimcher, SfJ. (19û2b). "Concentrations of ostdcin and . . phosphoprotein as a function of minerai content and age in cortical bone." Calaf.34: 382- 387.

Linde, A., Bhowm. Sf. and Buder. W.T. (19üû). "b~oncolIagenousproteins of dentin. .A re-examination of proteins from rat incisor dentin utilinng techniques to avoid artifrtcts." J_2 55 : 593 1- 5932.

Linde, .A., B ho-, !vf. and Butler. W.T. ( 1981 ). "Son-mflagenous proteins of rat dentine. Evidence that phosphoprotein is not covalwtly bound to collageo." Biochim. 667: 341-33.

Linde, PI.. Jontell, M.. Lundgren. T..Nilson. B. and Svanberg. C. (1982)."~oucolIagenous proteins of rat compact bone." I_ 25 8 : 1698- 1705.

Linde. .4. (1981a). The Morphology of Dentine and Dentinogenesis. -. -4. Linde. Boca Raton, CRC Press. 1: 1- 19.

Linde, A. (1981b). Lipids in dentinogenesis. .A- Linde. Boca Raton. Horida. CRC Press. II: 99-106.

Lmde. A. (1989). "Dentin matnx proteins: Composition and possible functious in calcification." AMLRCL 224: 154-166.

Linde. .4. and Lussi, -4. (1989a). "blineral induction by pol yanionic dentin and bone proteins at physiological ionic conditions." Cono.2 1: 197-203.

Linde, A.. Lussie, .4. and Crenshaw. M.A. (1989b). "kiinerai induction by immobilized polyanionic proteins." Calcif 44 40: 286-295.

Linde, A. and Goldberg, M. (1993). ''Dmtinogenais.*' Crit4(5) 40: 679-728.

MacBeath. J.R.E. Shackleton, D.R.and Hulmes, DJ.S. (1993). 'Tyrosine-nch acidic matrix protein accelerates collagen fibril formation in vitro." J. Blol. 15 : 19826- 19832.

.'MacDougail. M.. Zelchner, D.M.and Slavkin, H.C.( 19û5). "Production and characterization of antiùcniies against murine dentine phosphoprotein." Biochem. 232: 493-500- .bhgioire, H., Joffre, A. and Hartmann, DJ. (1988). "Lx>calization and synthesis of type III collagen and fibronectin in human reparative dentine. Immunoperoxidase and immunogold staining."

Maier, G-D., Lechner, J.H. and Veis. A. (1983). 'The dynamics of formation of a collagen-phosphophoqn cornplex oonjugate in relation to the passage of the mineralization front in rat incisor dentine." .J5.m.L Chem. 258: 1450-1455.

Majesica. RJ. and Wuthier, R.E. (1W5)."Studies on matri.\: vesicles isolated from cbck epiphyseai cartilage. .4ssociation of pyrophosphatase and ATPase activities with alkaline phosphatase." Scta 391: 51-60.

Mer, GD.. Lechner, J.H. and Veis. A. (1983). The dynamics of formation of a coliagen-phosphophosa conjugate in relation COthe passage of the mineraiization front in rat incisor dentin." J2b.U&m 258: 1150- 1455.

Mone. J.D.,Ttitelbaum. G.I., Griffin, G.L.. Senior, R..M. and Kahn. -4.J. (1982a). "Recruiunent of osteocIast precursors by purified bone matrix constiments."J. 92: 227-238.

MaIone, J.D., TeiteIbaum, S.L., Griffin, G.L.. Senior, R.M. and Kahn, AJ. (1982b). "Recniitmenr of osteoblast precursors by purified bone matrix constituents." J. 92: 227-230.

.Mandell, S.M. and Sodek. J. (1982)."Metabolism of collagen tjpes 1, III. auci i in the estradiol-stimulated uterus." JJbLQcm 257 57: 526û-5273.

Mann, K., Deutmiann, R.. Padsson, M. and Timpl. R. (1987). "Solubilizaîion of protein Bh14 from a basement membrane tumor with chelating agents and evidence for its idwti ty with osteonectin and SP.4RC." FERS 2 18 : 167- 172.

Mani, T.J., Trackman, P.C..Kagan, H.M., Eddy, R.L., Shows, T.B., Boyd, C.D. and Deak. S.B. (19%). 'The complete derived amino acid sequence of hiunan Iysyl osidase and assignrnent of the gene to chromosome 5." Mmk 12 : 242-2443.

Mark, M.P.. Rince, C.W., Oosawa. T.. Gay. S.. Bronckers, A.L.J.J. and Butler, W.T. ( 1987). "ïmmunohistochemid demonstration of a 44 Daphosphoprotein in developing rat bones." L 35: 707-715.

Mark, %{.P.. Butler. W.T., Prince, C.W., Finkelman, R.D. and Ruch, J.V. (1988a). "Developmental 44 expression of kDa bme phosphoprotein (osteopontin) and bone gamma-carboxggIutamc. - acid (Glal- containing protein (osteocdan) in cdcifying tissues of rat." Differentiabmi 37: 123- 136.

Uark, M.P.. Rince, C.W., Gay. S., Austin. RL., Bhown, M., Finkleman. RD. and Butler, W.T. (1988b). "4-4kDa bone phosphoprotein (osteopontin) antigenicity at ectopic sites in newborn rats: kidney and nervous tissues." CeU 25 1: 23-30.

Marsh. M.E. (1989). "Binding of caicium and phosphate ions to dentin phosphophoryn." Ibdmxu~28: 346-352.

Maniyama, K.. Mikawa, T. and -hi. S. (1984). ''Detecrion of calaum binding proteins by 4% autoradiography on nitrocellulose manbmne after sodium dodecyl sulfate gel electrophoresis." L 95: 511-519.

Mason. I.J., Taylor, A.. Williams, I.G., Sage. H. and Hogan, B.L.M. ( 1986a). "Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endderm. is related to an endorhelial œll 'culture shd' glycoprotein of Mr 13,000." FlMRO 5: 14651472. %on, IJ.. Murphy, D., Munke, M., Francke, tt.. Ellioc. R.W.and Hogan, B.L.M. (1986b). "Developmental and transformation-sensitive expression of the SP.4.RC gene on mouse chromosome 1 1." 5: 183 1-1837.

Masters, P..M.(1985). "ln vivo decornpsition of phosphoserine and serine in noncollagenous protein from human denùn." Calc. 37 37: 236-Bl. khuzawa. T. and .4nderson, H.C. (1971). "Phosphatase of epiphyseal cartilage studies by electron microscopie cytûchemical meth&." J. 12: 801-ûûû.

-McKee, M.D. and Nanci, .4. ( 19%). 'Qsteopon un and the bone remodelling sequene:. ColIoidal-gold . immunocytochemistry of an intedactial extracelluiar matris protein." AM. I\r .k. Jud, 7 60 : I77- 189.

