The role of keratan sulphate in the modulation of aggrecanase activity
Christopher James Poon
Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy
May 2005
Department of Paediatrics The University of Melbourne
i
Dedicated to my family – past and present
ii Abstract
Abstract
Arthritis is a debilitating disease of the joints caused by the accelerated breakdown of cartilage, resulting in painful, swollen joints. Cartilage protects the joint by absorbing the shock that would otherwise be transferred directly to the underlying bone. One crucial component of cartilage is a specialised molecule known as aggrecan. Aggrecan consists of a core protein with three globular domains (G1, G2 and G3) and is modified with over one hundred highly sulphated glycosaminoglycan chains. Two types of glycosaminoglycans are substituted along the length of the protein, chondroitin sulphate and keratan sulphate. The glycosaminoglycans impart a highly negative charge to the tissue, giving it the ability to retain water and resist compressive forces.
Aggrecan is lost from cartilage following cleavage by aggrecanases. Too little aggrecan in cartilage destabilises the structural integrity of the tissue and is associated with arthritis. Of the five known aggrecanase cleavage sites, it is cleavage within the interglobular domain (IGD) between the G1 and G2 domains at NITEGE373 - 374ARGSVI that directly contributes to loss of aggrecan function.
The chondroitin sulphate and keratan sulphate located between the G2 and G3 domains is responsible for maintaining the biomechanical properties of aggrecan. The role of keratan sulphate within the G1-G2 domain is unknown, but it is not thought to be essential for aggrecan function. However the literature suggests a possible role of keratan sulphate in facilitating aggrecanase cleavage of NITEGE373 - 374ARGSVI in the IGD. The aim of my project was to examine the role of keratan sulphate in aggrecanase-mediated cleavage of aggrecan in the IGD. Three major goals have been accomplished in this thesis: 1) Identification of a cell type capable of sustained keratan sulphate synthesis. 2) Expression of a recombinant G1-G2 protein substituted with keratan sulphate (rG1-G2). 3) Demonstration that endogenous N-linked keratan sulphate is sufficient to potentiate aggrecanase cleavage of rG1-G2 in the IGD.
iii Abstract
Cultured cells do not synthesise keratan sulphate. Therefore identifying a cell type, and culture conditions to maximise keratan sulphate synthesis, was a major undertaking. Conditions were identified which allowed for maximal keratan sulphate synthesis, albeit on a small scale, in primary bovine keratocytes. Using a Vaccinia virus expression system, recombinant G1-G2 was expressed in primary bovine keratocytes.
Analysis of the rG1-G2 revealed that it was substituted with 5 kDa of keratan sulphate. One important aspect of the study was that the keratan sulphate was all N-linked to the core protein. Subsequent aggrecanase digests, comparing substrates before and after removal of keratan sulphate, showed that aggrecanase cleavage was markedly more efficient when keratan sulphate was present. The results contained in this thesis add significantly to the established literature by providing a greater understanding of the mechanisms involved in aggrecanase-mediated cleavage of aggrecan and cartilage destruction. The results suggest that aggrecan substitution with N- linked keratan sulphate potentiates aggrecanase activity. The results from this study identify N-linked keratan sulphate as a possible target for the development of new drugs for the management of arthritis.
iv Declaration
Declaration
This is to certify that:
(i) the thesis comprises only my original work towards the PhD except where indicated in the Preface (ii) due acknowledgement has been made in the text to all other material used, (iii) the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.
Christopher James Poon
v Declaration
Preface
Rotary shadowing electron microscopy was done by Dr. Doug Keene, Shriners Hospital for Children, Portland, OR, USA.
Karena Last, Dr. Heather Stanton, and Clare Meeker prepared the conditioned medium used for aggrecanase digests.
The Vaccinia G1-G2 virus used throughout this project was generated by Dr. David McQuillan, LifeCell Corporation, Branchburg, NJ, USA.
The results contained in this thesis have recently been accepted for publication in the Journal of Biological Chemistry; PMID: 15849197 (Poon et al. 2005). This article is currently in press, refer to the appendix for more information.
vi Acknowledgements
Acknowledgements
First I must thank Dr. Amanda Fosang, not only for her excellent supervision over the years but also for her tireless support. Thank you for giving me the opportunity to work with you and for saving me from what would otherwise have been an ordinary existence.
Between the two of them, Karena Last and Dr. Heather Stanton have almost taught me everything I know today. My project would not have been possible without them and for that I am extremely grateful to both of them.
Special thanks to my editors: Amanda Fosang, Richard Wilson, Karena Last, Clare Meeker, Sue Golub, and Charlotte East.
I would also like to thank the Mizutani Foundation for Glycoscience and the Arthritis Foundation of Australia for financial support.
I must also thank the following people: Dr. David McQuillan for making the Vaccinia G1-G2 virus; Dr. Bernard Moss for generously donating Vaccinia B5R-GFP; Prof. Bruce Caterson for the 5D4 antibody; Dr. Anna Plaas for her assistance in setting up the FACE technique in our laboratory; and of course Prof. John Bateman and all the members of the Cell & Matrix Biology Research Unit for all the friendly advice they have given me.
Lastly, I would like to thank everyone who has been a part of the Arthritis Research group: Amanda Fosang, Karena Last, Heather Stanton, Chris Little, Sue Golub, Fraser Rogerson, Clare Meeker, Stephanie Gauchi, Charlotte East, Jessica Faggian, Kate Lawlor, and Anita Fernando.
vii Contents
Contents
Abstract ...... iii Declaration ...... v Preface...... vi Acknowledgements ...... vii Contents...... viii Figures...... xi Tables ...... xiii Abbreviations ...... xiv Chapter I: Introduction ...... 1 Cartilage ...... 2 The Extracellular Matrix ...... 2 Link module ...... 3 Hyalectans ...... 6 Versican...... 7 Neurocan ...... 10 Brevican...... 13 Aggrecan ...... 15 Keratan sulphate ...... 20 KS Synthesis and Elongation ...... 24 Sulphotransferases...... 25 Sialyltransferases and fucosyltransferases...... 26 In vitro variations in keratan sulphate proteoglycan synthesis and secretion ...... 28 Arthritis ...... 31 Osteoarthritis ...... 31 Rheumatoid Arthritis...... 32 Aggrecan Proteolysis...... 33 Zinc Metalloproteinases ...... 35 The Pro-peptide domain ...... 36 Metalloproteinase domain ...... 38 Hemopexin domain ...... 38 Disintegrin-like domain...... 39 Thrombospondin type I motif...... 39 MMPs ...... 40 The reprolysins ...... 43 ADAMs ...... 43 ADAMTS and the aggrecanases...... 44 ADAMTS-4 (Aggrecanase-1) ...... 48 ADAMTS-5 (Aggrecanase-2) ...... 48 Processing of ADAMTS-4 ...... 50 Keratan sulphate analysis ...... 52 Overcoming the charge separation ...... 53 Quantitation of the resolved carbohydrates ...... 54 Separation of labelled carbohydrates...... 55 FACE for glycosaminoglycan analysis ...... 56 Depolymerisation of KS ...... 56 Keratanase II (from Bacillus sp. Ks36), EC3.2.1...... 56 Endo-β-galactosidase (Escherichia freundii), EC 3.2.1.103...... 57 Neuraminidase and fucosidase ...... 57 Expression of recombinant proteoglycans...... 59 Vaccinia virus...... 61 The Vaccinia virus T7 RNA polymerase expression system...... 64 Project goals ...... 66 Chapter II: Materials & Methods...... 68 Materials...... 69 Methods...... 71 Keratocyte preparation ...... 71
viii Contents
Generation of vG1-G2...... 71 Vaccinia preparation...... 72 Expression of B5R-green fluorescent protein...... 73 Generation of recombinant G1-G2 ...... 73 Expression of 35S-methionine labelled G1-G2 ...... 73 Fluorography ...... 74 Analysis of recombinant and native G1-G2 by rotary shadowing microscopy ...... 74 Coupling of hyaluronan to EAH-sepharose...... 75 HA-sepharose affinity chromatography...... 75 High Performance Liquid Chromatography ...... 76 Glycosaminoglycan analysis ...... 76 35 Sample preparation for [ S]SO4-labelled glycosaminoglycans ...... 76 35 [ S]SO4 labelling of primary cultures: FGF-2 and serum dosage effect...... 77 Papain digestion...... 77 BioGel P-10 size exclusion chromatography...... 78 Cell proliferation in response to FGF-2 and FCS ...... 78 Antibodies used for immunoblotting...... 78 Western blotting ...... 80 Glycosidase digests ...... 80 Nitrous acid treatment ...... 80 Preparation of AMAC-labelled carbohydrates ...... 81 Carbohydrate electrophoresis ...... 81 Cartilage explant cultures ...... 81 Aggrecanase digests ...... 82 Silver stain...... 82 Chapter III: In search of a good host ...... 83 Introduction ...... 84 Results – Part I ...... 89 CCL-60...... 89 CRL-2048...... 91 SK-N-MC ...... 92 Results – Part II ...... 94 Keratan sulphate synthesis by primary keratocytes...... 98 Discussion ...... 107 Chapter IV: Optimising the expression of rG1-G2...... 111 Introduction ...... 112 Results ...... 115 Monitoring the response of cells to infection by Vaccinia ...... 115 HT-1080 cells produce rG1-G2...... 118 rG1-G2 expression by COS-7 cells ...... 119 Production of rG1-G2 in primary chicken keratocytes...... 120 Expression of rG1-G2 by chicken corneal explant cultures ...... 123 Expression of rG1-G2 by primary bovine keratocytes ...... 126 Discussion ...... 132 Chapter V: Purification of rG1-G2...... 137 Introduction ...... 138 Results ...... 139 Purification of bovine-expressed rG1-G2...... 139 Visualisation of rG1-G2 by rotary shadowing electron microscopy ...... 145 Discussion ...... 148 Chapter VI: Keratan sulphate analysis of rG1-G2...... 150 Introduction ...... 151 Specificities of keratan sulphate-degrading enzymes ...... 152 Keratanase II (Bacillus sp. Ks36), (EC 3.2.1) ...... 153 Endo-β-galactosidase (EC 3.2.1.103) ...... 153 Neuraminidase (EC 3.2.1.18) and Fucosidase (EC 3.2.1.51) ...... 154 Results ...... 155 Establishing standards ...... 155 Quantitation by FACE...... 155 Optimisation of fucosidase digests ...... 156
ix Contents
Comparison of the KS on rG1-G2 and native G1-G2...... 157 Analysis of FACE results ...... 159 N-glycosidase digestion of rG1-G2 ...... 161 Preliminary analysis of the keratan sulphate linkage region...... 162 Discussion ...... 164 Chapter VII: Characterisation of rG1-G2...... 167 Introduction ...... 168 Results ...... 171 Endogenous NITEGE373 neoepitope in conditioned medium...... 171 rG1-G2 cleavage by aggrecanase ...... 173 Effect of keratan sulphate on aggrecanase digestion – A closer look...... 178 Discussion ...... 181 Chapter VIII: Final Discussion...... 185 Purpose of this project...... 186 A brief history of keratan sulphate ...... 187 Discussion of experimental techniques ...... 188 Summary of Results ...... 189 Overview ...... 191 Future directions...... 193 Conclusion...... 196 References ...... 198 Appendix ...... 236
x Figures
Figures
Figure 1: Domain structure of the hyalectans...... 6 Figure 2: Versican splice variants ...... 8 Figure 3: Cartilage aggrecan aggregate ...... 15 Figure 4: Interaction of aggrecan with hyaluronan...... 17 Figure 5: General structure of keratan sulphate...... 20 Figure 6: Stages of aggrecan loss and cartilage destruction in a canine model of arthritis...... 31 Figure 7: Zinc metalloproteinase family...... 36 Figure 8: MMP prototype...... 40 Figure 9: Schematic of ADAMTS-4 ...... 48 Figure 10: Schematic of ADAMTS-5...... 48 Figure 11: Proposed model of ADAMTS processing...... 51 Figure 12: Structure of fluorophores used in FACE...... 54 Figure 13: Fluorophore labelling of carbohydrates ...... 54 Figure 14: Keratanase II products ...... 56 Figure 15: Endo-β-galactosidase products...... 57 Figure 16: Vaccinia virus ...... 61 Figure 17: Outline of Vaccinia T7 RNA polymerase expression system ...... 65 Figure 18: Glycosaminoglycan composition of proteoglycans secreted by CCL-60 cells in culture .....90 Figure 19: Glycosaminoglycan composition of proteoglycans secreted by CRL-2048 cells in culture .91 Figure 20: Glycosaminoglycan composition of proteoglycans secreted by SK-N-MC cells in culture .93 Figure 21: The effect of FCS and FGF-2 on cell proliferation...... 94 Figure 22: 5D4 blot of keratan sulphate from CCL-60...... 95 Figure 23: Quantitation of 5D4 intensity per cell from CCL-60 culture medium ...... 95 Figure 24: Primary keratocytes cell proliferation in response to FCS and FGF-2...... 99 Figure 25: 5D4 and MZ15 blot of primary keratocyte glycosaminoglycan...... 100 Figure 26: Quantitation of KS blot of glycosaminoglycans from primary keratocytes...... 101 Figure 27: Glycosaminoglycan synthesis by primary keratocytes grown in 0.4% FCS and increasing amounts of FGF-2...... 103 Figure 28: Glycosaminoglycan synthesis by primary keratocytes grown in 2% FCS and increasing amounts of FGF-2...... 104 Figure 29: KS synthesis in response to FGF-2 and FCS ...... 105 Figure 30: Vaccinia B5R-GFP infection of cells...... 117 Figure 31: HT-1080 cells expressing rG1-G2 ...... 119 Figure 32: Timecourse of rG1-G2 production in COS-7 cells ...... 120 Figure 33: rG1-G2 expression by primary chicken keratocytes ...... 122 Figure 34: Chicken cornea explant infections ...... 125 Figure 35: rG1-G2 expression in primary bovine keratocytes – Vaccinia dosage response...... 126 Figure 36: Timecourse of rG1-G2 expression in bovine keratocytes ...... 128 Figure 37: Analysis of the keratan sulphate substituted on rG1-G2...... 130 Figure 38: FGF-2 and its effect on rG1-G2 KS substitution ...... 131 Figure 39: Purification of rG1-G2 with HA-sepharose ...... 140 Figure 40: Purification of rG1-G2 with HPLC...... 142 Figure 41: Enhanced purification of rG1-G2 with HA-sepharose...... 143 Figure 42: Analysis of HPLC fractions ...... 145 Figure 43: Rotary shadowing electron microscopy of G1-G2...... 147 Figure 44: The structures of KSI, KSII, and KSIII...... 153 Figure 45: Quantitation of monosaccharides...... 156 Figure 46: FACE analysis of bovine corneal KS...... 157 Figure 47: FACE analysis of pig G1-G2 and rG1-G2 KS...... 158 Figure 48: N-glycosidase digestion of rG1-G2...... 162 Figure 49: Analysis of rG1-G2 keratan sulphate including the linkage region ...... 163 Figure 50: Relationship between keratanase digestion and detection of NITEGE373 ...... 171 Figure 51: Pig NITEGE373 neoepitope present in conditioned medium ...... 172 Figure 52: Aggrecanase cleavage of rG1-G2 after 24h ...... 173 Figure 53: 30h aggrecanase timecourse of rG1-G2 digestion ...... 174 Figure 54: 4h aggrecanase timecourse of rG1-G2 digestion ...... 176
xi Figures
Figure 55: Aggrecanase cleavage of rG1-G2 ...... 177 Figure 56: Aggrecanase cleavage of selectively deglycosylated rG1-G2...... 179
xii Tables
Tables
Table 1: Core protein classification of hyalectans...... 7 Table 2: CS glycosylation in versican splice variants ...... 8 Table 3: Matrix Metalloproteinases...... 42 Table 4: ADAMTSs ...... 47 Table 5: Sites of aggrecan cleavage by aggrecanases...... 49 Table 6: Culture conditions for the cell proliferation assay...... 99 35 Table 7: Culture conditions for [ S]SO4 labelling...... 102 Table 8: KS and CS synthesis by primary bovine keratocytes ...... 105 Table 9: CS and KS synthesis by primary bovine and chicken keratocytes ...... 106 Table 10: Primary keratan sulphate catabolic products of keratanase digestion ...... 155 Table 11: Analysis of pig G1-G2 and rG1-G2 KS – Summary of results ...... 161 Table 12: KS linkages of the G1 and IGD...... 168
xiii Abbreviations
Abbreviations ADAMTS A disintegrin and metalloproteinase with thrombospondin motifs AEBSF 4-(2-Aminoethyl)benzenesulphonyl fluoride hydrochloride AMAC 2-aminoacridone CS chondroitin sulphate CSPG chondroitin sulphate proteoglycan DMSO dimethyl sulfoxide DS dermatan sulphate DTT dithiothreitol EβG endo-β-galactosidase ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor ELISA enzyme-linked immunosorbent assay FACE fluorophore-assisted carbohydrate electrophoresis Fuc fucose G1-G2 The N terminal region of aggrecan spanning the first two globular domains and IGD Gal galactose Gal6S galactose-6-sulphate GalNAc N-acetyl galactosamine GlcNAc N-acetyl glucosamine GlcNAc6S N-acetyl glucosamine-6-sulphate GuHCl guanidine hydrochloride HA hyaluronan, also known as hyaluronic acid HLA human leukocyte antigens HPLC high performance liquid chromatography IGD interglobular domain KI keratanase I KII keratanase II KS keratan sulphate KSPG keratan sulphate proteoglycan met methionine MHC multihistocompatibility complex MMP matrix metalloproteinase MWCO molecular weight cut-off NeuAc neuraminic acid, also known as sialic acid OA osteoarthritis PBS phosphate-buffered saline PPO 2,5-Diphenyloxazole PVDF polyvinylidene fluoride
xiv Abbreviations
RA rheumatoid arthritis rG1-G2 recombinant G1-G2 SA sialic acid SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis vG1-G2 Vaccinia expressing G1-G2 vTF7-3 Vaccinia expressing bacteriophage T7 RNA polymerase
xv Chapter I: Introduction
Chapter I: Introduction
“Knowledge is of two kinds. We know a subject ourselves, or we know where we can find information on it.” - Samuel Johnson (1709 - 1784)
1 Chapter I: Introduction
Cartilage
Articular cartilage is found at the ends of long bones and serves to protect the joints against mechanical loading by acting as a shock absorber. It is a complex tissue produced and maintained by chondrocytes embedded in a dense network of triple helical collagen II fibres. These fibres are interspersed with large, negatively charged, proteoglycan aggregates of aggrecan bound to hyaluronan. The charge density of aggrecan draws water into the extracellular matrix and allows the tissue to maintain a high hydroscopic pressure. Healthy articular cartilage is able to withstand enormous amounts of pressure, with estimates between 10 – 20 MPa (100 – 200 atm) for physiologically stressful activities such as stair climbing (Grodzinsky et al. 2000). The tensile strength of the type II collagen network combined with the compressive resistance of the hyaluronan-aggrecan aggregates gives the tissue its resilience. Compressing cartilage causes the cells and extracellular matrix to be compressed and gives rise to hydrostatic pressure gradients and interstitial fluid flow. This temporary displacement of fluid from the cartilage extracellular matrix absorbs the pressure that would otherwise be transferred to the bone.
The Extracellular Matrix
The extracellular matrix is made up of a variety of different proteins, each consisting of multiple domains that are often repeated a number of times. The extracellular matrix proteins are thought to have evolved along with multicellular organisms 700 million years ago, which explains the high sequence homology across different species. As these organisms evolved and became more complex, so too did their corresponding ECM proteins; this came about as a result of exon shuffling, duplication, and recombination, among other evolutionary mechanisms. So from what is thought to have been a relatively small number of genes encoding domain-sized polypeptides, have come a huge number of extracellular proteins, each with different functions and consisting of similar domain structures (Doolittle 1995). Studies of the major domain structures have resulted in a greater understanding of the biological function of ECM proteins. Some of these components include:
2 Chapter I: Introduction
laminin-type epidermal growth factor-like domains, laminin G-like domains, fibronectin domains, fibrillin domains, nidogen-associated domains, Link domains, von Willebrand factor type A domains, coiled-coil oligomerisation domains, and hydroxyprolines and collagen triple helix domains. Hohenester et al. have recently done a thorough review of these domains (Hohenester et al. 2002).
Link module
The hyaluronan-binding proteins are also known as hyaladherins or hyalectans. Many of the hyaladherins have a common structural domain of approximately 100 amino acids in length, known as a Link module or a proteoglycan tandem repeat (Day et al. 2002). The proteins containing Link modules are involved in the assembly of extracellular matrix, cell adhesion, and migration (Barta et al. 1993; Kohda et al. 1996; Hohenester et al. 2002). The Link module (Kohda et al. 1996) was originally identified in the link protein isolated from cartilage (Perkins et al. 1989; Perkins et al. 1991). The link protein is made up of an immunoglobulin domain followed by two Link modules. A single Link module consists of two α-helices and two triple- stranded anti-parallel β-sheets. Link modules have been identified in a variety of different hyaladherins and while they all contain the basic Link module described above, the size of the Link module varies from protein to protein, for example tumour necrosis factor-stimulated gene-6, TSG-6, contains a single Link module and this alone, is sufficient for high affinity binding to hyaluronan. CD44 has an extended Link module consisting of a Link module with N- and C-terminal extensions, all of which are required for the proper function of the protein. Aggrecan has an even larger Link domain; its hyaluronan-binding region contains two contiguous Link modules and both are involved in hyaluronan-binding. The size of the HA-binding region is approximately proportional to the length of hyaluronan that is bound by it (Day et al. 2002). The variations in size of the identified Link modules has led to them being classified into classes A, B, or C. Class A Link modules consist of a single Link module and is capable of high affinity hyaluronan-binding. Class B modules contain additional N- and C-terminal extensions required for correct
3 Chapter I: Introduction
folding and activity such as that found in CD44. Class C Link modules consist of an N-terminal Ig domain followed by two contiguous Link modules; these are found in the hyalectan family (Seyfried et al. 2005). The entire class C Link module is sometimes referred to as a proteoglycan tandem repeat (PTR) and each of the three domains, the Ig domain and the two Link modules, are individually referred to as the A, B, and B’ loops respectively.
A family of four hyaluronan and proteoglycan-binding link proteins (HAPLN) have been discovered (Spicer et al. 2003). Expressed sequence tag databases were searched for cDNA clones encoding cartilage link protein (crtl1) and any related proteins. Four classes of link protein were identified and were labelled HAPLN1 – 4. Two of these had previously been described, HAPLN1 (crtl1) and HAPLN2 (bral1), however HAPLN3 and 4 had not been identified. Not only were these link proteins found in humans, they were also present in mouse, rat, and zebrafish, and were therefore suggested to be ubiquitously expressed in all vertebrates. Based on Northern analysis, HAPLN1 (crtl1) was expressed in small intestine and placenta as well as cartilage. HAPLN2 (bral1) and HAPLN4 were both expressed exclusively in the brain, and HAPLN3 was expressed in multiple tissues. Analysis of the genome revealed that each HAPLN gene was co-localised with a hyalectan gene. The pairings were as follows: HAPLN1:versican (CSPG2), HAPLN2:brevican (BCAN), HAPLN3:aggrecan (AGC1), and HAPLN4- neurocan (CSPG3).
Cartilage link protein interacts with aggrecan via its Ig domain (Heinegard et al. 1974), and it is this domain along with the signal sequence that have the lowest sequence identity between Link domains. This is suggested to be due to the different binding preferences of each link protein to their respective hyalectan. The predicted mass of these proteins range from 38 – 43 kDa.
The cartilage link protein is very similar to the G1 domain of aggrecan. In the proteins TSG-6 and CD44, a single Link module is present but both proteins are still able to bind to hyaluronan, however, with only a single Link module these proteins have specificity for hexasaccharide stretches of HA as
4 Chapter I: Introduction
opposed to the decasaccharide HA-binding of G1 and link protein (Hardingham et al. 1973; Hascall et al. 1974). It was once thought that the presence of conserved basic amino acids was responsible for mediating the binding to the negatively charged hexuronate groups of hyaluronan. However more recent studies found that the peptide binding experiments from which this theory was derived, did not occur through binding with specific residues but via non-specific interactions (Horita et al. 1994). Despite this, the presence of basic residues is still considered important for HA-binding.
Functional studies of link protein have been analysed with link protein knockout (crtl1) mice (Watanabe et al. 1999). The homozygous link protein knockout mice have a similar phenotype to cmd mice (refer to page 15 for more information), though less severe, and the heterozygotes have no discernable phenotype. For the most part, the link protein knockout is a lethal mutation and the majority of crtl1 mice die soon after birth from respiratory failure, however some survive and develop dwarfism and lordosis of the cervical spine (Watanabe et al. 1999). The major phenotypes are dwarfism, deformation of the cartilage-derived cranial bones, and reduced amounts of cartilage in which the chondrocytes are disorganised and the aggrecan is significantly reduced. From these observations, it would appear that while it is important in stabilising proteoglycan aggregate formation, the link protein also has an important role in chondrocyte differentiation, in particular, from prehypotrophic to hypertrophic chondrocytes (Watanabe et al. 1999; Watanabe et al. 2002).
5 Chapter I: Introduction
Hyalectans
Versican (cspg2), neurocan (ncan), brevican (bcan), and aggrecan (agc1) are a family of genes encoding proteoglycans which share structural and functional similarities. They all have a number of similar structural features: an N-terminal globular domain, commonly referred to as G1, made up of an immunoglobulin-type repeat and two link protein modules forming tandem repeats which mediate binding of the protein to hyaluronan; one or more central regions with multiple glycosaminoglycan-binding sites; and a C- terminal G3 domain consisting of alternatively spliced epidermal growth factor-like motifs, a lectin-binding module, a complementary regulatory domain (Iozzo 1998). This characteristic binding has led this family of four proteoglycans to be collectively known as hyalectans. Each hyalectan has its own distinctive protein structure (figure 1) and distribution pattern, and most importantly their own distinctive biological function. See Table 1 for the loci and physical characteristics.
Amino acid residues
0 500 1000 1500 2000 2500
Aggrecan (human)
Versican (human)
Neurocan (rat)
Brevican (bovine)
Ig-like Lectin-like
Link protein-like Complement regulatory protein-like
EGF-like
Figure 1: Domain structure of the hyalectans Adapted from (Yamada et al. 1994)
6 Chapter I: Introduction
Table 1: Core protein classification of hyalectans Human Core Human GAG Human Mouse Protein (kDa) type (number locus chromosome of chains) Aggrecan 220 CS/KS 15q26 7 (~100/~0-30) Versican 265-370 CS/DS (0-23) 5q13.2 13 Neurocan 136 CS (3-7) 19 8 Brevican 100 CS (1-3) 1q25-q31 3 Adapted from (Silbert et al. 2002)
Versican
Versican was originally identified by cDNA analysis of a human placental library. It has since been completely cloned in mice, cows, chickens, and of course, humans. Versican is the largest of the hyalectans, with a core protein size ranging from 265-370 kDa, and is made up of an N-terminal G1 domain and a C-terminal G3 domain separated by an extensive chondroitin sulphate region consisting of two large subdomains known as GAG-α and GAG-β. In the chicken, versican is sometimes referred to as PG-M. The G1 domain forms aggregates with the glycosaminoglycan, hyaluronan, whereas the G3 domain is made up of a C-type lectin domain, two epidermal growth factor (EGF) domains, and a complement regulatory region typical of the hyalectans. The chondroitin sulphate region separating the two globular domains, GAG-α and GAG-β, are encoded by two exons, exon 7 and 8 respectively (Wight 2002).
At least four splice variants of versican are known to exist; these are referred to as V0, V1, V2, and V3. The V0 splice variant is the full length proteoglycan and is made up of the hyaluronan-binding G1 domain, an extensive chondroitin sulphate region containing both GAG-α and GAG-β, and the C- terminal G3 domain containing a lectin domain, two EGF domains, and a complement regulatory domain. The V1 splice variant is missing the GAG-α domain, the V2 splice variant is missing the GAG-β domain, and finally, the
7 Chapter I: Introduction
V3 splice variant is missing both GAG-α and GAG-β domains. Figure 2 gives a schematic representation of the versican splice variants (Iozzo 1998).
V0 G1 GAG-α GAG-β G3
V1 G1 GAG-β G3
V2 G1 GAG-α G3
V3 G1 G3
Figure 2: Versican splice variants
In humans, the degree of CS glycosylation is as follows (table 2) (Wight 2002):
Table 2: CS glycosylation in versican splice variants Splice Variant Number of CS chains V0 17-23 V1 12-15 V2 5-8 V3 0
Depending on their originating tissue the total mass of the glycosaminoglycans can range from 25-60 kDa. Not only does the amount of chondroitin sulphate vary with the expressing tissue, but so too does the ratio of chondroitin-4-sulphate : chondroitin-6-sulphate.
Versican has been assigned a number of different biological functions, most prominent is its role in cellular proliferation and in cell-substrate adhesion. Versican has been reported to inhibit the binding of cells to various extracellular matrix components including collagen, fibronectin, and laminin via its G1 domain. The versican gene, cspg2, is expressed in a wide variety of tissues; it is most highly expressed in embryonic tissue like the lung, limb buds, aorta, cornea, and skeletal muscle. In adult tissue, it is expressed in the connective tissue of various organs, blood vessels, and in the proliferative
8 Chapter I: Introduction
zone of the epidermis (Wight 2002). Though the expression pattern is quite diverse, versican expression is most common in fast growing tissues and cells (Touab et al. 2002; Arciniegas et al. 2004; Sheng et al. 2005; Snow et al. 2005). This suggests a role in cellular proliferation. Further studies have demonstrated that addition of exogenous versican or over-expression of versican both increased cell proliferation (Yang et al. 1999). The G3 domain has been shown to be responsible for this activity, and it is probably via its two EGF-like domains, as demonstrated by the creation of a versican construct missing its G3 domain (Yang et al. 1999). However a further deletion of the CS-domain resulted in a G1 construct able to stimulate cell proliferation (Yang et al. 1999). It was concluded from the study that versican is able to stimulate cellular proliferation via two domains: G1, by reducing cell adhesion to various ECM compounds; and G3 via the EGF receptor pathway.
Besides its effect on adhesion and cellular proliferation, versican is able to affect other cellular processes. Versican is thought to block the migration of neural crest cells because cells generally do not enter tissues that express high levels of versican (Landolt et al. 1995; Yang et al. 1999; Wu et al. 2004). Versican also has a role in the development of the heart. In heart-defective mice (hdf), there is a mutation in the versican gene, resulting in a loss of migration of the endocardial cushion cells (Mjaatvedt et al. 1998). The V1 splice variant of the human aorta has been shown to be susceptible to cleavage at the E441-442A site by both ADAMTS-1 and ADAMTS-4 (Sandy et al. 2001).
In the nervous system the V2 splice variant is able to inhibit axonal outgrowth and migration and this effect can be partially reduced by chondroitinase treatment. This suggests that the V2 core protein as well as the attached CS chains are involved in this axonal inhibition. Versican is also thought to have other biological functions (Wight 2002). As well as forming large aggregates with hyaluronan, versican also interacts via its lectin domain with tenascin-R (Aspberg et al. 1995; Aspberg et al. 1997), and fibulin-1 and fibulin-2 (Aspberg et al. 1999; Olin et al. 2001) which are expressed at high levels in the heart valve.
9 Chapter I: Introduction
Neurocan
As the name suggests, neurocan is expressed almost exclusively in the central nervous system. This 136 kDa protein is made up of a number of protein modules that are common to all of the hyalectans, namely, a G1 and G3 globular domain separated by a central chondroitin sulphate region containing between 3 and 7 CS chains. However neurocan also contains additional sequences that do not have any obvious homology to any protein family. At its N-terminus, neurocan has a G1 domain that is structurally identical to all of the hyalectans. Composed of one immunoglobulin module and two link modules, neurocan is able to bind with a high affinity to hyaluronan.
Except for a single cysteine residue present in the chicken sequence, the approximately 600 amino acid central domain of neurocan is cysteine-free, but is rich in serine, threonine, and proline (Rauch et al. 2001). The central region of the protein is glycosylated with 3 - 7 chondroitin sulphate chains which are mostly sulphated at carbon 4 on GalNAc residues, although sulphation at carbon 6 also occurs. This phenomenon is especially evident in 7 day old rats, in which the level of chondroitin-6-sulphate (C6S) is approximately 20% and the median mass of chondroitin sulphate is 22 kDa. However in adult rats, the levels of C6S is dramatically decreased to less than 3%, while the median mass of chondroitin sulphate is, on average, 10 kDa higher, at about 32 kDa. Under the electron microscope, this region has a mucin-like appearance due to the presence of up to 40 O-linked oligosaccharides (Jentoft 1990; Rauch et al. 2001).
The C-terminal domain is made up of two EGF modules, a C-type lectin module, a sushi module, and an additional C-terminal sequence of 45 amino acids. In the rat and human sequence, an additional cysteine residue is present within the second EGF module as well at the extreme C-terminus and is thought to form disulphide bonds. Another feature is the accumulation of basic amino acids and histidine residues; these are thought to form a furin cleavage site (Rauch et al. 2001).
10 Chapter I: Introduction
Neurocan binds to a number of different extracellular matrix components. The most obvious of these is hyaluronan, which it binds to with link protein via its G1 domain. At the other end of the protein, the C-terminus is able to bind heparin, at least in vitro under physiological buffering conditions (Rauch et al. 2001). This interaction is further enhanced when chondroitin sulphate chains are removed prior to binding, though this has not been observed in vivo. The binding of FGF-2 to neurocan is also reduced after CS chains are first removed with chondroitinase. Neurocan is also known to interact with other molecules, particularly those involved in growth. Some of these include: amphoterin, a 30 kDa protein found in growth cones and migrating cells; the matrix proteins tenascin-C and tenascin-R (Grumet et al. 1994; Milev et al. 1998); as well as the neural cell adhesion molecules N-CAM, L1/Ng-CAM, and TAG-1/axonin-1 (Grumet et al. 1993; Friedlander et al. 1994; Milev et al. 1996; Retzler et al. 1996; Brummendorf et al. 1998; Oleszewski et al. 1999); and also the GPI-linked membrane molecule N-acetyl-galactosaminyl- phosphoryl-transferase (Balsamo et al. 1995; Li et al. 2000).
In the rat, neurocan accumulates in the brain from embryonic day 12 and peaks at postnatal day 7, subsequent proteolytic events and biological processes reduce this accumulated neurocan to lower concentrations (Oohira et al. 1994). As well as the high expression in the brain, neurocan has also been detected in the eye, using the monoclonal antibody, 1G2 (Oohira et al. 1994). Like its expression in the brain, the expression of neurocan in the eye varies too. From embryonic day 14 -16 of the rat, neurocan is expressed in the retina. From embryonic day 18 to postnatal day 3, the majority of the expression can be observed in the nerve fibre layer and the ganglion cell layer. Neurocan expression slowly decreases so that by postnatal day 42, the expression drops to a level that is almost undetectable (Inatani et al. 1999).
Neurocan is capable of interacting with many different molecules present in the central nervous system, and as such, it is likely to modulate these processes. It is expressed mainly during modelling and remodelling stages of development. Although it has been implicated in many biological processes, and in vitro, has been shown to inhibit neuronal adhesion and outgrowth,
11 Chapter I: Introduction conclusive proof of its biological role is yet to be discovered, but whatever its role, it is likely to be subtle, as demonstrated by the seemingly normal state of neurocan knockout mice, which are apparently viable, fertile, and have no anatomical brain abnormalities (Zhou et al. 2001).
12 Chapter I: Introduction
Brevican
Brevican is the newest member of the hyalectan family (Yamada et al. 1994) and so far, has only been detected in the central nervous system. It was cloned and sequenced in 1994 by two independent groups. Jaworski et al. cloned this hyalectan from postnatal day 12 rat brains using a probe made from the HA-binding domain of aggrecan and named this new protein brain enriched hyaluronan binding protein or BEHAB (Jaworski et al. 1994). At the same time, Yamada et al. identified a gene encoding the same protein by screening a bovine brain expression library with polyclonal antibodies that recognise multiple proteoglycan core proteins, this group named the protein, brevican (Yamada et al. 1994). It is for this reason that this proteoglycan is often referred to as BEHAB/brevican, however for convenience, it will be referred to as brevican here.
