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 O Copyright by Canne10 Domenicucci 1997 National Library Bibiiothéque nationale B*m of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services sewices bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AON4 OrtawaON KIAON4 Canada Canada Your iVe Vaire rehrefue Our !W Notre referwice The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seil reproduire, prêter, distribuer ou copies of ths thesis in rnicroform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fh, de reproduction sur papier ou sur format électronique. 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