.Wee, b1.D. and Naaci, .4. (1996). 'Qsteopontin at mïnerdized tissue interfaces in bone, teeth and osseointegrated implants: Cl trastructural distribution and implications for mineralized tissue formation, turnover and repair ." 33 33: 141 - 164.

.Mecha&. G.L., Banes, .i\.J., kiasauki, H. and Yamauchi. hl. ( 1%). Possible cullagen srnictural control of mineralization. wvofof. Tissues.. T. Butler. Birmingham. .Alabama, Ebsco Media: 98- 106. hlergenhagen. S.E. .Martin. G.R., Rim. .-\.A.. Wright. D.S.and Scott. D.B.(f%O). "Caicrf~cationin virro of implanteci coilagen." 43 ~3: 563-565. hienil. CR.Goldman, D. and Van Keuren. -ML.(1982). "Simplified silver procein detecuon and image enhancement methods in polyacrytamide gels." 3: 17-23.

Miller. A. (19%). "CoIlagen: the organic rnauix of bone." ph il.^ R,SD(Iu304 : 455-477. hliller. EJ. ( 19&lb). Rwnt information on the chemistry of the collageus. g.the. T. Buder: 80-93.

Miyauchi. A.. .Zlvarez J., Greenwood. E.M.,Teti. A., Grano, Lf.. Colucci, S., Zambonin-Zallone. .A., Ross. F.P.. Teiteibaum. S..tl., Cheresh, D.,et al. (1991). f.266: 20369-20374.

Sliyazaki, le.,Setoguchi. M.. k'oshida. S., Higuchi, Y.. .LUuzula. S. and Yamamoto, S. (1990). 'The mouse osteopontin gene. Expression in monocytic lineages and complete nucleotide sequene."LBmL Chem. 265: IU32- lU38.

Moore, ,M.A.. Gotoh, Y.,Rafldi. K.,and Gerstenfeld, L.C. (1991). "Charactaization of acDN.4 for chcken os teopontin: expression during bone development. osteoblast differentiation. and tissue dismbution." 30: 2501-2508,

Mundy. G.R. and Poser. I.W. (1983). ''Chexnotactic activity of y-carboxygiutamic acld containing protein in bone." 3535: 164168.

Mqhy-Cllrich, JE. Lighmer, V.A.. Aukbil, I., Yan, Y Z.. Erickson. H.P. and Hook. M. (199 1). "Focal achesion integnty is downregulated by the alternatively spiiced domain of human tenascin." UZdl BiaL 115: 1127-1136.

Muthukumaran, N., Sampath, T.K. and Reddi, A.H. (1985). "Cornparison of bone inductive proteins of rat and porcine borie matris." 13 1: 3741. Nagata, T.,Tdescan, R.. Goldberg, H.A.. hg,Q. and Sodek,J. (1989). "Sulfation of secreted phosphoprotein 1 (SPPl. Osteopontin) is associateci with mineraiid tissue formation." Biûchem. 165: 23-240.

Nagata. T.,Goldberg. H.A., Zhang, Q., Domenicucci, C. and Sodek, J. (1991 a). "Biospthesis of bone proteins by fetal porcine calvariae in vitro. Rapid association of sulfatai sialoproteins (secre ted phosp hoprotein 1 and bone sidoprotein) and chondroitin sulfate proteoglycan (CS-PG[II) with bone rninéral."Uatris 11: 86-100.

Nagata. T., Bellow. CG..Kasugai. S., Butler. W.T. and Scdek, J. (1991b). "Biosynthesis of bone proteins. SPP- 1 (secreted phosphoprotein- 1, osteopontin) BSP (bone sialoprotein) and SPARC (osteonectin) in association with mineralized tissue formation by fetaI rat calvaxiai ceils in culture." E?m&mJ 274: 5 13-520.

Nakamura, O., Gohda. E.. Ozawa. M., Senba. 1.. 'Lti yazaki, H.. Murakami. T.and Dalkuhara, Y. ( 1985).

"Immunoh s tochemical studies with a monoclonal anti body on the distri bution of phosphopboqli in predentin and dentin."Calc. 37 3f 49 1-500.

Sancollas, G.H.. hre. M., Perez, L.. Rcbardson. C. and Zawacki, S J. (1989). "Minerd phases of calcium phosphate." Anat. 224: 2N-21I.

Sarayanan. A.S.. Siegel, R.C. and >fartin. G.R.(1974). "Stabiiity and purification of Iysyl osidase." Ad. 162: 231-237.

Neame, P.J., Choi. H.C. and Rosenberg. L.C. (1989). 'The isolation and primaq structure of a 22-kDa extracelldar matris protein from bovine skin." J.264: 5473-5479.

Xeuhoff. \'. . Stamm, R. and Eibl, H. (1 9û5). 6 : 427448.

2leiun;rn. R*.F.and Seuman, hl. (1953). 'The nature of the minerai phase of bone." Clhem_ 53: 1.

Siyibxzi. C. and E~re.D.R. (1989). "Boue type \' collagen: chain composition and localization of a qpsin cleavage si te." Conn 20 20: 247-33.

Siyibizi. C. and Eyre. D.R. (1994). "Suucturai chzincteristics of aoss-1in)Üng sites in tjpe Y collagen of bone. Cham specificities and heterotypic links to type 1 collagen." Eiu. 224: 543-950.

Somura, S., 1-ills,-4.J.. Edwards, D.R., Hath, J.K. and Hogan, B.L.M. (1988)."Deveiopmentai expression of 2ar (osteopontin) and SP-AFlC (osteonectin) RKA as revealed by in siru hybridization." J. CelLEbi 106: U1440.

ûike, Y.,fimata. K., Shinomura, T.. Xbwa,K., and Suniki. S. (1980) Structural adysis of chick- ernbryo cartilage proteuglycan by selective degradation with chonciroitin 1yases (chondroitinases) and end&eta-i(keratanase). Biochem 19 1 : 193-2M.

Oldberg ,A., Fm.A. and Heinegard, D. ( 1986). "Cloningand sequence anal y sis of rat bone sialoprotein . . (osteopontin) cDXA reveals an kg-Gly-Asp dlbinding sequence." -aSCr. I S 4 83 : 88 19-8823.

Oldberg, .4., Fr-, A.. Heinegard, D.,Pierschbacher. LM.and Ruoslahti. E. ( 1988a). "identification of a bune siaioprotein receptor in osteosarcomacds." J. 263: 19433-19436.

Oldberg. A., Fm,A. and Heinegard. D. ( 19ûûb). 'The primary structure of a çell-binding bone sialoprotein." LBiol. Chem. 263: 19530- 19432. OIdberg, A.. Jirskog-Hed. B.. Axelsson, S. and Heinegard, D. ( 1989)."Regulation of bone sialoprotein (BSP) mRNA by stuosd hormones." J. 1O9 : 3 183-3 186.