Brevican is structurally similar to aggrecan, versican, and neurocan, in that it is composed of a G1 domain made up of an immunoglobulin-like domain, and two link protein-like domains, which together are able to interact with hyaluronan and link protein. The C-terminus, known as the G3 domain, is made up of an EGF-like domain, a lectin-like domain, and a complement regulatory protein-like domain. However, the central region spanning these two globular domains shows little homology to the other members of the hyalectan family. This 295 amino acid region makes this proteoglycan considerably shorter than the other members of this family. The central region is rich in glutamic acid residues including a stretch of eight consecutive amino acids thought to be involved in cationic binding. The core protein contains a total of 13 Ser-Gly or Gly-Ser dipeptides which can potentially act as GAG attachment sites, five of these sites are flanked by at least one acidic amino acid, which make them better GAG attachment sites, and four of these are found in the glutamic acid-rich central domain Ser-391, Ser-524, Ser-528, and Ser-539 (Seidenbecher et al. 1995). The full length core protein is 145 kDa however an N-terminally truncated 80 kDa form also exists (Yamada et al. 1994). Northern analysis of brevican isolated from bovine brain has revealed a single 3.3 kb transcript (Yamada et al. 1994), however analysis of rat brain
13 Chapter I: Introduction
revealed two transcripts of 3.6 kb and 3.3 kb (Seidenbecher et al. 1995). The 3.6 kb transcript had an 82% sequence identity with bovine brevican and is thought to be the rat ortholog, the 3.3 kb variant was found to be truncated downstream of the central domain. Its core protein of ~100 kDa undergoes proteolytic cleavage at an aggrecanase-like site (Yamada et al. 1995). This results in the loss of the N-terminal G1 domain and the central glycosaminoglycan domain containing up to 3 chondroitin sulphate chains; the cleavage of brevican in the central nervous system has been associated with synaptic loss (Yuan et al. 2002). In addition to this, brevican has been shown to exist in two forms: a soluble form, and as a glycosylphosphatidylinositol (GPI) anchored plasma membrane form (Seidenbecher et al. 1995). Chondroitin sulphate has since been shown to be attached to the core protein by its susceptibility to chondroitinase digestion (Yamada et al. 1994; Seidenbecher et al. 1995).
The biological role of brevican is still not completely clear however it has been shown to be involved in a number of different biological processes. It is upregulated in response to glial cell proliferation and migration in the developing and injured brain, and also in some rat brain tumours (Jaworski et al. 1996). During these periods of high expression, there is also an increased presence of two brevican cleavage products, a ~50 kDa N-terminal fragment and a ~90 -100 kDa C-terminal fragment (Nutt et al. 2001). This cleavage site has been determined to be Glu395 – Ser396 and evidence suggests that ADAMTS-4 is the protease responsible for this cleavage (Matthews et al. 2000).
14 Chapter I: Introduction
Aggrecan
Aggrecan monomer
HA
Figure 3: Cartilage aggrecan aggregate Multiple aggrecan monomers bind to hyaluronan (HA) to form an aggregate. Image courtesy of Dr. M. Mörgelin, University of Lund, Sweden.
Aggrecan was the first of the hyalectans to be described (Sajdera et al. 1969). This proteoglycan is the major proteoglycan of cartilage (Doege et al. 1991; Walcz et al. 1994), and along with the rest of the hyalectans, is expressed in the central nervous system as well (Schwartz et al. 1996; Iozzo 1998). Although the aggrecan from cartilage and the brain has the same core protein, they have vastly different functions, which are brought about due to differences in glycosylation (Schwartz et al. 1996). In the brain, aggrecan is not modified with keratan sulphate chains, and the degree of chondroitin sulphate substitution is also lower than in cartilage. Aggrecan’s core protein consists of three globular domains - G1, G2, and G3, separated by regions containing glycosaminoglycan attachment sites.
The importance of aggrecan in mammalian physiology has been established with the identification of an aggrecan gene knockout in mice. These cartilage matrix deficiency (cmd) mice, when homozygous for this perinatal lethal mutation, are characterised by dwarfism, an abnormally short trunk, limbs, tail and snout, and have protruding tongues and cleft palates (Rittenhouse et al. 1978). The heterozygotes with a single functioning aggrecan gene, appear normal at birth but die shortly afterwards from respiratory failure (Watanabe et
15 Chapter I: Introduction
al. 2002). The aggrecan gene of the cmd mice contains a 7 bp deletion in exon 5; the exon encoding the B loop of the G1 domain; this deletion results in a premature stop codon further downstream in exon 6. Examination of the homozygous mice found no aggrecan in the cartilage, but there appeared to be normal levels of collagen II and link protein (Kimata et al. 1981). The lack of aggrecan core protein from cartilage prevents the formation of normal cartilage aggregates. Unlike wild-type mice with abundant amounts of ECM, the cartilage of cmd mice is grossly different. Their cartilage contains very little matrix and is tightly packed with chondrocytes. Analysis of the cartilage by electron microscopy showed abnormally thick collagen fibrils and bundle- formations of the fibrils (Kobayakawa et al. 1985). This observation supports the theory that as well as forming the highly hydrophobic gel of the cartilage ECM, aggrecan also assists in collagen fibril maintenance.
In contrast to the homozygotes, the cmd heterozygous mice are apparently normal at birth, though slightly dwarfish. This dwarfism is pronounced 28 days after birth. Another abnormal phenotype becomes apparent approximately one year after birth. It is around this time that the cmd heterozygous mice display abnormalities in their spines. The misalignment of the cervical and thoracic spine affects their movement and the mice eventually starve to death through lack of movement (Watanabe et al. 2002). It should be noted that the joints of the cmd heterozygous mice appear to be normal, with the pathological effects confined to the spine. However this is thought to be due to the higher gravitational loading of the spine compared with the joints.
Aggrecan has a core protein size of about 230 kDa, whereas fully glycosylated aggrecan is 2300 kDa. The interaction of aggrecan with hyaluronan is via the G1 domain, and this interaction with the link protein is irreversible under physiological conditions.
The G1 domain has been found to consist of three motifs, which are referred to as loop A, B, and B’. The B and B’ loops are also known as the proteoglycan tandem repeat (PTR) or the link module (Kohda et al. 1996).
16 Chapter I: Introduction
The A loop is similar to the immunoglobulin fold and its function is to interact with and stabilise the binding by the B and B’ loops to hyaluronan (Figure 4).
Figure 4: Interaction of aggrecan with hyaluronan Adapted from Heinegard D, Oldberg A. Glycosylated matrix proteins. In: Royce PM, Steinmann B, eds. Connective Tissue and Its Heritable Disorders: Molecular, Genetic, and Medical Aspects. New York, NY: Wiley-Liss; 1993:193
The G2 domain is also made up of PTR loops but in contrast to the G1 domain, the G2 domain has no binding capacity for either link protein or hyaluronan (Fosang et al. 1989).
There is a relatively inflexible, rod-like domain connecting the G1 and G2 domains. When viewed under rotary shadowing electron microscopy, this domain is consistently linear and has no observable tertiary structure (Paulsson et al. 1987). This interglobular domain is thought to retain its rigidity due to the presence of keratan sulphate chains that are substituted throughout this region. The interglobular domain is of particular interest to researchers because this region is sensitive to proteolysis during cartilage degradation. It has been known for some time now that these cleavage events do not occur at random sites along the length of the protein. In fact, examination of the catalytic products in arthritic joints consistently shows
17 Chapter I: Introduction
similarly sized GAG-bearing fragments in the synovial fluid, and increased ratios of short G1-bearing fragments retained in the cartilage (Sandy et al. 2001). The interglobular domain contains key cleavage sites for two families of proteases: matrix metalloproteinases and aggrecanases. Aggrecan fragments beginning with Ala374 have been found in the synovial fluids of patients with various joint diseases (Lohmander et al. 1993), these fragments carry chondroitin sulphate chains as well as the majority of keratan sulphate chains and this results in the complete loss of glycosaminoglycans from the remaining aggrecan fragment still complexed with HA and link protein.
The third globular domain is found at the C-terminus of aggrecan and consists of the following motifs: an epidermal growth factor-like domain, a lectin-like domain, and a complement regulatory protein-like domain. The EGF-like domain is made up of two modules known simply as EGF1 and EGF2, though these dual modules have only been found in human aggrecan; other species have a single EGF-like domain. Alternative splicing results in most transcripts without an EGF-like domain, and only a quarter of transcripts are known to contain a single EGF domain, and even fewer have both EGF1 and EGF2 modules (Day et al. 2004). Based on their role in other proteins and more recently on expression studies, the most likely role of the G3 domain is in modulating cellular proliferation and metabolic activity or as a binding site for various proteins. The possibility that the G3 domain modules have a role in intracellular translocation and secretion has been examined (Day et al. 1999). A portion of the adjacent chondroitin sulphate-rich domain and an individual module of the G3 domain was expressed in mammalian cells. The results showed that any of the three modules that make up the G3 domain was required for intracellular translocation and secretion.
Recently analysis of the G3 domain and in particular, the lectin-like domain, found that it is able to bind to tenascin-C at a site containing fibronectin type III repeats (Day et al. 2004). Analysis of the G3 splice variants found that each splice variant had different preferences for various ligands, including tenascin- C, tenascin-R, fibulin-1, and fibulin-2. The EGF domain enhanced binding to the ligands, and both EGF1 and EGF2 further enhanced this binding.
18 Chapter I: Introduction
Alternative splicing of the G3 domain was proposed as a mechanism for modulating interactions and extracellular matrix assembly.
Aggrecan is substituted with two types of glycosaminoglycan, keratan sulphate and chondroitin sulphate. The region spanning G2-G3 contains the bulk of the glycosaminoglycan chains with a keratan sulphate-rich region followed by an extensive chondroitin sulphate-rich region consisting of two domains known as CS1 and CS2; the CS-rich region can be substituted with as many as 100 chondroitin sulphate chains in cartilage. The degree of glycosylation within the G2-G3 region varies greatly with different species and is the least well conserved region of aggrecan. The KS-rich region is made up of hexapeptide repeats which correspond to the number of KS chains present. There is also KS present in the IGD, but to a far lesser degree.
Sequencing of a cDNA clone encoding the KS-rich region revealed the presence of a hexapeptide sequence: E-(E/L)-P-F-P-S, repeated 23 times in bovine aggrecan (Antonsson et al. 1989). Sequencing of the KS-rich region in 11 other species found that this hexapeptide sequence was repeated to varying degrees with each species ranging from 13 in human aggrecan to 4 in murine aggrecan (Barry et al. 1994; Walcz et al. 1994). Chicken aggrecan does not appear to have a KS-rich domain with any recognisable motifs (Chandrasekaran et al. 1992; Chandrasekaran et al. 1993). The number of hexapeptide repeats appears to be proportional to the amount of KS present in the KS-rich region; this sequence therefore correlates to an O-linked KS- attachment site.
Like the KS-rich domain, the CS-rich regions are made up of repeating sequences which are due to polymorphisms present in the region encoding the CS domains (Roughley 2001). The CS1 domain contains tandem repeats of 19 amino acids that are repeated up to 33 times, though repeats of 25 to 29 are most common in humans (Doege et al. 1991; Roughley et al. 2002). The number of repeats varies across different species, with 15 present in the rat and 29 in human aggrecan. The CS2 domain contains much larger repeats of 100 amino acids, and has larger spaces between the CS attachment sites.
19 Chapter I: Introduction
Keratan sulphate
The basic structure of keratan sulphate was determined by the 1950s. It was characterised as a polymer of N-acetylactosamine[-3Galβ1-4GlcNAcβ1-] with sulphation at C6 at either hexose, giving rise to disaccharides that are unsulphated, monosulphated, and disulphated. (Funderburgh 2000; Funderburgh 2002).
KSI
NeuAc2-3/6 (SO4 ) SO4 SO4 SO4 SO4 NeuGc2-3/6 6 6 6 6 6 Galα 1-3 Fuc -Gal-4GlcNAc-(3Gal-4GlcNAc)8 - 34 -(3Gal-4GlcNAc)10 - 12 -(3Gal-4GlcNAc)2 -2Man (SO )GalNAc1-3 4 6 6 Man-4GlcNAc-4GlcNAc-N-Asn (SO4 )GlcNAc1-3 3 NeuAc2-3Gal-4GlcNAc-2Man
KSII
(SO4 ) SO4 SO4 (SO4 ) SO4 6 6 6 6 6 NeuAc2-3/6 Gal-4GlcNAc-3Gal-4GlcNAc-(3Gal-4GlcNAc)3 - 11 3 Fuc 6 GalNAc-O-Ser/Thr 3 NeuAc2-3Gal
KSIII
SO4 6 (Gal-4GlcNAc)7-24 - 3Man-O-Ser/Thr Figure 5: General structure of keratan sulphate Figure adapted from (Krusius et al. 1986; Funderburgh 2000). NeuGc (N-glycolylneuraminic acid); NeuAc (N-acetylneuraminic acid); Gal (galactose); GalNAc (N-acetylgalactosamine); GlcNAc (N- acetylglucosamine); Man (mannose); Asn (asparagine); Ser (serine); Thr (threonine); Fuc (fucose).
There are two main classes of keratan sulphate which are categorised according to their linkage to the core protein. KSI includes all the N-linked KS molecules; though most commonly found in corneal proteoglycans, N-linked KS can also be found in the cartilage. The cornea has the highest concentration of KS in the body and has up to ten times more than that found in cartilage (Funderburgh 2000). Its presence in the cornea in the form of keratan sulphate proteoglycans, particularly keratocan and lumican, are
20 Chapter I: Introduction
critical in maintaining the interfibril spacings of collagen I and V, and to a lesser degree, other collagens. Disruption of the interfibril spacings due to loss of keratan sulphate, results in corneal clouding (Kurpakus Wheater et al. 1999; Kao et al. 2002; Michelacci 2003), this is especially evident in patients suffering from macular corneal dystrophy. In the diseased corneas, the integrity and function of the corneal stroma is severely compromised and is characterised by widespread cloudiness of the eye. This condition causes a disruption in glycosaminoglycan synthesis and results in downregulation of keratan sulphate as well as upregulation of chondroitin sulphate (Plaas et al. 2001). It is not surprising then, that much of the work on keratan sulphate biosynthesis has concentrated on corneal keratan sulphate. However the role of keratan sulphate has since been demonstrated in other tissues like the brain, where a possible third type of keratan sulphate is present (figure 5).
Following spinal cord injury, there is a dramatic increase in the expression of keratan sulphate proteoglycans surrounding the site of injury by a number of different cells including reactive microglia, macrophages, and oligodendrocyte progenitors (Jones et al. 2002; Krautstrunk et al. 2002). Observations of the rat brain during development have found that striatal proteoglycans have specific patterns of expression and modification. This was thought to have some influence on the outgrowth of dopaminergic neurons. To further elucidate the effect of KSPGs on neuronal development, embryonic rat mesencephalic cells were cultured on different glycosaminoglycan substrates and the adhesion and outgrowth of the cells was observed. Adhesion of the cells was generally quite poor, and the extension of the neuronal fibres varied according to the type of glycosaminoglycan used as the substrate. Further studies using slices of rat brain of different developmental stages found that the chondroitin sulphate-rich striatum mediated the adhesion and outgrowth of dopaminergic neurons, whereas the keratan sulphate-rich cortex seemed to have the opposite effect; it appeared to inhibit the outgrowth of the neuronal fibres (Mace et al. 2002). These fibrous outgrowths from neurons are commonly known as mossy fibres, and they exhibit a very specific pattern of innervation that is restricted to the stratum lucidum. CSPGs and KSPGs were used to determine their effect on mossy fibre outgrowth from sections taken of
21 Chapter I: Introduction
the hippocampus (Butler et al. 2004). Axonal outgrowth and regeneration of the normal innervation pattern was studied following treatment of the tissue with either chondroitinase or keratanase. Chondroitinase treatment of the tissue sections had no effect on mossy fibre outgrowth however mossy fibre outgrowth was observed after keratanase treatment, although this new growth was disorganised. Because phosphacan is the primary KSPG of the brain, and this KSPG is produced by astrocytes, it has been suggested that keratanase alters mossy fibre outgrowth by modifying astrocyte function (Butler et al. 2004).
Davies et al. (Davies et al. 1999) examined the migration of cells in the human endothelium of normal and wounded corneas. They showed using immuno- gold labelling, that under normal conditions, keratan sulphate labelling of corneal cells was high, whereas during the wound healing response, keratan sulphate labelling was low. Kavanagh et al. (Kavanagh et al. 2002) found that in cartilage, keratan sulphate is restricted to the inner layer of the perichondrium known as the chondrogenous layer and suggested that like in the central nervous system and cornea, keratan sulphate produced in the chondrogenous zones of cartilage serve to diminish cellular migration, maintaining the chondrogenous zones.
The importance of keratan sulphate is slowly becoming apparent in other tissues as well. Recent reports have identified the keratan sulphate proteoglycan, lumican, in the secretory epithelial cells of human prostate, where it was found associated with prostatic secretory granules. Though its role in the maintenance of the cornea has been established, as well as its role in tissue repair and cell migration (Vuillermoz et al. 2004), the role of lumican in the prostate is yet to be determined. It has been suggested to be involved in tumour suppression; marking the early stages of malignant transformation (Holland et al. 2004). This data has also been backed up in similar studies examining the effect of lumican on melanoma progression. In that study, mouse B16F1 melanoma cells were transfected with lumican cDNA before subcutaneous injection. Lumican expression was found to significantly decrease subcutaneous tumour progression by inducing apoptosis of the
22 Chapter I: Introduction
B16F1 cells (Vuillermoz et al. 2004). However, the greatest understanding of keratan sulphate has resulted from studies of the cornea.
Corneal KS proteoglycans are almost entirely of the KSI variety and to date is the most widely studied. KSII includes all the KS molecules linked to the core protein via GalNAc-O to either Ser or Thr. Type II KS is predominately found in load-bearing articular cartilage (Huckerby et al. 2000), although KSI is also present in cartilage on fibromodulin and can be fucosylated and sialylated. KSII is much more sulphated than KSI with sulphate groups found on GlcNAc as well as galactose and these chains can be modified with fucose groups and sialic acid capping. Another type of KS linkage has been found in the brain, with Man-O-Ser linkages to chondroitin sulphate proteoglycans and may constitute another class of KS (Funderburgh 2000; Funderburgh 2002).
Though it is found in many tissues, keratan sulphate is found only on a limited number of proteins (Funderburgh 2002). The post-translational modification of proteins with keratan sulphate is therefore likely to be dependant on the protein sequence alone.
N-linked KS (KSI) is very highly expressed in the cornea, where it is found substituted on three core proteins: lumican, keratocan, and mimecan. Analysis of these proteins led researchers to identify the consensus sequence: N-X-(T/S) for N-linked KS attachment (Funderburgh et al. 1991; Dunlevy et al. 1998), where ‘X’ is any amino acid except proline (Grogan et al. 2002). Although the N-linked KS appeared to be substituted on this consensus sequence, not all of the available sites were occupied in each of the corneal proteoglycans analysed. This suggests that the secondary structure as well as the consensus sequence is involved in N-linked KS substitution.
23 Chapter I: Introduction
KS Synthesis and Elongation
Synthesis and elongation of keratan sulphate begins in the rough endoplasmic reticulum. It is a complex process involving numerous transferase enzymes, many of which are yet to be identified, which add successive β-4-galactose and β-3-N-acetylglucosamine residues to the growing oligosaccharide. Not only are there enzymes for elongation, there are also modifying enzymes as well. Each type of enzyme serves a specific role, and often has very specific substrates. Among the list of modifying enzymes are: sialyltransferases, which are involved in sialic acid capping; sulphotransferases, which add sulphate groups to carbohydrates; and fucosyltransferases which are involved in fucosylation.
Keratan sulphate on human articular cartilage aggrecan undergoes various stages of structural modifications with age. Brown et al. (Brown et al. 1998) took articular cartilage samples from individuals of various ages ranging from 0 – 85 years of age. They found that from 0 – 9 years, there is increased α(1- 3)-fucosylation, α(2-3)-sialylation, as well as increased sulphation on the galactose residues. From ages 9 – 18 years, aggrecan keratan sulphate undergoes further fucosylation and galactose sulphation, there is also α(2-6)- sialylation. Beyond the age of 18, keratan sulphate undergoes no further changes.
Though far from being completely understood, four key enzyme families have been shown, in vitro, to be sufficient to produce keratan sulphate, though in its most basic structural form: β-1,3-N-acetylglucosaminyltransferase (iGNT); N- acetylglucosaminyl-6-sulphotransferase (GlcNAc6ST); β-1,4- galactosyltransferase (βGal-T1); keratan sulphate galactosyl-6- sulphotransferase (KS-Gal6ST) (Akama et al. 2001; Akama et al. 2002; Funderburgh 2002).
β4Gal-T1 is the most studied enzyme. It is widely distributed, and has a consistent expression level regardless of the cellular activity. Activity of this gene was monitored in the corneas of developing chickens. As expected,
24 Chapter I: Introduction
mRNA levels increased with certain stages of corneal development involving increased KS synthesis. However expression of β4Gal-T1 in corneal fibroblasts is also high, despite having relatively low levels of keratan sulphate synthesis (Cai et al. 1996).
Sulphotransferases
Sulphotransferases transfer sulphate from 3’-phosphodenosine 5’- phosphosulphate (PAPS) to specific residues of a given carbohydrate (Fukuda et al. 2001). Initial cloning of sulphotransferases was based on cDNA cloning using the amino acid sequence from purified enzymes. Heparan sulphate N-deacetylase sulphotransferase; chondroitin sulphate GalNAc 6-O- sulphotransferase; heparan sulphate GlcN 3-O-sulphotransferase; and galactosylceramide 3-O-sulphotransferase (Hashimoto et al. 1992; Eriksson et al. 1994; Fukuta et al. 1995; Honke et al. 1997; Shworak et al. 1997; Fukuda et al. 2001) were all cloned using this method. Subsequent sulphotransferases have been cloned based on sequence similarity to known sulphotransferases. A conserved sequence: X-X-R-D-P-Z-Z-Z-X; where X refers to a hydrophobic amino acid, and Z refers to any amino acid; has been identified in the cloned sulphotransferases (Ong et al. 1998).
In keratan sulphate, it is always C6 which is sulphated, and this sulphation occurs at either the galactose or N-acetylglucosamine group. There are currently two enzymes known to transfer sulphate groups to the galactose moieties: chondroitin-6-sulphotransferase (C6ST) and keratan sulphate galactose-6-sulphotransferase (KS-Gal6ST).
C6ST predominately adds sulphate groups to the N-acetylgalactosamine of chondroitin sulphate in vivo, however in vitro, this enzyme has the ability to modify the galactose group of keratan sulphate, albeit with a 10-30 fold lesser activity than with chondroitin sulphate. C6ST knockout mice have 90% loss of 6-sulphation of chondroitin sulphate, however keratan sulphate in the spleen was not affected (Uchimura et al. 2002). In humans, C6ST mutations result in severe chondrodysplasia with major involvement of the spine. Urine analysis
25 Chapter I: Introduction
showed a marked reduction of 6-sulphation, however keratan sulphate was apparently unaffected (Thiele et al. 2004). This enzyme is therefore unlikely to be involved in keratan sulphate sulphation in vivo.
KS-Gal6ST was cloned by Fukuta et al. and assigned to chromosome 11p11.1-11.2 by in situ hybridization; it is another enzyme able to modify galactose with sulphate. KS-Gal6ST has specific activity for keratan sulphate, and is expressed most strongly in the brain and cornea (Fukuta et al. 1997) which are also tissues with high keratan sulphate expression. GlcNAc tends to be more sulphated than Gal residues, especially in the cornea where they are almost exclusively 6-sulphated. GlcNAc6ST is a general name for the sulphotransferase responsible for sulphating GlcNAc residues. Different forms are expressed in different tissues; C-GlcNAc6ST is exclusively expressed in the cornea, whereas I-GlcNAc6ST is expressed mainly in the intestine.
Sialyltransferases and fucosyltransferases
There are likely to be in excess of 20 different sialyltransferases, each expressed in different tissues and at different developmental stages (Harduin- Lepers et al. 2001). All have a common function, and that is to transfer sialic acid to the terminal positions of oligosaccharides, however from numerous reports in the literature, it is clear that sialyltransferases are also involved in many other biological processes. One example of this is β-Galactoside α2,6- sialyltransferase, an enzyme strongly expressed in the liver, responsible for transferring α2,6-linked sialic acid residues to glycoproteins. Studies have shown that its expression is upregulated in hepatocarcinoma patients (Dall'Olio et al. 2004). Aberrant sialyltransferase activity has also been linked to Alzheimer's disease (Huang et al. 2004); it is thought that incorrectly sialylated glycoproteins may play a role in the onset of the disease.
Members of the sialyltransferases have three conserved regions; these are known as sialylmotif L (long), sialylmotif S (short), and sialylmotif VS (very short) (Tsuji et al. 1996; Geremia et al. 1997). To date, 15 human sialyltransferase cDNAs have been cloned and characterised based on these
26 Chapter I: Introduction
motifs (Harduin-Lepers et al. 2001). However the molecular structure and their mechanism of action is not well understood; it was only as recent as 2004 that the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, was reported (Chiu et al. 2004).
Nine human fucosyltransferases have been cloned to date: two α1,2- fucosyltransferases; one α1,3/4-fucosyltransferase; five α1,3- fucosyltransferases; and one α1,6-fucosyltransferase. As well as modifying glycoproteins and proteoglycans with fucose, fucosyltransferases are involved in the synthesis of fucosylated cell surface oligosaccharides known as LewisX structures, which serve as ligands for L-, P- and E-selectins, which in turn, mediate inflammation via selectin-dependent leukocyte recruitment (Homeister et al. 2001). Fucosyltransferases are therefore intensely studied for their role in a number of different inflammatory conditions. Depending on the type of fucosyltransferases involved, fucose residues can be transferred to either the galactose or N-acetylglucosamine of Galβ1,3GlcNAc or Galβ1,4GlcNAc chains. However if there is an adjacent sialic acid residue present such as in SAα2,3Galβ1,3GlcNAc or SAα2,3Galβ1,4GlcNAc chains, only the N-acetylglucosamine is modified.
The fucosyltransferases are expressed in different tissues throughout the body depending on the type of enzyme. To summarise, fucosyltransferases 1, 2, and 3 are expressed in the epithelial cells of tissues such as in colorectal and stomach tissues, the mammary gland, lung, and uterus (Narimatsu et al. 1996; Narimatsu et al. 1998). Fucosyltransferase 4 is also expressed in multiple tissues including myeloid tissue (Gersten et al. 1995). Fucosyltransferase 7 expression is restricted to leukocytes and high endothelial venule cells (Kimura et al. 1997). Finally, fucosyltransferases 9 is expressed in neuronal and glial cells, the stomach, kidneys, and blood cells (Kudo et al. 1998; Kaneko et al. 1999; Nakayama et al. 2001).
27 Chapter I: Introduction
In vitro variations in keratan sulphate proteoglycan synthesis and secretion
Keratan sulphate was first identified in extracts of cornea in 1939 by Suzuki (Suzuki 1939). Since then, the vast majority of keratan sulphate studies have concentrated on the cornea due to its important role in maintaining corneal transparency (Beales et al. 1999; Long et al. 2000; Carlson et al. 2003). The corneal stroma makes up 90% of the thickness of the human cornea, and is the tissue with the highest abundance of keratan sulphate, this is followed by skeletal tissue and the brain.
The major components of the corneal stroma are collagen I, V, VI, XII, the CSPG decorin, and a number of KSPGs (Hassell et al. 1992; Carlson et al. 2003; Funderburgh et al. 2003). Keratocytes make up the majority of the population of cells of the cornea and it is these cells which express the KSPGs and collagen (Carlson et al. 2003). In their natural state, keratocytes are quiescent and express KSPGs with long, highly sulphated keratan sulphate chains. However, in response to corneal trauma, keratocytes become active and migrate to the site of the wound and begin to divide (Funderburgh et al. 2001). During this wound healing response, keratocytes develop a myofibroblastic morphology, expressing F-actin stress fibres and at the same time keratan sulphate expression is greatly reduced (Funderburgh et al. 2001; Carlson et al. 2003; Funderburgh et al. 2003). Cell migration is linked to a decrease in keratan sulphate and an increase in chondroitin sulphate and heparin sulphate (Davies et al. 1999).
Carlson et al. examined the expression of KSPGs by keratocytes following corneal injury in mice and observed normal keratocyte function at the site of corneal scarring returning after 12 weeks post-injury (Carlson et al. 2003). This same phenomenon of reduced keratan sulphate production in keratocytes occurs in culture as well, and in multiple species; subculturing by trypsin treatment also appears to inhibit KS expression. As an example, a comparison of KSPGs secreted by chick corneas and corneal fibroblasts was
28 Chapter I: Introduction
reported to produce proteoglycans with significantly smaller amounts of KS in corneal fibroblasts (Schrecengost et al. 1992). Keratan sulphate makes up as much as 47% of the total glycosaminoglycan content of the cornea, conversely, keratocytes grown in culture produce less than 3% keratan sulphate (Beales et al. 1999). It should be noted that while the keratan sulphate chains are drastically shortened and much less sulphated, the overall expression of KS-linked core proteins remains the same (Funderburgh et al. 2003).
There have been a number of studies to date examining the effect different culture conditions have on keratan sulphate expression. Beales et al. reported prolonged high KS expression in bovine keratocytes in response to low or serum-free culture medium (Beales et al. 1999). Another group (Long et al. 2000) discovered that the addition of FGF-2 to primary keratocytes maintained KSPG secretion albeit with reduced KS, for several days longer than in the standard 10% FCS medium. Nakazawa et al. examined keratocytes grown in a three dimensional collagen matrix, while the collagen matrix on its own, increased keratan sulphate synthesis only slightly, combining the collagen matrix culture system with CG medium, a serum-free medium, was reported to maintain keratan sulphate synthesis at levels similar to those of the control primary keratocytes (Nakazawa et al. 1995).
While there are lots of studies reporting the downregulation of keratan sulphate in response to pathological changes like wound healing or tissue culture, very few studies go into any detail as to why this effect should occur. Down regulation of key keratan sulphate-sulphotransferase enzymes is known to occur in vitro (Nakazawa et al. 1998) and it has also been suggested that because the loss of keratan sulphate synthesis is usually associated with inflammation, proinflammatory cytokines may have a role in the downregulation of keratan sulphate biosynthesis (Funderburgh 2002).
This brings me to my project. One aspect of my project involved the expression of recombinant aggrecan G1-G2 modified with keratan sulphate. However, as already explained, under standard tissue culture conditions, cells
29 Chapter I: Introduction very quickly stop expressing ‘normal’ levels of keratan sulphate. Therefore, a large proportion of my project was spent identifying a type of cell capable of maintaining high levels of KS synthesis, and once a cell was identified, optimising culture conditions to also prolong KS synthesis.
30 Chapter I: Introduction
Arthritis
Figure 6: Stages of aggrecan loss and cartilage destruction in a canine model of arthritis From (Visco et al. 1993)
As of 2001, 13.67% of the Australian population suffered from long term arthritis or a similar form of joint disease (Australian Bureau of Statistics). Arthritis is more than a single disease; it is a complex collection of more than 100 different conditions affecting the joints. Two of the most common forms of arthritis are osteoarthritis, and rheumatoid arthritis.
Osteoarthritis
Osteoarthritis is the most common of the more than 100 forms of arthritis and occurs more frequently with age, affecting females more than males. Unlike rheumatoid arthritis, which is an autoimmune disease affecting multiple tissues, osteoarthritis only affects the joints.
Osteoarthritis is a disease affecting the cartilage, bone, synovium, and joint capsule and is characterised by synovial joint damage. The affected joints are typically painful, with reduced mobility after a period of inactivity. The pathological signs are: focal areas of articular cartilage destruction, capsular thickening, and varying degrees of mild synovitis. Bone cysts can also develop due to the increased pressure on the bone from the lack of cartilage. The disease can be initiated by an instability or change in the shape of the joint however osteoarthritis is not just the mechanical degeneration of the
31 Chapter I: Introduction
joints. Instead, it is a complex biological and mechanical disorder leading to joint degeneration and then osteoarthritis. It has been suggested that due to our relatively recent evolutionary adoption of an upright posture, certain joints have not completely adapted to their new position and are therefore susceptible to osteoarthritis. These joints include the hips, the knee, the cervical and lumbar facet joints, and the hand. Symptomatic osteoarthritis is more prevalent in the knee than in the ankle, this is due to differences in the biochemistry and in gene expression levels; the higher proteoglycan content of the ankle joint is thought to help protect it from osteoarthritis. This disease can be initiated with any number of pre-existing conditions and although osteoarthritis is far from being completely understood, much progress has been made, for example it is known that in osteoarthritic knee cartilage there are elevated levels of biglycan, decorin, fibromodulin and lumican. This increase in small proteoglycans is thought to interfere with the proper repair process in knee cartilage. (Hascall et al. 2002).
Rheumatoid Arthritis
Rheumatoid arthritis is characterised by chronic synovial inflammation and pannus formation involving large and small joints. The general clinical signs of affected joints are painful swollen joints with stiff, limited movement. The joints themselves have a slightly elevated temperature compared with unaffected joints and there is very little redness; loss of muscle strength is also common. As well as the joints, other parts of the body are also affected in rheumatoid arthritis. In the lungs of affected individuals, the membranous lining surrounding the lungs, known as the pleura, commonly develop lesions, and nodules also develop in the lungs. In 20% of patients, rheumatoid nodules also appear on the elbows, finger joints, Achilles’ tendon, and sacral prominences. Other clinical signs include vasculitis, pericardial effusions, and osteoporosis. Patients can also experience gastrointestinal, ocular, renal, and neurological complications.
The primary age of onset is 55 years, however the disease can manifest itself at any time from the age of 20. The worldwide prevalence of adult rheumatoid
32 Chapter I: Introduction arthritis is 1%, but there are racial differences; Asians and Africans appear to be much less affected than Caucasians. Also of interest is that RA occurs predominantly in women rather than in men with a ratio of 2:1 to 3:1 in most studies. It is because of this difference that hormonal levels are thought to have some part to play in initiating RA.
Rheumatoid arthritis is a polygenic disease. The multihistocompatibility complex (MHC) located on 6p21.3 is the most extensively studied component of the human genome due to its association with autoimmune diseases. This is especially the case with rheumatoid arthritis. Although there are a number of candidate genes associated with rheumatoid arthritis, the most extensively studied are the polymorphic human leukocyte antigens alleles (HLA) within the MHC. The region associated with HLA gives rise to the strongest and best characterised genetic predisposition to rheumatoid arthritis. It is thought that two fifths of the genetic contribution to rheumatoid arthritis is accounted for by genes in the HLA region. Genes encoding both the HLA class I and class II molecules have been associated with RA. (Firestein et al. 2000).
Aggrecan Proteolysis
One of the early signs of arthritis is the loss of aggrecan from joint cartilage. Cleavage of aggrecan occurs primarily within the interglobular domain. This releases the bulk of the proteoglycan containing the keratan sulphate-rich region and the chondroitin sulphate-rich regions from the cartilage. Loss of aggrecan from the extracellular matrix decreases the water-binding capacity of the cartilage and as more aggrecan is lost, the hydroscopic pressure is reduced to the point where it is no longer able to withstand compressive forces. The weakened tissue eventually fibrillates and can be completely lost. Aggrecan is degraded by two families of proteases: matrix metalloproteinases (MMP), and aggrecanases. It was not until aggrecan was cloned and sequenced (Doege et al. 1991; Walcz et al. 1994), that the “aggrecanase” cleavage sites were identified. “Aggrecanases” were stimulated in bovine nasal cartilage with either retinoic acid (Ilic et al. 1992; Hughes et al. 1997), or interleukin 1α (Sandy et al. 1991; Loulakis et al. 1992), and the cleavage
33 Chapter I: Introduction products were purified and sequenced. “Aggrecanases” were found to be active at a total of five sites; one primary cleavage site was within the IGD, and four were in the CS-rich G2-G3 region. These cleavages were later attributed to ADAMTS enzymes.
34 Chapter I: Introduction
Zinc Metalloproteinases
The zinc metalloproteinases encompass a large family of enzymes, including the MMPs and aggrecanases, that are involved in a wide range of remodelling and developmental processes. Amino acid analyses have shown that they all share a short zinc-binding HEXXH motif in their catalytic domain. These zinc metalloproteinases can be further categorised into subfamilies according to the sequences flanking the HEXXH motif. These are divided into two families; the gluzincins and the metzincins.
The gluzincins are made up of the thermolysin family, the endopeptidase- 24.11 family, the angiotensin-converting enzyme family, and the aminopeptidase family. The consensus sequence for the gluzincins is HEXXH…….E, in which the two histidines and the last glutamate residues co- ordinate with a Zn2+ ion.
The other type of zinc metalloproteinases have been classified the metzincins. This group is also made up of four families of proteases; the astacin family, the serratia family, the reprolysin family, and the MMP family. The metzincins differ slightly in that their zinc-binding motif is extended. This motif includes three conserved histidine residues which form the basis of the zinc-binding domain; the consensus for the zinc-binding motif is HEXXHXXGXXH. The zinc metalloproteinases have a modular structure, and share a number of similar domains. A summary of the zinc metalloproteinases is shown in figure 7.
35 Chapter I: Introduction
Zincins Inverzincins
Astacin Serratia Reprolysin Matrixin family family family family
Gluzincins Metzincins
Figure 7: Zinc metalloproteinase family
A brief description of the major domains follows:
The Pro-peptide domain
The metzincins are expressed as zymogens, meaning they are expressed as inactive, latent enzymes, containing an N-terminal prodomain. It is thought that the inclusion of this prodomain alters the folding of the enzyme, maintaining it in its latent form. The N-terminal domain of the metzincins contain a signal sequence to allow the protein to be secreted via the secretory pathway. Immediately following this sequence is a propeptide domain. The propeptide domain maintains the zymogen in its inactive form through a mechanism known as the cysteine switch.
The two most conserved domains of the proMMPs are the zinc-binding motif of the catalytic domain and the cysteine switch motif of the propeptide domain. Among the MMPs the cysteine switch motif is particularly well conserved with a consensus sequence of: P-R-C-G-(V/N)-P-D-(V/L)- (A/G).(Van Wart et al. 1990). The cysteine switch of the reprolysins are similar
36 Chapter I: Introduction
but do not appear to be as well conserved as in the MMP family, but they are still activated with the same mechanism (Birkedal-Hansen 1995; Morgunova et al. 1999; Gomis-Ruth 2003).
Conserved histidine residues of the zinc-binding domain co-ordinate around a zinc atom and form the catalytic site. However in the presence of an intact pro-domain, the conserved cysteine residue folds over and forms a complex with the zinc atom. This results in a slight conformational change which results in the active site being blocked, therefore the zymogen remains in its latent form. The mode of activation of zymogens was not fully revealed until crystal structures of proMMPs were examined (Morgunova et al. 1999). In the case of proMMP-2, loops within the propeptide domain act as a kind of bait for proteases. When adequate cleavage has occurred, the prodomain structure breaks down and exposes the catalytic site, allowing water molecules to enter and hydrolyse the zinc-cysteine complex. This results in a conformational change, switching the latent enzyme ‘on’. However, it should be noted that there are multiple methods of activation, not all of them requiring proteolytic cleavage of the prodomain. Chemical treatment disrupting the zinc-cysteine complex is also known to activate proMMPs (Gomis-Ruth 2003).
Aggrecanases are also expressed as latent enzymes and are therefore thought to be similarly activated. They must be cleaved of their prodomain before they become biologically active. Until recently, the enzyme responsible for the prodomain cleavage in vivo, remained unclear. In vitro, furin was shown to process proADAMTS-4 to its active form (Gao et al. 2002). The
consensus furin cleavage site is R-Xn-R (where n=0, 2, 4, or 6 residues). Furin’s important role in proADAMTS-4 processing to its active form was demonstrated when furin inhibitors and RNA interference prevented proADAMTS-4 processing (Wang et al. 2004).
37 Chapter I: Introduction
Metalloproteinase domain
Also known as the catalytic domain, it contains the zinc-binding motif H-E-X- X-H-X-X-G-X-X-H. The active site contains zinc and water, which are required for hydrolysis of substrates. The three conserved histidine residues of the zinc-binding motif co-ordinate with a zinc atom. Immediately downstream of the third zinc-binding histidine of the catalytic domain is an absolutely conserved methionine residue found in all reprolysins and MMPs. This methionine makes up the so-called "met-turn"; a tight turn arranged in a right- handed screw formation that loops around to face the zinc-binding motif which helps to co-ordinate with zinc (Seals et al. 2003).
Hemopexin domain
Found only in MMPS, the hemopexin domain is named after hemopexin. It is a glycoprotein found freely circulating in serum and has a very strong binding capacity for haem. Once bound, the hemopexin-haem complex is transported to the liver where the haem is broken down for iron recovery. Many MMPs have a hemopexin-like domain at their C-terminal ends, downstream of a short hinge region. Based on crystal structures of a variety of different MMPs, the hemopexin-like domain has been described as having a 4-bladed β- propeller fold with a disulphide bond between the first and fourth blade which stabilises the structure (Visse et al. 2003). The hemopexin domain does not bind haem like the glycoprotein, but it is known to have a variety of molecular and protein ligands. For example, in tumours, MMP-9 has been correlated with tumour growth and metastasis. Examination of its hemopexin-like domain found that it was able to bind to gelatin and that a recombinant hemopexin-like domain was able to act as a MMP-9 antagonist (Roeb et al. 2002). The hemopexin domains of some MMPs are also able to bind to tissue inhibitor of metallopeptidases (TIMPs).
38 Chapter I: Introduction
Disintegrin-like domain
The disintegrin-like domain, found only in the ADAMTS family of proteinases, share some sequence similarity to the snake venoms – the disintegrins (Kuno et al. 1997). The disintegrins are a family of small peptides approximately 70 amino acids in length found in the venom of many varieties of snake. They are characterised by the presence of multiple cysteine residues as well as an Arg- Gly-Asp sequence, commonly known as the RGD sequence (Calvete et al. 1991). The disintegrins act to inhibit fibrinogen interactions with platelet receptors expressed on the glycoprotein Iib-IIIa complex (Dennis et al. 1990; Williams et al. 1990). In short, the disintegrins inhibit platelet aggregation by blocking binding of integrins to the platelet surface. Integrins contain the RGD sequence which acts as a docking site for many adhesion proteins (Ruoslahti et al. 1986; D'Souza et al. 1991). The disintegrins also contain this sequence and it is via this region that it acts as an agonist, binding to the integrin glycoprotein Iib-IIIa receptor on the surface of platelets. However, it should be noted that while the ADAMTS family of proteases all have a disintegrin-like domain, so far none have been demonstrated to have any disintegrin activity.
Thrombospondin type I motif
The thrombospondin type I motif of the ADAMTSs was originally identified as a 55 amino acid module in thrombospondin 1 and 2. The TSP1 motifs share two highly conserved regions: W-S-X-W and C-S-V-T-C-G. The TSP1 motif is thought to play an important role in protein and glycoconjugate ligand binding. In ADAMTS-4, the W-S/G-X-W sequence has been implicated in the binding of thrombospondin with sulphated GAG chains including heparin, heparin sulphate, and chondroitin sulphate. The sequence C-S-V-T-C-G binds to CD36, a thrombospondin receptor. As a whole, the TSP1 motif has been shown to be critical for aggrecan binding and cleavage (Tortorella et al. 2000). ADAMTS-4 binds to aggrecan via this motif to the GAG chains. Enzymatic deglycosylation of the GAG chains demonstrably lowers ADAMTS-4 binding and cleavage of aggrecan.
39 Chapter I: Introduction
MMPs
To date, more than 25 matrix metalloproteinases have been described mainly in vertebrates. Each of these MMPs cleave various substrates, though their specificities often overlap. Together, the MMPs are able to cleave many extracellular proteins. The MMPs are made up of highly homologous domains, and all have a conserved zinc-binding motif (completely conserved residues are in bold): H-E-X-G-H-X-X-G-X-X-H-X, and a sequence known as the “Met turn” X-X-M-X-P (Sternlicht et al. 2001).
SH
Pre Pro Catalytic Zn Hemopexin
Figure 8: MMP prototype
All MMPs have a number of conserved domains (figure 8). They all have an N-terminal “pre” domain containing the signal sequence, which is cleaved after entering the endoplasmic reticulum. This domain is followed by the propeptide domain, or “pro” domain; this sequence keeps the enzyme in its inactive form until it is removed. Finally there is the catalytic domain containing the zinc-binding region followed by a short hinge region and a hemopexin domain. The catalytic domain and the C-terminal domain dictate substrate specificity of the enzyme via an active site cleft, sub-site pockets, and substrate-binding exo-sites (Overall 2001). Aggrecan is cleaved by MMPs 1 (Flannery et al. 1993; Fosang et al. 1993), 2 (Fosang et al. 1992), 3 (Fosang et al. 1991; Flannery et al. 1992), 7 (Fosang et al. 1992), 8 (Fosang et al. 1993), 9 (Fosang et al. 1992; Flannery et al. 1993), 10 , 12, 13 (Fosang et al. 1996), and 14 (Fosang et al. 1998). These cleave primarily within the
interglobular domain at the site …DIPEN341 – F342FGVG… MMPs have been implicated in arthritis due to the presence of elevated levels of MMP cleavage products in the synovial fluid of arthritis patients (Yoshihara et al. 1995; Ahrens et al. 1996; Yoshihara et al. 2000; Giannelli et al. 2004; Tchetverikov et al. 2004).
40 Chapter I: Introduction
The MMP family includes: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and a few unclassified MMPs. These are presented in table 3.
41 Chapter I: Introduction
Table 3: Matrix Metalloproteinases Enzyme MMP Human locus Collagenases Interstitial collagenase; MMP-1 11q22-q23 collagenase 1 Neutrophil collagenase; MMP-8 11q21-q22 collagenase 2 Collagenase 3 MMP-13 11q22.3 Collagenase 4 (Xenopus) MMP-18 NA Gelatinases Gelatinase A MMP-2 16q13 Gelatinase B MMP-9 20q11.2-q13.1 Stromelysins Stromelysin 1 MMP-3 11q23 Stromelysin 2 MMP-10 11q22.3-q23 Stromelysin 3 MMP-11 22q11.2 Matrilysins Matrilysin 1; Pump-1 MMP-7 11q21-q22 Matrilysin 2 MMP-26 11p15 Membrane-type MMPs Transmembrane MT1-MMP MMP-14 14q11-q12 MT2-MMP MMP-15 15q13-q21 MT3-MMP MMP-16 8q21 MT5-MMP MMP-24 20q11.2 GPI anchor MT4-MMP MMP-17 12q24.3 MT6-MMP MMP-25 16p13.3 Others Macrophage elastase MMP-12 11q22.2-q22.3 No trivial name MMP-19 12q14 Enamelysin MMP-20 11q22.3 XMMP (Xenopus) MMP-21 ND CA-MMP MMP-23 1p36.3 CMMP (Gallus) MMP-27 11q24 Epilysin MMP-28 17q21.1 Adapted from (Visse et al. 2003)
42 Chapter I: Introduction
The reprolysins
The proteinases that make up the adamalysins were originally characterised from snake venom metalloproteinases, the first of which was adamalysin II, identified from the Eastern diamond rattlesnake, Crotalus adamanteus, hence the classification - adamalysin. This family of proteins is also known as ADAMs (a disintegrin and metalloproteinase) or MDCs (metalloproteinase-like disintegrin-like and cysteine-rich proteins). This family of enzymes are zinc metalloproteinases, and have been classified as reprolysins. The snake venom metalloproteinases are capable of digesting various extracellular matrix components.
The adamalysin family of metalloproteinases encompass the snake venom metalloproteinases (SVMP), the ADAMs (a disintegrin and metalloproteinase), and the most recently described ADAMTSs (a disintegrin and metalloproteinase with thrombospondin repeats). Members of this family of SVMPs were identified in non-hemorrhagic and hemorrhagic venoms. They were characterised by their ability to degrade a variety of different extracellular matrix components resulting in tissue necrosis. All members of the adamalysin family have a prerequisite zinc and calcium-dependent catalytic domain; the SVMPs have been subdivided into four classes according to their structural domains (Gomis-Ruth 2003):
NI/PI: Signal peptide, pro-domain, catalytic domain NII/PIII: NI/PI + disintegrin-like domain NIII/PIII: NII/PIII + cysteine-rich domain NIV/PIV: NIII/PIII + C-terminal lectin domain
ADAMs
The ADAMs are a related family of cell surface proteins containing a disintegrin and metalloproteinase domain. The ADAMs are found primarily in mammals and some lower order eukaryotes. They have a similar structural makeup as the class NIII/PIII snake venom metalloproteinases but they have
43 Chapter I: Introduction
an additional EGF-like domain as well as transmembrane domains and a cytoplasmic tail. Unlike the SVMPs, not all members of the ADAM family have complete zinc-binding sites. At last count, there were a total of 39 known ADAMs; 22 of these contain complete zinc-binding motifs, namely ADAMs 1, 8-10, 12, 13, 15, 16, 17, 19, 20, 21, 24, 25, 26, 28, 30, 33, 34, 37, 38, and 39. Expression analysis has indicated that many of the ADAMs are testis-specific, or are expressed primarily in the testis. These are ADAMs 2, 3, 5, 6, 16, 18, 20, 21, 24, 25, 26, 29, 30, and 34 – 39 (http://www.people.virginia.edu/~jw7g/Table_of_the_ADAMs.html).
The ADAMs are cell surface proteins and contain disintegrin domains and cysteine-rich domains. With these two domains, they are able to adhere to a number of different substrates. The disintegrin domain allows for integrin- mediated cell adhesion, while the cysteine-rich domain has been shown to interact with heparan sulphate proteoglycans (White 2003). Together, the disintegrin and cysteine-rich domains have been shown to bind to fibronectin as well as laminin (White 2003). It is through this interaction that allows some ADAMs to proteolytically release the ectodomain of membrane-anchored cell surface proteins. It is thought that ADAMs use their adhesion domains to bind to membrane-anchored substrates and then cleave them with their catalytic domain (Gomis-Ruth 2003), allowing for the regulation of many developmental functions. This shedding of adhesion molecules from the plasma membrane has led to the alternative name, ‘sheddase’ or ‘secretase’ (Primakoff et al. 2000).
ADAMTS and the aggrecanases
The ADAMTS family of proteinases (table 4) are a closely related group of proteins, however, unlike the membrane-bound ADAMs, the ADAMTS proteins are secreted, and contain no transmembrane domains, but do contain additional thrombospondin-like domains (Kuno et al. 1997). Several members of the ADAMTS family of proteinases have been implicated in aggrecan breakdown (Flannery et al. 1999), particularly ADAMTS-4 (figure 9)
44 Chapter I: Introduction
and ADAMTS-5 (figure 10), also known as aggrecanase-1 and aggrecanase-2 respectively.
Aggrecanases are characterised by their ability to cleave aggrecan at
glutamyl bonds within the interglobular domain (table 5) at the Glu373 – Ala374 site. However, they also cleave at four additional sites further downstream within the G2-G3 domain. In bovine aggrecan, these cleavage sites are as follows: Glu1480 – Gly1482, Glu1667 – Gly1668, Glu1771 – Ala1772, and Glu1871 –
Leu1872 (Loulakis et al. 1992; Sandy et al. 1995). Initially, two enzymes were identified with this ability to cleave the IGD, and these were named aggrecanase-1 and aggrecanase-2. It wasn’t until these enzymes were cloned and sequenced that they were found to belong to a family of zinc metalloproteinases known as the ADAMTSs (a disintegrin-like and metalloproteinase (reprolysin-type) with thrombospondin type 1 motifs). There are currently 19 members of the ADAMTS family of zinc-binding metalloproteinases; the first of these, ADAMTS-1, was described in 1997, though the activities of many of these were known prior to their cloning; a prime example of this being ADAMTS-4, otherwise known as aggrecanase-1. Like the MMPs, many ADAMTS enzymes have similar, overlapping functions; this is thought to be due to their evolution from ancestral genes via exon shuffling and duplication. The ADAMTS family of enzymes all share a number of similar domains, namely: an amino-terminal pre domain containing the signal peptide; a pro-peptide domain; a catalytic metalloproteinase domain containing the zinc-binding motif; a disintegrin-like domain; and one or more thrombospondin type 1 motifs. Aggrecanase-1 and aggrecanase-2 both have these domain structures and were designated as ADAMTS-4 (Tortorella et al. 1999) and ADAMTS-5 (Abbaszade et al. 1999) respectively. To date, ADAMTS-1 (Kuno et al. 2000; Rodriguez-Manzaneque et al. 2002), ADAMTS- 8 (Collins-Racie et al. 2004), ADAMTS-9 (Somerville et al. 2003), and ADAMTS-15 (Yamaji et al. 2001) have also been shown to cleave aggrecan at the characteristic aggrecanase cleavage sites in vitro.
The biological role of ADAMTS-1 has been explored with the creation of ADAMTS-1-null mice by gene targeting (Shindo et al. 2000). Mutant mice
45 Chapter I: Introduction
were smaller than normal and had malformed organs and adipose tissue. The females also exhibited reduced fertility. ADAMTS-1 was therefore presumed to be necessary for normal growth, fertility, and organ morphology and function.
Because of its similar sequence to ADAMTS-4 and ADAMTS-5, ADAMTS-8, a previously uncharacterised proteinase, was hypothesised to be able to cleave aggrecan at the aggrecanase-susceptible sites. With an anti-NITEGE373 neoepitope antibody, Collins-Racie et al. (Collins-Racie et al. 2004) showed that aggrecan was susceptible to cleavage by ADAMTS-8 at the E373-374A aggrecanase site. mRNA transcripts were identified in both normal and osteoarthritic cartilage.
46 Chapter I: Introduction
Table 4: ADAMTSs Gene name Protein name Alternative Human Locus Known substrates names ADAMTS1 ADAMTS-1 METH-1; 21q21 Aggrecan; versican aggrecanase-3 V1 ADAMTS2 ADAMTS-2 PCINP 5q35 Procollagen I, II, and III N- propeptides ADAMTS3 ADAMTS-3 KIAA0366 4q21 Procollagen II N- propeptide ADAMTS4 ADAMTS-4 Aggrecanase-1; 1q23 Aggrecan; KIAA0688 Brevican; versican V1; fibromodulin; decorin; carboxymethylated transferring ADAMTS5 ADAMTS-5 Aggrecanase-2; 21q21 Aggrecan ADAMTS-11 ADAMTS6 ADAMTS-6 5q12 ADAMTS7 ADAMTS-7 15q24 ADAMTS8 ADAMTS-8 METH-2 11q25 Aggrecan ADAMTS9 ADAMTS-9 KIAA1312 3p14 Aggrecan; versican ADAMTS10 ADAMTS-10 19p13 ADAMTS12 ADAMTS-12 5q35 ADAMTS13 ADAMTS-13 vWFCP 9q34 von Willebrand factor ADAMTS14 ADAMTS-14 10q21 Procollagen I N- propeptide ADAMTS15 ADAMTS-15 11q25 Aggrecan ADAMTS16 ADAMTS-16 5p15 ADAMTS17 ADAMTS-17 15q24 ADAMTS18 ADAMTS-18 16q23 ADAMTS19 ADAMTS-19 5q31 ADAMTS20 ADAMTS-20 12q12
Adapted from (Porter et al. 2005)
47 Chapter I: Introduction
ADAMTS-4 (Aggrecanase-1)
Pro Catalytic Disintegrin TSP1 Cysteine- Spacer
Pre like rich
Figure 9: Schematic of ADAMTS-4
Until the first aggrecanase was cloned and sequenced in 1999 (Tortorella et al. 1999), the uncharacterised enzyme/s involved in the cleavage of aggrecan at the characteristic Glu373 – Ala374 site were referred to as glutamyl endopeptidases or simply as “aggrecanase". Aggrecanase-1 was purified from interleukin-1-stimulated bovine nasal cartilage. TIMP-1, a tissue inhibitor of metalloproteinases which binds to the active site of a number of MMPs, ADAMs, and ADAMTSs (Nagase et al. 2003); and an anti-TIMP-1 antibody, were used to form a TIMP-1/anti-TIMP-1 antibody/aggrecanase-1 complex. The final purified aggrecanase was obtained following affinity purification. NH2-terminal sequencing revealed aggrecanase-1 to be substantially homologous to the ADAMTS family of metalloproteinases. Aggrecanase-1 was subsequently designated as ADAMTS-4. ADAMTS-4 is widely expressed in many different tissues including the joints, ovary, spinal cord, uterus, brain, and heart. ADAMTS-4 has been shown to be dramatically induced with interleukin-1 (Pratta et al. 2003), and type β transforming growth factor (Moulharat et al. 2004).
ADAMTS-5 (Aggrecanase-2)
Pro Catalytic Disintegrin TSP1 Cysteine- Spacer TSP1
Pre like rich
Figure 10: Schematic of ADAMTS-5
Aggrecanase-2 was cloned and sequenced soon after aggrecanase-1 (Abbaszade et al. 1999). The deduced amino acid sequence taken from the sequenced cDNA showed substantial homology to the murine ADAMTS-1 and the previously cloned human ADAMTS-4, however, ADAMTS-5 contained an
48 Chapter I: Introduction additional domain at the C-terminus; a TSP1 submotif. Both aggrecanases cleave aggrecan at the same sites; in the interglobular domain, and in the CS- rich domain. In our laboratory, ADAMTS-5 has been demonstrated to be the major aggrecanase in mice (Stanton et al. 2005).
Table 5: Sites of aggrecan cleavage by aggrecanases Aggrecan IGD CS-rich region Bovine Glu373- Glu1480- Glu1667- Glu1771- Glu1871- Ala374 Gly1481 Gly1668 Ala1772 Leu1872 Human Glu373- Glu1545- Glu1714- Glu1819- Glu1919- Ala374 Gly1546 Gly1715 Ala1820 Leu1920 Mouse Glu373- Glu1279- Glu1467- Glu1572- Glu1672- Ala374 Gly1280 Gly1468 Ala1573 Leu1673 Rat Glu373- Glu1274- Glu1459- Glu1564- Glu1664- Ala374 Gly1275 Gly1460 Ala1565 Leu1665
49 Chapter I: Introduction
Processing of ADAMTS-4
Unlike members of the ADAM family, the ADAMTS proteases do not contain any transmembrane domains and are therefore not cell-surface bound. As well as the initial activation of proADAMTS by furin-mediated cleavage of the prodomain, a number of mechanisms are also known to occur before aggrecanase is in its final form, though the intermediate forms also have activity. Since its initial characterisation (Tortorella et al. 1999), ADAMTS-4 has been found to have a number of truncated, but still active forms which ranged in size from the 100kDa proADAMTS-4 to the furin-activated 68kDa ADAMTS-4, as well as a 53kDa and 40kDa form. These are commonly referred to as p100 (proADAMTS-4), p68 (prodomain removed), p53 (prodomain and spacer domain removed), and p40 (prodomain, spacer, and cysteine-rich domains removed) (Tortorella et al. 2000). Although these different furin-cleaved forms still show aggrecanase activity, their proteolytic activities vary. For example examination of the aggrecanase activity of recombinant ADAMTS-4 revealed that the p68-ADAMTS-4 cleaved aggrecan at multiple sites within the CS-rich as opposed to TEGE373 - 374ARGS (Tortorella et al. 2000), whereas p53-ADAMTS-4 has very high proteolytic activity for E373 - 374A and E1480 - 1481G (Kashiwagi et al. 2004). A general theory of ADAMTS-4 processing has been proposed (Gao et al. 2004) and is the suggested mode of activation for other aggrecanases, primarily ADAMTS- 1, 4, 5, and 9, however this is yet to be proven. A summary is presented in the following figure.
50 Chapter I: Introduction
Furin Intracellular p100
Pro ADAMTS-4 III IV I II Syndecan-1 Syndecan-1
MT4 - MT4 - MMP MMP CS CS ADAMTS-4 HS HS ADAMTS-4 p53 p68 ADAMTS-4 Extracellular ADAMTS-4
Adapted from Gao et al.. 2004 ADAMTS-4 p40
Figure 11: Proposed model of ADAMTS processing I – The intracellular proADAMTS-4 (p100) has its prodomain cleaved with furin, forming the activated p68. II – The p68 intermediate associates with GPI-MT4-MMP (glycosylphosphatidyl inositol-anchored membrane type 4 matrix metalloproteinase) and moves to the cell surface where its spacer domain is removed to form the p53 intermediate. III – This p53 form has been found associated with the heparan sulphate and chondroitin sulphate chains of the proteoglycan, syndecan-1. IV – Finally, the p40 form is released after cleavage of the spacer and cysteine-rich domains.
51 Chapter I: Introduction
Keratan sulphate analysis
Aggrecan is a proteoglycan, and as a proteoglycan, its core protein is substituted with multiple glycosaminoglycan chains of chondroitin sulphate and keratan sulphate. Keratan sulphate substitution has been examined in cartilage. Work by Barry et al. (Barry et al. 1995) involved the detailed analysis of the hyaluronan-binding region of aggrecan spanning G1 and parts of the interglobular domain in bovine cartilage. This work focused on the types of carbohydrate substituted within this region and in particular whether the keratan sulphate chains were N or O-linked. Up to four possible KS attachment sites were identified, with the possibility of N and O-linkage. A comparison of immature and mature bovine cartilage found that mature bovine aggrecan contained additional KS. One of these sites was found within the aggrecanase cleavage site NITEGE, with the presence of KS at either N368 or T370. The appearance of the KS chain in mature aggrecan was suggested to influence aggrecanase cleavage.
Further analyses of the effect of glycosylation on aggrecan cleavage were carried out by Pratta et al. (Pratta et al. 1997; Pratta et al. 1999). Starting with full length aggrecan (~250 kDa), cleavage at the aggrecanase site was monitored after treatment with different deglycosidases. After removal of the KS chains, aggrecanase digestion at the Glu373 - Ala374 site was not detected, however cleavage still occurred at the sites within the CS-rich domain, though in a different pattern than usual. Removal of CS chains slowed down the rate of cleavage at Glu373 - Ala374.
With such a vast array of possible glycoforms of both KS and CS proteoglycans, and their role in ECM remodelling, it is clear that while protein analysis is very important, analysis of the glycosaminoglycans attached to these proteins is equally important. Until recently, carbohydrate analysis was the domain of specialised labs with expertise in the use of techniques such as gas chromatography, nuclear magnetic resonance spectroscopy, and various chromatographic methods (Chaplin et al. 1986). Then in 1991, pioneering work by Jackson et al. (Jackson 1991; Jackson 1993; Jackson 1994; Jackson
52 Chapter I: Introduction
1994; Jackson et al. 1994) gave the field of glycobiology a simple and inexpensive analytical technique. This technique involved the use of fluorescent tags attached to the reducing end of carbohydrates, followed by their resolution on high percentage polyacrylamide gels. This technique was called fluorophore-assisted carbohydrate electrophoresis (FACE) and addressed the three main problems associated with carbohydrate analysis: 1) Not all carbohydrates are charged and are therefore difficult to resolve on gels. 2) Even if carbohydrates were resolved, often the technique used was not very sensitive and samples could not be accurately quantitated. 3) Carbohydrates are relatively small in comparison to other molecules and are difficult to resolve with any technique.
Overcoming the charge separation
Any kind of electrophoresis requires the product of interest to be charged, otherwise samples will remain unresolved. Not all carbohydrates are charged, some are completely neutral. So to overcome this problem a number of negatively charged fluorescent tags are used. Carbohydrates that have been tagged with these fluorescent tags have an overall negative charge, regardless of the carbohydrate’s original charge state. This allows for migration through an electrophoretic gel. Each of the four commonly used fluorescent tags imparts different resolving properties to the carbohydrate, so a different tag can be used to suit any given carbohydrate. The four fluorescent tags used are: 1) disodium 8-aminonaphthalene-1,3,6- trisulphonate (ANTS); 2) 2-aminoacridone (AMAC); 3) potassium 7-amino-1,3- naphthalene disulphonate (ANDA); 4) sodium 4-amino naphthalene sulphonate (ANSA) (figure 12). ANTS, ANDA, and ANSA are generally used for oligosaccharide profiling, whereas AMAC is used for monosaccharide and disaccharide profiling.
53 Chapter I: Introduction
Figure 12: Structure of fluorophores used in FACE
Quantitation of the resolved carbohydrates
The FACE labelling reaction is stoichiometric, so quantitation is possible. Analysis of carbohydrates requires accurate quantitation of the resolved products. There are a wide variety of semi-quantitative stains for both DNA and proteins, however, a good general purpose stain is yet to be developed for carbohydrates. In the past, quantitation at the level required for carbohydrates has always required the use of radiolabels, but containment of the radioactive waste and treatment of the PAGE gels for autoradiography also proved to be a problem. These issues are solved by using fluorescent tags, and with the right equipment, can be detected at picomolar concentrations.
Figure 13: Fluorophore labelling of carbohydrates
FACE involves a process known as reductive amination as shown in figure 13 (Starr et al. 1996). (Step I) shows the equilibrium of the reducing sugar, switching between the hemi-acetal and the aldehyde form of the sugar. By
54 Chapter I: Introduction
adding a fluorescent tag, in this case ANTS, it quickly reacts with the aldehyde, forming a Schiff base (Step II), although the formation of this Schiff base occurs very quickly, it is thermodynamically unfavourable, and reverts back to the open aldehyde form. Adding the reducing agent, sodium cyanoborohydride (Step III) allows the stable amine to form, containing the attached fluorescent tag.
Separation of labelled carbohydrates
The carbohydrates that are labelled for analysis are relatively small molecules, monosaccharides range in size from 180 – 300 Da, and oligosaccharides rarely exceed a few thousand Da (Starr et al. 1996). So for adequate separation, high percentage polyacrylamide gels, up to 40%, are used. Due to their small size, the molecular conformation and their charge also influence the way carbohydrates migrate. Unfortunately, it has proven to be difficult to accurately predict how far a given carbohydrate will migrate on a gel, though in general, carbohydrates with the greatest charge density tend to migrate the furthest.
Fluorophore-assisted carbohydrate electrophoresis has been adapted for use in many types of carbohydrate analyses. Recently, FACE was tested for its possible applications in analysing the muropeptide subunits comprising bacterial peptidoglycan; a task usually performed with reverse-phase high- performance liquid chromatography (HPLC) (Li et al. 2004). A comparison of the two techniques was performed on the peptidoglycans of E. coli. FACE was determined to be as qualitative and quantitative as HPLC, with the added ability to be used as a possible in vitro enzyme assay to detect metabolic by- products. Recently, FACE was adapted for analysis of glycosaminoglycans (Calabro et al. 2000; Calabro et al. 2000; Calabro et al. 2001; Plaas et al. 2001; Plaas et al. 2001).
55 Chapter I: Introduction
FACE for glycosaminoglycan analysis
Depolymerisation of KS
For optimal analysis and resolution on FACE gels, carbohydrates need to be depolymerised into disaccharides. There are many different enzymes available and specific types of carbohydrate can be analysed by choosing the appropriate substrate-specific enzyme (Calabro et al. 2000; Calabro et al. 2001; Plaas et al. 2001; Plaas et al. 2001; Plaas et al. 2001).
For the analysis of keratan sulphate, four main hydrolytic enzymes are useful for the structural analysis of KS by FACE: keratanase II, endo-β- galactosidase, neuraminidase, and fucosidase. However, another common keratan sulphate-degrading enzyme is keratanase I, which is similar to endo- β-galactosidase in that it cleaves Galβ1-4GlcNAc bonds. Unlike endo-β- galactosidase, keratanase I requires that the GlcNAc be sulphated. Keratanase I was not used for FACE analysis in these studies.
Keratanase II (from Bacillus sp. Ks36), EC3.2.1
Keratanase II has a preference for mono or disulphated disaccharides. Two disaccharide products are possible: Gal6S-GlcNAc6S, or Gal-GlcNAc6S. The disaccharide products are illustrated in the figure below (figure 14). Keratanase II is unable to cleave tetrasaccharides to any significant rate, therefore tetrasaccharides may be present as a product of keratanase II digestion.
Figure 14: Keratanase II products
56 Chapter I: Introduction
Endo-β-galactosidase (Escherichia freundii), EC 3.2.1.103
Endo-β-galactosidase has a preference for less sulphated regions of keratan sulphate, cleaving at either unsulphated or monosulphated disaccharides. The two possible products are: GlcNAc-Gal, or GlcNAc6S-Gal. These are illustrated in figure 15.
Figure 15: Endo-β-galactosidase products
Neuraminidase and fucosidase
As well as sulphation, carbohydrate chains can be modified with sialic acid (also known as neuraminic acid), and fucose. With more than 50 naturally occurring forms, the sialic acids are a family of 9-carbon monosaccharides (Angata et al. 2002). In humans the most common form of sialic acid is N- acetylneuraminic acid, and in other mammals another form containing an hydroxyl group at the N-acyl position, N-glycolylneuraminic acid, is also expressed. Sialic acid is most commonly found as a single terminal residue attached via either an α2,3; α2,6 or α2,8 glycosidic linkage to galactose or N- acetylgalactosamine of cell surface oligosaccharide chains. Neuraminidases cleave sialic acid from oligosaccharide chains. Though they are commonly used in FACE analysis, neuraminidases are of particular interest to researchers studying the influenza virus. The surface of the virus is bound with neuraminidase; it uses this enzyme to release replicated virus particles from the surface of the host cell. Many anti-influenza drugs are neuraminidase inhibitors, and prevent the release of newly formed influenza virus particles from budding off the cell surface. There are many different neuraminidases, however, all hydrolyse the glycosidic linkage of sialic acid and the oligosaccharide chain.
57 Chapter I: Introduction
Fucosidases release fucose residues from carbohydrates by hydrolysing the α1,2; α1,3; or α1,6 linkages. The expression of the fucosidases is wide-spread and activity has been found in many different tissues throughout the body from hair roots (Kido et al. 1987) to the eye (Kamei 1998).
58 Chapter I: Introduction
Expression of recombinant proteoglycans
Much of the knowledge in the field of glycobiology has been gained from studying mammalian cells. The structure and synthesis of glycoproteins in eukaryotic cells is reasonably well understood. These post-translational glycosylation events are not strictly under direct control of the nucleus, but are known to occur at a number of key organelles instead (Lechner et al. 1989). In the case of N-glycans, after translation of the core protein, it is sent to the endoplasmic reticulum and the golgi apparatus for trimming, processing and glycosylation. Prokaryotes lack these structures, and it is for this reason that bacteria are not suitable for the expression of proteoglycans.
Bacteria such as Escherichia coli are most commonly used to produce recombinant proteins because of their rapid growth and high yield. Unfortunately, many prokaryotic cells are not equipped for the post- translational modifications necessary to produce proteoglycans. Eukaryotic cells are therefore used; a number of different types of eukaryotic cells are routinely used to express proteins requiring post-translational modifications: yeast, insect, or mammalian cells. The usual method involves transfection of the eukaryotic cell with a vector containing the gene encoding the protein of interest. However, transfection requires careful optimisation for any given cell type and results are generally poor with low transfection efficiencies and low protein production.
It is possible to synthetically glycosylate proteins; this procedure has been attempted a number of times with varying degrees of success. Proteoglycan synthesis performed in vitro is a complex procedure and while the results are adequate, only proteoglycans with relatively simple glycosaminoglycan chains can be produced (Grogan et al. 2002). N-linked glycosylation has been successfully carried out on purified tumour necrosis factor receptor (TNFR) – the glycosylated form has been shown to have therapeutic properties (Grogan et al. 2002). In this series of experiments, the purified glycoprotein, TNFR, was exposed to two types of transferase – galactosyltransferase and sialyltransferase, and resulted in tumour necrosis factor receptor with partial
59 Chapter I: Introduction
amounts of sialylation. Further studies have concentrated on producing modified cells capable of producing larger amounts of glycosylating enzymes (Weikert et al. 1999). This group modified Chinese hamster ovary cells to overexpress human β1,4-galactosyltransferase and/or α2, 3-sialyltransferase, the proteoglycans produced by these cells contained significantly higher levels of glycosaminoglycans than their respective controls. Another method of producing proteoglycans is the chemoenzymatic approach (Unverzagt et al. 2002). This procedure involves exposing a synthetic peptide containing an N- linkage site, to alternating commercial glycosyltransferases. However, this technique is completely synthetic and is not applicable to the study of native glycosaminoglycan structures. The same also applies to a similar technique, where N-linked glycans are successively added to a growing chain of glycosaminoglycans using chemistry based on 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (Merrifield 1969; Carpino et al. 1972).
An alternative method for expressing complex proteins including proteoglycans is the Vaccinia expression system. This method has many advantages over other methods: 1) Vaccinia is equipped with all the genes required for replication in the cytoplasm of the host, 2) Vaccinia is able to infect and replicate in a wide range of mammalian cells, 3) The efficiency of infection is high.
60 Chapter I: Introduction
Vaccinia virus
Figure 16: Vaccinia virus Image by Dr. Milan V.Nermut of the National Institute for Biological Standards and Control. Herts, U.K.
Vaccinia is an orthopoxvirus of the family Poxviridae. It differs from other eukaryotic DNA viruses because it replicates entirely in the cytoplasm of the cell rather than in the nucleus. Vaccinia viruses encode a multisubunit RNA polymerase, which is similar to those found in eukaryotes, as well as all the other enzymes necessary to synthesise capped, methylated, and polyadenylated mRNA. In fact, Vaccinia is equipped with all the proteins required for transcription of the genes contained within its own genome (Moss 1990).