Otsubo. K.. KatZ E.P., Mechanic. G.L.and Yamauchi. M. (1992). "Cross-linking connectivity in bone cuilagen fibrils: The COOH-terminal locus of free aldehyde." hchmu~~3 1: 39442.

Otsuka, K., Yao, K.L.,Wasi, S., Tung, P.S., Aubin, J.E., Sodek. J. and Tennine, J.D. (LW)."Biosynthesis of osteonectin by fetai porcine calvarial cells in vitro." J. 259: 9805-9812.

Otsuka, K.. Pitaru, S., Overall, C.M., Aubin, J.E. and Sodek, J. (1988). "Biochemical cornparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis." 66: 167-176.

Overdl, C.M. (1987). "A microtechnique for dialysis of small volume solutions with quantitative recoveries." Ad5hh~.165: 208-214.

Overall. CM.and Sodek, J. (lm."Initial charactexization of a neutral rnetalloproteinase. active on native 3.4- collagen fragments, synthesized by ROS 17 '2.8osteoblastic exils. periodontal fibroblasts. and identifieci in gingival crevicular fluid."l. 66 : 127 1- 1282.

Overall, M. and Limeback. H.(1988). "ïdentificationand characterization of enamel proteinases isolated from developing enamel." I3hchmJ 256 : 945-972.

Paglia, L., Wilczek, J., Diaz de Leon, L., Martin, G.R., Horlein. D. and >ldier, P. (1979). "Inhibition of procollagen cd-free s)mthesis by amino- terminal extension peptides." 18 : 5030-5034.

Paglia, L.-M., Wiestner, M., Duchene, M.. Ouelette, LA, Horlein, D., hiartin, G.R. and hldler. P.K. (1981). 20: 3323-3327.

Palamara, J., Phakey , P.P., Rachinger, W.A. and Orams, HJ. (198 1). 'The dtrastructure of enamel from the kangaroo. .Macropus giganteus; Tubular enamel and its black spots defect." Aus.LL~29 : 643 - 652.

Patarca, R., Freanan, G.J., Singh. R.P.. Wei, F.Y.,Durfee, T.. Blattner. F.. Regmer, D.C., Kozak, C.A., -Mock, B.A.. Morse in, H.C.,et al. (19û9). "Structural and functiod snidies of the early T lymphocyte activation (Eta- 1) gene." J.1 70: 145- 162.

Pearson. C.H. and Gibson. GJ. (1982). "Proteoglycans of bovine periodontd ligament and shn. Occurrence of different hy brid-sul phated galactosaminog1ycans in distinct proteogl ycans." Biochem. 20 1 : 27-37.

Pearson, C.H.,Winterbttom, K., Fackre, D.S.,Scott, P.G.and Carpenter, .M.R. (1983). 'The 3&-tenniaal amino acid sequenœ of bovine skin protedermatan sulfate." J.258: 15 10 1- 15 104.

Pearson. CH.and Pringle, G.A. (1986). "Chemical aud immmochemical characteristics of proteoglycans in bovine gingiva and dental pdp." Arch.3 1: 541-548.

Penke, B.. Ferenczi, R. and Kovacs, K. (1974). "t4new acid hydrol ysis method for determining tryptophan in peptides and proteins." Anal. 6 0 : 45-50.

Pettigrew, D.W., Ho, G.H., Sadek. J., Bmette, D.M. and Wang, H-hl. (1978). "Effect of oxygen tension and indometbacin on production of collagenase and neutral proteinase enz)mes and th& latent forms b y porcine gingival explants in culture." Arch.23: 767-777.

Pettigrew. D.W.,Wang, H.M.,Sodek, J. and Brunette, DM.(1981). "Spthesis of collagenolytic enzymes and their inhibi tors by gingivai tissue in vitro." J. 16 : 637-645. Pokric. B. and Pucar, 2. (1979). "fiecipitacion of calcium phosphates under conditions of doubIe diffusion in . . coiiagen and gels of gelatin and agar." CaInf.27: 171-176.

Poole. A.R.. Pidoux, 1. and Rosenberg, L. (1982). "Role of proteogiycam in endochondral ossification: immunofluorescent locdization of link protein and proteqlycan monomer in bovine fetal gnphyseal growth plate." I. 92 : 249-260.

Pmle, A-R., Pidoux, 1.. Reiner, A., Choi. H. and Rosenberg, L.C. (1984). "Association of an exrracelluiar protein (chondrocaicin) with the calcification of cartilage in endochondrai bone formation." J. C'eilBiaL 98: 54-65.

Poole, A-R., Matsui. Y., Hinek, A. and Lee, E.R. (1989). "Cartilage m#xomoIecdes and the caicification of cartilage matrirr." Anat. 224: 167- 179.

Aie, P.A., Otsuka. A.S.. Poser, J.W., fistaponis. 1. and Raman. N. (1976). "Characterization of a gamma- - - a-boxyglutamicacid-containing protein from bone." -- SSci, 73: 1447- 1151.

Price, P.A. and Baukol. S.4. (1980)."la Dihydrosyvitamin D3 increases spthesis of the vitamin K dependent bone protein by osteosarcorna celIs." J. 255 : 1 1660-1 1663.

fice, P..4. and Williamson. >$.K.(1981). "Effects of warfarin on bone. Studies on the vitamin K-dependent protein in rat bone." J.-Cha~ 2 5 6 : 127% 12759.

Rice, P..\., Cnst, M.R. and Otawara, Y.(1983). "liatrix Gla-pmein; a new y-carbosyglutamic aadcontaining protein which is associated with the organic mauix of bone." Bi

Price, P..\. (1988). 'Role of vitamin K-dependent proteins in bone metabolisrn." Xnrt 88: û65-883.

Rice, P..4. (1992). Gla-concaining proteins of mineraiized tissues. Tissues. H. Slavkin and P. A. Rice. Sew 1-ork. Elsevier: 169-176.

Prince, C.W., Oosawa. T.. Butler. N..T., Tomana, .Li.. Bhown. AS.. Bbow-n, hl. and Schrohenioher. R.E. (1986). "Isolation. cbaracterization and biosqnthesis of a phosphorylated glycoprotein from rat bone." L Biol. 262: 290-2907.

Prince, C.W. and Butter, W.T. (1987). "1.3-Dihydroxyvilamin D3 regdates the bios)athesis of osteopontin. a bonederived ceil attachent protein, in donal osteoblast-like os teosarcorna œiis." 7: 305-313. fina, C.W., Oosawa. T., Butler. W.T., Tomana. M., Bhown, AS., Bhomn. M. and Schrohdoher. R.E. (IW."Isolation, characterization, and biospthesis of a phosphorylated glycoprotein from rat bone." JLBkUhn262: 290-2907.

Frince, C.W. (1989). "Secondriry stmcture predictions for rat osteopontin." Conn.2 1: 15-20.