Poxviruses undergo a number of key events in their replication cycle, namely: virus entry, regulated gene expression, DNA replication, virion assembly, and virus dissemination (Moss 1990). The first stage of the replication cycle is entry of the virus into the cell. This has been shown to occur via direct fusion to the cellular plasma membrane (Chang et al. 1976). Following fusion, the components of the viral envelope become incorporated into the plasma membrane. This release of phospholipid is accompanied by the release of 50% of the virion protein (Joklik 1964) – marking an event known as the first
61 Chapter I: Introduction
stage of uncoating, during which, Vaccinia virus cores are released to the cytoplasm and transcription occurs (Munyon et al. 1966; Kates et al. 1967; Dahl et al. 1970), producing polyadenylated mRNA containing a methylated cap structure at the 5’ end (Boone et al. 1977). This capping and methylation is carried out by a 127 kDa multifunctional enzyme complex made up of a 97 and 33 kDa subunit (Martin et al. 1975); these modifications have been demonstrated to be important for binding to the ribosome (Muthukrishnan et al. 1978). mRNA synthesis gradually ceases, marking the end of the first stage of uncoating and the beginning of the second stage of uncoating. The time at which this second stage begins is dependant on the number of virus protein cores present in the cytoplasm but usually occurs by 2 hours after infection. During this stage, the viral genome becomes DNase-sensitive and DNA replication begins shortly after the viral-encoded synthesis of DNA polymerase. Finally, the late stage of replication involves processing of structural proteins, and assembly of the first of two infectious virus forms, the Intracellular Mature Virus (IMV), a process that is poorly understood with multiple theories being examined. Three to four hours following infection, DNA and protein-rich “factories” appear, and it is from these nucleus-sized structures that the maturing viral particles form (Griffiths et al. 2001).
The IMV appears as a brick-shaped structure consisting of an outer membrane (or membranes) enclosing an inner membrane-like palisade layer containing the viral DNA (Moss 1990). The IMV is converted to the second infectious form, the Extracellular Enveloped Virus (EEV), when trans-Golgi network (TGN)-derived viral membranes are wrapped around the IMV (Schmelz et al. 1994). Traditionally, there have been two competing theories of Vaccinia virus assembly; both have arisen due to different preparation techniques. Dales et al., believe that the IMV acquires its membrane via de novo viral membrane assembly, resulting in viral particles surrounded by a single bilayer (Dales et al. 1981). Another group claim the Vaccinia virus membranes are derived from the virally-modified smooth-membraned domain of the endoplasmic reticulum and are therefore surrounded by cisternal membranes as opposed to simple bilayers (Roos et al. 1996; Salmons et al. 1997; Locker et al. 1999). However, this argument has recently been settled
62 Chapter I: Introduction with cryo-electron tomography of the virus, which allows for high resolution tomographic reconstructions of both the internal and external structures of the Vaccinia virus (Cyrklaff et al. 2005). Cyrklaff et al. showed that Vaccinia virus is made up of a single-layered outer membrane (~5-6 nm thick) which is removed during internalisation, revealing the intracellular mature virus surrounded by two layers (~18 – 19 nm thick). The inner layer closest to the core was consistent with a lipid membrane, whereas the outer layer was described as a discontinuous palisade layer with paracrystalline patches of spikes. New structures were also discovered during their study, the most notable was what appeared to be pore-like formations which spanned the inner membrane; these were theorised to facilitate the export of RNA during early stages of infection.
63 Chapter I: Introduction
The Vaccinia virus T7 RNA polymerase expression system
The Vaccinia virus T7 RNA expression system was developed as a method for the efficient expression of recombinant proteins that require post- translational modifications such as proteoglycans. It takes advantage of the high transcriptase activity and stringent promoter specificity of the virus (Fuerst et al. 1986; Elroy-Stein et al. 1989). The method itself is relatively straightforward (figure 17) – cells are infected with two varieties of Vaccinia virus. The first is the recombinant Vaccinia virus, vTF7-3 (available from ATTC, accession number VR-2153) which, when used to infect eukaryotic cells, results in high levels of expression of T7 bacteriophage RNA polymerase. At the same time, the cells are simultaneously co-infected with Vaccinia virus containing the cDNA encoding the protein of interest under the control of a T7 promotor, and also containing a leader sequence containing the encephalomyocarditis (EMC) untranslated region (UTR); this leader sequence has been found to greatly increase the translation efficiency by facilitating cap-independent ribosome-binding (McQuillan et al. 2001).
The Vaccinia virus T7 expression system has been used to express a number of different proteins. One classic example was its use in producing recombinant decorin glycoforms (Ramamurthy et al. 1996). Decorin is a small chondroitin sulphate proteoglycan that is ubiquitously expressed in the ECM of many different tissues and in vitro, it has been shown to interact with multiple proteins and membrane receptors including fibronectin, transforming growth factor-β, collagen type I, type II, and type VI, among others (Ramamurthy et al. 1996). It was the aim of that series of studies to produce a recombinant decorin glycoform that was more representative of the natural form, as opposed to the harshly extracted and often denatured decorin substrates that were more commonly used as substrates for experiments. By using the Vaccinia virus T7 RNA polymerase, recombinant decorin glycoforms were produced in chemical amounts and examination of the expression proteoglycan found that unlike the extracted decorin, the recombinant decorin maintained its extensive secondary structure, and was therefore suggested to make a more useful substrate for its functional studies. Recombinant biglycan,
64 Chapter I: Introduction a chondroitin sulphate proteoglycan, has also been produced using the same technique (Hocking et al. 1996). A number of different glycoforms were produced, ranging from the unglycosylated core protein, to one with an intermediate level of glycosylation with a total mass of 28 kDa, to a 40 kDa highly glycosylated form.
Recombinant Vaccinia virus TF7-3 G1-G2
T7 RNA polymerase
G1-G2 mRNA
G1-G2 Figure 17: Outline of Vaccinia T7 RNA polymerase expression system Adapted from (McQuillan et al. 2001)
65 Chapter I: Introduction
Project goals
Aggrecan is a member of the hyalectan family of proteoglycans and is expressed primarily in cartilage. Its function in the central nervous system is thought to be a biochemical one, where it has been implicated in the growth and direction of neural fibrous outgrowths. In contrast, the biological function of cartilage aggrecan is structural and is much more glycosylated with extended regions of chondroitin sulphate and keratan sulphate glycosaminoglycans. Disruption of the shock-absorbing quality of cartilage occurs when aggrecan is proteolytically cleaved. This primarily aggrecanase- driven cleavage can reach a point where fibrillation of the tissue occurs, and can result in severe joint damage. The biological function of keratan sulphate on aggrecan is still not known. Substituted primarily on cartilage aggrecan, keratan sulphate substitution levels are far exceeded by the amount of chondroitin sulphate substitution. Though no chondroitin sulphate exists in the IGD, keratan sulphate is substituted in this domain, with one keratan sulphate chain attached quite close to the aggrecanase cleavage site in mature cartilage (Barry et al. 1992). Age-related effects have been observed in aggrecanase-mediated aggrecan cleavage, with increased cleavage observed with older, more KS-substituted aggrecan (Pratta et al. 2000). Combined with other evidence studying the effect of GAGs on aggrecanase, it seems increasingly likely that the keratan sulphate present in the IGD of aggrecan has a functional role in cleavage by aggrecanases.
It is the aim of this project to examine the keratan sulphate of the interglobular domain and to examine its role in aggrecanase-mediated cleavage. The single greatest problem facing those studying keratan sulphate is that cells grown in vitro using standard tissue culture techniques quickly stop synthesising keratan sulphate. To facilitate the study of cleavage at the aggrecanase site, a Vaccinia virus T7 RNA polymerase expression system modified to express the human aggrecan G1-G2 domain was used to produce recombinant G1-G2 modified with keratan sulphate.
66 Chapter I: Introduction
One of the major tasks of this project was identifying an appropriate type of cell as well as optimal growth conditions capable of sustaining keratan sulphate synthesis long enough for G1-G2 synthesis using the Vaccinia T7 RNA polymerase expression system. Once identified, the Vaccinia expression system had to be optimised for expressing chemical amounts of recombinant G1-G2. The next major task was establishing a purification protocol and once purified, fluorophore-assisted carbohydrate electrophoresis was used to characterise the microstructure of the keratan sulphate present on the purified G1-G2. Finally aggrecanase cleavage studies were carried out on the keratan sulphate-substituted G1-G2.
67 Chapter II: Materials & Methods
Chapter II: Materials & Methods
“It is common sense to take a method and try it. If it fails, admit it frankly and try another. But above all, try something.” -Franklin D. Roosevelt (1882 - 1945)
68 Chapter II: Materials & Methods
Materials
Product Supplier 1,9-dimethylmethylene blue chloride SERVA, Germany 2-aminoacridone Molecular Probes, USA 3’-Sialyl-Lewis-a tetrasaccharide Sigma, Australia 35S-methionine Amersham, Australia 35 [ S]SO4 Amersham, Australia α(1-3,4) Fucosidase Prozyme, USA AEBSF Merck, Australia Ammonium acetate Merck, Australia Anti-mouse HRP Dako Corporation, Denmark Anti-rabbit HRP Dako Corporation, Denmark Benzamidine-HCl Sigma, Australia Bio-Gel P-10 BioRad, USA Biosep-SEC S4000, 300 x 7.8 mm Phenomenex, USA analytical column Cell line CCL-60 ATCC: CCL-60 Cell line COS-7 ATCC: CRL-1651 Cell line CRL-2048 ATCC: CRL-2048 Cell line HT-1080 ATCC: CCL-121 Cell line SK-N-MC ATCC: HTB-10 Chondroitinase ABC (protease free) MP Biomedicals, Australia DMEM (cysteine, methionine-free) Invitrogen, Australia DMEM/Ham’s F12 (50:50) Thermo Trace, Australia E-64 Roche Diagnostics Australia EAH-Sepharose Pharmacia, Australia ECL Plus Amersham, Australia EDTA Merck, Australia Emulsifier Safe Packard, Australia Endo-β-galactosidase Seikagaku, Japan Fibroblast growth factor - 2 Sigma, Australia Foetal calf serum HyClone, USA Hyaluronan Sigma, Australia Interleukin-1α Genzyme Diagnostics, USA Keratanase I Seikagaku, Japan Keratanase II Seikagaku, Japan Microcon 3,000 & 30,000 MWCO spin Millipore, Australia columns N-caproic acid Merck, Australia N-ethyl-N´-(3-dimethylaminopropyl) Sigma, Australia carbodi-imide hydrochloride Neuraminidase Sigma, Australia
69 Chapter II: Materials & Methods
N-glycosidase F Roche Diagnostics Australia Nitrocellulose Osmonics, USA NP 40 Sigma, Australia Pepstatin Roche Diagnostics Australia PPO Sigma, Australia Prestained precision standards BioRad, USA Retinoic acid ICN Biochemicals, USA Silver nitrate Merck, Australia X-ray film Kodak, Australia
70 Chapter II: Materials & Methods
Methods
Keratocyte preparation
Corneas were removed from the eyes of freshly slaughtered bovine steers and rinsed in PBS containing 100 U/ml penicillin and 100μg/ml streptomycin, then incubated in a solution of 0.25% w/v trypsin and 0.5mM EDTA for 20 min at 37°C. Endothelial and epithelial cell layers were removed by gentle scraping and after further rinsing in PBS, the tissue was cut into small pieces and digested in DMEM/Ham’s F12 (50:50) containing 2 mg/ml collagenase II and antibiotics at 37ºC. After 16 hours, the digest was passed through a 70µm cell strainer to remove undigested tissue debris. The cells were collected by centrifugation, washed once with DMEM/F12 containing 10% FCS, then washed twice in fresh DMEM/F12 media. The cells were counted and seeded at a density of 1.11 x 105 / cm2 in DMEM/F12 medium containing 2% FCS and antibiotics.
Chicken keratocytes were prepared using a modified method. The corneas of 14 day old chicken embryos were peeled off and the scleral rims were removed. After rinsing in PBS containing 100 U/ml penicillin and 100μg/ml streptomycin, the corneas were given a 30 min collagenase treatment using DMEM/F12 containing antibiotics and 2 mg/mL collagenase II, to remove the epithelial and endothelial cell layers. The media was then replaced with fresh collagenase and left shaking overnight at 37°C. After 16 hours, the digested tissue was then treated as described above.
Generation of vG1-G2
A construct for producing Vaccinia virus expressing G1-G2 (vG1-G2) was made in-house by Dr Amanda Fosang by subcloning cDNA encoding a human G1-G2 fragment of aggrecan (Mercuri et al. 1999) into the pTM1 vector (McQuillan et al. 2001) at the SacI and XhoI cloning sites. The G1-G2 insert was repositioned in the vector so that the initiating ATG codon was part of the NcoI cloning site in the pTM1 polylinker. A forward primer Agg96-2 (5’-
71 Chapter II: Materials & Methods
AAACACGATAATACCATGACCACTTTACTCTGG-3’) which includes a 5’ sequence complementary to a region surrounding the NcoI site in the polylinker (italics), a 3’ sequence complementary to the G1-G2 insert (bold) and an ATG codon (underlined) common to both, was used in a PCR reaction with reverse primer Agg96-1 (5’-GAGATGGCTCTGTAATGGAA-3’) to amplify a 500bp fragment. This fragment contained a 15bp 5’ overhang that extended into the G1-G2 insert, beyond a unique NsiI site. In a separate PCR reaction, forward primer pTM1SL.1 (5’-TATAAGATACACCTGCAAAG-3’) and reverse primer pTM1SL.2 (5’-CATGGTATTATCGTGTTTTT-3’) were used to amplify a 257bp region of the pT-cam1 polylinker immediately adjacent to, and terminating at the ATG codon of the NcoI site. This 257bp fragment contained a central KpnI site. Splicing by overlap extension PCR was used to generate a 760bp fragment from the 500bp and 257bp templates with the pTM1SL.1 and Agg96-1 primer pairs. The 760bp fragment was digested with KpnI and NsiI then ligated into the KpnI and NsiI restriction sites of the pT-cam1-G1-G2 plasmid, in place of a 560bp cassette. The construct was sent to LifeCell, USA where it was sequenced then used to produce a recombinant Vaccinia virus encoding human G1-G2 under control of the T7 Promoter as previously described (McQuillan et al. 2001).
Vaccinia preparation
Correct preparation of Vaccinia is vital for efficient infection of cells. During storage, virus particles form aggregates, reducing the available plaque- forming units. It is therefore necessary to break up these aggregates prior to infection. Vaccinia TF7-3 and G1-G2 viral stocks were thawed at 37°C and sonicated twice for 30 sec, with chilling on ice for 2 min between each sonication. An aliquot each of vTF7-3 and vG1-G2 were combined with an equal volume of 0.25% w/v trypsin, 0.5mM EDTA and incubated at 37°C for 20 min with vortexing every 5 min. DMEM/F12 was used to dilute each viral stock if it was required.
72 Chapter II: Materials & Methods
Expression of B5R-green fluorescent protein
A Vaccinia virus expressing a GFP-labelled membrane protein B5R (Ward et al. 2001; Ward et al. 2003), was used to determine the degree of infection and also the level of recombinant protein expression. Primary keratocytes were isolated from fresh bovine corneas and seeded into 2 chambered slides with 300,000 cells in DMEM/F12 (50:50) containing 2% FCS and antibiotics. The cells were incubated overnight at 37°C, then infected with Vaccinia B5R-GFP at a concentrations up to 100 pfu/cell. After 6 hours the media was replaced with fresh DMEM/F12 (50:50) containing antibiotics and incubated for a further 18 hours. Fluorescence was imaged with a Leica Leitz Diaplan camera at 200x magnification.
Generation of recombinant G1-G2
Cells seeded in DMEM/F12 medium containing 2% FCS and antibiotics at a density of 1.11 x 105 / cm2 were incubated at 37ºC for up to 5 hours or until the cells had attached. The media was replaced with 55.5μL/cm2 DMEM/F12 containing 2% FCS and antibiotics and flasks were inoculated with 5 pfu/cell of both vTF7-3 and vG1-G2. The cells were incubated for 6 hours at 37ºC and after this time, the media was replaced with 111 μL/cm2 of fresh DMEM/F12 containing antibiotics. The cells were then allowed to incubate for a further 4 days before the media was harvested.
Expression of 35S-methionine labelled G1-G2
The expression of newly synthesised rG1-G2 was monitored by the addition of 35S-methionine to culture medium. Confluent cell monolayers cultured in DMEM/F12 with 2% FCS and antibiotics were infected with vTF7-3 alone (control), or co-infected with vTF7-3 and vG1-G2, for 6 hours at 37°C. The media was replaced with DMEM deficient in methionine and cysteine. 35S- labelled methionine (10 μCi/ml) was added to the wells and the infection continued at 37°C for various times up to 4 days. Aliquots of media were precipitated overnight at -20°C with 3 volumes of 96% ethanol, 100 mM ammonium acetate and the precipitate recovered by centrifugation at 14,000 x
73 Chapter II: Materials & Methods
g for 15 min. The pelleted, radiolabelled samples were analysed by non- reducing SDS-PAGE and fluorography.
For corneal explant infections, corneas were either scratched with a needle or cut into quarters prior to inoculating the corneas with 5 pfu/cell based on the average number of keratocytes per cornea. Cultures were allowed to incubate for 5 days at 37°C in medium supplemented with 2% FCS and 35S- methionine. Cultures incubated at room temperature or at 4°C contained half the normal concentration of bicarbonate to maintain the correct pH. After 5 days, media was collected, and the corneal explants were extracted with 10 volumes of 4M GuHCl for 3 days at 4°C. Both the collected media and the corneal extracts were precipitated with 3 volumes of chilled ethanol for at least 1 hour at -20°C. The precipitate was collected by centrifugation at 14,000 x g and dissolved in sample buffer prior to analysis by SDS-PAGE.
Fluorography
Fluorography is used to help visualise radiolabelled proteins resolved on a SDS-PAGE gel. Particles emitted by the labelled proteins excite the 2,5- Diphenyloxazole (PPO) present in the gel and visible light is emitted, which is captured on X-Ray film. 35S-methionine-labelled proteins were resolved by SDS-PAGE and the gel was fixed at 10°C for an hour in a solution of 45% methanol, 10% acetic acid. The gel was rinsed briefly in water and given 2 x 30 min washes in dimethyl sulfoxide (DMSO), followed by up to 3 hours in 22.2% (w/v) PPO, then rinsed in water. After 30 min the gel was dried on filter paper and exposed to X-ray film at –70°C for up to one week.
Analysis of recombinant and native G1-G2 by rotary shadowing microscopy
Recombinant or native G1-G2 was purified and shipped to Dr. Doug Keene, Shriners Hospital for Children, Portland, USA, for analysis by rotary shadowing as described previously (Mercuri et al. 1999). Briefly, the proteins were dialysed against 0.1M ammonium bicarbonate. Each sample (100μg/ml)
74 Chapter II: Materials & Methods
was mixed with glycerol to a final concentration of 70% glycerol (v/v) and nebulized with an airbrush onto freshly cleaved mica. The 100μl sample was dried in vacuum and rotary shadowed with carbon/platinum using an electron beam gun at an angle of 6° relative to the mica surface in a Balzers BAE 250 evaporator. The replica was then backed with carbon at 90°. The replica was floated onto distilled water and picked up onto bare 600-mesh copper grids. Photomicrographs were taken using a Phillips 410 electron microscope operated at 80KV using a 30 micron objective aperture.
Coupling of hyaluronan to EAH-sepharose
Like all the hyalectans, aggrecan binds to hyaluronan via the G1 domain at its N-terminus. This characteristic is exploited in hyaluronan-sepharose chromatography and allows aggrecan and related proteins to be quickly purified from tissue extracts or in this case, culture medium. Hyaluronan was partially cleaved by dissolving 100 mg umbilical cord hyaluronan in a 10 mL solution of 0.2M sodium acetate pH 6.8, 1mM sodium ascorbate, 1μM copper sulphate. The solution was left rolling at room temperature overnight and then dialysed against distilled, deionised water. After dialysis, the solution was freeze-dried then combined with ~7.5 mL EAH-sepharose, then made up to a final volume of 20 mL with distilled, deionised water pH 4.5. This solution was thoroughly mixed before adding 140 mg N-ethyl-N’-(3-dimethyl aminopropyl)- carbodiimide hydrochloride pH 5.3 and left rolling slowly overnight at room temperature. The coupling reaction was stopped by adding 1 mL acetic acid then left rolling for a further 6 hours at room temperature. Finally the HA- sepharose was washed with 100 mL each of: 1M sodium chloride; 1M sodium chloride, 0.1M Tris-hydrochloride pH 8.1; 50 mM sodium formate pH 3.1; and distilled, deionised water. The HA-sepharose was stored in 0.5M sodium acetate pH 5.8, 0.05% azide at 4°C.
HA-sepharose affinity chromatography
Harvested media was centrifuged briefly at 3000 x g to remove cellular debris and passed through a 2 mL HA-sepharose column equilibrated in PBS. The
75 Chapter II: Materials & Methods
column was then washed with 5 bed volumes of PBS and protein that remained bound to the column was eluted with 4M GuHCl, 50mM sodium acetate pH 5.8. In some cases, PBS washes were followed by additional washes of 5 bed volumes each of 0.5 M NaCl and 1 M NaCl prior to eluting with guanidine-hydrochloride. Collected fractions were desalted with 30 kDa molecular weight cut-off spin columns before analysis by SDS-PAGE.
High Performance Liquid Chromatography
High performance liquid chromatography was used to further purify rG1-G2. The sample was applied directly to a BioSep-Sec-S 4000 size exclusion HPLC column, and eluted under dissociative conditions into tubes containing protease inhibitors (to a final concentration of 10mM EDTA, 5mM benzamidine-HCl, 1μM pepstatin, 5μg/ml E-64 and 0.1M caproic acid). Fractions containing G1-G2 were identified by Western blotting with 1C6 and 5D4 monoclonal antibodies. Fractions containing 1C6 immunoreactivity were pooled and reapplied to the same column. Fractions containing high molecular weight 5D4 immunoreactivity co-migrating with 1C6 immunoreactivity were pooled and desalted on Millipore spin columns for further analysis. Protein concentration in the pooled fractions was estimated by spectroscopy at 278nm and the concentration of sulphated glycosaminoglycan, in this case exclusively keratan sulphate, was estimated by the 1,9- dimethylmethylene blue dye-binding assay (Farndale et al. 1982).
Glycosaminoglycan analysis
35 Sample preparation for [ S]SO4-labelled glycosaminoglycans CCL-60, CRL-2048, and SK-N-MC cells were routinely cultured in DMEM with 10% FCS. The cells were seeded in 80 cm2 flasks at a density to give confluent monolayers. Cells were counted so that each of the three cell lines was seeded at approximately equal numbers. After 5 hours of incubation at 35 37°C, the media was replaced with DMEM containing [ S]SO4 (67μCi/mL) and 10% FCS, and then allowed to incubate overnight at 37°C. After
76 Chapter II: Materials & Methods
35 approximately 16 hours of [ S]SO4 labelling, the culture medium and cells were collected and papain-digested to generate individual glycosaminoglycan chains. The digest was centrifuged at 3000 rpm and the supernatant collected. The sample was precipitated in 4 volumes of chilled ethanol overnight at 4°C, centrifuged for 15 min at 3000 rpm. The pellets were dried and then suspended in an appropriate buffer.
35 [ S]SO4 labelling of primary cultures: FGF-2 and serum dosage effect
Primary keratocytes were cultured with DMEM/F12 in 6-well plates containing an appropriate concentration of FCS and FGF-2. The cultures were initially seeded in 4 mL media per well. After 24 hours of incubation at 37°C, the 35 media was changed and replaced with 3 mL fresh media containing [ S]SO4 to a concentration of 55 μCi/mL and fresh FCS and FGF-2. The cultures were allowed to incubate at 37°C for a further 3 days and the media was collected and precipitated with 4 volumes of chilled ethanol. The remaining cells were trypsinised and counted. After 24 hours, the precipitate was papain-digested (100 μg/mL) at 60°C overnight. After digestion, each of the six samples was centrifuged at 14,000 rpm for 5 min, and the supernatant was freeze-dried and suspended in an appropriate buffer.
Papain digestion
A 5x papain digestion buffer was prepared containing: 50 mM EDTA, 50 mM cysteine, and 1 M sodium acetate pH 5.8. Cells were scraped in their accompanying culture medium and 5 x papain digestion buffer was added to the collected cell scraping / tissue culture medium to make it the concentration equivalent to 1x. Papain was then added to a total concentration of 100 μg / mL. The sample was then incubated at 60ºC for up to 24 hours.
77 Chapter II: Materials & Methods
BioGel P-10 size exclusion chromatography
35 [ S]SO4-labelled proteoglycans were analysed for their chondroitin sulphate and keratan sulphate content by gel filtration chromatography. Samples were dissolved in 500 μL 0.5M ammonium acetate pH6.8 and applied to a 100 mL column of BioGel P-10 (exclusion limit 20,000 Da) equilibrated in 0.5M ammonium acetate pH 6.8. Aliquots of each fraction were analysed by scintillation counting and the void peak was collected and freeze-dried. The dried void samples were split into 2 or 3 equal fractions and digested with either keratanase I or chondroitinase ABC, or treated with nitrous acid to depolymerise heparan sulphate. Each digest was then reapplied to the column and the proportion of keratan sulphate to chondroitin sulphate was determined with scintillation counting.
Cell proliferation in response to FGF-2 and FCS
FGF-2 and FCS concentration was examined for their effect on cellular proliferation. Nine hundred thousand cells were suspended in 24mL DMEM:F12 (50:50), giving a concentration of approximately 37,500 cells/mL. 2 mL of this cell suspension was pipetted into 12 wells (6-well plates). FCS and FGF-2 was added to each well to the appropriate concentration. The cultures were incubated at 37°C for 5 days, and the media was collected from each well. The remaining cells from each of the 12 wells were trypsinised and counted. The collected media was precipitated in 3 volumes of cold ethanol and kept at 4°C for 24 hours. The precipitate was then papain-digested (100 μg/mL) for 24 hours at 60°C before Western analysis for keratan sulphate.
Antibodies used for immunoblotting
Monoclonal antibodies 1C6 (Caterson et al. 1986), 3H1 (Rauch et al. 1991), and I22 (Funderburgh et al. 1982) were from the Developmental Studies Hybridoma Bank established under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The antibody MZ15 was a gift from the Kennedy Institute of Rheumatology, London, UK. The monoclonal antibody, 5D4, was a gift from
78 Chapter II: Materials & Methods
Prof. Bruce Caterson, University of Wales, Cardiff. The anti-NITEGE antibody was generated in-house as previously described (Mercuri et al. 1999).
A rabbit polyclonal antibody, called anti-IGD, raised against a synthetic peptide acetyl-CPDMELPLPRNITEG-amide by Quality Controlled Biochemicals Inc, (MA, USA). The peptide sequence was chosen to elicit an antibody that would recognise the interglobular domain, but not the NITEGE neoepitope. The antibody reacted well with human antigen by Western blotting and ELISA assay, but did not recognise the pig IGD which has a valine residue at 361, in place of methionine.
Antibodies were used at the following concentrations/dilutions:
1C6 1:500 5D4 1:10,000 I22 1:500 3H1 1:500 MZ15 1:500 anti-IGD 10 μg/mL anti-NITEGE 10 μg/mL anti-mouse HRP 1:10,000 anti-rabbit HRP 1:10,000
Dot blotting Dot blotting was used to quickly probe fractions for either keratan sulphate (5D4) or G1-G2 (1C6). Prepared samples were vacuum-immobilised onto nitrocellulose, and the membrane was blocked in 5% skim milk (prepared in PBS) for 1 hour. The membrane was then probed with an appropriate antibody in a solution of 0.5% skim milk for up to 3 hours, or alternatively overnight at 4°C. Membranes were washed for 30 min with PBS containing 0.1% TWEEN 20. Then membranes were probed with an appropriate
79 Chapter II: Materials & Methods
secondary antibody for 1 hour in 0.5% skim milk, before developing with ECL Plus.
Western blotting
Samples for Western blotting were made up in sample buffer using a 5 x stock solution consisting of: 62.5 mM Tris-HCl, pH 6.8; 10% glycerol; 2% SDS; 0.00125% bromophenol blue (w/v). DTT to a concentration of 20 mM was also included if necessary to reduce antigens. Samples were boiled for 5 min before resolving on a 7.5% polyacrylamide gel. The samples were then transferred to PVDF membrane overnight at 100 mA in a buffer of 25 mM Tris-HCl, 192 mM glycine, 20% methanol. Membranes were probed as described above for dot blotting.
Glycosidase digests
Glycosidase digests were routinely carried out as follows. Up to 25 μg of glycosaminoglycan was incubated for at least 16 hours at 37°C in digestion buffer (0.1 M ammonium acetate, 0.5 mM AEBSF, 3.4 μg/mL pepstatin, 20 μg/mL E-64, 5 mM benzamidine) with either 5 - 10 mU keratanase I (pH 7.4), 0.5 - 1 mU keratanase II (pH 6), 0.5 - 1 mU endo-β-galactosidase (pH 6), 10 mU chondroitinase ABC (pH 8), 0.1 mU fucosidase (pH 5), or 10 mU neuraminidase (pH 6). For N-glycosidase digests, samples were dissolved in 125 mM Tris, pH 6, 1% SDS, 0.6% NP-40 and boiled for 5 min. After cooling on ice, 1 U N-glycosidase F was added and the digest incubated for up to 24 hours at 37°C.
Nitrous acid treatment
Nitrous acid treatment selectively cleaves heparan sulphate. The freeze-dried samples were dissolved in a buffer of 75 μL 0.4M GuHCl / 5 mM sodium
acetate pH 6.5, 50 μL 2.5 M sulphuric acid, 312.5 μL 5.5 M NaNO2. After 10
min at room temperature, the solution was neutralised with 75 μL 1M Na2CO3.
80 Chapter II: Materials & Methods
Preparation of AMAC-labelled carbohydrates
2-Aminoacridone, Hydrochloride (AMAC) was prepared by dissolving 25 mg in a solution of 850 μL DMSO, 150 μL glacial acetic acid. Aliquots were stored at –70°C until required. Between 4 – 8 μg of glycosidase-digested keratan sulphate was passed through Microcon YM-3 spin columns (Molecular weight cutoff of 3 kDa) and the recovered saccharides were freeze-dried and dissolved in 10 μL AMAC. After 15 min at room temperature, 10 μL of freshly prepared 1 M sodium cyanoborohydride was added. The samples were then allowed to incubate overnight at 37°C before the addition of 20 μL 37.5% glycerol.
Carbohydrate electrophoresis
The FACE gels used for carbohydrate analysis were either purchased from Glyko (later ProZyme) or prepared fresh with 20% polyacrylamide resolving gels prepared in 45 mM tris-acetate pH 7, 2.5% glycerol, and an 8% polyacrylamide stacking gel prepared in 45 mM tris-acetate pH 7, 4.4% PEG 8000. A tris-borate-EDTA running buffer (89 mM Tris, 89mM borate, 2.4 mM EDTA) was chilled on ice before use. Up to 6 μL of AMAC-labelled keratan sulphate was analysed per well along with a set of monosaccharide standards made up of a solution of fucose, galactose, N-acetyl glucosamine, galactose-6 sulphate, and N-acetyl glucocosamine-6 sulphate, at concentrations of 0.05, 0.1, 0.15, 0.2, and 0.25 nmol / μL. respectively. No more than 0.5 nmol carbohydrate were analysed at any time. Gels were run at 500 V for up to 90 min. Gel images were captured using either a Storm phosphorimager or a BioRad ChemiDoc system.
Cartilage explant cultures
Conditioned medium was harvested from explants of pig articular cartilage (2.75 mg) cultured for 4 days in the presence of 1 µM retinoic acid and 10 ng/mL IL-1α in serum-free DMEM as described previously (Fosang et al. 2000). The medium was concentrated 9-fold on Millipore 10 kDa MWCO centrifugal filter units, and used as a source of aggrecanase activity.
81 Chapter II: Materials & Methods
Aggrecanase digests
rG1-G2 (5 µg) was treated with or without glycosidases, then digested with 1 µL of concentrated aggrecanase-containing conditioned medium at 37°C for times as given, in buffer containing 50 mM Tris-HCl, 100 mM NaCl, 10 mM
CaCl2, 0.5 mM AEBSF, 3.4 μg/mL pepstatin, 20 μg/mL E-64, 5 mM benzamidine, pH 7.5. Following digestion, aggrecanase activity was inhibited by the addition of 10 mM EDTA and 2 mM 1,10-phenanthroline and the samples analysed by SDS-PAGE and Western blotting. The anti-NITEGE polyclonal neoepitope antibody was used to detect the G1 fragment derived from aggrecanase cleavage.
Silver stain
After electrophoresis, 7.5% Laemmli gels were fixed for at least 30 min in a solution of 30% ethanol, 10% acetic acid, followed by 30 min or more in 10% ethanol. After fixing, gels were rinsed thoroughly in distilled, deionised water for 30 min, then reduced for 20 min at room temperature in a solution of 5μg/mL DTT. The solution was then replaced with 0.05% silver nitrate and incubated at room temperature for 20 min. The gel was then rinsed briefly in distilled, deionised water before developing in a solution of 3% sodium carbonate, 0.02% formaldehyde; 5 mL of 2.3M citric acid was added to stop the reaction. After 30 min, the citric acid was removed and replaced with 10% glycerol and allowed to incubate at room temperature for at least 30 min before the gel was scanned and dried.
82 Chapter III: In search of a good host
Chapter III: In search of a good host
“Basic research is what I am doing when I don't know what I am doing.” - Wernher von Braun (1912 - 1977)
83 Chapter III: In search of a good host
Introduction
Proteoglycans play a major role in tissue hydration and load-bearing. The structures responsible for this activity are carbohydrate side-chains known as glycosaminoglycans. The major proteoglycan of cartilage is aggrecan, which has both chondroitin sulphate and keratan sulphate substituted along its core protein. Keratan sulphate is found in varying quantities throughout the body, but it is concentrated in the cornea, the brain, and cartilage in the form of keratan sulphate proteoglycans. In cartilage, the primary source of keratan sulphate is aggrecan, though proportionally, the degree of chondroitin sulphate substitution on the same protein is far greater. The degree of keratan sulphate substitution within the keratan sulphate-rich region on aggrecan varies according to the number of hexapeptide repeats, and the number of hexapeptide repeats varies markedly between species. The varying number of keratan sulphate chains does not appear to affect the biological function of aggrecan. Therefore, the keratan sulphate of the KS-rich region, which makes up the majority of the total aggrecan keratan sulphate, does not have any obvious biological role.
Analysis of the HA-binding region and the flanking interglobular domain of bovine aggrecan has revealed that as the animal matures, the IGD is modified with keratan sulphate (Barry et al. 1995). Initial reports showed that completely deglycosylated aggrecan was more susceptible to aggrecanase cleavage (Pratta et al. 1997; Pratta et al. 1999). Subsequent studies demonstrated an age-related effect of keratan sulphate on aggrecanase cleavage, in which the more keratan sulphate-modified aggrecan is more susceptible to aggrecanase cleavage than the less keratan sulphate-modified immature cartilage aggrecan (Pratta et al. 2000). In another study, the addition of highly sulphated glycosaminoglycans inhibited aggrecanase- mediated cleavage of aggrecan in bovine articular cartilage explant cultures (Munteanu et al. 2002). What has not been demonstrated yet is which region of keratan sulphate-containing aggrecan is responsible for this effect on aggrecanase. However, it is likely that if there is a cleavage-enhancing property imparted on aggrecanase by keratan sulphate, it is the keratan
84 Chapter III: In search of a good host
sulphate that is present within the interglobular domain. It is the aim of this series of studies to further examine the role of keratan sulphate within the interglobular domain and determine its role in aggrecanase-mediated cleavage.
In order to show this, different glycoforms of G1-G2 need to be compared for their susceptibility to aggrecanase digestion. By comparing three varieties of G1-G2: a baculovirus-expressed unglycosylated G1-G2 (Mercuri et al. 1999; Mercuri et al. 2000), a recombinant G1-G2 substrate with intermediate amounts of keratan sulphate, and the native fully glycosylated G1-G2 extracted from pig, it may be possible to determine the effect of keratan sulphate on aggrecanase-mediated cleavage. From the available literature exploring the role of glycosylation on aggrecanase, the keratan sulphate of the interglobular domain is expected to enhance aggrecanase cleavage (Pratta et al. 2000). It is the aim of my work to assign a biological function to the keratan sulphate of aggrecan’s interglobular domain. Two of the three glycoforms are available; the baculovirus-expressed G1-G2 created in insect cells is completely unglycosylated and has a core protein size approaching 100 kDa. Its susceptibility to MMP and aggrecanase digestion has been demonstrated to be similar to the native substrate (Mercuri et al. 1999; Mercuri et al. 2000). The native G1-G2 was purified from a trypsin-treated pig cartilage extract; the trypsin digestion cleaves aggrecan immediately downstream of the G2 domain. The resulting fragment contains the entire G1- G2 domain and is highly substituted with up to 50 kDa of keratan sulphate. However, a G1-G2 substrate with an intermediate amount of keratan sulphate is unavailable. As discussed previously, a Vaccinia virus T7 RNA polymerase expression system was chosen as the method used to produce recombinant G1-G2 because of its high efficiency and the freedom to use a wide variety of mammalian cells.
The first step in expressing recombinant G1-G2 was choosing an appropriate cellular host for the Vaccinia expression system, one capable of sustained keratan sulphate synthesis. This chapter is presented in two parts. The first part describes the initial screening of three cell lines for their basal levels of
85 Chapter III: In search of a good host
keratan sulphate synthesis. The second part describes the effect of basic fibroblast growth factor (FGF-2) on glycosaminoglycan synthesis by primary bovine keratocytes as well as a representative cell line, CCL-60.
Although it has been described in the literature previously that cell lines synthesise little or no keratan sulphate, exact amounts are difficult to gauge. 35 Three cell lines were screened using [ S]SO4-labelling to determine the proportion of keratan sulphate to chondroitin sulphate synthesised in culture. SK-N-MC, CCL-60, and CRL-2048 were chosen as candidate hosts. SK-N- MC (Biedler et al. 1973) is a cell line that was originally established in September 1971 from the supra-orbital area of a neuroepithelioma of a 14 year old girl (ATCC). Having a neurogenic origin, SK-N-MC was considered a good candidate for a neural, KS-producing cell line. The remaining two are corneal cell lines. CCL-60 (Leerhoy 1965; Phillips et al. 1966; Rhim et al. 1967; Farris et al. 1999) was established from rabbit corneas, whereas CRL- 2048 (Gospodarowicz et al. 1977; Bethea et al. 1984) was established from the explants of adult bovine corneas. Because the cornea has the highest concentration of keratan sulphate anywhere in the body, both of the corneal cell lines were also considered good candidate hosts.
35 Each candidate cell line was screened using [ S]SO4-labelling, which, when added to culture medium, is incorporated by the cells into newly synthesised glycosaminoglycans. Relative proportions of keratan sulphate to chondroitin sulphate can be determined by tracking the degree of digestion by either keratanase or chondroitinase using size exclusion chromatography.
Keratan sulphate production is reduced significantly in cells once they are cultured. While the specific mechanism for this change in synthesis is yet to be identified, it has been suggested that this reduction in keratan sulphate synthesis is linked to cellular morphological changes to a fibroblast-like appearance, mimicking conditions observed during wound healing in which keratan sulphate production is temporarily halted. Current data suggests that this down regulation of keratan sulphate proteoglycans occurs at both the protein level as well as the glycosylation level (Long et al. 2000). This same
86 Chapter III: In search of a good host phenomenon occurs in vitro when cells are cultured under standard tissue culture conditions, namely, being cultured in medium containing foetal bovine serum and subculturing by trypsinisation (Fini et al. 1990). Keratan sulphate expression by keratocytes in vitro has been closely linked to the stroma; keratocytes that are removed from the stroma rapidly lose the ability to secrete keratan sulphate, and what little keratan sulphate that is secreted, is truncated and undersulphated (Long et al. 2000).
The cornea is perhaps most reliant on proper keratan sulphate synthesis and expression because of its role in maintaining corneal transparency. Three keratan sulphate proteoglycans are expressed in the cornea: lumican, keratocan, and mimecan. Mice homozygous for a null mutation affecting lumican develop bilateral corneal opacification as a result of disruption of collagen fibril morphology. However, mice lacking a functional decorin gene, the corneal chondroitin sulphate and dermatan sulphate proteoglycan, had unaffected corneas (Danielson et al. 1997; Chakravarti et al. 1998). It is therefore no surprise that efforts have been made to study the expression of keratan sulphate in corneas, primarily in bovine and chicken corneas. Culturing cells in the presence of low concentrations of serum have been demonstrated to maintain keratan sulphate expression levels for longer than when they are cultured in the presence of 10% serum (Beales et al. 1999). In previous studies, a low serum concentration in culture medium was found to result in higher keratan sulphate synthesis. Recently, Long et al. hypothesised that mitogens may have a positive effect on KS synthesis. A range of mitogens were tested, and basic fibroblast growth factor-2 (FGF-2) was found to significantly increase KS synthesis but this effect was observed only in primary cultures of cells. Keratan sulphate concentrations were initially maintained at levels equivalent to those found in vivo, however, after a period of 7 days in culture, a substantial decrease in keratan sulphate production was observed. Under standard conditions, Nakazawa et al. have shown that keratan sulphate expression is greatly reduced in chick keratocytes after being cultured for seven days with serum (Nakazawa et al. 1998). Recent studies have found that the addition of FGF-2 to culture medium helped to maintain the keratocyte morphology as well as maintain keratan sulphate
87 Chapter III: In search of a good host expression levels for longer periods than usual (Jester et al. 1996; Nakazawa et al. 1996; Long et al. 2000).
The second part of this chapter investigates cell culture conditions which would maintain high levels of keratan sulphate. Basic fibroblast growth factor– 2 (FGF-2) and low concentrations of foetal calf serum was added to primary bovine keratocyte cultures as well as a representative cell line, CCL-60, (introduced earlier in this chapter) and KS production was observed.
88 Chapter III: In search of a good host
Results – Part I
CCL-60
35 The rabbit corneal cell line, CCL-60, was [ S]SO4-labelled for 16 hours. Following complete protein digestion of the collected sample by papain, the remaining sample containing both the labelled glycosaminoglycans and the 35 free, unincorporated [ S]SO4 was applied to a column of BioGel P10 for size exclusion chromatography. The gel filtration resulted in the separation of the
glycosaminoglycans (eluting as a void peak, Vo, at approximately fraction 5) 35 from the free [ S]SO4 (eluting at fraction 30, Vt). The proportion of keratan
sulphate present at Vo was determined by keratanase I treatment followed by size exclusion chromatography (Figure 18). The lack of any detectable 35 [ S]SO4 at the Vt peak at fraction 6 in figure 18 demonstrates the lack of keratan sulphate synthesis by this cell line.
In contrast to the keratanase I digestion, chondroitinase ABC digestion
resulted in almost a complete shift of the Vo peak (figure 18) from fraction 7 to fraction 39, indicating that the glycosaminoglycan content of CCL-60 is composed almost entirely of chondroitin / dermatan sulphate. The remaining chondroitinase ABC-resistant peak representing less than 5% of the total GAG content, remained undigested and was therefore assumed to be an alternative GAG, possibly heparan sulphate. To confirm whether this was true, the labelled GAGs were treated with nitrous acid to determine whether any heparan sulphate had been synthesised and secreted by the CCL-60 cells. The results show that nitrous acid treatment failed to generate smaller GAG
fragments (figure 18). The lack of conversion of any portion of the Vo peak to
Vt by nitrous acid treatment indicates that CCL-60 cells are unable to synthesis heparan sulphate in cell culture. The results shown in figure 18 show that the glycosaminoglycans synthesised by this cell line are exclusively chondroitin / dermatan sulphate and that they do not synthesise significant amounts of either keratan sulphate or heparan sulphate.
89 Chapter III: In search of a good host
CCL-60: Keratanase I
Vo Vt 30000 29 mL 25000 20000 15000 cpm 10000 5000 0 1 4 7 101316192225283134374043464952 Fraction
CCL-60: Chondroitinase ABC
20000 78.5 mL 18000 16000 14000 12000 10000 cpm 8000 6000 4000 30.5mL 2000 0 1 4 7 101316192225283134374043464952 Fraction
CCL-60: Nitrous Acid
25000 27.5 mL 20000
15000
cpm 10000
5000
0 1 4 7 101316192225283134374043464952 Fraction
Figure 18: Glycosaminoglycan composition of proteoglycans secreted by CCL-60 cells in culture
90 Chapter III: In search of a good host
CRL-2048
The bovine corneal cell line, CRL-2048 was also examined for glycosaminoglycan synthesis. Samples were prepared as described
previously. As shown (figure 19), the Vo peak at fraction 7 was completely resistant to digestion by keratanase I, indicating a complete lack of keratan
sulphate being synthesised by CRL-2048 cells, however when the Vo was 35 treated with chondroitinase ABC all the [ S]SO4 was shifted from the Vo to the Vt. This was identical to the results seen for the CCL-60 cell line - the bovine corneal cell line, CRL-2048, synthesised insignificant levels of keratan sulphate. The only glycosaminoglycan produced by the CRL-2048 cell line was chondroitin / dermatan sulphate.
CRL-2048: Keratanase I
V V 5000 o t 29 mL 4000 3000
cpm 2000 1000 0 1 4 7 101316192225283134374043464952 Fraction
CRL-2048: Chondroitinase ABC
4000 75.5 mL 3500 3000 2500 2000 cpm 1500 1000 500 0 1 4 7 101316192225283134374043464952 Fraction
Figure 19: Glycosaminoglycan composition of proteoglycans secreted by CRL-2048 cells in culture
91 Chapter III: In search of a good host
SK-N-MC
Having a neurogenic origin, the SK-N-MC cell line is from tissue that is naturally rich in both chondroitin sulphate as well as keratan sulphate, though 35 not to the same degree as either the cornea or cartilage. [ S]SO4-labelled glycosaminoglycans were prepared as described previously for the corneal
cell lines. The Vo was collected and digested with keratanase I or chondroitinase ABC (figure 20). Gel filtration of the keratanase-digested
sample resulted in no observable Vt peak, indicating a lack of keratan sulphate in the glycosaminoglycan pool. However, chondroitinase ABC digestion of the Vo sample resulted in a complete conversion of the Vo peak to
Vt. The results show that in the neurogenic cell line, SK-N-MC, keratan sulphate synthesis was at levels undetectable with this analytical technique, and that chondroitin / dermatan sulphate makes up the vast majority of the synthesised glycosaminoglycans.
92 Chapter III: In search of a good host
SK-N-MC: Keratanase I
Vo Vt 3500 29 mL 3000 2500 2000
cpm 1500 1000 500 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 Fraction
SK-N-MC: Chondroitinase ABC
2000 75.5 mL
1500
1000 cpm
500
0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 Fraction
Figure 20: Glycosaminoglycan composition of proteoglycans secreted by SK-N-MC cells in culture
The results shown in figures 18 – 20 show that in cell lines originating from corneal and neural tissues, keratan sulphate synthesis is undetectable with 35 [ S]SO4 labelling, and that the only type of glycosaminoglycan synthesised and secreted is chondroitin / dermatan sulphate. The results also show that a 35 small amount of [ S]SO4-radiolabel incorporated into CCL-60 proteoglycans is refractile to digestion by chondroitinase ABC, keratanase and nitrous acid treatment.
93 Chapter III: In search of a good host
Results – Part II In part I, it was established that under standard tissue culture conditions, namely, DMEM with 10% FCS, the corneal cell lines CCL-60, CRL-2048, and the neurogenic cell line SK-N-MC do not synthesise keratan sulphate that is detectable with size exclusion chromatography. In part II, FGF-2 and serum concentrations were examined for their effects on keratan sulphate synthesis in a representative corneal cell line, CCL-60, as well as primary bovine keratocytes.
To determine whether either FGF-2 or serum can be used to enhance keratan sulphate biosynthesis in culture, CCL-60 cells were cultured with increasing concentrations of both FCS and FGF-2. In figure 18, CCL-60 cells were shown to secrete only chondroitin sulphate proteoglycans. By using this cell line for this series of experiments, any resulting keratan sulphate can be attributed to changes in either FGF-2 or FCS.
CCL-60 Cell counts
1000000
800000 0 ng/mL FGF-2 600000 0.1 ng/mL FGF-2 400000 1 ng/mL FGF-2 10 ng/mL FGF-2 200000 Number of Cells of Number 0 0.4 2 10 % FCS
Figure 21: The effect of FCS and FGF-2 on cell proliferation
A series of cultures of the rabbit corneal cell line, CCL-60, were seeded in 6- well dishes with FCS concentrations of 0.4, 2, and 10% FCS, as well as 0, 0.1, 1, or 10 ng/mL FGF-2 (figure 21). Cells were incubated for 5 days and monitored for proliferation and keratan sulphate expression. The results of the cell counts show that it is the concentration of FCS rather than FGF-2 that
94 Chapter III: In search of a good host
influences cell proliferation, with greater cell proliferation with higher serum levels. Media was collected and papain-digested for immunoblotting.
ng/mL FGF-2 00.11 10 0.4
% FCS 2
10
Figure 22: 5D4 blot of keratan sulphate from CCL-60
A portion of each digested sample was immobilised on nitrocellulose and the keratan sulphate was detected by dot blot using the keratan sulphate antibody, 5D4. The results show that FGF-2 is capable of stimulating KS expression as reported by Long et al. (Long et al. 2000). The results also show that the level of keratan sulphate appeared to be at its maximum when 0.1 ng/mL FGF-2 was used in culture. Regardless of the concentration of FCS, it appeared that any amount of FGF-2 increased the amount of KS above that of the baseline level in the absence of FGF-2 as shown in the first column of figure 22. The concentration of FCS did not appear to affect the amount of KS greatly.
CCL-60
80 60 0 ng/mL FGF-2 40 0.1 ng/mL FGF-2 20 1 ng/mL FGF-2 0 Relative Intensity Intensity Relative 0.4210 10 ng/mL FGF-2 % FCS
Figure 23: Quantitation of 5D4 intensity per cell from CCL-60 culture medium
Scanning dot blots gives a useful semi-quantitative estimate of the relative amount of KS in one sample compared with another. The scanned dot blot was quantitated and the results corrected for cell number so that the amount
95 Chapter III: In search of a good host
of KS was expressed per cell rather than as a proportion of the total glycosaminoglycan pool. The results are summarised in figure 23. When the KS synthesis was expressed as intensity per cell, the results show that lower serum concentrations helped to maintain higher levels of keratan sulphate synthesis, and that as the serum concentration increased, the keratan sulphate synthesis per cell dropped. When the FGF-2 was used at a concentration of 0.1 ng/mL, the KS synthesis was maintained at higher levels than other concentrations. Together, these results show that FGF-2 used at a concentration of 0.1 ng/mL was able to most efficiently maintain keratan sulphate synthesis in vitro, and that concentrations above this do not further enhance KS synthesis. In fact, it appears that increasing the concentration of FGF-2 beyond 0.1 ng/mL decreases the amount of keratan sulphate. The same applies to the concentration of FCS; high concentrations of FCS greatly enhance cell proliferation (figure 22), but the newly divided cells do not add to the pool of synthesised KS. Although the KS synthesis appears to be most efficient on a per cell basis in the presence of 0.4% FCS, the total cell count remains low and the total amount of keratan sulphate also remains low. Increasing the concentration of FCS, much like FGF-2, appears to decrease the amount of keratan sulphate, however it must be noted that this is on a per cell basis (figure 23). When the keratan sulphate levels are not corrected for cell number (figure 22), the amount of keratan sulphate appears to be similar with different FCS concentrations, at in the case of 0.1 ng/mL FGF-2. It can therefore be concluded from these sets of experiments that for optimal KS synthesis, 2% FCS combined with 0.1 ng/mL of FGF-2 is required.
5D4 is a notoriously sensitive antibody and Western blots are not indicative of 35 chemical amounts of keratan sulphate. [ S]SO4 labelling was therefore used in a larger scale experiment in order to quantitate the increased keratan sulphate synthesis stimulated by the combined use of FGF-2 and low FCS concentrations. In an 80 cm2 flask, CCL-60 cells were cultured with 2% FCS 35 and 0.1 ng/mL FGF-2 and [ S]SO4. After 16 hours of incubation, cells and media were harvested and papain-digested followed by size exclusion chromatography to detect KS. However, keratan sulphate was not detected. So it was concluded that while the combination of 2% FCS and 0.1 ng/mL is
96 Chapter III: In search of a good host
effective at maintaining keratan sulphate at levels that can be detected with the monoclonal antibody 5D4, using this strategy to boost keratan sulphate synthesis in cell lines to those found in vivo is ineffective due to the already low levels of keratan sulphate being produced in the cells. This is in agreement with published results (Nakazawa et al. 1995; Nakazawa et al. 1996; Nakazawa et al. 1997; Nakazawa et al. 1998; Long et al. 2000) which suggest that the loss of KS synthesis in cultured cells is irreversible.
Finally, it is also important to remember that the 5D4 epitope is a highly sulphated tetra- or hexasaccharide region of keratan sulphate, so not only are 5D4 blots indicative of the amount of keratan sulphate present, they are also indicative of the degree of sulphation on the keratan sulphate chains. Because of its high degree of sulphation, the 5D4 epitope is also resistant to digestion by keratanase but not keratanase II. With this in mind, 5D4 blots that give poor immunoreactivity may not be lacking keratan sulphate, but may be undersulphated. In figure 22, supplementing CCL-60 with 0.1 ng/mL FGF-2 resulted in the greatest 5D4 immunoreactivity, whereas 1 and 10 ng/mL FGF- 2 resulted in the poorest immunoreactivity. There are two interpretations for this result; the first being that changes in FGF-2 concentration may affect keratan sulphate content and the second is that the FGF-2 does not affect keratan sulphate content but does affect the sulphotransferase activity. Overall, the very low levels of keratan sulphate produced by cell lines makes them unsuitable for the production of keratan sulphate-bearing G1-G2.
97 Chapter III: In search of a good host
Keratan sulphate synthesis by primary keratocytes
By examining keratan sulphate synthesis in cell lines and then examining the effect of varying concentrations of FCS and FGF-2 on KS synthesis, it became apparent that under normal cell culture conditions, regardless of their originating tissue, cell lines do not synthesise keratan sulphate beyond micro- scale quantities. Keratan sulphate synthesis could be modified by altering the concentration of FCS and FGF-2 (figures 22 & 23), however these conditions do not appear to reverse the already down-regulated state of keratan sulphate synthesis that is common to cell lines.
Because keratan sulphate expression in cell lines is low and apparently irreversible, cell lines were eliminated as possible hosts for the Vaccinia T7 RNA polymerase expression system. Cells from fresh bovine corneas were chosen as the next most suitable type of cell to infect. These primary keratocytes, having been freshly isolated from corneas, were assumed to still retain high levels of keratan sulphate synthesis. Having come from the corneal stroma, primary keratocytes express high amounts of keratan sulphate proteoglycans (Funderburgh et al. 1987; Nakazawa et al. 1995; Funderburgh et al. 1996; Nakazawa et al. 1996; Nakazawa et al. 1997; Nakazawa et al. 1998; Funderburgh 2000; Long et al. 2000; Funderburgh et al. 2001; Funderburgh 2002; Funderburgh et al. 2003). They also have the advantage of not containing any endogenous aggrecan, so purifying recombinant aggrecan G1-G2 and sorting it from bovine aggrecan is of no concern.
Keratocytes were isolated from the eyes of freshly slaughtered steers and seeded in culture medium containing increasing concentrations of FCS and FGF-2 according to the table below (table 6).
98 Chapter III: In search of a good host
Table 6: Culture conditions for the cell proliferation assay Culture % FCS ng/mL FGF- 2 1 0.4 0 2 0.4 0.1 3 0.4 1 4 0.4 10 5 2 0 6 2 0.1 7 2 1 8 2 10 9 10 0 10 10 0.1 11 10 1 12 10 10
Primary keratocytes Cell counts
800000 700000 600000 0 ng/mL FGF-2 500000 0.1 ng/mL FGF-2 400000 300000 1 ng/mL FGF-2 200000 10 ng/mL FGF-2
Number of cells of Number 100000 0 0.4210 % FCS
Figure 24: Primary keratocytes cell proliferation in response to FCS and FGF-2
After 5 days of incubation, cells were trypsinised and counted to determine the effect of FCS and FGF-2 on cell proliferation (figure 24). Increasing the concentration of either FCS or FGF-2 increased the rate of cell proliferation. At a concentration of 0.4% FCS, cells did not proliferate much more than the initial seeding, however at 10% FCS cells became over-confluent and were difficult to count.
99 Chapter III: In search of a good host
ng/mL FGF-2 00.11 10 0.4
% FCS 2
10 Figure 25: 5D4 and MZ15 blot of primary keratocyte glycosaminoglycan
Based on dot blot analysis using a combination of two keratan sulphate antibodies, 5D4 and MZ15 (figure 25), keratan sulphate content was greatest when serum levels were at 0.4% or 2%. Because the different serum concentrations increased cell proliferation at higher FCS concentrations, analysis of KS by primary keratocytes by scanning densitometry, was normalised so that the KS in the figure (figure 26) is expressed as KS produced per cell. From the graph, it appears that primary keratocytes respond to both FCS and FGF-2 similarly to the established corneal cell line, CCL-60. When cell cultures were supplemented with 10% FCS, cells divided much quicker than when serum levels of 0.4% or 2% were used, however this increase in cell proliferation did not increase the amount of KS produced. The effect of FGF-2 on KS synthesis appeared to vary somewhat, but looking at total keratan sulphate secretion (figure 25) the amount of KS synthesis increased with the amount of FGF-2 added to culture medium.
Like CCL-60 cells, primary keratocytes originate from the corneal stroma where there is a naturally high concentration of keratan sulphate. However, unlike the corneal cell line which produce trivial amounts of keratan sulphate, the keratan sulphate synthesis of the primary keratocytes was assumed to still be quite close to those found in vivo, as reported by other researchers (Nakazawa et al. 1995; Funderburgh et al. 1996; Nakazawa et al. 1996; Nakazawa et al. 1997; Long et al. 2000). By supplementing the cells with low concentrations of FCS and concentrations of FGF-2 up to 10 ng/mL, KS secretion by these cells, based on Western blot analysis, was maintained at levels above those of cells cultured under standard conditions of 10% FCS. Although the addition of 0.1 ng/mL FGF-2 in culture enhances KS secretion
100 Chapter III: In search of a good host with any concentration of FCS, using concentrations of FGF-2 above this, did not appear to further increase KS secretion. The exception was when the FCS concentration was at 2%, where the KS production kept increasing with added FGF-2; in this case there was almost a two-fold increase in KS secretion with 10 ng/mL FGF-2 compared with cultures maintained without FGF-2.
Primary keratocytes
50 40 0 ng/mL FGF-2 30 0.1 ng/mL FGF-2 20 1 ng/mL FGF-2 10 10 ng/mL FGF-2 Relative intensity 0 0.4 2 10 % FCS
Figure 26: Quantitation of KS blot of glycosaminoglycans from primary keratocytes
The results of anti-KS blotting of a representative cell line, CCL-60 (figure 22), and primary keratocytes (figure 25) show that low concentrations of FGF-2 combined with low concentrations of serum increase KS synthesis. However, in order to quantitate the proportion of keratan sulphate produced by cultured 35 primary keratocytes, [ S]SO4 labelling was required.
35 [ S]SO4 labelling was performed using freshly isolated bovine keratocytes, and the culture conditions being examined were 0.4% and 2% FCS, as well as 0.1, 1, and 10 ng/mL FGF-2. The initial cell seeding was carried out differently to previous labelling experiments. In this round of experiments, primary keratocytes were seeded so as to account for the increased cell proliferation due to increasing serum levels. Cells cultured in the presence of 0.4% FCS were seeded with 150,000 cells in 6-well dishes, whereas cells cultured with 2% FCS were seeded with 75,000 cells (table 7). This was to avoid over-confluent cell populations. Wells were seeded with the appropriate
101 Chapter III: In search of a good host
number of cells and the appropriate amount of FCS and FGF-2, and were allowed to incubate at 37ºC overnight to ensure the cells had adequately attached. After approximately 18 hours the media was removed and fresh 35 media, containing the same supplements and 55 μCi/mL [ S]SO4 was added. After 5 days media was collected and prepared for size exclusion chromatography as previously described.
35 Table 7: Culture conditions for [ S]SO4 labelling Culture No. cells seeded % FCS ng/mL FGF-2 1 150,000 0.4 0.1 2 150,000 0.4 1 3 150,000 0.4 10 4 75,000 2 0.1 5 75,000 2 1 6 75,000 2 10
Void volumes containing the intact glycosaminoglycan pools were collected after an initial round of gel filtration, and each sample was divided into two equal portions for either keratanase I or chondroitinase ABC digestion. Samples were then fractionated once more to determine the degree of digestion with keratanase or chondroitinase and therefore the proportion of KS to CS in each sample.
Figure 27 shows the results of the keratocytes cultured in 0.4% FCS with 0.1, 1, or 10 ng/mL FGF-2. The amount of keratan sulphate produced by cells cultured with 0.1, 1, and 10 ng/mL FGF-2 was 12, 25, and 16% respectively, while the amount of chondroitin sulphate was 84, 75, and 78% respectively. If we assume that the glycosaminoglycans produced by keratocytes is primarily KS and CS/DS, the two numbers combined should naturally add up to 100%. Though the amount of CS/DS produced is not particularly important, it also makes for a good indication of the amount of KS present, that is, if we were to take the difference of 100% to be the proportion of KS present in each cell. This ‘backup’ glycosidase digestion allowed for a more accurate determination of the glycosaminoglycan content of each culture condition.
102 Chapter III: In search of a good host
0.4% FCS
Vo Keratanase I Vt Chondroitinase ABC 18000 16000 Vo Vt 14000 12000 10000
cpm 8000 0.1 6000 4000 12% 84% 2000 0 18000 16000 14000 12000 10000 cpm 1 8000 25% 75% 6000 4000 2000 0
30000
25000
20000
cpm 10 15000 ng/mL FGF-2 10000 16% 78%
5000
0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 1 4 7 101316192225283134374043464952 Fraction Fraction Figure 27: Glycosaminoglycan synthesis by primary keratocytes grown in 0.4% FCS and increasing amounts of FGF-2
103 Chapter III: In search of a good host
This was especially the case with cells cultured with 2% FCS in figure 28, in which the keratanase I digests returned unusually high degrees of digestion, with the KS proportions for 0.1, 1, and 10 ng/mL FGF-2 returning 51, 43, and 27% KS. Because in vivo proportions have been estimated to be closer to 40% KS in the cornea (Nakazawa et al. 1995; Nakazawa et al. 1996; Nakazawa et al. 1997; Long et al. 2000), these results were considered to be overestimates of the KS being synthesised in these cells. Fortunately, the chondroitinase ABC digests appeared to return more reasonable results with 76, 69, and 81% CS for 0.1, 1, and 10 ng/mL FGF-2 respectively. If the chondroitinase-resistant glycosaminoglycans are primarily KS, then this would leave 24, 31, and 19% KS.
2% FCS Keratanase I Chondroitinase ABC 3000
Vt Vo Vt 2500 Vo 2000
1500 0.1 cpm 51% 76% 1000
500
0
5000
4000 43% 69% 3000 1 cpm
2000
1000
0 8000
7000
6000 5000 10 cpm 4000 27% 81% ng/mL FGF-2 3000
2000
1000
0 1 4 7 101316192225283134374043464952 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 Fraction Fraction Figure 28: Glycosaminoglycan synthesis by primary keratocytes grown in 2% FCS and increasing amounts of FGF-2
104 Chapter III: In search of a good host
40 0.4% FCS 2% FCS 30
20 % KS
10
0 0.1 1 10 ng/mL FGF-2
Figure 29: KS synthesis in response to FGF-2 and FCS
Table 8: KS and CS synthesis by primary bovine keratocytes
Culture Conditions % KS (KS % CS % FCS FGF-2 (ng/mL) calculated from %CS) 0.4 0.1 13 (16) 84 0.4 1 25 (25) 75 0.4 10 14 (22) 78 2 0.1 51 (24) 76 2 1 43 (31) 69 2 10 27 (19) 81
The graph and table above summarise the results of the column chromatography and show the effect of different serum and FGF-2 concentrations on KS expression in primary bovine keratocytes. We can conclude from these experiments that low concentrations of serum combined with low concentrations of FGF-2 support KS expression in primary bovine keratocytes. KS content has been graphically presented in figure 29, and presented again in table 8. In table 8, the %KS has been presented twice, the first number refers to the %KS calculated from the degree of keratanase I digestion, while the number in parentheses refers to the %KS calculated from the proportion of chondroitinase ABC-resistant glycosaminoglycan. Although the proportion of KS calculated using each method correlated very well among the 0.4% FCS cultures, the calculated %KS for cells cultured in 2% FCS using both methods correlated poorly. This suggests that there is an
105 Chapter III: In search of a good host over-estimate of either KS or CS in the samples derived from 2% FCS cultures. The power of enzyme digestions is the specificity of the enzymes, and the specificities of keratanase I and chondroitinase ABC are well described. However, from the present studies it is not possible to determine whether discrepancies in the proportions of KS and CS are due to overestimates of keratan sulphate or chondroitin sulphate.
From these experiments, the culture condition most favourable for KS synthesis is 2% FCS and 1 ng/mL FGF-2, though the amount of secreted KS with 0.1 ng/mL FGF-2 is also quite high, and economically, is probably most efficient. Low concentrations of FCS (0.4%), combined with 1 ng/mL FGF-2 also results in high levels of KS production on a per cell basis, however from figure 25, primary keratocytes do not respond well to such low serum levels.
Table 9: CS and KS synthesis by primary bovine and chicken keratocytes %FCS ng/mL Bovine Chicken FGF-2 keratocytes keratocytes %KS %CS %KS %CS 0.1% HS 1 14 82 29 61 2% FCS 0 14 85 26 63 2%FCS 0.1 13 85 30 60
The results presented in table 8 show that the culture conditions that result in the most efficient expression of keratan sulphate proteoglycans was by supplementing primary bovine keratocytes with 2% FCS and 0.1 ng/mL FGF- 2. To further examine KS expression, sulphate labelling was repeated using primary bovine keratocytes and primary chicken keratocytes and the use of horse serum (HS) was introduced as an alternative to FCS, as suggested in a previous study (Long et al. 2000). From the results (table 9), it was apparent that the keratan sulphate-promoting effect of FGF-2 and low serum concentrations in medium may be more subtle than previously thought. In all conditions, the new expression of keratan sulphate was unchanged despite changing the type of serum used, and despite the addition of FGF-2, unlike the previous experiments.
106 Chapter III: In search of a good host
Discussion
Using a Vaccinia virus T7 RNA polymerase expression system I plan to express recombinant G1-G2 glycoforms that are modified with intermediate levels of keratan sulphate, in a type of cell capable of sustained keratan sulphate synthesis over the course of the Vaccinia incubation period. However, the biggest problem facing KS researchers is that cells grown in culture stop producing ‘normal’ amounts of KS soon after seeding to monolayer (Nakazawa et al. 1995; Funderburgh et al. 1996; Nakazawa et al. 1996; Nakazawa et al. 1997; Nakazawa et al. 1998; Long et al. 2000).
This phenomenon is thought to apply to all keratan sulphate-producing cells, though the degree to which this occurs has only been well studied in corneal cells due to its critical role in maintaining corneal transparency by keeping collagen fibres aligned. Corneal tissue is known to contain the highest concentration of keratan sulphate in the body, followed closely by cartilage and neural tissue. In order to identify a cell line capable of synthesising sustained levels of KS in vitro, three cell lines: CCL-60, CRL-2048, and SK-N- MC. were examined for their KS production under standard tissue culture conditions (i.e. DMEM 10% FCS). This chapter examined the types of GAG produced by the three chosen cell lines, each originating from the cornea and the brain. It may seem odd to have not included a cartilage-derived variety of cell in these studies. The idea of using chondrocytes was considered but it was decided that baseline levels of aggrecan would be produced continually by these cells, and purifying recombinant human aggrecan fragments from host-derived, full length aggrecan would be too difficult.
Examination of the keratan sulphate produced by each cell line was 35 performed by [ S]SO4-labelling to allow newly synthesised 35 glycosaminoglycans to be labelled. The level of incorporation of [ S]SO4 into the newly synthesised glycosaminoglycans varied between each cell line. The 35 greatest amount of incorporated [ S]SO4 was detected in the CCL-60 sample. This variation in the extent of radiolabelling most likely reflects differences in biosynthetic rates between the cell lines. To a much lesser
107 Chapter III: In search of a good host extent, variations in sample recovery after ethanol precipitation may contribute to small differences in the level of radioactivity. The proportion of keratan sulphate to chondroitin / dermatan sulphate; the two most likely types of GAGs present; was determined by the degree of susceptibility to either keratanase or chondroitinase. No keratan sulphate was detected in any of the cell lines examined, only chondroitin / dermatan sulphate. Analysis of the
CCL-60 GAGs, showed that approximately 5% of the Vo was resistant to keratanase and chondroitinase ABC. It is possible that this material represents sulphated protein, or the linkage region of the chondroitin sulphate / dermatan sulphate chains that is sulphated and attached to the protein core. However, another explanation, albeit, far less likely because of the heterogeneity of KS, is that this undigestable material may represent KS that is fully disulphated, which would render it resistant to keratanase I. Cell lines were therefore deemed unsuitable hosts for recombinant G1-G2 expression, at least, under ‘normal’ tissue culture conditions.
Various culture conditions and mitogens have been investigated for their effect on glycosaminoglycan synthesis in cultured cells. Keratan sulphate studies are generally performed using bovine or chick corneas due to the high keratan sulphate synthesis by keratocytes. However, through mechanisms that are not completely understood, keratan sulphate synthesis is greatly down regulated in response to wound healing as well as standard tissue culture conditions, this therefore makes studying keratan sulphate in cells difficult. To help maintain keratan sulphate levels close to those found in vivo, many researchers have investigated the effect of altering culture conditions on KS synthesis, such as the addition of mitogens in culture medium.
The aim of this series of experiments was to determine whether cells could be cultured in such a way so as to express reasonably high levels of keratan sulphate in proportions similar to those found in vivo (~40%). In the initial series of experiments, a number of different cell lines were examined for KS expression under normal tissue culture conditions, all of these were found to express little or no KS. Primary cells were therefore chosen as likely
108 Chapter III: In search of a good host candidates for all future experiments. Primary cells from fresh bovine corneas were used, not chondrocytes, for the reasons given earlier.
Similar studies in KS expression have found that primary cells grown in the presence of FGF-2 and low FCS concentrations are able to produce proportionately more KS than cells grown under normal tissue culture conditions. It was the aim of this study to confirm these findings by culturing primary bovine keratocytes in the presence of variable concentrations of both FGF-2 and FCS. One of the first experiments was to monitor cell proliferation in response to increasing concentrations of FCS and FGF-2. The results of the cell proliferation experiments of CCL-60 cells (figure 21) and primary bovine keratocytes (figure 24) show that cells proliferate much more in the presence of either or both of these supplements. There are some deviations in the cell proliferation results, particularly in figure 21, 10% FCS. In those particular cultures, there was a decrease in cell proliferation with increasing concentrations of FGF-2. It may be that the cells divided too quickly and lifted off the tissue culture dish; this would explain the unexpected results, it also agrees with the results observed by Long et al. when higher concentrations of FGF-2 were used (Long et al. 2000). However, newly divided cells do not contribute to the pool of synthesised KS. This result was not surprising because it has been reported many times by other researchers. The consensus is that cell lines or recently harvested cells that have undergone a single division, lack the ability to synthesise normal, full length keratan sulphate. Whether it is due to a wound healing response or a change in cell morphology, or a dramatic down-regulation of a necessary gene is unknown. Whatever the case may be, the decrease in keratan sulphate synthesis seen in my experiments agrees with what has been published previously.
Because cell lines have already been subcultured many times, it was perhaps not surprising that the degree of KS synthesis in these cells was very low and only able to be detected by Western blot with the monoclonal antibody, 5D4. Unlike cell lines, primary cells, when grown in the presence of FCS and FGF- 2, were able to express KS at quite high levels. As a result of these experiments, culture conditions have been found which most efficiently
109 Chapter III: In search of a good host enhance KS expression in primary bovine keratocytes, namely, 2% FCS and 0.1 ng/mL FGF-2, although repeating these experiments subsequently resulted in much more subtle effects with very little changes with any medium supplement.
It remains to be seen whether these culture conditions are able to induce Vaccinia-infected cells to maintain high keratan sulphate levels given the system-wide changes they undergo in response to infection. One of the key findings by Long et al. (Long et al. 2000) during their experiments with FGF-2, was that stimulation of keratan sulphate proteoglycans required a minimum of 2 to 4 days for any significant results. If this is indeed the case, then it is possible that FGF-2 may not prove to be a useful supplement for Vaccinia infections, where media can be harvested as soon as 24 hours post inoculation.
110 Chapter IV: Optimising the expression of rG1-G2
Chapter IV: Optimising the expression of rG1-G2
“The best scientist is open to experience and begins with romance - the idea that anything is possible.” - Ray Bradbury (1920 - )
111 Chapter IV: Optimising the expression of rG1-G2
Introduction
In the previous chapter, SK-N-MC, CCL-60, and CRL-2048 cells were examined for keratan sulphate synthesis. These cell lines originated from corneal and neural tissue, tissues with naturally high concentrations of keratan sulphate. These cells are therefore equipped with all the components necessary for keratan sulphate synthesis, however previously published reports indicate a dramatic decrease in keratan sulphate synthesis once cells are cultured, and this phenomenon is especially pronounced when cells are propagated by subculturing (Nakazawa et al. 1995; Funderburgh et al. 1996; Nakazawa et al. 1996; Nakazawa et al. 1997; Long et al. 2000). Cell lines were therefore not expected to synthesise normal amounts of keratan sulphate, but the degree of this inhibition was unknown. Sulphate-labelling showed that keratan sulphate substitution was incomplete in each cell line. Keratan sulphate was not detectable with scintillation counting and was only detectable by Western blotting with the high affinity antibody, 5D4. I concluded that chondroitin sulphate was the only type of glycosaminoglycan produced by these cell lines under the standard conditions in which they were cultured.