Ringle. G.A. ( 1985). Production, Characterization and Lse of Mmodonal Anti bodies to Fbteodermatan Sulfate., University of Alberta.

Ringîe, G.A. and Dodd, C.M.(1990). "Immunoelectron micmclocalization of the cure protein of decorin near the d and e bands of tendon collagen fibrils by use of monoclonal antibodies." Q~Q&IXL 38: 1405-141 1. Rockop, DJ-.Kivirikko. KI., Tuderman, L. and Gumian, S.A. (1979). 'The ùiosynthesis of collagen and its disordgs." Nm3,tgFnP.W 30 1 : 13-23 and 77-85.

Prout, R.E.S. (1%9). "A mctbd for separalion and estimation of chondroitin sulphate isomers by electrophoresis on cellulose acetate papa." 17 7 : 157- 158.

Provencher, S.W. and Glockner, J. (1981). "Estimation of globuiar protein secondaq strucfure from àrcular dicksm."~Q&III& 20: 33-37.

Rahima. M., Tsay, T.G., Andujar. M. and Veis, A. (1988). "LocaIization of phosphophoryn in rat incisor den tin using immunocytwhemical techniques." J. 36 : 153- 157.

Raines, E.W., Lane, T.F.. I~eIa-Arispe,M.L.. Ross, R. and Sage, E.H. (1992). 'The extracellular gl ycopiprein SPmCinteracts with platelet-deriveci grow th factor (PDGF)-AB and -BB and inhi bi ts the . . binding of PDGF to its receptors." Proc..Sa. 1.i SA 89: 1281-1285.

Rao, L.G., ?;g, B., Brunette, D.M. and Heersche, J.X.M. (1977). "Parathjroid hormone- and prostaglandin El- response in a selected population of bone cells after repeated subculture and srorage at -80'C." 100: 1233-1241.

Rao, L.G., Wang, HM,Kalliecharan. R.. Heersche. J.S.Xl. and Sodek. J. (1979). "Specific immunohistochemicd ldizatioa of type 1 collagen in porcine periodontal tissues using the peroxidase-labelled antibody techique." HimdxmJ 1 11: 73-82.

Raynal, C., Delmas, PD. and Chenu. C. (19%). "Bone siaioprotein stimulates in vitro hne resorption." Fndocrinaloa. 137 : 2337-2354.

Reichert. T., Siorkel, S., Becker, K. and Fisher, L.N'. (1992). 'The roIe of osteonectin in human tmth . . devdopment: an irnmunological study." 50 : 468472.

Reiaholt. F.P., Hdtenby, K., Oldberg, A. and Heinegard. D. (1990). "Osteopontin - a possible anchor of osteoclasts to boue." Roc. 87: 4473475.

Rennard, S.I.. Berg, R., hiartin. G.R.,Foidart, J.11.and Robe!, P.G.(1980). "Enzyme-linked immunoassay (ELiSA) for connecti ve tissue components." Anal. 1 O4 : 205-2 11.

Rey, C. and Glimcher, 543. ( 1992). Short rage organizatioa of the Ca-P minera1 phase in bone and enamel ; changes with age and maturation. a.H. Slavkin and P. Rice. Amsterdam, Elsevier Science Publishers B.V.: 5- 18.

Ritchie. H.H., Hou, H.. Veis, A. and Butler, W.T. (1994). "CIoning and sequace detennination of rat dentin sialoprotein. a novel dentin protein." J~ML~~R269: 3698-3702.

Ritchie, H.H.,Rnero, G., Hou, H. and Butler, W.T. (1995). "%lolecuiar amiysis of rat dentin sialoprotein (dsp)." Connect.33 : 73-79.

Robison, R. (1923). The possible significance of hexose phosphoric esters in ossification." Biochem 17: 286-293.

Rohde. H., Nowack. H., Becker, U. and Timpf ,R. (1976). "Radioimmunoassap for the aminoterminal peptide of promllagenpa 1 (1)-chain."- 11: 135-145.

Rohde, H. and Tïimpl, R. (1979). "Structure of antigenic detenninants in the N-terminal region of denuntosparactic shetp procoIlagen type 1." Bu&xdi 17 9 : 643-647. Romberg. R.W.. Werness, P.G., Lollar. P.. Riggs. B.L. and Mann. KG. (1985). "Isolation and characterîzation of native adult osteonectin."J. 260 : 2728-2736.

Rosenberg, L.C.,Choi, H.U., Tang, L.H.. Johnson, T.L.,Pal, S., Webber. C., Reiner. .4. and Poole. -4.R. ( 1985). ''Isolation of dermatan sulfate protmgl ycans from mature bovine articular cartilages." ,L&QL Chem. 260: 6304a313.

Ross, F.P., Chappel, J.. AIvarez J.I., Sauder, D., Butler. W.T., Farach-Carson, h1.C.. %fintz. RA, Robey. P.G..Teitelbaum, SL. and Cheresh, D.A. (1993). "Interactions between the bone matnx proteins osteopontin and bone sialoprotein and the osteoclast integrin a, & potw tiate bueresorption." J..JhL 5268: 9Kl1-99û7.

Ruosiahti, E. ( 1989). "Proteoglycans in ce11 regdation. (Review)." J. 264: 13369- 13372.

Sage, H.. Johnson, C. and Bernstein, P. (1%). "Characterization of a novel senun albumin-binding glycoprotein secreted by endothelid cells in culture." LJ~AEQL259: 3993W.

Sage, H., Tupper. J. and Bramson. R. (1986). "Endothelial ceIl injur) in vitro is associated with increased secretion of an .% 43,000 glycoprotein ligand." J.127 : 373-387.

Sage, H., Vernon. R.B., Funk, S.E.. Eventt, E.A. and .bgelIo, J. (1989). *'SP.UK, a secreted protein associated wi th ceIl uiar proiiferation, inhibits cet 1 spreading in rirro and exhi bi ts Gaz+-dependait binding to the exffa~eIluiarmatris." J,109: 34 1-356.

Sage, E.H.and Vernon. R.B.(1994). "Regdation of angiogenesis by extracellular niauix: the growth and the glue." J. of Hjpamsh 12 (su ppi): S 1G-S i 52.

Sajdera, S.lV.and Hasdl, V .C. (1969). 'Proteinpolysaccharide cornplen from bovine nasal cartilage. .A cornparison of low and high shear extraction procedures." J. 242: 77-87.

Salonen. J.. Dornenicucci, C., Goldberg, HA.and Sodek, J. (1990). "Imrnunohistochemical locaiization of SPARC (osteonectin) and denatured coIlagen and their relationship to remodelling in rar dental tissues." 4rch. 35: 337-346.

Sato, S.. Rahemtulla, F., Prince, C.W., Tomana, Si. and Butier, W.T. (I985a)."Proteoglycaas of adult bovine compact bone." Connect,14: 65-75.