Low concentrations of serum and FGF-2 were later explored for their effect on keratan sulphate synthesis. Tests performed on CCL-60 cells found that both of these supplements were able to promote keratan sulphate synthesis, though only on a very small scale, detected only by Western blot. On larger 35 scale assays using [ S]SO4 labelling, keratan sulphate was not detected in cell lines.
This is the reason primary keratocytes were used. Having never been passaged, the levels of keratan sulphate were expected to be closer to those found in vivo. After a series of labelling experiments, culture conditions were found which most efficiently promoted keratan sulphate synthesis: 2% FCS and 0.1 ng/mL FGF-2. However, it was uncertain whether these conditions would be sufficient to maintain keratan sulphate synthesis in primary cells when infected with Vaccinia.
112 Chapter IV: Optimising the expression of rG1-G2
In this chapter, experiments to optimise the expression of recombinant G1-G2 are described. Initial testing and optimisation steps involved a number of different types of cells. As well as the cells screened in chapter III, two additional types of cell are introduced in this chapter, COS-7 and HT-1080; all were used to optimise the Vaccinia T7 RNA polymerase expression system.
The first series of experiments involved the use of a modified Vaccinia virus expressing the membrane protein, B5R, fused to a green fluorescent protein (B5R-GFP) (Ward et al. 2001). This allowed a rapid, visual readout of recombinant protein expression and was used to determine conditions for optimal infection. The GFP-tagged recombinant protein was detected immediately, without the need for post-infection processing, protein isolation, SDS-PAGE or Western blotting. Cells expressing recombinant B5R-GFP were visualised by fluorescence microscopy.
After the initial experiments with Vaccinia B5R-GFP, the Vaccinia T7 RNA polymerase expression system was used for rG1-G2 expression. Again, a variety of different types of cells were infected, primarily to explore the extent of keratan sulphate substitution present on each resulting rG1-G2 substrate. Bovine and chicken primary keratocytes, and COS-7 cells, were all included as possible hosts for the expression of rG1-G2. Although cell lines had already been eliminated as keratan sulphate-producing cells, a recent conference abstract reported the expression of 5D4-positive recombinant aggrecan in COS-7 cells (Hering et al. 2002). COS-7 cells were therefore included, so as not to exclude a possible cell line capable of keratan sulphate proteoglycan synthesis.
During the testing phase of the Vaccinia T7 expression experiments, when culture conditions were being optimised for keratan sulphate expression, culture conditions matching those determined in the previous chapter were applied to the expression of rG1-G2. One of the conditions tested was 2% FCS and 0.1 ng/mL FGF-2, which was included throughout the course of infection. However, FGF-2 and low concentrations of serum failed to increase keratan sulphate substitution on rG1-G2 (assessed by a size shift following
113 Chapter IV: Optimising the expression of rG1-G2 keratanase digestion). In light of these results, the addition of this growth factor to culture medium during Vaccinia infections was discontinued, and other variables were examined to enhance keratan sulphate substitution on rG1-G2 as well as to enhance the yield of rG1-G2.
Analysis of the degree of keratan sulphate substitution on rG1-G2 expressed in bovine and chicken keratocytes, and COS-7 cells, revealed that rG1-G2 contained the greatest amount of keratan sulphate when expressed in primary bovine keratocytes, and that neither the FCS nor the addition of FGF-2 was able to promote keratan sulphate substitution above the ~5kDa found on rG1- G2 expressed in primary bovine keratocytes supplemented with only 2% FCS.
114 Chapter IV: Optimising the expression of rG1-G2
Results
Monitoring the response of cells to infection by Vaccinia
A number of different types of cells were prepared for infection with Vaccinia B5R-GFP (Ward et al. 2001) to gauge the response of each cell type to infection by the virus. Besides the GFP component present in its genome, the Vaccinia B5R-GFP virus is normal in all aspects and is functionally indistinguishable from its wild-type counterpart (Ward et al. 2001). Infection with Vaccinia B5R-GFP was therefore considered to be a good way to assay the response of cells to infection by Vaccinia TF7-3 and Vaccinia G1-G2, as well as to determine whether one type of cell was more efficient at expressing recombinant protein than another. It should be noted that these Vaccinia B5R- 35 GFP experiments were done while [ S]SO4-labelling was still being used to determine keratan sulphate levels in different cell types (chapter III), hence the inclusion of cell lines in the experiment.
The recombinant protein expressed by these cells was visualised by fluorescence microscopy at a magnification between 100 and 200 times. Each type of cell: CCL-60, CRL-2048, SK-N-MC, and primary bovine and chicken keratocytes, was infected with Vaccinia B5R-GFP at a concentration of 4 and 20 pfu/cell (figure 30). The purpose of the concentrations was to determine the minimum amount of virus required to still efficiently infect the cells but prevent them from dying too quickly. In each case, a low concentration of Vaccinia was sufficient for B5R-GFP expression and infecting cells with a concentration of 20 pfu/cell appeared to give indistinguishable results.
Of the three cell lines tested, the rabbit corneal cell line, CCL-60, appeared to most efficiently express B5R-GFP. The neuroblastoma cell line, SK-N-MC, also appeared to express B5R-GFP, though not to the extent of the CCL-60 cells; it appeared that SK-N-MC cells are either less susceptible to infection by Vaccinia, or the synthesis of B5R-GFP is inhibited. It is interesting to note that while the primary bovine keratocytes were able to express the fluorescent protein very well and perhaps of all the cells tested was the most susceptible
115 Chapter IV: Optimising the expression of rG1-G2 to infection by Vaccinia (as seen by the large amount of cellular debris and extensive blebbing from the cell surface), the bovine corneal cell line, CRL- 2048, very inefficiently expressed B5R-GFP. In figure 30, the CRL-2048 cells showed very little fluorescence, but remained largely intact as seen by light microscopy (not shown). The primary chicken keratocytes also expressed the GFP fusion protein. These cells are the only avian cells tested and from the figure, it would appear that the chicken keratocytes remain intact over the course of the incubation period, suggesting that a longer incubation period may be possible with chicken keratocytes, and hence a greater amount of secreted recombinant protein available for harvesting.
116 Chapter IV: Optimising the expression of rG1-G2
Figure 30: Vaccinia B5R-GFP infection of cells CCL-60, CRL-2048, and SK-N-MC, as well as the primary keratocytes from bovine and chicken corneas, were infected with Vaccinia B5R-GFP and recombinant protein expression was determined by fluorescence.
117 Chapter IV: Optimising the expression of rG1-G2
Having demonstrated that Vaccinia is capable of infecting a variety of different cells, the next step was to infect cells with Vaccinia TF7-3 and Vaccinia G1- G2 to produce rG1-G2. Following this, adjustments to culture conditions were made to enhance the yield of rG1-G2.
HT-1080 cells produce rG1-G2
The first experiment was designed to test the Vaccinia T7 RNA expression system and to confirm that the recombinant virus would express recombinant G1-G2. Keratan sulphate substitution of rG1-G2 was not a concern at this point. HT-1080 cells were used for this initial experiment; these cells are a human fibrosarcoma cell line and do not synthesise keratan sulphate.
Three different viral loads were used to inoculate HT-1080 cultures, each with an appropriate Vaccinia TF7-3-only control. To ensure that rG1-G2 was expressed, slightly higher than normal concentrations of Vaccinia were used to infect the cells; the concentrations were 10, 20, and 40 pfu/cell. Cultures were inoculated and allowed to incubate for 2 days before analysis by SDS- PAGE and fluorography (figure 31). The results show that cells infected with vTF7-3 and vG1-G2 produced a unique 35S-labelled protein migrating at approximately 110 kDa on SDS gels. In contrast, the control cells infected only with vTF7-3 did not synthesise the 110 kDa band. The 110 kDa size is consistent with the size expected for the unglycosylated rG1-G2.
Irrespective of the dose of virus used to inoculate the HT-1080 cultures, the same amount of rG1-G2 was produced. This suggests that inoculations of 10 pfu/cell or less are sufficient for rG1-G2 production, at least in the HT-1080 cell line. Because HT-1080 cells are not known to synthesise keratan sulphate, the rG1-G2 in the figure represent protein that is completely devoid of keratan sulphate, and results in a sharp band migrating at approximately 110 kDa.
118 Chapter IV: Optimising the expression of rG1-G2
vTF7-3 pfu/cell 10 2020 40 40 80 vG1-G2 pfu/cell 10 20 40
220 -
97.4 -
66 -
46 -
Figure 31: HT-1080 cells expressing rG1-G2 HT-1080 cells were infected with increasing amounts of either Vaccinia TF7-3 (T7 RNA polymerase) alone as a control, or with Vaccinia TF7-3 and Vaccinia G1-G2 to produce rG1-G2. 35S-methionine was included in the culture medium to label newly synthesised proteins and cells were incubated for 48 hours.
rG1-G2 expression by COS-7 cells
After reports of 5D4-reactive recombinant aggrecan expressed in COS-7 cells (Hering et al. 2002), COS-7 cells were grown in monolayers and co-infected at 5 pfu/cell for a period of 4 days. Samples were taken every 12 hours for analysis by SDS-PAGE. Keratan sulphate was examined by keratanase digestion. The results show that rG1-G2 began to be secreted by 24 hours (figure 32A) and appeared to peak after 48 hours. By 60 hours, the amount of accumulated rG1-G2 had decreased; no rG1-G2 was detected beyond this point. These results are also presented as a graph in figure 32B. No keratan sulphate was detected at any point, as determined by a keratanase-induced shift in size on polyacrylamide gels (figure 32A), although the rG1-G2 was 5D4-positive (data not shown).
119 Chapter IV: Optimising the expression of rG1-G2
TF7-3 TF7-3 TF7-3 TF7-3 TF7-3 A TF7-3 G1-G2 TF7-3 G1-G2 TF7-3 G1-G2 TF7-3 G1-G2 TF7-3 G1-G2 -+ -+ -+-+ -+ -+ -+-+ -+ -+ 250 -
150 -
100 -
75 -
12h 24h 36h 48h 60h
B 25
20
15
10
5 Relative Pixel Density Pixel Relative
0 2030 40 50 60 70 80 90 100 Time (hours)
Figure 32: Timecourse of rG1-G2 production in COS-7 cells (A) Confluent monolayers of COS-7 cells were infected with Vaccinia TF7-3 alone as a control, or co- infected with Vaccinia TF7-3 and Vaccinia G1-G2. Cells were incubated in the presence of 35S- methionine for 4 days and samples were digested with keratanase I, keratanase II, and endo-β- galactosidase (+), and compared with the undigested rG1-G2 (-). (B) The rG1-G2 accumulated at each time point was plotted against relative pixel density.
Production of rG1-G2 in primary chicken keratocytes
Unlike HT-1080 cells which do not synthesise keratan sulphate, primary chicken keratocytes were shown in the previous chapter to produce up to 30% keratan sulphate (table 9). Accordingly, primary keratocytes were isolated from the corneas of 14 day old chicken embryos and co-infected with Vaccinia TF7-3 and Vaccinia G1-G2.
120 Chapter IV: Optimising the expression of rG1-G2
Experimental results presented in figures 30 and 31 suggested that low concentrations of Vaccinia were sufficient for rG1-G2 expression. Therefore primary chicken keratocytes were infected with viral loads lower than those previously used. Concentrations ranging from 1 pfu/cell to 90 pfu/cell were used to inoculate five cultures of primary chicken keratocytes (figure 33A). A single culture inoculated only with Vaccinia TF7-3 served as a control. After 48 hours of incubation samples were resolved with SDS-PAGE and visualised by fluorography.
Figure 33 shows that the amount of rG1-G2 produced was proportional to the amount of Vaccinia used to infect the cells (figure 33B); this linear relationship between viral load and protein expression was not seen when HT-1080 cells were infected (figure 31). The observed size of the labelled protein was very similar to the HT-1080-expressed rG1-G2 and was approximately 110 – 120 kDa. However in comparison to the rG1-G2 expressed by the HT-1080 cells, the rG1-G2 bands of the primary chicken keratocytes appeared to be broader, suggesting that the rG1-G2 expressed by the chicken keratocytes may have been glycosylated. However, keratanase digestion of this substrate resulted in no change in migration by SDS-PAGE, indicating a lack of keratan sulphate substitution, despite supplementing the cells with 2% FCS and 0.1 ng/mL FGF-2 during culture (figure 33C).
When infected with low concentrations of Vaccinia, a single sharp band resolving at 100 kDa were observed (figure 33A, indicated with an arrow); these were particularly prominent in the cultures inoculated with 1 and 3 pfu/cell of Vaccinia. It was possible that this band may have been an unglycosylated form of rG1-G2, however, Western blots showed the band was 1C6-negative (data not shown), and therefore not rG1-G2.
121 Chapter IV: Optimising the expression of rG1-G2
A TF7-3 TF7-3/G1-G2 pfu/cell 180 1 3103090
215 -
d te 100 - ges i se nd 'a U K C 71 -
B 140 120 100 80 60 40
Relative pixel density pixel Relative 20 0 0 20406080100 pfu/cell
Figure 33: rG1-G2 expression by primary chicken keratocytes (A) Primary chicken keratocytes were infected with increasing amounts of Vaccinia TF7-3 and Vaccinia G1-G2. A single Vaccinia TF7-3-infected culture was included as a control. Cells were cultured in the presence of 35S-methionine and supplemented with 2% FCS and 0.1 ng/mL FGF-2 and incubated for 48 hours. The unknown 100 kDa protein is marked with an arrow. (B) The fluorograph was quantitated with scanning densitometry and the quantitated bands were plotted against relative pixel density (C) rG1-G2 was digested with keratanase I, keratanase II, and endo-β-galactosidase and compared with the undigested rG1-G2 by SDS-PAGE.
122 Chapter IV: Optimising the expression of rG1-G2
Expression of rG1-G2 by chicken corneal explant cultures
With a potential of producing up to 30% keratan sulphate, the rG1-G2 expressed in chicken keratocytes was expected to be substituted with keratan sulphate. In an attempt to provide a more natural environment for the primary keratocytes to synthesise keratan sulphate, corneal explants rather than cells were investigated for infection. Initial infections of whole corneas resulted in no rG1-G2 being produced; this was presumably due to a lack of penetration of the Vaccinia into the tissue. A new experiment was designed using corneal explants that had either been cut into quarters or scratched with a needle prior to inoculation with Vaccinia. The cultures were allowed to incubate for an extended period to ensure that as many cells as possible were infected and allowed to express rG1-G2. The explants were incubated for 5 days before the media was harvested. Tissue was collected and protein was extracted for 2 days with 4M GuHCl to ensure that any rG1-G2 not secreted into media would still be collected for analysis. Media (M) and corneal extracts (E) of both the cut corneas and the scratched corneas were run along with a corresponding T7 control on a polyacrylamide gel. The result (figure 34A) shows that cutting the corneas into quarters prior to infection gives the best yield, however the size of rG1-G2 (~100 kDa) did not appear to be any larger than the rG1-G2 produced in infected chicken keratocytes, indicating that there was very little glycosylation.
One final experiment was performed to try to increase the amount of keratan sulphate synthesised by the chicken keratocytes; corneas were again cut into quarters and infected. Three cultures were incubated at three different temperatures: 37°C, room temperature, and ~4°C, in an attempt to slow down the metabolic processes of the cells. The reasoning being that rG1-G2 synthesis by Vaccinia occurs at a very fast rate and it was thought that more time was required for adequate keratan sulphate substitution on the synthesised protein. Similar experiments had been done in the past, each examining the effect of altered culture conditions on keratan sulphate synthesis. The first report examined glycosaminoglycan synthesis in bovine keratocytes (Bleckmann et al. 1979). A number of different culture conditions
123 Chapter IV: Optimising the expression of rG1-G2 were looked at, including lowering the incubation temperature, however this resulted in reduced glycosaminoglycan synthesis. Another report examined keratan sulphate synthesis in chicken corneal explants (Midura et al. 1989). In this paper, the authors reported that chicken corneas incubated for 20 hours at 5°C produced keratan sulphate at levels similar to those found in normal corneal tissue.
The media from explant cultures at 37°C, room temperature, or 4°C were collected and treated with keratanase I, keratanase II, and endo-β- galactosidase (+). These samples were analysed by SDS-PAGE and compared with the corresponding undigested rG1-G2 substrate (-). A sample from figure 33A (cells) was included for comparison (figure 34B). The results show that the yield from infected monolayers is far greater than with infected explants, and that there is no rG1-G2 produced by the cells at temperatures at or below room temperature. Although the rG1-G2 produced in explants appears to be slightly larger than that expressed in monolayers (figure34B – cells vs. 37°C), keratanase digestion of either rG1-G2 did not result in a change in migration on polyacrylamide gels.
From infections of chicken keratocytes and corneal explants, it was clear that no culture condition would be suitable for the synthesis of keratan sulphate- substituted rG1-G2. The use of chicken keratocytes in producing glycosylated rG1-G2 was therefore discontinued and another type of cell was investigated.
124 Chapter IV: Optimising the expression of rG1-G2
TF7-3 TF7-3 TF7-3 G1-G2 TF7-3 G1-G2 A MEMEMEME B - + - + - + - + k’ase 250 - - 250
150 - - 150
- 100 100 - - 75 75 -
Cells 37°C RT 4C° 50 - Cut
Cut Scratched Figure 34: Chicken cornea explant infections (A) rG1-G2 was expressed in chicken corneal explants that had been either cut into small pieces (cut) or scratched with a needle (scratched) prior to inoculation. Media (M) and corneal extracts (E) were collected after 5 days and resolved on SDS-PAGE gels and visualised by fluorography. (B) rG1-G2 expression was compared in cut corneas at three different temperatures, 37˚C, room temperature, and 4˚C. After 5 days corneal extracts were digested with keratanase I, keratanase II, and endo-β- galactosidase (+), alongside an undigested substrate (-). As a control, a sample from figure 33 was included for analysis by SDS-PAGE and fluorography.
125 Chapter IV: Optimising the expression of rG1-G2
Expression of rG1-G2 by primary bovine keratocytes
Primary bovine keratocytes were the next cell type to be infected. Keratocytes were isolated and infected with vTF7-3 and vG1-G2. Again, two types of experiment were designed to test for the expression rG1-G2: a Vaccinia dose experiment where increasing amounts of Vaccinia were used to infect cells; and a timecourse experiment, where cultures were inoculated with a fixed concentration of Vaccinia and then allowed to incubate for periods up to 4 days.
TF7-3 pfu/cell 0 2110 5 20 10 30 15 40 20 G1-G2 pfu/cell 0 1 5 10 15 20
250 -
150 -
100 -
75 -
123 4 5 6 78910 11 Figure 35: rG1-G2 expression in primary bovine keratocytes – Vaccinia dosage response Primary bovine keratocytes were infected with increasing amounts of Vaccinia TF7-3 and Vaccinia G1-G2 (lanes 2-11). A single uninfected culture was included as a control (lane 1). Cells were cultured in the presence of 35S-methionine and incubated for 40 hours.
126 Chapter IV: Optimising the expression of rG1-G2
To determine an appropriate viral load for rG1-G2 expression, primary bovine keratocytes were infected with increasing amounts of Vaccinia from 1 pfu/cell of Vaccinia TF7-3 and Vaccinia G1-G2 to 20 pfu/cell and the cultures incubated for approximately 40 hours. The amount of rG1-G2 expressed was proportional to the amount of Vaccinia used to inoculate the cultures; this was similar to the rG1-G2 expressed in primary chicken keratocytes (figure 35). After 40 hours incubation, cultures inoculated with 1 pfu/cell did not produce detectable levels of rG1-G2, a minimum of 5 pfu/cell appeared to be required for expression of rG1-G2 40 hours post-inoculation.
A timecourse of rG1-G2 production over 4 days was done to determine an optimal incubation period for maximal amounts of rG1-G2 production. From the results shown in figure 30 and 35, a concentration of 5 pfu/cell is adequate for the production of rG1-G2 over a period of 40 hours. This concentration was used in the timecourse experiment. Primary bovine keratocytes were co- infected with 5 pfu/cell of Vaccinia TF7-3 and Vaccinia G1-G2 and incubated for up to 96 hours. Every 12 hours samples were collected for analysis by SDS-PAGE (figure 36A). Results showed that when co-infected with 5 pfu/cell of Vaccinia TF7-3 and Vaccinia G1-G2, secretion of rG1-G2 began 24 hours post-inoculation, and continued to accumulate until the final time point at 96 hours (figure 36B). The timecourse experiment demonstrated the need for incubation periods greater than 24 hours for maximal amounts of rG1-G2 to be produced in these primary cells.
127 Chapter IV: Optimising the expression of rG1-G2
2 2 2 2 2 2 2 2 -G -G -G -G -G -G -G -G A 1 1 1 1 1 1 1 1 /G /G /G /G /G /G /G /G -3 -3 -3 -3 -3 -3 -3 -3 7 7 7 7 7 7 7 7 TF7-3 F TF7-3 F TF7-3 F TF7-3 F TF7-3 F TF7-3 F TF7-3 F TF7-3 F T T T T T T T T
250 -
150 -
100 -
75 -
12h 24h 36h 48h 60h 72h 84h 96h
B 25
20
15
10
5 Relative Pixel Density Pixel Relative
0 2030 40 50 60 70 80 90 100 Time (hours)
Figure 36: Timecourse of rG1-G2 expression in bovine keratocytes (A) Primary bovine keratocytes were infected with 10 pfu/cell Vaccinia TF7-3 alone as a control, or co-infected with 5 pfu/cell of Vaccinia TF7-3 and Vaccinia G1-G2 for 4 days in the presence of 35S- methionine. Samples were analysed by SDS-PAGE and fluorography. (B) The fluorograph was scanned using densitometry and each time point was plotted against relative pixel density.
Keratan sulphate modification was examined for each time point by comparing the keratanase-digested rG1-G2 to the undigested rG1-G2 (figure 37A). The results show that 24 hours post inoculation there was very little difference between the keratanase-digested (+) and undigested rG1-G2 (-), indicating only a slight amount of keratan sulphate was present. However the degree of keratan sulphate substitution appeared to increase by 36 hours with a total of approximately 5 kDa keratan sulphate. This level of glycosylation remained stable to the end of the timecourse. To examine the response to individual enzymes, rG1-G2 was digested with either chondroitinase ABC,
128 Chapter IV: Optimising the expression of rG1-G2 endo-β-galactosidase, keratanase II, or keratanase I (figure 37B). As expected, rG1-G2 was completely resistant to digestion by chondroitinase ABC, indicating a complete lack of chondroitin sulphate. However, a shift in migration was observed after digestion with the three keratanases; the greatest change in size was seen after digestion with endo-β-galactosidase, suggesting that the region closest to the linkage region of KS may be less sulphated than the terminal ends of the KS chains. This would explain why endo-β-galactosidase digestion was able to release more KS than the keratanase digestions, which prefer more sulphated regions of KS.
These results demonstrating the presence of 5 kDa of keratan sulphate substituted on rG1-G2 represent the first report of a recombinant protein substituted with chemically-detectable amounts of keratan sulphate.
129 Chapter IV: Optimising the expression of rG1-G2
K’ase -+-+-+-+-+-+-+ A 250 -
150 -
100 -
75 -
24h 36h 48h 60h 72h 84h 96h
d te C es B ig A nd h' U C EGβ KII KI B
1 2 3 45 Figure 37: Analysis of the keratan sulphate substituted on rG1-G2 (A) Keratan sulphate substituted on rG1-G2 at each stage of the timecourse was determined by digestion with a cocktail of keratanases (keratanase I, keratanase II, and endo-β-galactosidase). (B) rG1-G2 was digested with chondroitinase ABC (Ch’ABC), endo-β-galactosidase (EβG), keratanase II (KII), or keratanase I (KI).
Finally I examined the effect of FGF2 on KS synthesis. Experiments in chapter III showed that keratan sulphate synthesis on endogenous proteoglycans was enhanced by adding low concentrations of FGF-2 to culture medium. Figure 38 shows the rG1-G2 produced in bovine keratocytes supplemented with 2% FCS and 0.1 ng/mL FGF-2. Keratanase digestion shows that the amount of substituted keratan sulphate was approximately 5 kDa, similar to the rG1-G2 produced by cells not supplemented with FGF-2. Thus, FGF-2 supplements were unable to significantly increase keratan sulphate substitution on rG1-G2 in the presence of Vaccinia virus infection.
130 Chapter IV: Optimising the expression of rG1-G2
k’ase - + 220 -
97.4 -
Figure 38: FGF-2 and its effect on rG1-G2 KS substitution rG1-G2 expressed in primary bovine keratocytes was cultured in medium supplemented with 35S- methionine, 2% FCS and 0.1 ng/mL FGF-2. rG1-G2 was digested with keratanase I, keratanase II, and endo-β-galactosidase (+) and compared with the undigested rG1-G2 (-).
131 Chapter IV: Optimising the expression of rG1-G2
Discussion
In the previous chapter, cell lines and primary cells were screened for their ability to synthesise keratan sulphate on endogenous proteoglycans; the 35 results of [ S]SO4-labelling were agreeable with reports in the literature indicating that cell lines or cells that had been extensively cultured produced very little keratan sulphate (Midura et al. 1989; Nakazawa et al. 1995; Funderburgh et al. 1996; Nakazawa et al. 1996; Nakazawa et al. 1997; Long 35 et al. 2000). Conclusions from [ S]SO4-labelling were that primary corneal cells were the most appropriate type of cell for the expression of keratan sulphate-substituted rG1-G2.
Keratan sulphate substitution on recombinant, rather than endogenous proteins, has been investigated in this chapter. The incubation period was an important consideration throughout this chapter. Not only was keratan sulphate synthesis highly dependent on the incubation period, but so too was the rG1-G2 production. From the timecourse experiments, extended periods were optimal for the greatest yield of rG1-G2, however long incubation periods impact on keratan sulphate synthesis (Long et al. 2000). So a fine balance was required. From the results of the timecourse experiment performed on primary bovine keratocytes, shown in figure 36, rG1-G2 continued to accumulate to 96 hours, and it is likely that rG1-G2 may have continued to accumulate beyond this time. However, because keratan sulphate synthesis is drastically reduced after a few days in culture (Long et al. 2000), rG1-G2 produced after 96 hours may not be able to sustain keratan sulphate synthesis levels to substitute 5 kDa of keratan sulphate on rG1-G2. The incubation period of 96 hours is three times longer than that used to produce recombinant biglycan using the same type of expression system, which served as a prototype for the production of rG1-G2 (Hocking et al. 1996). Biglycan is a small proteoglycan substituted with either chondroitin sulphate or dermatan sulphate chains, depending on the tissue in which it is expressed. For the expression of biglycan, an incubation period of 30 hours was sufficient for the production of glycosylated biglycan substituted with up to 40 kDa of chondroitin sulphate. Unlike keratan sulphate, chondroitin sulphate
132 Chapter IV: Optimising the expression of rG1-G2 synthesis is not affected by extended periods of tissue culture, therefore the high levels of glycosylation detected on recombinant biglycan is expected.
Prior to rG1-G2 production, a collection of different cell lines and primary cells were infected with Vaccinia B5R-GFP. Although the results had no bearing on rG1-G2 production or yield, they did give a good indication of the response of each type of cell to infection by Vaccinia. While the cell lines screened in chapter III had been shown to be unsuitable for keratan sulphate synthesis, they were included because their keratan sulphate levels had yet to be confirmed and were therefore not yet eliminated as possible hosts. The neuroblastoma cell line, SK-N-MC, did not appear to express high levels of the fluorescent protein following infection by Vaccinia; after incubation with the virus, cells lost their fibroblastic-like appearance, and appeared much rounder with very few cellular processes. The results did not change when cultures were inoculated with a large viral dose, suggesting that the type of cell may influence the expression of B5R-GFP.
Of much more interest were the four remaining cell lines, all originating from the cornea. The two groups of primary cells were bovine and chicken keratocytes. Both were capable of producing the fluorescent protein. Judging by the amount of blebbing and the change from a fibroblastic to a spherical morphology, the primary bovine keratocytes appeared to be more susceptible to Vaccinia infection than the chicken keratocytes, however there did not appear to be any noticeable difference in fluorescence, suggesting that the expression of B5R-GFP in both types of primary cell were equally efficient.
In contrast to the primary bovine keratocytes, their cell line counterpart, the CRL-2048 cells proved to be completely inefficient at expressing B5R-GFP, with very little fluorescent protein detected with any viral concentration. Interestingly, bovine keratocytes that had been passaged once, also did not express high levels of B5R-GFP (results not shown). The results of the Vaccinia B5R-GFP infections showed that even inoculations containing very low numbers of viral particles were efficient at infecting cells and producing recombinant protein. After testing the Vaccinia expression system with HT-
133 Chapter IV: Optimising the expression of rG1-G2
1080 cells to confirm that rG1-G2 could be expressed, primary chicken keratocytes and primary bovine keratocytes were infected with Vaccinia and the rG1-G2 was analysed. Infected chicken keratocytes showed a proportional increase in rG1-G2 with the viral dose; this accumulation in recombinant protein was not apparent when cells were infected with Vaccinia B5R-GFP. B5R is a 42 kDa membrane protein of Vaccinia. It is required for the conversion of intracellular mature virus (IMV) to extracellular enveloped virus (EEV), and its expression occurs along with 200 other viral genes (Mathew et al. 2001). Therefore the amount of B5R is related to the number of viral particles present. In contrast, rG1-G2 is an entirely foreign protein which is secreted and its expression is only dependent on the presence of T7 RNA polymerase. This may explain the discrepancies between the two dose- related effects of the Vaccinia-expressed proteins.
Another product produced in chicken keratocytes with rG1-G2, was a protein resolving as a sharp 100 kDa band just below the rG1-G2 band (figure 33). Initially, the sharp 100 kDa band was thought to be an unglycosylated form of rG1-G2 and the larger, more diffuse 110 – 120 kDa band was thought to be the glycosylated form of rG1-G2. If true, these results would have been similar to those reported for the production of recombinant biglycan, a chondroitin sulphate proteoglycan (Hocking et al. 1996). In their study, recombinant biglycan was present as a series of glycoforms, with proteins containing very high levels of chondroitin sulphate substitution to very little substitution. In this chapter, analysis of the proteins produced after infecting primary chicken keratocytes, showed that the sharp, 100 kDa band was not 1C6-positive and was therefore not rG1-G2, while the larger 110 – 120 kDa was confirmed as rG1-G2. However subsequent keratanase digests showed that the rG1-G2 was not substituted with keratan sulphate. This was surprising given that primary chicken keratocytes produce up to 30% keratan sulphate on endogenous proteoglycans in culture (chapter III, table 9). One explanation for this is an incompatibility of the chicken keratocytes with the Vaccinia expression system.
134 Chapter IV: Optimising the expression of rG1-G2
Initially, the lack of keratan sulphate substitution on chicken keratocyte- expressed rG1-G2 was thought to be due to the manner in which the keratocytes were cultured. Keratan sulphate synthesis in chicken corneal explants has been reported (Nakazawa et al. 1995) therefore attempts were made to infect corneal explants with Vaccinia. Notwithstanding the very low yield, the rG1-G2 was resistant to keratanase, indicating a complete lack of keratan sulphate.
Soon after screening of primary chicken keratocytes for glycosylated rG1-G2, a report of recombinant aggrecan that was 5D4-positive was presented at a conference (Hering et al. 2002). In the conference abstract, Hering et al. reported the production of 5D4-positive, recombinant aggrecan in COS-7 cells, indicating the presence of keratan sulphate. Intrigued by the possibility of a cell line that was capable of keratan sulphate production, testing of the cell line with Vaccinia was initiated. Initial experiments showed that rG1-G2 production in this cell line was as effective as chicken keratocytes. While the rG1-G2 produced in the cell line was 5D4-positive, the keratan sulphate was resistant to keratanases and the rG1-G2 showed no change in size by SDS- PAGE. As the 5D4 epitope is highly sulphated and keratanase II readily cleaves keratan sulphate of this type, a change in mass, as shown by a shift in migrating position on gels, was expected. However, because there was no size difference, I concluded that only minuscule amounts of keratan sulphate were present. The degree of KS substitution was not suitable for my needs.
Finally, primary bovine keratocytes were examined for rG1-G2 production. Keratan sulphate synthesis by keratocytes has been extensively studied by Funderburgh et al. (Funderburgh et al. 1982; Funderburgh et al. 1987; Funderburgh et al. 1991; Funderburgh et al. 1996; Funderburgh 2000; Long et al. 2000; Funderburgh et al. 2001; Funderburgh 2002; Funderburgh et al. 2003). In 2000, this group showed that additions of FGF-2 to culture medium helped to maintain keratan sulphate synthesis at levels higher than cultures maintained without FGF-2 (Long et al. 2000). However in this chapter, I show that irrespective of whether FGF-2 was included in the culture medium or not, primary bovine keratocytes were capable of substituting rG1-G2 with keratan
135 Chapter IV: Optimising the expression of rG1-G2 sulphate. The degree of keratan sulphate substitution was consistently at 5 kDa. This is the first report of a recombinant protein substituted with significant amounts of keratan sulphate.
136 Chapter V: Purification of rG1-G2
Chapter V: Purification of rG1-G2
“Philosophers say a great deal about what is absolutely necessary for science, and it is always, so far as one can see, rather naive, and probably wrong.” Richard Feynman (1918 - 1988)
137 Chapter V: Purification of rG1-G2
Introduction
The steps taken to produce optimal yields of keratan sulphate-substituted recombinant G1-G2 were covered in chapter IV. Briefly, a number of different types of cells were screened for their ability to modify rG1-G2 with keratan sulphate. Primary bovine keratocytes were the only type of cell able to produce rG1-G2 with keratan sulphate. The amount of substituted keratan sulphate was estimated to be approximately 5 kDa. In this chapter, I describe the procedures required to purify a large preparation of rG1-G2 as well as the visualisation of the purified recombinant protein by rotary shadowing electron microscopy.
138 Chapter V: Purification of rG1-G2
Results
Purification of bovine-expressed rG1-G2
For keratan sulphate analysis and protease cleavage studies, larger amounts of rG1-G2 were required. It was demonstrated in chapter IV that rG1-G2 expressed in bovine keratocytes were substituted with keratan sulphate. Accordingly, a large scale infection of primary bovine keratocytes was incubated for 4 days in medium supplemented with 2% FCS. Media was collected and passed through an HA-sepharose column and unbound proteins in the flowthrough fraction was kept for analysis. The column was then washed with 5 column volumes of PBS and finally, the rG1-G2 bound to the column was eluted with 4M GuHCl. After desalting or dialysis of each fraction, the proteins were resolved by SDS-PAGE and analysed by silver stain, and Western blotting with the monoclonal antibodies 1C6 and 5D4 (figure 39).
The silver stain shows the proteins eluted with each step of the purification process. Lane 1 shows that a large amount of protein of approximately 75 kDa did not bind to the column, and after washing with PBS (lane 2) much of the remaining protein was removed, leaving behind the bound proteins which were then eluted with 4M GuHCl (lane 3). However, as well as the rG1-G2 running unusually high at 150 kDa, a significant amount of the 75 kDa protein was still present (figure 39, lane 3). Immunoblotting with antibody 1C6 (lanes 4-6) recognising the aggrecan G1 and G2 domains confirmed that the larger band was rG1-G2, while the smaller 75 kDa band was 1C6-negative. Immunoblotting for keratan sulphate with 5D4 (lanes 7-9) showed that the rG1-G2 was modified with keratan sulphate, however there remained a faint smearing of 5D4-reactive material. This smearing was most strongly 5D4- reactive from 150 - 100 kDa, however it continued beyond this point down to ~50 kDa. A distinct 75 kDa band was not identified, however since a number of keratan sulphate proteoglycans are present in the cornea, it was important to remove keratan sulphate-immunoreactivity that was not associated with G1-G2. Size exclusion HPLC was used to further purify the rG1-G2.
139 Chapter V: Purification of rG1-G2
d nd nd h d d h bou bou sh boun n lute n lute luted U Was E U Wa E Un Was E
250 -
150 -
100 - 75 -
123 4 5 6789 Silver stain 1C6 5D4
Figure 39: Purification of rG1-G2 with HA-sepharose A large scale preparation of rG1-G2 was done in primary bovine keratocytes and partially purified with HA-sepharose. Media was collected and passed through the HA-sepharose column (unbound). The column was washed with PBS (wash) and bound proteins were eluted with 4M GuHCl (eluted). The unbound, wash, and eluted samples were analysed by silver stain (lanes 1-3), and Western blot with 1C6 (lanes 4-6) and 5D4 (lanes 7-9).