Sato, S., Rahemhilla, F., Prince, C.W.. Tomma, M.and Butler. W.T. (198%). "Acidic glycoproteins from bovine compact bone." 14 : 5 1-64.

Sauk, J.J., Gay, R., Miller, EJ. and Gay. S. (1980). b'hunohistochemical localization of Type III collagen in the deutin of patients with osteogenesis imperf'ecta and hereditaq opalescent dentine." LQmL Pathol. 9: 210-220.

Schagger, H. and Von Jagow, G. (1987). 'Tricine-sodium dodecyl sulfate pol yacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa" Anal. 166 : 368-379.

Schiueter, RJ. and Veis, A. (1964). 'Tbe macromoleculm organization of dentine matrix collagen. II. Periodate degradation and dmhydrate cross-linking." 3 : 1657.

Schmid, TM.,Mayne. R., Bruns, R.R. and Linseamayer, T.F.( 1%). "Molecular structure of short chan (SC) cartilage by electron micn>scopy." J. 8686: 186- I9 1. SchwarzbaLIer, J. and Spencer. C.S. (1993). 'The Caenorhabditis elegans homoIog of the extracellular calcium binding pmttin SPARC/ostewectin affects nematode body morpholog and mobility." Mdli~L W(W1 -952).

Scott, P.G.and Veis. A. (1976a). 'The cyanogen broxrtide peptides of bovine soluble and insoluble collagens: 1. Characterizahon of peptides from soluble Type 1 collagen by sodium ddecylsulphate plyacrylamide gel electrophoresis." Connect.4 : 1107- 1 16.

Scott, P.G. and Veis, A. (1976b). 'The cyanogen bromide peptides of bovine soluble and insoluble collagens. II. Tissue smc aoss-tiaked peptides of insoluble skin and dentine collagen." 4 : 1 17-129.

Scott, P.G.and Pearson, C.H.(1978). "Selective soiubilization of type 1 coilagen from calf skin by cathepsin D." fiiochem.6: 1197-1199.

Scott. P.G.,Wincerbottm. K.. W.C.51.. Edwards, E. and Pearson, C.H.(1986). ".4 role for disuiphide bridges in the protein core in the interaction of protedermatan su1 phate and collagen." Bicrhem. Bior>hvs, 138: 1348-1353.

Senga. D.R.. Winh. D.F. and Hynes. R.0.(1979). 'Transformed mammdian cells semete specific protcins and phosphoproteins." 16 : 885-893.

Senger. D.R.. PM.C.,4., Gracey. C.F., Papadopodos. .A. and Tenen. D.G.( 1988). "Secreted phosphoprotein assaiated with neoplastic transfomation: dose homology with plasma proteins cleaved during blood coagulation." 4 8 : 577)-5774.

Senger. D.R..Pd, C..4. and Papadopoulos. A. (1989a). "Eievated expression of secreted phosphoprotein 1 (osteopontin 2ar) as a conseqwnce of neoplastic uansformation."~9: 1291- 1300.

Senger. D.R.. Peruzzi. C.A.. Papadopoulos. -4. and Tenen. D.G. (1989b). "Purification of human milk protein close1y similar to -or-seaeted phosphoprotein and osteopontin." 996 96: 43- 38.

Seyedin. SM.,Segarini, P.R..Rosen. DM..Tompson. A.Y..Ben% H. and Graycar. J. (1987). "Mlqe- in&cing factor-$ is a unique protein structuraIly and functionalIy related to transforming growth factor- @." J.262: 1936-1949.

Shackleton. D.R. and Huimes, D.J.S.(19%). 'Purification of lysyl oxidase from piglet skin by selective interaction with Sephacryl 5-200." EuxbnJ 266 : 9 17-919.

Sbapiro, H.S.,Cbm, J.. Wrana. J.L.. Zhang. Q.. Blum. hl. and Sdek. 1. (1993). "*Chamct&zationof porcine bone sialoptein: primary structure and cellular expression." :Clanix 13 : 43 1-W.

Shen, X.. Roberts. E.. Peel, S.A.F. and Davies, J.E. (1993). Cells 33: 251-272.

Shepard. N. and Mitchell. N. (19ûS). 'Zn trastnicttxrai modifications of proteoglycans coinadent aith minerdization in local regions of rat growth plate." J. 67 : 45455461.

Shuaga, H.. Min. W., VanDusen. WJ.,Clayman, M.D.. Miner, D.. TareIl. C.H.. Shhtie. J.R.. Fo~mao. J.W.. Pqsiedj. C., Neilson. E.G.,et al. (1992). '4nbibition of daum o.date aystal growth in vitro b y uropootin, a new member of the aspartic acid-rieh protein superfamily." Rnc.Yd,AzU& 89 : 426430. Sbunleworth, C.A. and Sdley.J.W. (1986). Periodontd Ligament. TissueResearch. D. -4. Hall and D. S. Jackson. London. Academic bs.10: 2 1 1-2-48.

Siegel. R.C. (1975). "Biosynthesis of coliagen crosslinks: increased activity of purified lysyl osidase with . . feconstimted collagen fibrils." Roc_ 7 Sa : 112640.

Siegel. R.C. (1979). "LysyI oxidase." Int 8: 73- 1 18.

Silbert, JE, Palmer, ME.,Humphnes, DE and Silbert, C.K.(1986). "Formation of dermatan sulfate by cuitured human siun fibrobiasts. Effects of suifate concentration on proportions of c&rmawchondroitin.'*J. 26 f : l33W- lMlû.

Smith. 4J.and Leaver. AG. (1978). 'The effets of peridte degradation and collagenase digestion on the mganic matrix of hiuniui dentine." Arch 23 : 53-92.

Sodek. J. and hiandeIl, S -51. ( 1982). "Collagen metabolism in rat inüsor predentine in vivo: Syuthesis and maturation of type 1, a 1 (1) trimer. and type V collagens." Eb&mui~2 1 : 20 1 1-2015.

Sdek. J.. Domenicucci. C.. Zung, P., Kuwata, F. and Wasi, S. (1986). Bios)nthesis. properties and . .. distribution of osteonectin. [. S. lesicles..Ai. .Amsterdam. Eisevier: 135- 141.

Saiek. J., Overdl. C.M.. Wrana. J.L., 'Liaeno. M. and Kubota, T. (1988a). 'Liotecular mechanisms of remodelling in the periodontium: regdation by transforming gowth factor-$. -. -. -. J. ishikawa .knsterdam. Elsevier.

Sodek, J. ( 1989). Collagen turnover in penodontal ligament. Biologt of -. -4.L. Sonon and C. J. Burstone. Boca Raton, CRC Press: 157- 181.

Sodek. J.. Zhang, Q.. Goldberg. H..Z., Domenicuu5. C., bugai. S.. Wr~ia.J.L.. Shapiro. H. and Chen. J. ( 1991). Non-collagenous bone proteins and their role in substrate-indud bioactivit?. lkhn~ -. -. J. E. Davies. Toronto, Lniversity of Toronto Press: 97-1 10.