The fraction containing rG1-G2 required two rounds of HPLC to adequately purify rG1-G2 from the 75 kDa product and other contaminants. The results are presented in figure 40. The semi-purified rG1-G2 from HA-sepharose chromatography was fractionated by size exclusion chromatography on a BioSep-Sec-S 4000 HPLC system. The HPLC profile from the first round of chromatography is shown in figure 40A. Four distinct peaks eluted from the column, with one major peak eluting at approximately fraction 20 (28 min). Western blotting with antibody 1C6 was used to determine where rG1-G2 had eluted (figure 40B). rG1-G2 was shown to elute in fractions 20 to 26, corresponding to the second peak of the HPLC profile (figure 40A). Western blotting for keratan sulphate with 5D4 (figure 40C) showed that there were still substantial amounts of smaller keratan sulphate contaminants in the 1C6- reactive fractions.
Fractions 19 – 26 in figures 40B and 40C were pooled and refractionated on the same column to further purify rG1-G2 (figure 40D). After the second round of chromatography, two main peaks were present. Western blotting with 1C6
140 Chapter V: Purification of rG1-G2
(figure 40E) showed rG1-G2 concentrated within the first peak in fractions 21 – 23. Blotting for keratan sulphate with 5D4 (figure 40F) showed that each of the two resolved products were 5D4-reactive. The faster migrating keratan sulphate products that were 1C6-negative were not completely removed by the second round of HPLC fractionation. 5D4 immunoreactivity was still present as a smear from 75 – 150 kDa (figure 40F). This purification procedure was unable to produce sufficiently pure rG1-G2.
One of the goals of my project was to characterise the keratan sulphate microstructure of rG1-G2, so it was important that any endogenous corneal keratan sulphate be removed. Therefore, modifications to the purification procedure were required.
141 Chapter V: Purification of rG1-G2
Fraction A) 5 10 15 25 30 35 40 45
Fraction
D) 5 10 15 20 25 30 35 40 45 Absorbance (278 nm) Absorbance (278 nm)
10 20 30 40 50 60 10 20 30 40 50 60 Time (Min) Time (Min)
18 19 20 21 22 23 24 25 26 20 21 22 23 24 B) -250 E) -250 -150 -150 -100 -100 -75 -75
-50 -50
18 19 20 21 22 23 24 25 26 20 21 22 23 24 C) -250 F) -250 -150 -150
-100 -100 -75 -75
-50 -50
Figure 40: Purification of rG1-G2 with HPLC After partial purification of rG1-G2 with HA-sepharose, the eluted sample was further purified using size exclusion HPLC. After the first round (A) fractions were analysed by SDS-PAGE and Western blotting with 1C6 (B) and 5D4 (C). Fractions 19-26 were pooled and refractionated on the same column (D), fractions were again analysed by Western blot using 1C6 (E) and 5D4 (F)
142 Chapter V: Purification of rG1-G2
Previous attempts to purify rG1-G2 to homogeneity were unsuccessful. Because HA-sepharose was the faster and more efficient of the two chromatographic techniques used, efforts were made to reduce the level of non-specific binding of proteins to HA-sepharose.
To increase the purity of rG1-G2 eluted from HA-sepharose, two salt washes were included following the PBS wash. After the initial PBS washes, 5 column volumes of 0.5M NaCl were passed through the column, followed by 5 column volumes of 1M NaCl.
l l l l l l C l C C l C C l C a C H a C H a C H N a u N a u N a u M N G M N G M N G .5 M M .5 M M .5 M M 0 1 4 0 1 4 0 1 4 250 - 250 - 150 - 150 - 100 - 100 - 75 - 75 -
50 - 50 - 37 - 37 -
A) 5-D-4 B) 1-C-6 C) Silver stain
Figure 41: Enhanced purification of rG1-G2 with HA-sepharose The purification of rG1-G2 with HA-sepharose was modified to reduce non-specific binding of contaminant proteins. After passing harvested media through the HA-sepharose column, the column was washed with 0.5M NaCl, and 1M NaCl, before eluting with 4M GuHCl. Samples were analysed by Western blot using (A) 5D4 and (B) 1C6, and by (C) silver stain
Samples were analysed by Western blotting with 1C6 and 5D4 to confirm the presence of rG1-G2 in the eluted fraction and to determine the amount of keratan sulphate in each fraction. From the results of the 5D4 blot (figure 41A), the majority of the keratan sulphate eluted with the 0.5M NaCl wash, including all of the contaminating keratan sulphate products seen in figures 40C and F. The identity of the rG1-G2 band resolving at ~120 kDa was confirmed by 1C6 (figure 41B); this band was also positive for 5D4 reactivity,
143 Chapter V: Purification of rG1-G2 and contained none of the smaller contaminating keratan sulphate products seen in the 0.5M NaCl wash. Silver staining (figure 41C) showed general staining of protein of all sizes eluting with the 0.5M NaCl wash and a strongly staining rG1-G2 band migrating at ~120 kDa in the 4M GuHCl elution. The diffuse doublets migrating at ~60 kDa was keratin, and was likely introduced during analysis.
From these results, it was clear that the modified HA-sepharose method was able to purify rG1-G2 from harvested media considerably. To further purify the rG1-G2, HPLC was used to remove any remaining protein contaminants (figure 42A). rG1-G2 eluted from the column first at fraction 28, appearing as the major peak, and a number of smaller peaks followed.
Analysing the fractions with 1C6 and 5D4 showed that rG1-G2 eluted as the main peak, and was present in fractions 28 - 30 (Figure 42B). Immunoblotting with 5D4 showed that fractions 28 – 33 were all positive for 5D4 (figure 42C); the presence of three additional fractions was not surprising given the very high sensitivity of the 5D4 antibody. Fractions also appeared to have slight 5D4 immunoreactivity at 75 – 100 kDa, however these represent only a very small proportion of the total KS. Because most of the 1C6 and 5D4-positive signal was detected in fraction 28-30, these fractions were pooled for further analysis.
144 Chapter V: Purification of rG1-G2
A Fraction 20 25 30 35 40 45 50 278 λ Absorbance nm Absorbance
30 40 50 60 70 Time (Min)
B 1C6 C 5D4 25 26 27 28 29 30 31 32 33 25 26 27 28 29 30 31 32 33 250 - 250 - 150 - 150 - 100 - 100 - 75 - 75 -
50 - 50 -
37 - 37 -
25 - 25 - Figure 42: Analysis of HPLC fractions (A) To further purify the rG1-G2 eluted from the HA-sepharose column, the recovered sample was fractionated on a size exclusion HPLC column under dissociative conditions. Fractions eluted from the HPLC column were resolved by SDS-PAGE and analysed by Western blot using 1C6 (B) and 5D4 (C).
Visualisation of rG1-G2 by rotary shadowing electron microscopy
Previous studies in our laboratory comparing native pig laryngeal G1-G2 with recombinant G1-G2 produced in baculovirus (G1-G2bac) (Mercuri et al. 1999) have shown that G1-G2 expressed in this system does not substitute keratan sulphate chains on the core protein, they also showed that the recombinant G1-G2 is shorter when measured by rotary shadowing electron microscopy. Having prepared highly purified recombinant G1-G2 that is 5D4-positive and therefore almost certain to contain keratan sulphate chains, it was of interest to compare my rG1-G2 glycoform with the previous G1-G2 preparations. Samples of three G1-G2 glycoforms: a preparation from native pig aggrecan, baculovirus-expressed G1-G2 and the rG1-G2 described here in this thesis
145 Chapter V: Purification of rG1-G2 were shipped to Dr. Doug Keene (Portland, Oregon, USA) for analysis by rotary shadowing (figure 43).
Unlike Kleinschmidt electron microscopy which is useful for imaging highly glycosylated aggrecan but is poor at visualising protein structures, rotary shadowing is a valuable technique for visualising proteins and was therefore used to visualise each G1-G2 glycoform. The native pig G1-G2 carries approximately 40 kDa of keratan sulphate, whereas G1-G2bac has no keratan sulphate at all (Mercuri et al. 1999). In each of the pictures, two individual G1- G2 monomers are shown. The measurements refer to the distance from the centre of one globular domain to the other.
Regardless of the amount of keratan sulphate substituted, the interglobular domain appears as a rod-like, inflexible structure, it is this characteristic that makes it possible for accurate measurements of this domain. The native G1-
G2 had an average length of 37 nm. In contrast, the G1-G2bac had a measured length of 19 nm. Interestingly, the rG1-G2 had an intermediate length of 22 nm, and together with the 5D4 immunoreactivity, suggests that the rG1-G2 does indeed carry at least some keratan sulphate. The results suggest that the degree of keratan sulphate substitution in the interglobular domain may be proportional to the length of the interglobular domain.
146 Chapter V: Purification of rG1-G2
37nm 22nm 19nm A) B) C)
Native G1-G2 rG1-G2 G1-G2bac
Figure 43: Rotary shadowing electron microscopy of G1-G2 Rotary shadowing electron microscopy was used to directly visualise three G1-G2 glycoforms: (A) native G1-G2 prepared from pig laryngeal cartilage, containing approximately 40 kDa of keratan sulphate; (B) rG1-G2 which had been expressed in primary bovine keratocytes using the Vaccinia TF7-3 RNA expression system, and containing approximately 5 kDa of keratan sulphate (chapter IV & V); and (C) G1-G2 expressed using a baculovirus expression system, containing no keratan sulphate. Each picture shows two G1-G2 monomers.
147 Chapter V: Purification of rG1-G2
Discussion rG1-G2 was generated in primary bovine keratocytes using a Vaccinia T7 RNA polymerase expression system. Tests showed that the protein was substituted with 5 kDa of keratan sulphate. This chapter describes the steps taken to purify a large scale preparation of rG1-G2 to homogeneity.
The hyaluronic acid-binding properties of aggrecan were used to purify rG1- G2 from harvested culture medium using an HA-sepharose column. Initial attempts to purify rG1-G2 resulted in a heterogeneous mixture of the 120 kDa rG1-G2 as well as lesser amounts of an unknown keratan sulphate-containing 75 kDa protein, which was negative for 1C6. Western analysis for keratan sulphate with 5D4 was used primarily to identify contaminating keratan sulphate products, and therefore determine the level of purity of recovered rG1-G2. Using the basic HA-sepharose method, a wide range of contaminating keratan sulphate products, most likely from endogenous stromal keratan sulphate proteoglycans such as lumican and keratocan, were eluted with rG1-G2.
Attempts to remove the contaminants using size exclusion HPLC were not successful, therefore modifications to the HA-sepharose method were made to reduce non-specific binding of proteins to HA-sepharose. By adding two sequential salt washes of increasing concentration, I was able to purify rG1- G2 to near-homogeneity. Subsequent HPLC fractionation removed any remaining contaminants, as shown by 5D4 Western analysis (figure 42C). In addition to the strong 5D4-immunoreactivity of the 120 kDa rG1-G2, weak immunoreactivity was also seen as far down as 75 kDa. However, due to the extraordinary sensitivity of the 5D4 antibody, and the very weak signal returned, the smaller keratan sulphate-containing proteins represent only a tiny proportion of the total 5D4 immunoreactivity.
Having purified the rG1-G2 to homogeneity, rotary shadowing electron microscopy was used to visualise the protein and compare it with two other glycoforms, one expressed with baculovirus containing no keratan sulphate,
148 Chapter V: Purification of rG1-G2 and the other native G1-G2 purified from extracts of pig laryngeal cartilage. By comparing the three glycoforms, I was able to show that one effect that keratan sulphate has on G1-G2 is to lengthen the interglobular domain. From the rotary shadowing, it appears that the length of the interglobular domain is proportional to the amount of keratan sulphate. It is unknown whether this is due to differences in the number of keratan sulphate chains, or whether it is due to differences in chain length. The rotary shadowing also confirms that the rG1-G2 preparation was highly purified, as shown by the lack of any contaminating proteins.
149 Chapter VI: Keratan sulphate analysis of rG1-G2
Chapter VI: Keratan sulphate analysis of rG1-G2
“The most exciting phrase to hear in science, the one that heralds new discoveries, is not 'Eureka!' (I found it!) but 'That's funny ...' “ - Isaac Asimov (1920 - 1992)
150 Chapter VI: Keratan sulphate analysis of rG1-G2
Introduction
Recombinant G1-G2 was expressed in primary bovine keratocytes and purified using a combination of HA-sepharose and HPLC. The purified rG1-G2 was different to the native G1-G2; the native trypsin-generated pig G1-G2 was modified with approximately 30 - 40 kDa of keratan sulphate (Mercuri et al. 1999), whereas the rG1-G2 expressed in bovine keratocytes was substituted with only 5 kDa of keratan sulphate.
The primary aim of this project is to determine whether keratan sulphate affects aggrecanase-mediated cleavage of aggrecan. Another important aim is to determine whether there is any structural component of the keratan sulphate chains that mediates this possible interaction. Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) is the most efficient technique to study carbohydrate structures. Not only is it simpler and faster than alternative carbohydrate analytical techniques, it is also relatively sensitive, capable of detecting samples at nanomolar concentrations. It was therefore important that the rG1-G2 was highly purified because any contaminating keratan sulphate present on endogenous corneal proteoglycans could potentially be detected and skew the results.
Having purified rG1-G2 from primary bovine keratocytes, the next step was to analyse the keratan sulphate on the rG1-G2 core protein. This was accomplished using two keratan sulphate-specific enzymes, keratanase II, and endo-β-galactosidase. These enzymes cleave highly sulphated disaccharides or less sulphated disaccharides respectively, allowing the overall degree of sulphation to be determined. Other types of keratan sulphate modifications can be examined with other enzymes. Two enzymes that will be used are neuraminidase, which preferentially cleaves neuraminic acid (more commonly known as sialic acid) and fucosidase, which cleaves fucose residues. The four enzymes combined allow for the identification of the most common components of keratan sulphate chains – disaccharide sulphation, sialic acid capping, and fucose modifications. A more thorough description of the individual enzyme specificities is described below.
151 Chapter VI: Keratan sulphate analysis of rG1-G2
Specificities of keratan sulphate-degrading enzymes
Keratan sulphate is primarily made up of repeating subunits of Galβ1- 4GlcNAc polymerised by β1-3 linkages. Sulphation occurs at the C6 position on Gal and/or GlcNAc. In the first chapter, a diagram of corneal and articular cartilage keratan sulphate was shown. For convenience, it has been included in this chapter (figure 44). The figure shows the two most common forms of keratan sulphate: KSI, which includes the KS chains linked to the core protein via Asn residues, and KSII which includes KS chains attached to the core protein via GalNAc-O-Ser or GalNAc-O-Thr.
Keratan sulphate chains are commonly made up of two branches, a C-6 branch constituting the majority of the KS, and a C-3 branch consisting only of a few residues (as shown in figure 44). This biantennary structure is standard for both KSI and KSII. Analysis of KS chains from a variety of different species have found that sialylation occurs at the terminal ends of both C-6 and C-3 branches, however, the degree of sialylation of the C-3 branch varies between species and is often incomplete (Funderburgh 2000). In bovine corneal KS, 70% of the chains are terminated with sialic acid, and the remaining 30% are modified with βGalNAc or α-Gal (Tai et al. 1996; Tai et al. 1997). α-linked fucose is present throughout KSII chains on sulphated GlcNAc, but not within four hexose moieties of the non-reducing terminus (Brown et al. 1996). In contrast, tracheal cartilage KS is not fucosylated and is modified only with α2- 3-linked sialic acid at the terminal ends (Nieduszynski et al. 1990; Dickenson et al. 1991).
152 Chapter VI: Keratan sulphate analysis of rG1-G2
KSI
NeuAc2-3/6 (SO4 ) SO4 SO4 SO4 SO4 NeuGc2-3/6 6 6 6 6 6 Galα 1-3 Fuc -Gal-4GlcNAc-(3Gal-4GlcNAc)8 - 34 -(3Gal-4GlcNAc)10 - 12 -(3Gal-4GlcNAc)2 -2Man (SO )GalNAc1-3 4 6 6 Man-4GlcNAc-4GlcNAc-N-Asn (SO4 )GlcNAc1-3 3 NeuAc2-3Gal-4GlcNAc-2Man
KSII
(SO4 ) SO4 SO4 (SO4 ) SO4 6 6 6 6 6 NeuAc2-3/6 Gal-4GlcNAc-3Gal-4GlcNAc-(3Gal-4GlcNAc)3 - 11 3 Fuc 6 GalNAc-O-Ser/Thr 3 NeuAc2-3Gal
KSIII
SO4 6 (Gal-4GlcNAc)7-24 - 3Man-O-Ser/Thr Figure 44: The structures of KSI, KSII, and KSIII Figure adapted from (Krusius et al. 1986; Funderburgh 2000). NeuGc (N-glycolylneuraminic acid); NeuAc (N-acetylneuraminic acid); Gal (galactose); GalNAc (N-acetylgalactosamine); GlcNAc (N- acetylglucosamine); Man (mannose); Asn (asparagine); Ser (serine); Thr (threonine); Fuc (fucose). The linkage regions to the core protein are highlighted in bold.
Keratanase II (Bacillus sp. Ks36), (EC 3.2.1) Keratanase II specifically hydrolyses the β1–3 glucosaminidic linkages of keratan sulphate chains. For cleavage to occur, the GlcNAc residue must be sulphated at the 6 position. Therefore keratanase II cleavage of KS results in two main disaccharide products: Galβ1-4GlcNAc6S, and Gal6Sβ1- 4GlcNAc6S. Keratanase II does not efficiently cleave tetrasaccharides, therefore as well as the two disaccharide products, tetrasaccharides may also be present. More complex structures are also possible in which the disaccharides are modified with other residues such as sialic acid or fucose.
Endo-β-galactosidase (EC 3.2.1.103)
Endo-β-galactosidase specifically hydrolyses the internal β1-4 galactosyl linkages of keratan sulphate chains. Cleavage of KS by this enzyme requires that the Gal residue remain unsulphated, although sulphation may occur on
153 Chapter VI: Keratan sulphate analysis of rG1-G2 the GlcNAc residue. Hydrolysis of keratan sulphate by endo-β-galactosidase results in the following products: GlcNAc-Gal and GlcNAc6S-Gal. As with the keratanase II products, more complex products are also possible in which other residues are present, such as sialic acid.
Neuraminidase (EC 3.2.1.18) and Fucosidase (EC 3.2.1.51)
Neuraminidase catalyses the hydrolysis of α2-3, α2-6, and α2-8 glycosidic linkages of terminal sialic residues in oligosaccharides. The fucosidase used was α(1-3/4) fucosidase. α(1-3/4) fucosidase catalyses the hydrolysis of α1-3 or α1-4 linkages of fucose residues in oligosaccharides.
154 Chapter VI: Keratan sulphate analysis of rG1-G2
Results
Establishing standards
Fluorophore-assisted carbohydrate electrophoresis resolves carbohydrates according to their conformation, charge, and mass. The resolution of carbohydrates is therefore difficult to predict. Initially, commercially-available bovine corneal keratan sulphate was used to become familiar with the techniques and to confirm that the specific hydrolase products described in the literature (Plaas et al. 2001) could be identified. Commercial KS was digested with keratanase II, endo-β-galactosidase, and neuraminidase, labelled with AMAC, and analysed with FACE. In collaboration with Dr. Anna Plaas who pioneered the technique of FACE analysis for keratan sulphate, many of the products resulting from keratanase II and endo-β-galactosidase digestion were able to be identified. The main products identified were a mixture of monosulphated and disulphated di-, tri- and tetrasaccharides. These are summarised in table 10.
Table 10: Primary keratan sulphate catabolic products of keratanase digestion Keratanase II Endo-β-galactosidase Gal-GlcNAc6S GlcNAc-Gal SA-Gal-GlcNAc6S GlcNAc6S-Gal Gal6S-GlcNAc6S SA-Gal-GlcNAc6S-Gal
Quantitation by FACE
The carbohydrates for analysis are derivatised with 2-aminoacridone by reductive amination; the stoichiometry of the labelling reaction is 1:1 so resolved products can be quantitated based on a set of mass standards. Quantitation is based on manual densitometric readings made from captured images. Monosaccharide bands were manually selected and their mass values plotted against relative pixel densities. Based on multiple experiments, it was found that carbohydrates at concentrations in the range of 0.01 - 0.5 nmoles can be quantitated with reasonable accuracy, as reported previously in the literature (Calabro et al. 2001; Plaas et al. 2001). Because the
155 Chapter VI: Keratan sulphate analysis of rG1-G2 fluorophore labelling of carbohydrates is stoichiometric, the technique is quantitative, and any combination of monosaccharides or oligosaccharides that resolve from each other on gels can be used as standards after stoichiometric tagging at their reducing ends. Figure 45 shows a variety of different carbohydrate standards at the upper end of the scale from 0.1 to 0.5 nmoles.
25 GlcNAc6S
20 Gal6S Fuc - 15 Gal - GlcNAc GlcNAc - 10 Gal pixel density 5 Fuc Gal6S - 0.1 0.2 0.3 0.4 0.5 GlcNAc6S - nmoles monosaccharide
Figure 45: Quantitation of monosaccharides The stoichiometric labelling of samples for FACE enables accurate quantitation by scanning densitometry.
Optimisation of fucosidase digests
Among the enzymes used in FACE analysis, fucosidase was initially the most unreliable. Based on the suggested incubation times as well as the digest conditions published in the literature (Plaas et al. 2001), unfortunately fucosidase digestion did not appear to be very efficient. It was not until a fucosidase timecourse was performed using a Lewis-a tetrasaccharide (α- NeuNAc-(2 → 3)-β-D-Gal-(1 → 3)-(α-L-Fuc-[1 → 4])-D-GlcNAc), that the required incubation time of 72 hours was determined (figure 46). As shown in figure 46, after 24 hours, the fucosidase digestion is only partially complete, and it is only after 72 hours that all of the fucose residues have been released from the Lewis-a tetrasaccharide. This extended incubation period of 72 hours was used for all subsequent fucosidase digestions.
156 Chapter VI: Keratan sulphate analysis of rG1-G2
0 24 72h
- Fuc Lewis a - - SA-Gal-GlcNAc
Figure 46: FACE analysis of bovine corneal KS Lewis-a tetrasaccharide was digested with fucosidase for 0, 24, or 72 hours. The products were labelled with AMAC for FACE analysis. After 72 hours, the Lewis-a tetrasaccharide was completely digested, releasing all of the substituted fucose moieties.
Comparison of the KS on rG1-G2 and native G1-G2
Two of the three available G1-G2 substrates for aggrecanase studies are substituted with keratan sulphate; pig G1-G2 is substituted with up to 40 kDa of keratan sulphate, and rG1-G2 expressed in primary bovine keratocytes is substituted with approximately 5 kDa of keratan sulphate. The keratan sulphate of both species is indistinguishable by Western blot using 5D4 (chapter V), hence the need for FACE to identify differences in the microstructure. rG1-G2 expressed by baculovirus does not carry any keratan sulphate chains and therefore cannot be analysed by FACE (Mercuri et al. 1999).
Keratan sulphate from rG1-G2 and pig G1-G2 was digested with keratanase II and endo-β-galactosidase. The digested products were then filtered through 3 kDa molecular weight cut-off spin columns to recover saccharide products in the filtrate and remove the core protein with attached linkage region in the retentate. Aliquots of the saccharide products were then digested with neuraminidase to identify sialic acid capping. The samples were then analysed by FACE. The results are shown in figure 47.
157 Chapter VI: Keratan sulphate analysis of rG1-G2
N + N I I + N G G + + N G a) I β β b) β β K KI E E KII KII EG E
Fuc Gal GlcNAc * Gal-GlcNAc6S-Gal GlcNAc6S-Gal Gal-GlcNAc6S-Gal
SA-Gal-GlcNAc6S-Gal SA-Gal-GlcNAc6S Gal6S GlcNAc6S *
* Gal6S-GlcNAc6S 12 3 4 567 89
Figure 47: FACE analysis of pig G1-G2 and rG1-G2 KS Keratan sulphate from purified rG1-G2 (a) and native pig G1-G2 (b) was digested with keratanase II, endo-β-galactosidase, and neuraminidase. The products were labelled with AMAC and resolved on Glyko monocomposition gels. Non-specific bands have been marked with asterisks.
The results show that the keratan sulphate of rG1-G2 is simple and contains very little sialic acid. From the keratanase II digestion (lane 2), there were two main products, the monosulphated Gal-GlcNAc6S and the disulphated Gal6S- GlcNAc6S. There was also a very faint SA-Gal-GlcNAc6S band resolving just above the Gal6S marker, indicating a very small degree of sialic acid capping present on the ends of the keratan sulphate chains. In lane 3, with an additional neuraminidase digestion, the faint sialic acid-containing carbohydrates from lane 2 have disappeared, proving that there was indeed, a small amount of sialic acid present, although the low concentration of these bands made it difficult to quantitate.
Endo-β-galactosidase digests (lane 4) showed a single main product identified as GlcNAc6Ss-Gal and three non-specific bands marked with asterisks (figure 47). As in the keratanase II digestion, there was a very faint band resolving a little further than the GlcNAc6S-Gal band identified as SA-Gal-GlcNAc6S-Gal (marked with an arrow). This SA-Gal-GlcNAc6S-Gal band disappeared after neuraminidase digestion (lane 5), converting to Gal-GlcNAc6S-Gal (also marked with an arrow) and sialic acid. These products were not able to be
158 Chapter VI: Keratan sulphate analysis of rG1-G2 quantitated in the rG1-G2 sample but are clearly visible in the pig sample in lanes 8 and 9.
FACE analysis on pig G1-G2 keratan sulphate gave clearer results because the amount of keratan sulphate available was much greater. In keratanase II digestions, three main bands appeared (lane 6); the monosulphated disaccharide Gal-GlcNAc6S, the monosulphated trisaccharide SA-Gal- GlcNAc6S, and the disulphated disaccharide Gal6S-GlcNAc6S. The presence of sialic acid was proven with a subsequent neuraminidase digestion (lane 7) with the conversion of the SA-Gal-GlcNAc6S band to Gal-GlcNAc6S and sialic acid (which resolves as three distinct bands; a doublet and a diffuse band resolving quite high on the gel).
As well as a number of unidentified fainter bands, three main bands resulted from endo-β-galactosidase digestion (lane 8), two of which have been identified as the monosulphated disaccharide GlcNAc6S-Gal, and the monosulphated tetrasaccharide SA-Gal-GlcNAc6S-Gal. The additional neuraminidase digestion resulted in a monosulphated trisaccharide Gal- GlcNAc6S-Gal and a monosulphated disaccharide GlcNAc6S-Gal.
Analysis of FACE results
The microstructure of keratan sulphate substituted on native pig G1-G2 and recombinant human G1-G2 was analysed by comparing the ratio of the products. Two ratios were calculated. The first was the ratio of monosulphated disaccharides to disulphated disaccharides in keratanase II digests. This gives an indication of the degree of sulphation. The second result was the ratio of sialic acid to total number of disaccharides. Assuming that sialic acid capping is complete, this ratio gives an indication of the average length of the keratan sulphate chains. Quantitation of the amount of unsulphated disaccharides in rG1-G2 was not possible, this suggests that the KS chains of rG1-G2 were made up primarily of disaccharides containing at least one sulphate group.
159 Chapter VI: Keratan sulphate analysis of rG1-G2
The pig G1-G2 keratan sulphate was slightly more sulphated than the rG1-G2 keratan sulphate, with a monosulphated to disulphated disaccharide ratio of 1 : 1.1. In comparison, the ratio in the rG1-G2 keratan sulphate was 1 : 0.9. The degree of sialic acid capping in the rG1-G2 keratan sulphate was very low, with an estimated ratio of 1 sialic acid residue per 64 disaccharides. The degree of sialic acid capping in the pig G1-G2 keratan sulphate was approximately three times greater, with a ratio of 1 : 24.
If we assume that each keratan sulphate chain is capped with a sialic acid residue, then from the results shown in table 11, the KS chains on rG1-G2 are approximately three times longer than the KS on native G1-G2 extracted from pig cartilage. Alternatively, the results may suggest that the keratan sulphate modifications on rG1-G2 are incomplete, with much less sialic acid capping present. Bovine aggrecan is estimated to have approximately 5 KS substitution sites in the G1 and interglobular domains, and each has a length of up to 15 disaccharides (Barry et al. 1995), constituting up to 40 kDa in mass. The rG1-G2 in the present study has approximately 5 kDa of keratan sulphate and with the possibility of 5 KS substitution sites, we would not expect each KS chain on rG1-G2 to be made up of more than a few disaccharides. Using a molecular mass of 463 for a monosulphated KS disaccharide, a single keratan sulphate chain on rG1-G2 could not be longer than 10 – 11 disaccharides in length. If all 5 KS substitution sites were occupied, then each KS chain would not be expected to exceed 2 – 3 disaccharides in length. However, if only a few sites are substituted, the KS chains would of course be slightly longer. If we assume that each KS chain is capped with sialic acid, then it is clear that the ratio of sialic acid : disaccharides can only range from 1 : 2 to 1 : 10. Because a ratio of 1 : 64 was calculated, we can therefore assume that sialic acid capping on the KS chains is incomplete. Fucosidase digests were also done, however no fucosidase-derived products were detected, indicating a lack of fucose on the rG1-G2 keratan sulphate. Fucosidase digests of native pig KS also revealed no fucose.
160 Chapter VI: Keratan sulphate analysis of rG1-G2
Table 11: Analysis of pig G1-G2 and rG1-G2 KS – Summary of results
SO4 per disaccharide Sialic acid capping
Mono(SO4) : Di(SO4) SA : Disaccharides
Pig G1-G2 KS 1 : 1.1 1 : 24 rG1-G2 KS 1 : 0.9 1 : 64
N-glycosidase digestion of rG1-G2
Earlier studies (Barry et al. 1995) established that the G1-G2 domain of aggrecan contained multiple KS-attachment sites, one of these was at Asn368, very close to the NITEGE373 ↓ 374ARGSV cleavage site. An N-linked KS chain at this site was suggested to have a possible influence on aggrecanase cleavage. One aim of this project was to investigate this possibility. Corneal keratan sulphate is exclusively N-linked (Funderburgh 2000; Funderburgh 2002), so the keratan sulphate on rG1-G2 expressed in primary bovine keratocytes was also expected to be N-linked.
Digestion of rG1-G2 with a variety of different keratanases (figure 37B) has shown that rG1-G2 is substituted with approximately 5 kDa of keratan sulphate. To determine whether rG1-G2 contained N-linked keratan sulphate, rG1-G2 was digested with N-glycosidase or with a cocktail of keratanases containing keratanase I, keratanase II, and endo-β-galactosidase followed by N-glycosidase digestion. The samples were resolved on SDS-PAGE gels and probed with the anti-G1-G2 antibody, 1C6 (figure 48A). The antibody, 1C6, was used in Western blotting to show the mass of the core protein after digestion with each glycosidase treatment. Undigested, the rG1-G2 resolved at approximately 120-130 kDa. After N-glycosidase treatment the core protein was approximately 5 kDa smaller. The same result was seen after digestion with a cocktail of keratanases and N-glycosidase, indicating that the N-linked carbohydrates present on the core protein are entirely N-linked. To confirm whether the carbohydrates substituted on rG1-G2 is N-linked keratan sulphate, rG1-G2 was digested with N-glycosidase and then resolved on SDS-PAGE gels. Keratan sulphate was probed with 5D4 (figure 48B) and a
161 Chapter VI: Keratan sulphate analysis of rG1-G2
~130 kDa band was detected in the undigested sample. After N-glycosidase digestion, there was a very faint signal corresponding to a 5 kDa smaller protein. From the Western blot, comparing the undigested (-) with the digested (+) rG1-G2, it appears that the keratan sulphate substituted on rG1- G2 is exclusively N-linked.
- + A B K 250 - C N N 150 - 250- 100 - 150- 75 - 100- 50 - 75- 37 -
50- 25 -
Figure 48: N-glycosidase digestion of rG1-G2 A) rG1-G2 (~0.5 µg) was digested with N-glycosidase (N); a cocktail of keratanases containing keratanase I, keratanase II, and endo-β-galactosidase followed by N-glycosidase (KN). A portion (~0.2 µg) of each sample was resolved by SDS-PAGE along with an undigested control (C) and analysed by Western blot with the anti-G1-G2 antibody, 1C6. B) rG1-G2 (~0.5 µg) was digested with N- glycosidase F for 20 hours. A portion (62.5 ng protein) of both the digested (+) and undigested control (-) was resolved by SDS-PAGE and analysed by Western blot with the anti- keratan sulphate antibody, 5D4.
Preliminary analysis of the keratan sulphate linkage region
Figure 47 shows the results for FACE analysis of the keratan sulphate chain interior and capping structures. The keratanase-resistant linkage structures associated with these chains, shown in bold in figure 44, were discarded with the Microcon retentate and excluded from the analyses. In a preliminary experiment to examine the entire KS chain, including the linkage structures, the products of keratanase II and endo-β-galactosidase were not passed through Microcon columns. Instead, the entire sample was digested with neuraminidase or fucosidase.
162 Chapter VI: Keratan sulphate analysis of rG1-G2
FACE analysis of the entire keratan sulphate structures is shown in figure 49. The most noticeable difference was the appearance of intense sialic acid bands after neuraminidase digestion. Previous FACE analysis of the capping structures and interior keratan sulphate chain region detected small amounts of sialic acid on the rG1-G2 KS (figure 47), and it was only when the linkage structures were also included (figure 49), that substantial amounts of sialic acid were detected.
Since the KS of rG1-G2 is N-linked (figure 48) and is a branched, biantennary structure similar to the structure shown in figure 44, then the C-3 branch is a possible source of the sialic acid, although it does not explain why there was such an abundance. Therefore, there may also be other sialylated carbohydrates present on the core protein.
N + G β β EG E Sialic acid
Sialic acid
Figure 49: Analysis of rG1-G2 keratan sulphate including the linkage region KS chains including linkage structures were analysed by FACE after digestion with EβG, with or without subsequent digestion with neuraminidase.
These results show that rG1-G2 is substituted with keratan sulphate chains which are slightly less sulphated than the keratan sulphate of native G1-G2. However despite this, both native and recombinant G1-G2 KS species were substantially sulphated, having undetectable levels of unsulphated disaccharides. Other keratan sulphate modifications on rG1-G2 were incomplete, namely, fucosylation and sialylation.
163 Chapter VI: Keratan sulphate analysis of rG1-G2
Discussion rG1-G2 was expressed in primary bovine keratocytes; keratanase digests of the purified rG1-G2 showed that it was substituted with ~5 kDa of keratan sulphate. To gain further information about the keratan sulphate chains, fluorophore-assisted carbohydrate electrophoresis was used. If differences in aggrecanase-mediated cleavage are observed between the two G1-G2 substrates, results of the FACE analyses may help to determine whether there is any particular characteristic of the keratan sulphate chains that impart aggrecanase-inhibiting or enhancing properties.
FACE analysis of keratan sulphate from rG1-G2 and native G1-G2 keratan sulphate showed that in both cases the amount of sulphation was high, with only very low levels of unsulphated disaccharides detected. The native pig G1-G2 keratan sulphate was modified with slightly more sulphate groups than the rG1-G2 keratan sulphate. For the rG1-G2 keratan sulphate, the monosulphated : disulphated disaccharide composition was 1 : 0.9, whereas this ratio in the pig G1-G2 keratan sulphate ratio was 1 : 1.1. The slightly less sulphated keratan sulphate of rG1-G2 may have been a result of the Vaccinia expression system and the less than optimal cellular environment for keratan sulphate synthesis. The same can be said of the amount of sialic acid capping on the rG1-G2 keratan sulphate which was approximately three times less than the prototypical native G1-G2 keratan sulphate of pig laryngeal cartilage. The very low levels of sialic acid on the rG1-G2 keratan sulphate also made quantitation difficult. Therefore the final sialic acid levels should not be considered very accurate and likely have errors associated with them. Nevertheless the general conclusions made from the FACE analysis are still valid.
The initial FACE analysis was based on the keratanase II and endo-β- galactosidase-sensitive products, which constitute only the KS from the C-6 branch. This meant that the C-6 branches contained very little sialic acid capping. However, there appeared to be an abundance of sialic acid on the KS linkage region, indicating extensive sialic acid capping on the C-3 branch.
164 Chapter VI: Keratan sulphate analysis of rG1-G2
The abundance of sialic acid on the endo-β-galactosidase-resistant carbohydrates may in part, originate from the C-3 branches of keratan sulphate. Although this branch has a β1-4 galactosyl linkage for endo-β- galactosidase, the close proximity of sialic acid has been shown to interfere with cleavage by this enzyme (Nieduszynski et al. 1990; Plaas et al. 2001). However, the intensity of the sialic acid bands suggests other sources of sialic acid, possibly from other carbohydrates substituted elsewhere on the core protein.