Sodek. J.. Chen, J.. Kasugai. S., Nagata, T., Zhang, Q., Shapiro. H.S..U'rana. J.L. and Goldberg. HA. (lm). Sidoproteins in bone remodelling. The F3- of T~~E+lovemen~nd Craniofactai 2, Davidovitch. Birmingham, .Mabarna. Ebsco 'Liedin: 127- 136.

Sodek. J.. Chen. J., Kasugai. S .. Sagata, T., kg,Q., McKee, MD.and Sanci, .A. ( lW2b). Eludatrng the functions of hne sialoprotein and osteopontin in bone formation. -. -. H. Slavkin and P. Rice. .Amsterdam. Elsevier: 297-306.

Sodek. J., Chen. 3.. Sagata T.. bugai, S.. Todescan Jr., R.. Li. I.W.S. and lum. R.H. (1995). Regdation of Osteopontin Expression in Ostmblasts. v.D. T. Denhardt. W. T.Butler, A. F. Chambers and D. R. Senger. Sew 1-ork.The New York Xcademy of Sciences. 760: 223-251.

Somennan, MJ., Rince. C.W., Sauk. J.J.. Foster, R.A. and Butler, W.T. (1987). "Mechaaism of fibroblast amchment to bone extraceUuiar rnacrix: Role of a 44 kilodalton bone phosphoprotein." J3meMm~ Bes, 2 : 259-265. Sornerman, -M.J.. Fisher, L.W., Foster. R.4. and Sauk.-. J.J. (1988). "Human boue sialoproteins i and ll enhance fibroblast attachment in vitro." Calnf.43 : 5û-53. Sm-, M.J.. Prince, C.W..Butler. W.T ., Foster, R.A.. Moehring, J.M. and Sauk, J.I. (1989). "Ceil attachment activity of the 44 kDa bone phosphoprotein is not restricted to bone cefls." Matrix 9: 49- 54.

Spector. .4.R. and Glimciier, blJ. (1972). 'The extraction and characterizarion of soluble anionic proteins from bone." Bi6chem, 26 3 : 593-603.

Spccttor. AR. and Gh&er, M.J. (1973). 'The identification of O-phosphoserine in the soluble anionic phosphoproteins of bone." Rlocfum.303: 360-362.

Starkey. P.M..Barrett, .\.J. and Butteigfi, MC.( lm.'The degradation of articula coilagen by neutrophil proteinases." 4 8 3 83: 386-397. . . Stassen, F.L.H. (1976)."Properties of highly purifieci lysyl oxidase from embryonic chick cartilage." Riochim. 4 3 8 : 49-60.

Stein, G-S..Lan. J.B. and Owen. TA.(1990a). "Bone ce11 differentiation: a functiondly coupied relationshp between expression of cell-growth and tissue-speaîïcgens ." Ciirr Op in T~BQL2 : 10 18- 1027.

Stein, G.S.. Lian, J.B. and Owen, T.-4. (1990b). bbReIati~~hipof ceIl growth to the regdation of ussue- specific gene expression during osteoblast differentiation." E1SER.L. 4 : 3 1 1 1 -3 123.

Steinfort, J. (1990). The possible role of noncollagenous mauis components in dentin minerdization. The rat incisor as a model. .buterdam, Luiversit): of .-terdam: 1- 11 1.

Steinfort, J., Van de Stadt. R. and Beertsen, W. (1%). "Ideutification of new rat dentin proteogiycans utilizing C 18 chromatography ." 2 6169 : 22397-22U);L.

Stenner. D.D., Tracy. R.P., Riggs. L.B. and Mann, KG. (1986). "Human ptatelets contain and secrete osteonmin. a major protein of mineralized bone." Proc..83 83: 689263%.

Stetler-Stevenson, W.G. and Yeis, A. (1986). 'Type 1 coilagen shows a specific binding affinity for bovine dentin phosphophoqn."Calc. 38: 135-141.

Stetler-Stevenson, W.G. and Veis. -4. (1987). "Bovine dentin phosphophorp: Calcium ion binding propedes . - of a hi& moIecular weight preparation." Calcrf_ 40 : 97- 102.

Stevens. F.C..Waish, M.. Ho, H.C., Seng Teo. T.and Wang. J.H. ( 1976). "Cornparison of calcium-binding proteins. Bovine kart and brain protein activators of cyclic nucleotide phosphdesterase and rabbit skeletd muscle troponm C." J. BioL 25 1: 44954500.

Sullivan. K.A. and Kagan. HM.(1982). "Evidence for suucniral similarities in the multiple forrns of aortic and cdagelpyl oxidase and a catalytidy quiescmt amie prorein." lRiol ZS 7 : 13520- 13526.

Superû-Furga. -4.. Rocchi. M.. Schafer, B.W. and Gitzelmann, R. ( 1993). "Cornplaentary DX.4 sequence and chromosoma1 mapping of human proteoglycan-binding d-adhesion prorein (derniatopontin)." Genomics 17 : 463467.

Swanson. GJ., Nomura, S. and Hogan, B.L.M. (1989). "Distribution of expression of 2.- (osteopontin) in the embryonic mouse innerear revealed by in situ hybridization." Hearing 4 11 169- 178.

Syftestad, G.T. and LTrist,MX. (1982). %me aging."ChAdmp 162: 288-297. Tm Cate, A.R. (1989). Bone. Oral:Devv R. W.Reinhardt. Sr. Louis. Missouri, C.V. Mosby Co.: 115138. Tenenbaum, H. and Heersche,. . J.M.N. (1982). '*Differenliationof osteoblasts and formation of minerdi zeù bone in vitro." Calclf,Tissue 34: 76.

Tennine. J.D.and Conn, K.LM(1976). "Inhibition of apatite formation by phosphorylated metabdites and .. macromolecules." Calnf.2 2 : 1.19- 157.

Termine, J.D.,Belcourt, A.B., Christuer, P J.. Conn, KM.and NyIen, M.C. ( 1980). "Properties of dissociatively extracted fetal (00th matrix proteins. II. Separation and purification of fetal bovine dentin phosphoprotein." J.255: 9769-72.

Termine, J.D., Belcourt, AB.. COM. K.LMand Kleinman, H.K.(1981a). "Xfinerai and collagen-binding proteins of fetal calf bone." J_ 256: 10103-l(UOS.

Termine. J.D., Kleinman, H.K..Whitson. S.W., Conn, KM.,McGarvey, ML. and Martin. G.R. (198 1b). "Osteonectin, a bone-specific protein linking minerd to wllagen." QU26: 99- 105.

Tennine, J.D.. Robe?, P.G., Fisher, L.W.. Shimokawa. K.. Drum, .M..%, Corn. KM.,Hawkins. G.R.. Cruz, J.B. and Thompson. K.G. (1984). "Osteonectin, bone proteogl ycan. and phosphophorp defects in a form of bovine osteogenesis irnperfecta."PIoc.dS.&UA 8 1: 2213-2217.

Thomas. M.and Laver, AG. (1977). 'The less acidic glycoproteins of the organic matrix of human dentine." 22: 535-549.

Towbin, H., Staehelin, T. and Gordon, J. (1979). "EIectrophoretictransfer of proteins from polyaqlamide gels -. to nitrocellulose sheets: Procedure and some applications." Proc_Satl.a. 1 S 4 76: 4350439.

Trackman, P.C.,Pratt, X..Ll.. Urolanski, A.. Tang, S.S., Offner, G.D.. Trosier, R.F. and Kagao, HAI. (199 1). "CIoning of rat aorta Iysyt oxidase cDKA: complete donsand predicted amino acid sequence (correction)." Biochem. 30 : 8282.

Traub, UV.,Jodaikin. A.. Arad. T., C'eis, A. and Sabsay, B. (1%). "Dentin phosphophoqn binding to collagen fib~ls.",liiaozT 12: 197-201.

Triffit. J.T. and Owen, ME.(1973). "Studies on bone matrix glywproteins; Incorporation of [1-l-C] glucosamine and plasma [KIglycoprotein into rabbit cortical bone." Biochem. 136 : 125- 134.

Triffit. J.T., Gevauer, L., Ashton, B.A. and Owen, M.E.(1976). "Origin of plasma a2HS-glycoprotein and its accumulation in bone." Llanire. 26 2: 226-227.

Triffit, J.T., Owen, M.E., Ashton, B.A. and Wilson, J.U. (lm)."Plasma disappearance of rabbit a2HS- . - glycoproteins and its uptake by bone tissue." 2626: 155-161. . . Triffit. J.T. (1980). The organic matrix of bone tissue. e.SiPbvsroloPv.R- Urist. Philadelphia. J.B. Lippincott Co.: 45-82.

Tnffit. J.T. (1987). 'The spial proteins of bone tissue." Cllia 72: 39948.

Tung. P.S.,Domenicucci, C.. Wasi, S. and Sodek, J. (lm)."Specific immunohistochemicai locaiization of osteonectin and coilagen types 1 and III in fetal and adult porcine dental tissues." UWQ&IIL 33: 531-540.

Tyree, B. (1989). 'The partial degradation of osteonectin by a bone-derived metalloprotease enhances binding to type 1 collagm." J.4: Res.4:. Lrist. M.R. and Strates. B.S.(1971). "Bone morphogenetic protein." L~ent.~55: 1392-1-106. ti'rist, .M.R., Delange. RJ. and Finermazl, G.A.M. (1983). "Bone ceIl differentiation and growth factors." 680-686.

L'rist, kf.R.,Huo. Y.K..BrowneU. A.G., Hohl, W.,M., Suyske. J., Lieue, -4.. Tempst, P., Hwhpller. hl. and DeLange. RJ.(1984). "*F'urification of ùovine bone morphogenetic protein by hydroxyapatite chromatography." W-7 81i371-375. van der Rest. M., Rosenberg. L.C., OIsen, B.R. and Poole. .4.R. (1%). "Chondrocalcin is identical CO the C- propeptide oi trpe D procollagen."Riochem. 297: 923-925.

Vtis. -4. and Pq.A. (1967). 'The phosphoprotein of the dentin matrix." 6: 2409-2316.

Veis, .4., Spector. AR.and Zamosàanyk, H. (1972).'The isolation of an WTA-soluble phosphoprotein from mineralizing bovine dentine." 257 : XU-413.

Veis. A. (1989).Biochemical srudies of vertebrate tooth minerdization. S. Xiann, J. Webb and R. J. P. Williams. Weinhelrn. Federal Republic of Gennany ,VCH \'eriagsgeseIlschaft mbH: 189-222.

Veis. -4. (1992). -4cidic Roteios as Reguîators of Biomineralization in \*ertebrates.a ~f TmthSlovf 2- Davidovitch. Birmingham, Aabama, EBSCO Media Press: 1 15- 119.

C'eis. .A. (1993). "3fineral -matrix interactions in bone and dentin. cementun."1. of Borie 8 : S93- S497.

Vener. K., Fisher, L.W., hiiatz. K.P., Kopp, J.B.. Tumss. S.. Tennine. J.D. and Roky, P.G. (1991). "Os teogenesis imperfecta: changes in noncoliagenous proteins in bone." 6 : Res. 6: - 505.

Vogel, KG.and Fisher, L.W. ( 1986). "Cornparisons of antibody reactivi t? and enzyme sensitivity ktween smali proteogl ycans from bovine tendon. bone and cartilage." J. 2 6 11: 1 1334- 1 1340.

Vogel. KG.. Pauisson. .Li. and Heinegard, D. (1%). "Specific inhibition of type 1 and type II collagen fibdlogenesis by the small proreoglycan of tendon." Biochem, 223: 39-59,

Vol pin, D. and \'eis. A. ( 193)."Cvanogen bromide peptides from insoluble skin and dentin binecollagens." Biochemistr\. 1 2 : 1452-1W.

Wang, HM,'Janda. C'.. Rao, L.G., Melcher, A.H., Heersçhe, J.N.M. and Sdek, J. (1980). "Specific immunohistochemical localization of type iII collagen in porcine periodontal tissues using the peroxidase anti-perioxidase methd"J.. 2 8 : 12 15- 12î3.

Wasi, S ., Tung, P., Otsda, K.. Y ao. K.L.. Sodek, J. and Termine, J.D. ( 1983). "Synthesis of an osteonectin- . . like protein by various comective tissue cells in culture." Calclf.3 5 : 652.

Wasi, S.. Otsuka, K.. Yao, EX., Tung. P.S.,Aubin, JE, Sodek, J. and Tennine, J.D. (1984). "An osteonectin-like protein in porcine pcriodontal ligament and its synthesis by periodontal ligament fibroblasts." Gad. RhhnCgllBiol.2:47047û.

Weber, G.F.. .4shkar, S., Glimcher, MJ. and Cantor, H. (1996). "Receptor-ligand interaction between Cm and osteopontin (Eta- 1)." Saence 27 f : 509-5 12. Weber. K. and Osbom. .M.(1975). . TheProtetns.H. Neurath. R L. Hill and C. L. Boeder. New York. Academic Press. 1: 179-223.

Wciner. S. (19%). "Organization of organic matrix components in minetafized tissues." Amc~Zool.24 : 945- 951.

. *. Weiner. S. and Pnce, P.A. (1986). "Disaggregation of btme into qstals." Calaf. 39 : 365-375.

Weiner. S. and Traub, W. ( 1989)."Crystal size and organization in bone." Connect.'Iïssue 2 1 : 259-265.

Weiner, S., Arad, T.,Ziv, V. and Traub, W.(1992). The mineraiized collagen fibril: .A building block for many vertebrate skeletal tissues. Chemistrv;md.H. Siavkïn and P. Rice. Amsterdam, Elsevier Science Riblishers B.V.: 93- 102.

Weistner. .M., Krieg. T.. Horlein, D.. Glanville. R., Fietzek, P. and Muller, P. (1979). 'lnhibiting effect of pocollagen peptides on collagen biosqnthesis in fibroblast cuitmes." J.-Chrn, 2 54 : 70 16-7023.

Wheater. P.R., Burkitt, H.G. and Daniels, V.G.(1979). The skeietai tissues. : A m.Edinburgh. Longmm Group Limited: 12% 144.

White, S.W., Huimes. D.J.S., 'cliller. A. and Timmins, P.A. (1977). "Collagen-mineral aual relatiouship in calcifieci turkey leg tendon by ,Y-ray and neutron diffraction." 3&uœ 266 : 42 1-125.

7n1'hitson. S.Ur..Hamson. W.,Map, Xi.K., Bowers. D.E.. Fisher, L.W., Gehron-Robey, P. and Tennine. . . J.D.(1984). "Fetaf bovine bone ceils sjnthesize bone-sWc matrix proteins." 3. Ceil99: 607- 614.

Wong, H.J.. Aubin, J.E., Wasi, S. and Sodek. J. (19û5). 'Tell adhion to low-MI proteins extractable from mineralid and soft connective tissues." Biochem 2 32 1 19- 123.

Wong, H.J.. Aubin, J.E.. N-asi. S. and Sodek. J. ( 1986). "Cellular adhesion io low 5k connlxtive tissue mauin proteins." $33: -33:.

Wrana, J.L.. Maeno. Xi., 1-ao. K.L..Hawq l yshyn, B.. Domeaicum, C. and Sodek. J. ( lm)."Differentiai effects of TGF-f3 on the sqnthesis of extracellular matrix proteins by nonnal fetal rat calvarial hne ceil populations." J. 106 : 9 15-924.

Wrana. J.L., Zhang. Q. and Sodek. J. (1989). "Full length cDSA sequence of porcine secreted phosphoprotein-1 (SPP-1, osteopontin)." Su&c AridsBes, 17: 10 1 19.

Wrana, J.L..Kubota, T., hg,Q., Ovd,C.M.. Aubin. J.E., Butler, W.T. and Sodek. J. (1991). "Reguiation of transformation-sensitive secteteci phosphoprotein (SPP- 1 *Osteopontin)expression by transforming growth factor-fi. Cornparisons with SP.4RC cxpmsion." Riochem. 27 3 : 523-531.

Wu, CH., Donovan, C.B.and Wu, G.1'. (1986). "Evidence for prdranslaaonal regulation of coltagen sjnthesis by procollagen propeptides." f,Biol,26 1: 10482- 10484.

Wu. Y., Ri&, C.B.. Lincecum. J.. Trackman, P.C., Kagm. H.M.and Fosta, J.A. (1992). "Characterization and developmental expression of chick aortic lysyl oxidasc." J. Biol.r)ipm. 267 : 21199-25206.

Wu, Y.V., Cluskey, JE., Wl.L.H. and Friedman. .M. (1971). '%me optical poperues of S-8-(4 Pyndylethy1)-L-Cysteine and its Wheat Gluten and Saum Aibumin Derivatives." Rjolhem CeILBiaL 49: 1042-1049. Wuthier, R,( 198 1). Roposed mechanism of matrix vesicle fonnation and vesicle-mediated mineralizatioo. . .. of 0A. Asesrdes.. E. Bonucci and B. de Bemard. %filan.Italy, Wichtig Editore: 103- 109.

Yarnauchi, $1.. Chandler, G.S. and Katz, E.P. (1992). Collagen cross-linking and rnineralization. Chemu~ and.H. Slavkin and P. A. Rice. Yew York. Elsevier: 39-16.

Y-, K.L., Tdescan Jr., R. and Sodek, J. (1994). 'Temporai changes in matris protein synthesis and mR'?i.4 expression diaing mineraiized tissue fonnation b y adul t rat bone marrow cells in cdcure." L.Ebm _Miner. 9: 231-240.

Yoon. K.. Buenaga, R. and Rûdan. G..4. (1987). 'Tissue specifiaty and developmentd expression of rat osteopontin." 148: 1129-1136.

Young. IL.1.F.. Bolauder. M.E.. Day. A..\.. Ramis. CI.. Robey. P.G.. Yamada, Y. and Tennine. J.D.( 1986). "Osteonectin &VA: disuibution in normal and transformed cells." Nucleic 14 : +ü?349?.

Young, M.F..Kerr, J.M.. Termine, J.D., Wewer, C.M., Wang. MG.. SfcBride. 0.W. and Fisher. L.W. (19%))."cDN.4 cloning. mKX.4 distri bution and heterogeneitu. chromosornal localization, and RPLF anaiysis of human osteopontin (OPX)."Gcmmia 7 : 49 1-502.

Young, M.F., Kerr, I.M.. Ibaraki, K.. Heegaard, .\..Li. and Robey. P.G. (1992). "Strucnue. expression. and regdation of the -or noncollagenous matrix proteins of bone." C1In.28 1 . 175- 294.

Youngho. K.. Boyd. C.D. and Csiszar. K. (1995). "A new gene with sequence and structurai simil&r) ro the gene encoding human 1y syl osidase." J. 2 7 0 : 7 176-7 182.

Zanetti. M.. De Bernard, B., Jontell, Xi. and Linde. A. ( 1981 ). "Ca2+ tinding studies of the phosphoprotein from rat incisor dentine." Errr.1 13 : 52 1-545.

Zhang. Q.. DomenicucO. C.. Goldberg. H.A.. Wrana. J.L. and Sodek. I. ( 1990). "Characterization of secreted phosphoprotein I (SPPI. osteopontin). boue sialoprotein (BSP) and a 23 kDa plyxprotein from fetal porcine bone. Evidence that the 23 kDa protein 1s derived from SPPI ." J. 2 6 5 : 7583- 7589.

Sbang, Q., Wrana. J.L. and Sodek. J. (1992)."Characteritarion of thr promoter region of the porcine opn (osteopontin. secreted phosphoprotein 1) gene. Identification of positive and oegative regdatory elements and a 'silent' second promoter." Eur. 2 0 7 : 649-659.

Zhou. H. k'., Takib, H.,Fujisawa. R., bfinino. M., and Kuboki. Y. ( 1995) "S tirnuiation by bone * - siaioprotein of calcification in ostwblast-like MQT3-El fells." Calnf 56 56: W47.

Zuog. P.. DomeninicS. C.,Wasi. S.. Kuwau, F. and Sodek. J. (1986). "Osteonectin is a minor component of mineralized connective tissues in rat." Biochem. 64: 356-362. IMAGE EVALUATION TEST TARGET (QA-3)

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