The fact that samples digested with N-glycosidase, or N-glycosidase and keratanase, migrated at the same positions on the gels suggests that the KS attachment to rG1-G2 is exclusively N-linked (figure 48). Previous keratanase digestions (figure 37B) showed a size shift of approximately 5 kDa; digestion with N-glycosidase showed a similar shift in size. Subsequent digestion with keratanase did not reduce the mass any further, therefore the N-linked carbohydrates substituted on rG1-G2 are indeed keratan sulphate and not another type of carbohydrate. A 5D4 Western blot of the N-glycosidase- digested rG1-G2 returned a weak signal and further confirming that the N- linked oligosaccharides were keratan sulphate chains. Native G1-G2 from bovine cartilage has both N and O-linked keratan sulphate chains (Barry et al. 1995). The fact that rG1-G2 appears to have no O-linked keratan sulphate suggests that the presence of KS attachment motifs along the core protein is not sufficient for O-linked KS substitution. Additionally, it seems likely that the type of cell in which keratan sulphate is synthesised influences the type, and linkage, of KS substitution. Corneal cells do not express proteoglycans containing O-linked keratan sulphate but chondrocytes do. Therefore if chondrocytes were used to express rG1-G2 it is possible that O-linked KS would be present as well as N-linked KS.
This chapter shows that rG1-G2 is substituted with N-linked keratan sulphate which is similar, but not identical in its structure, to native G1-G2 from pig laryngeal cartilage. In addition to being substantially shorter than the prototypical native G1-G2 keratan sulphate, the rG1-G2 keratan sulphate is also slightly less sulphated, and contains significantly less sialic acid capping.
165 Chapter VI: Keratan sulphate analysis of rG1-G2
The fact that the recombinant G1-G2 is substituted only with N-linked keratan sulphate presents a unique opportunity to examine the effect of N-linked keratan sulphate on cleavage, rather than examining the effect of both O and N-linked keratan sulphate as originally planned using multiple glycoforms of G1-G2. Therefore comparisons with the native G1-G2 and baculovirus- expressed G1-G2 are superfluous. The next chapter describes aggrecanase digestions of rG1-G2 and the experiments conducted to characterise the effect of N-linked keratan sulphate on aggrecanase cleavage at the NITEGE373-374ARGSVI site.
166 Chapter VII: Characterisation of rG1-G2
Chapter VII: Characterisation of rG1-G2
“Study the past if you would define the future” - Confucius (551 BC - 479 BC)
167 Chapter VII: Characterisation of rG1-G2
Introduction
The interglobular domain of aggrecan contains attachment sites for N- and O- linked oligosaccharides and keratan sulphate. Whether keratan sulphate is O- or N-linked is highly tissue dependent. While both N- and O-linked keratan sulphate are expressed in cartilage, the level of substitution, as well as the overall expression of keratan sulphate on core proteins in other tissues can vary greatly. One example is the cornea, in which the keratocytes are only capable of substituting N-linked keratan sulphate.
In 1995, Barry et al. (Barry et al. 1995) did an analysis of the keratan sulphate linkages in the N-terminal region of aggrecan including the G1 globular domain and the N-terminal portion of the IGD of both immature and mature bovine cartilage aggrecan. These findings are summarised in table 12:
Table 12: KS linkages of the G1 and IGD Sequence or residue Region of Expression in immature substituted (Bovine substitution or mature cartilage sequence) O-linked Thr42 G1 domain; A loop In mature cartilage. N-linked Asn220 G1 domain; B loop Present in both, but shorter in immature cartilage. N-linked Asn314 G1 domain; B’ loop Minor substitution site present in mature cartilage. May instead be substituted with a complex oligosaccharide. O-linked 352TIQTVT457 (2 IGD between the Two KS chains present in chains) MMP and this sequence. Chains aggrecanase shorter in immature cleavage sites cartilage. N- or O-linked IGD adjacent to the In mature cartilage. 368NITEGE373 aggrecanase Linkage either on Asn368 or cleavage site Thr370.
From these results, in bovine aggrecan, there is potential for 3-4 O-linked and 1-3 N-linked keratan sulphate chains substituted on and around the G1 and IGD. Because the rG1-G2 in this project was expressed in corneal cells, only N-linked keratan sulphate was expected to be substituted on the protein. In
168 Chapter VII: Characterisation of rG1-G2 chapter VI, figure 48, N-glycosidase digestion of rG1-G2 confirmed that the protein was substituted with only N-linked keratan sulphate.
Notwithstanding the two possible N-linked keratan sulphate chains on the B and B’ loops of the G1 domain, there is only one possible site for N-linked keratan sulphate, Asn368 in the interglobular domain. This is also the most likely site for interactions with aggrecanases cleaving at the NITEGE373 374ARGSVI site.
The biological function of the keratan sulphate substituted along the length of aggrecan has remained relatively unknown. However recently Pratta et al. (Pratta et al. 2000) compared aggrecanase-mediated cleavage of aggrecan that had been keratanase-digested or not. Their results showed a marked decrease in cleavage at the NITEGE373 - 374ARGSVI site when aggrecan without its full complement of keratan sulphate was used as a substrate. Similar results were obtained when these researchers compared cleavage of aggrecan from mature and immature cartilage; immature cartilage has very little keratan sulphate and is a poor aggrecanase substrate, whereas mature aggrecan has more keratan sulphate and is a better substrate for aggrecanase. They concluded that keratan sulphate on aggrecan enhanced aggrecanase cleavage at NITEGE373 - 374ARGSVI.
Barry et al. established that there is an increase in keratan sulphate substitution within the G1 domain and the IGD with age (Barry et al. 1995). Pratta et al. reported an increase in the aggrecanase-derived G1-NITEGE neoepitope with age and suggested that it was due to an increase in keratan sulphate substitution. The idea of GAGs influencing aggrecanase-mediated cleavage is not a new one; a number of studies have reported a dose- dependent effect of glycosaminoglycans on aggrecanase activity (Sugimoto et al. 1999; Vankemmelbeke et al. 2001; Munteanu et al. 2002). With this in mind there is clearly a precedent for further examination of the role of keratan sulphate in aggrecanase cleavage in the interglobular domain.
169 Chapter VII: Characterisation of rG1-G2
I have produced a recombinant G1-G2 protein in primary bovine keratocytes, in which the 5 kDa of substituted keratan sulphate was entirely N-linked (chapter VI). This chapter describes experiments that examined the effect of N-linked keratan sulphate on aggrecanase cleavage at the NITEGE373 - 374ARGSVI site. The NITEGE neoepitope generated by aggrecanase cleavage was monitored with an anti-NITEGE neoepitope antibody. I was therefore able to study the role of N-linked keratan sulphate on rG1-G2 cleavage by aggrecanase.
170 Chapter VII: Characterisation of rG1-G2
Results
The aim of the experiments described in this chapter was to determine whether N-linked keratan sulphate had a role in enhancing aggrecanase activity; the experiments are a refinement of the work of Pratta et al. (Pratta et al. 2000) who showed that removing both O- and N-linked keratan sulphate results in less cleavage at the aggrecanase site of the IGD. However, these experiments presented a theoretical challenge for the following reason: by monitoring aggrecanase activity using the NITEGE373 neoepitope as the marker, keratanase treatment is reported by Pratta to diminish aggrecanase activity, yet keratanase treatment is known by us and others to enhance detection of the NITEGE373 neoepitope by Western blotting. Therefore, the results of the experiments depend on the balance between keratanase- enhanced detection of the NITEGE373 neoepitope and keratanase-diminished generation of the NITEGE373 neoepitope as represented schematically in figure 50.
Figure 50: Relationship between keratanase digestion and detection of NITEGE373 Schematic diagram showing the relationship between the detection of NITEGE373 neoepitope that is enhanced by keratanase digestion and the creation of NITEGE373 neoepitope that is reduced by keratanase digestion.
Endogenous NITEGE373 neoepitope in conditioned medium
The source of aggrecanase activity for these experiments was conditioned medium from cultured pig cartilage, stimulated with IL-1 and retinoic acid, as described previously by our laboratory (Fosang et al. 2000). The IL-1 and
171 Chapter VII: Characterisation of rG1-G2 retinoic acid stimulates the release of soluble aggrecanases into the medium, however the conditioned medium also contains detectable quantities of pig NITEGE373 neoepitope. The first experiment was therefore to analyse the pig conditioned medium for the presence of detectable levels of exogenous NITEGE373 and to determine whether the detection of NITEGE373 neoepitope was enhanced by keratanase digestion of the conditioned medium.
Pig conditioned medium was incubated with (+) or without (-) endo-β- galactosidase. The samples were analysed by Western blot with an anti- NITEGE antibody (figure 51). The figure shows that conditioned medium contains significant amounts of the NITEGE373 neoepitope, and that it is only unmasked when the keratan sulphate has been removed by keratanase. This also suggests that the anti-NITEGE373 antibody does not recognise the keratan sulphate-substituted aggrecan present in the pig cartilage conditioned medium.
EGβ - + 250 - 150 - 100 - 75 -
50 -
37 -
25 -
Figure 51: Pig NITEGE373 neoepitope present in conditioned medium Conditioned medium from cultured pig cartilage stimulated with IL-1 and retinoic acid was incubated with (+) or without (-) endo-β-galactosidase and analysed by Western blot using an anti-NITEGE antibody.
The results shown in figure 51 reveal that keratanase treatment of conditioned medium unmasks exogenous pig NITEGE373 in the conditioned medium. This would be expected to give false positives in experiments where conditioned medium, as a source of enzyme, was keratanase-digested after aggrecanase digestion. To avoid this complication, I decided that aggrecanase-digested
172 Chapter VII: Characterisation of rG1-G2 samples not previously treated with keratanase would not be given any subsequent keratanase treatments.
rG1-G2 cleavage by aggrecanase
Although the anti-NITEGE373 antibody appears to recognise the keratanase- digested pig NITEGE373 epitope better than the native epitope, it was unknown whether aggrecanase-digested rG1-G2 would be detected with its keratan sulphate intact. Recombinant G1-G2 was incubated with (+) or without (-) a cocktail of three keratanases: keratanase I, keratanase II, and endo-β-galactosidase to ensure complete digestion of the keratan sulphate. After boiling the digests to denature the keratanases, the two substrates were incubated with aggrecanase for the indicated length of time - either 0 or 24 hours (figure 52).
K’ase A + - + -
250 - B 150 - Aggrecanase digestion 100 - 40 75 - 35 30 25 20 50 - 15 10
Relative density Relative 5 37 - 0 + keratanase - keratanase 24 hours aggrecanase 0 24h Figure 52: Aggrecanase cleavage of rG1-G2 after 24h (A) rG1-G2 was incubated with (+) or without (-) a cocktail of keratanases (keratanase I, keratanase II, and endo-β-galactosidase) and then digested with aggrecanase for 24 hours. A zero time incubation was included as a negative control. Samples were analysed by Western blot with an anti-NITEGE373 antibody.(B) Scanning densitometry of the 24 hour NITEGE bands revealed a 3-fold difference in the detected signal between the (+) and (-) keratanase samples.
The results show that the rG1-G2 which is only partially glycosylated, can easily be detected by Western blot with the anti-NITEGE373 antibody, whether the substrate is keratanase-digested or not. The results shown in figure 52
173 Chapter VII: Characterisation of rG1-G2 also suggest that rG1-G2 substrates with their keratan sulphate chains still intact (-) are more susceptible to digestion by aggrecanase and generate more NITEGE fragments. Further testing was done to confirm these results, beginning with a series of timecourse experiments over various periods of time. Figure 53 shows the results of a timecourse experiment examining aggrecanase digestion of keratanase-digested or undigested rG1-G2 over 30 hours. After six hours, the rG1-G2 that still had its keratan sulphate intact was cleaved more readily than the keratanase-digested rG1-G2.
0 6 30h A C + - + - + - k’ases 250 - 150 - 100 - 75 -
50 -
37 -
B Aggrecanase digestion
35 30 25 20 15 10
Relative density Relative 5 0 6h + 6h - 30h + 30h - Incubation time / keratanase
Figure 53: 30h aggrecanase timecourse of rG1-G2 digestion (A) rG1-G2 was either digested with (+) or without (-) a cocktail of keratanases (keratanase I, keratanase II, and endo-β-galactosidase), then digested with aggrecanase for 0, 6, or 30 hours and then analysed by Western blot with an anti-NITEGE antibody. A control containing only conditioned medium “C” was included. (B) The bands were quantitated and plotted against their relative density.
174 Chapter VII: Characterisation of rG1-G2
Figure 54 shows the results of another timecourse over a period of 4 hours. Although the results are not as clear as in those of figure 52, these results also suggest an aggrecanase-enhancing role for keratan sulphate. As early as 30 min into aggrecanase digestion there was less NITEGE373 epitope generated in the keratanase-digested sample (figure 54, 0.5h +) compared with the glycosylated rG1-G2 (figure 54, 0.5h -). Again, the results suggest that the presence of keratan sulphate within the interglobular domain promotes aggrecanase activity.
The main point from figures 52 – 54 is that keratanase-digesting rG1-G2 decreases its susceptibility to aggrecanase cleavage. This is consistent with the results of Pratta et al. (Pratta et al. 2000) and elaborates on those results, showing that N-linked keratan sulphate alone, is sufficient to potentiate aggrecanase activity. However this result does not appear to be completely reproducible; in 9 out of 11 experiments the results were as those shown in figures 52 – 54, but on the other 2 occasions they were not. Experiments were done under various conditions of enzyme and substrate concentration, so the rate of reactions may vary from one experiment to another.
These results suggest that the effect of keratan sulphate substitution in the IGD may be to delay, or retard, the rate of cleavage at the NITEGE373 - 374ARGSVI bond, so that at early time points, the effects of delayed cleavage are more obvious by Western blot, but after an extended incubation time, the enzyme has had adequate time to digest the substrate.
175 Chapter VII: Characterisation of rG1-G2
A 00.5 1h K’ase + + - + - 250 - 150 - 100 - 75 - 50 -
37 -
25 -
B Aggrecanase digestion
35 30 25 20 15 10
Relative density Relative 5 0 0.5h + 0.5h - 1h + 1h - Incubation time / keratanase
Figure 54: 4h aggrecanase timecourse of rG1-G2 digestion (A) rG1-G2 was incubated with (+) or without (-) keratanase I for 18 hours then digested for 0, 0.5, or 1 hour with aggrecanase. The samples were analysed by Western blot with anti-NITEGE antibody. (B) The bands were quantitated and plotted against their relative density.
Overall, the results shown in figures 52 – 54 indicate that rG1-G2 substituted with N-linked keratan sulphate is more susceptible to aggrecanase digestion than the keratanase-digested rG1-G2. It appears that for the majority of the time, rG1-G2 substituted with keratan sulphate is more susceptible to aggrecanase digestion than the keratanase-digested rG1-G2.
To ensure that the pig NITEGE373 present in the conditioned medium remains masked, it is important that it is not keratanase-digested. Because of this, the
176 Chapter VII: Characterisation of rG1-G2
(-) keratanase/rG1-G2 samples remain glycosylated following aggrecanase digestion, whereas the (+) keratanase samples do not. This means that for keratanase-digested substrates incubated with aggrecanase, less NITEGE373 epitope will be generated, but it will be easier to detect (figure 52). Conversely, rG1-G2 not keratanase-digested may yield more NITEGE373 epitope, but because it is more difficult to detect, the amount of neoepitope may be underestimated. Therefore, it follows, that a decrease in NITEGE373 epitope in (+) samples, relative to (-) samples is also likely to be underestimated, and therefore that the real differential between the abundance of aggrecanase products before and after keratanase digestion may be more than it appears by NITEGE373 Western blotting.
To address this issue, aggrecanase-digested samples of (+) and (-) keratanase-digests were probed with another antibody (anti-IGD) which was raised against the sequence CPDMELPLPRNITEG and therefore recognised the IGD but not the NITEGE373 neoepitope just downstream. Duplicate samples were probed with both antibodies and the results were the same with anti-NITEGE373 and anti-IGD (figure 55).
Anti-IGD Anti-NITEGE KI + - + - 150 - 100 - 75 -
50 -
37 -
25 -
Figure 55: Aggrecanase cleavage of rG1-G2 rG1-G2 was digested with keratanase I (+) or not (-) followed by aggrecanase digestion for 6 hours with aggrecanase and analysis by Western blot with either anti-IGD or anti-NITEGE373 antibodies.
The anti-IGD blot detected two products with a mass of approximately 110 and 60 kDa respectively. The 60 kDa band corresponds to the G1-NITEGE373
177 Chapter VII: Characterisation of rG1-G2 product and the 110 kDa band corresponds to undigested rG1-G2. The results of the anti-IGD blot were similar to the anti-NITEGE373 blots, that is, rG1-G2 substituted with keratan sulphate chains was more susceptible to digestion by aggrecanase than deglycosylated rG1-G2. These differences were therefore authentic and not simply due to preferential binding of the antibody to the glycosylated G1-NITEGE373 neoepitope.
Effect of keratan sulphate on aggrecanase digestion – A closer look
Having established that one likely role of keratan sulphate in the interglobular domain may be to facilitate aggrecanase-mediated cleavage; the next step was to attempt to gain further insights into this property by selectively digesting the keratan sulphate with specific glycosidases and examining aggrecanase cleavage.
There are at least four keratan sulphate attachment sights in the N-terminal region of the interglobular domain (table 12), and N-glycosidase digests of rG1-G2 show that all of the keratan sulphate is N-linked (figure 48). The experiments so far have indicated that the keratan sulphate chains, as a whole, act to enhance aggrecanase cleavage, however, whether there is any particular component of the keratan sulphate chains that is responsible for this activity is unknown. rG1-G2 was digested with separate glycosidases with different specificities for sulphated substrates in an attempt to help formulate a possible mechanism for the enhanced aggrecanase cleavage.
In figure 56A, rG1-G2 was digested with three separate keratan sulphate- degrading enzymes, all of which had previously been used, though not separately. Conditioned medium alone (C) and rG1-G2 not digested with any keratanases (UD) were both included as controls. The results show that digestion with any of the three glycosidases gave indistinguishable results, each giving rise to less aggrecanase cleavage than the substrate containing intact keratan sulphate.
178 Chapter VII: Characterisation of rG1-G2
A B C UD KI KII EGβ N-glycosidase C - + 250 - 150 - 250 - 100 - 150 - 75 - 100 - 50 - 75 - 50 -
37 - 37 -
Figure 56: Aggrecanase cleavage of selectively deglycosylated rG1-G2 (A) rG1-G2 was either undigested (UD), or digested with keratanase I, keratanase II, or endo-β- galactosidase. The rG1-G2 was then digested with aggrecanase for 24 hours and the products analysed by Western blot with an anti-NITEGE antibody. A control (C) containing only conditioned medium was included. (B) rG1-G2 was incubated with (+) or without (-) N-glycosidase for 21 hours. The samples were then digested with aggrecanase for 4 hours and resolved by SDS-PAGE with a control (C) containing only conditioned medium. The samples were analysed by Western blot and probed with an anti-NITEGE antibody.
Keratanase I and endo-β-galactosidase specifically cleave mono or unsulphated keratan sulphate. The resulting NITEGE blots are similar to the NITEGE373 blot of the keratanase II-digested rG1-G2, which cleaves mono and disulphated keratan sulphate. No obvious differences were observed after either keratanase I, keratanase II, or endo-β-galactosidase digestion, suggesting that the extent to which each of the keratanases trims back the keratan sulphate chain is sufficient to diminish the capacity for aggrecanase to cleave the core protein.
Digestion with any of the three keratanases reduces the length of the chains but leaves a short oligosaccharide stub attached to the core protein; N- glycosidase in comparison removes the entire keratan sulphate chain back to the attachment site of the core protein. rG1-G2 was digested with N- glycosidase to selectively remove N-linked keratan sulphate within the interglobular domain. The selectively deglycosylated rG1-G2 was then digested with aggrecanase and analysed by Western blot with an anti- NITEGE antibody. The results show that removing the N-linked keratan
179 Chapter VII: Characterisation of rG1-G2 sulphate from the interglobular domain reduces the susceptibility of rG1-G2 to aggrecanase cleavage (figure 56B).
180 Chapter VII: Characterisation of rG1-G2
Discussion
This chapter examined aggrecanase-mediated cleavage of rG1-G2 in the presence and absence of keratan sulphate. From examinations of the G1 domain of bovine aggrecan (Barry et al. 1995), there are a number of O and N-linked keratan sulphate attachment sites along the interglobular domain. Increasing amounts of glycosylation also occur with age, one such site is on the Asn368 residue of the NITEGE373 sequence. Leading on from this study, Pratta et al. (Pratta et al. 2000) demonstrated an age-related increase in aggrecan cleavage at the NITEGE373 site. This increased cleavage of steer aggrecan compared with calf aggrecan seemed to agree with experiments comparing aggrecanase digestion of keratanase-digested aggrecan with undigested aggrecan. A different series of experiments looked at the effect of exogenous GAGs on aggrecanase-mediated cleavage of aggrecan (Munteanu et al. 2002). The results of their study demonstrated that keratan sulphate had no inhibiting effect on aggrecanase activity, whereas other highly sulphated glycosaminoglycans were able to inhibit aggrecanase. The results of the study suggests that while aggrecanases are able to bind directly to GAGs via their thrombospondin type I motifs (Tortorella et al. 2000), direct interaction with aggrecanase by keratan sulphate may not have any effect on aggrecanase activity. Because the C-terminal aggrecanase cleavage sites are cleaved before the NITEGE373 site, it suggests that aggrecanase may preferentially bind to chondroitin sulphate and similar GAGs over keratan sulphate; this also helps to explain the results of Munteanu et al. (Munteanu et al. 2002). Therefore, if the keratan sulphate of the interglobular domain increases the activity of aggrecanase as suggested by Pratta et al. (Pratta et al. 2000), the mechanism is unlikely to involve only the direct interaction of the keratan sulphate with aggrecanase.
The studies presented in this chapter indicate that rG1-G2 with its keratan sulphate intact, is a better substrate for aggrecanases than G1-G2 lacking its keratan sulphate. Since all the keratan sulphate on rG1-G2 is N-linked, this suggests that the N-linked keratan sulphate chain of the NITEGE373 sequence is sufficient to potentiate aggrecanase cleavage in the interglobular domain.
181 Chapter VII: Characterisation of rG1-G2
Although the results described in this chapter are consistent with the hypothesis that keratan sulphate enhances aggrecanase activity, there are likely to be other factors involved. In this chapter, I mentioned that for the majority of the time, rG1-G2 with its keratan sulphate chains intact is more susceptible to cleavage by aggrecanase, however, occasionally, the opposite result would be seen, where the keratanase-digested substrate was more susceptible to cleavage by aggrecanase. On two occasions out of a total of eleven aggrecanase digests, the keratanase-digested rG1-G2 appeared to be more susceptible to aggrecanase digestion than its glycosylated counterpart. This suggests that whatever influence the N-linked KS has on cleavage it is likely to be subtle, merely giving the G1-G2 a greater predisposition to cleavage than the less glycosylated substrate.
Because pig conditioned medium was used throughout these experiments as a source of aggrecanase, a certain level of endogenous pig G1-NITEGE373 was expected, and as shown in figure 51, the detection of this neoepitope was dramatically enhanced after the conditioned medium had been treated with keratanase. Not only did this demonstrate the preference of the anti- NITEGE373 antibody for the keratanase-digested substrate, it also clearly highlighted the need to avoid exposing rG1-G2 aggrecanase digests to keratanases to eliminate the possibility of false positives. However, by eliminating this possibility, it created a new problem, and that is, by comparing the keratanase-digested rG1-G2 and the glycosylated rG1-G2 with the anti- NITEGE373 antibody without a final keratanase digestion of the glycosylated substrate after aggrecanase digestion, the anti-NITEGE373 antibody favours the rG1-G2 lacking keratan sulphate chains. Therefore, anti-NITEGE373 Western blots of non-keratanase-digested samples appear to be less intense than the keratanase-digested sample. This also means that the differential in cleavage of the glycosylated and the deglycosylated rG1-G2 is likely to be even greater than what is seen by Western blot.
While there are probably other influencing factors involved in determining the efficiency of aggrecanase cleavage, the role of keratan sulphate within the interglobular domain cannot be ignored and requires further study. The results
182 Chapter VII: Characterisation of rG1-G2 in this chapter have clearly demonstrated that the presence of N-linked keratan sulphate within the interglobular domain enhances aggrecanase- mediated cleavage.
Munteanu et al. (Munteanu et al. 2002) have shown that exogenous keratan sulphate does not inhibit aggrecanase when included with substrates. The most obvious mechanism by which keratan sulphate could inhibit aggrecanase would be by directly binding to aggrecanase and altering the catalytic domain, or by interfering with the aggrecanase/G1-G2 interaction. Because exogenous keratan sulphate does not inhibit aggrecanase, it does not mean that it can not enhance it; Munteanu et al. did not rule this out. I have completed some preliminary experiments looking at the effect of exogenous keratan sulphate or chondroitin sulphate on aggrecanase- mediated digestion at the NITEGE373 site. Essentially, I was repeating the experiments described by Munteanu et al., except that I was specifically looking for aggrecanase enhancement. Although only very preliminary, the exogenous keratan sulphate or chondroitin sulphate did not appear to enhance or inhibit aggrecanase activity. Together, these experiments suggest that exogenous keratan sulphate does not directly affect aggrecanase, however the results in this chapter clearly show that the endogenous N-linked keratan sulphate substituted in the interglobular domain has an aggrecanase- enhancing effect. Therefore, for keratan sulphate to have any effect on aggrecanase, it must be substituted on the core protein, not dispersed in the medium. Thus, it may be, that it is the effect keratan sulphate has on the IGD rather than on aggrecanase that gives this aggrecanase-enhancing property. From the results of the rotary shadowing electron microscopy (figure 43), we know that the IGD is physically extended in response to keratan sulphate substitution; the more keratan sulphate that is substituted, the longer the interglobular domain is. Pratta et al. reported that aggrecan from mature cartilage is more susceptible to cleavage at the NITEGE373 site than aggrecan from immature cartilage (Pratta et al. 2000). We can hypothesise from these results that this is due to the differences in IGD length. One theory that supports the data in published reports as well as the results in this chapter is that keratan sulphate acts to physically expose the interglobular domain,
183 Chapter VII: Characterisation of rG1-G2 extending it to a state that is more accessible to aggrecanase digestion. However, even if exogenous keratan sulphate has no direct inhibiting or enhancing effect on aggrecanase, we know that ADAMTS-4 is capable of binding glycosaminoglycans via its thrombospondin motif and that this domain is critical in aggrecan recognition and cleavage (Tortorella et al. 2000).
Alternatively, aggrecanase may use the keratan sulphate as a docking mechanism to help bind to specific domains of aggrecan; this would explain why exogenous keratan sulphate has no effect on aggrecanase. Exogenous keratan sulphate may bind to aggrecanase, but this binding does not bring the aggrecanase any closer to the IGD. Exogenous keratan sulphate may therefore compete with the endogenous keratan sulphate for aggrecanase- binding, however this is unlikely to have any significant effect on cleavage, as proven by Munteanu et al. (Munteanu et al. 2002). From what has been published previously, aggrecanase preferentially cleaves aggrecan within the CS-rich domain rather than the IGD (Tortorella et al. 2000; Tortorella et al. 2002), so it is possible that this docking mechanism may apply primarily to chondroitin sulphate chains and not keratan sulphate. On the other hand, there may not be a preference for one type of glycosaminoglycan or the other, only a preference for charge. If this were the case then you can easily see why there would be preferential cleavage in the CS-rich domains over the IGD; the much higher proportion of CS and therefore the higher local concentration of negatively charged sulphate groups are more efficient at binding the thrombospondin domain of aggrecanase. Regardless of the specifics behind aggrecanase-glycosaminoglycan-binding, formulating a valid theory of aggrecanase cleavage is complicated, it may be that cleavage is influenced by a combination of these two theories. Direct visualisation of this cleavage event via crystallography or atomic microscopy may provide more insight into how keratan sulphate may modulate aggrecanase cleavage in the IGD.
184 Chapter VIII: Final Discussion
Chapter VIII: Final Discussion
“It's a job that's never started that takes the longest to finish” J. R. R. Tolkien (1892 - 1973)
185 Chapter VIII: Final Discussion
Purpose of this project
Aggrecan is a critical component of cartilage; its ability to draw water into the tissue enables cartilage to absorb the shock that would otherwise be transferred to the joint during movement. Aggrecanase-mediated cleavage of aggrecan within the IGD is a major catalytic event that clears aggrecan from cartilage. As a consequence, the biomechanical properties of cartilage can be compromised.
The main objective of this project was to gain further insight into the factors that influence cleavage of aggrecan at the NITEGE373 ↓ 374ARGSV site. There have been reports that keratan sulphate enhances cleavage at this site. A number of N- and O-linked keratan sulphate attachment sites are present in the IGD, and a single keratan sulphate chain in the NITEGE sequence is attached either to the N368 or T370. Aggrecanase cleaves at the E373 ↓ 374A bond and the very close proximity of the keratan sulphate to this site suggests that it may have some influence on cleavage. The number and length of keratan sulphate chains increase with age. This may have consequences for age-related aggrecanase-cleavage.
Exploring this idea were Pratta et al. (Pratta et al. 2000) who did two things. The first was to compare aggrecanase-mediated digestion of aggrecan from immature and mature cartilage. The second was to compare the efficacy of aggrecanase digestion on substrates that had or had not been treated with deglycosidases. They found a positive correlation between glycosaminoglycan content and aggrecanase activity, and conversely, they found that deglycosylating aggrecan with keratanase and chondroitinase ABC decreased cleavage by aggrecanase. It was the aim of my project to further investigate the influence of keratan sulphate in the IGD on cleavage at the NITEGE373 ↓ 374ARGSV site.
186 Chapter VIII: Final Discussion
A brief history of keratan sulphate
Having been discovered more than 60 years ago (Suzuki 1939), one may wonder whether there is any more to learn about keratan sulphate, particularly over the course of a PhD. Maybe we are not able to learn much more about the structure of keratan sulphate or even its biosynthesis, but there is still plenty to learn about its role in biological systems. Keratan sulphate has an important role in the central nervous system. Here keratan sulphate, which is primarily substituted on phosphacan, is used to regulate the growth of mossy fibre outgrowth (Mace et al. 2002). In the cornea, keratan sulphate is responsible for maintaining the interfibril spacing of collagen fibres, thereby maintaining corneal transparency. However, things get more complicated in cartilage; cartilage does not contain neurons nor is it transparent; keratan sulphate on cartilage aggrecan does not have an apparent biological function. The majority of the keratan sulphate present on aggrecan is within a domain known as the keratan sulphate-rich region. It is thought that the number of keratan sulphate chains substituted within this region is proportional to the number of hexapeptide repeats made up of the sequence Glu-Glu/Lys-Pro- Phe-Pro-Ser (Barry et al. 1994). The reason one is led to believe that the keratan sulphate has no obvious biological role is the difference in the number of hexapeptide repeats between species, from 23 in bovine to as little as 4 in murine aggrecan. This vast difference in keratan sulphate substitution between species does not appear to have any effect on the function or structural integrity of cartilage, which implies a secondary role, at least, for the keratan sulphate-rich region. So far, no studies have examined the keratan sulphate of the interglobular domain.
Keratan sulphate synthesis has been extensively studied in the cornea due to its extreme importance in maintaining corneal transparency. While it is true that keratan sulphate synthesis dramatically decreases when keratocytes are isolated and cultured, attempts have been made to prolong the synthesis of keratan sulphate for extended periods. Work by Funderburgh et al. (Funderburgh et al. 1996; Long et al. 2000; Funderburgh et al. 2003) and Nakazawa et al. (Nakazawa et al. 1995; Nakazawa et al. 1996; Nakazawa et
187 Chapter VIII: Final Discussion al. 1997; Nakazawa et al. 1998) among others, has established that keratan sulphate synthesis can be prolonged by culturing keratocytes with low concentrations of serum. Supplementing cells with basic fibroblast growth factor has also been shown to help prolong keratan sulphate synthesis. So with this in mind, primary keratocytes can be cultured under specific conditions which allow for the synthesis of keratan sulphate proteoglycans, albeit, for a limited time. Long et al. (Long et al. 2000) observed a marked reduction in keratan sulphate synthesis over a week, despite supplementing cultures of primary keratocytes with low concentrations of serum and FGF-2, although, it should be pointed out that the reported levels of keratan sulphate synthesis were still significantly higher than those found in cell lines.
Discussion of experimental techniques
A number of relatively new techniques were used in this project, the most notable being FACE analysis of keratan sulphate. FACE has been used for some time in carbohydrate analysis (Jackson 1991; Jackson 1993; Jackson 1994; Jackson 1994; Jackson et al. 1994). Although FACE has been used to analyse glycosaminoglycans, there was very little being done to analyse keratan sulphate with FACE. Not only was it because of the relatively recent development of this technique, it was also because of the lack of keratan sulphate standards, which makes identifying samples difficult. Dr. Anna Plaas was one of the pioneering researchers who developed FACE analysis for keratan sulphate (Plaas et al. 2001; Plaas et al. 2001). Plaas was able to identify many of the keratan sulphate products visible on FACE gels, and this in turn has made the keratan sulphate analysis in this thesis possible. The advantage of this technique over traditional analytical methods is the speed and sensitivity of FACE, as well as the large number of samples that can be screened at once.
The Vaccinia expression system was another important technique used in this project. One of the advantages of the Vaccinia expression system is its high efficiency and the freedom to use many different types of cell. Because a type of cell had yet to be chosen in the early stages of this project, it was important
188 Chapter VIII: Final Discussion that I had the flexibility to choose whatever type of cell was most appropriate for keratan sulphate synthesis, rather than worry about conflicts with the expression system. In theory, rG1-G2 could be expressed in any type of avian or mammalian cell, allowing us to customise the type of keratan sulphate to be substituted. However the major disadvantage to using a Vaccinia virus is its infectious nature. Because all mammalian cells can be infected with high efficiency, it also means that humans are at risk. In Australia, government legislature dictates that all users must be immunised against smallpox and the immunisation must be current. Smallpox vaccinations have not been a standard procedure for more than 20 years so the importation, TGA approvals, vaccine storage, and administration of the vaccine by a person approved to give smallpox vaccinations, in this day and age, are major logistical hurdles for using Vaccinia virus.
Summary of Results
In chapter III, keratan sulphate synthesis was examined in freshly isolated primary bovine keratocytes. Cells were cultured in medium supplemented with different concentrations of serum and FGF-2. Primary keratocytes cultured with low concentrations of serum and FGF-2 contained high levels of newly synthesised keratan sulphate (up to ~30%). In contrast, the same experiment performed on a variety of cell lines revealed undetectable amounts of newly synthesised keratan sulphate. Having identified conditions that support keratan sulphate synthesis in primary keratocytes, the next stage of the project was to use the Vaccinia expression system to express recombinant G1-G2 in the primary keratocytes.
In chapter IV, initial attempts to express rG1-G2 were successful and examination of the keratan sulphate revealed a total of 5 kDa of keratan sulphate substituted on the core protein. After careful optimisation, a method was devised for the production of maximum yields of rG1-G2, however keratan sulphate substitution was limited to a maximum of 5 kDa. This was not surprising given the extended incubation period required for infected cells to produce maximum yields of rG1-G2. Infected cells do not start to produce
189 Chapter VIII: Final Discussion rG1-G2 until 24 hours post inoculation, and it is within this period that keratan sulphate synthesis is most efficient.
After optimising the yield of rG1-G2 produced by the cells, purification of the rG1-G2 was the next challenge; this is described in chapter V. HA-sepharose affinity chromatography was used as a quick method of isolating rG1-G2 from the harvested media. By including two rounds of salt washes prior to eluting the rG1-G2 from HA-sepharose, I was able to remove the vast majority of the contaminating protein. Subsequent HPLC was sufficient to remove any remaining contaminants and resulted in a pool of purified rG1-G2 for analysis. A sample of purified rG1-G2 was compared with baculovirus-expressed G1- G2, and native pig G1-G2, using rotary shadowing electron microscopy. The results show that the length of the core protein was approximately proportional to the amount of keratan sulphate substituted. Expression (chapter IV) and purification (chapter V) of keratan sulphate-substituted rG1- G2 required the most attention and a significant portion of my project was spent on these two important aspects, however characterisation of rG1-G2 was relatively straightforward.
Analysis of the keratan sulphate substituted on rG1-G2 (chapter VI) using FACE revealed that it was highly sulphated, though not quite as sulphated as the keratan sulphate of native G1-G2 purified from pig cartilage. Results of FACE analysis also revealed that the keratan sulphate of rG1-G2 contained less sialic acid capping than native keratan sulphate and fucosidase digests also showed a lack of fucosylation. In comparison with the KS of bovine corneal KS and human articular cartilage KS (Plaas et al. 2001), the KS substituted on rG1-G2 is most similar to bovine corneal KS, in that it contains a significant proportion of mono- or disulphated disacchardies and very little unsulphate disaccharides. The KS of rG1-G2 also lacks fucose and has little sialic acid similar to bovine corneal KS. On the other hand, although the proportion of mono- and disulphated disaccharides are similar to KS from human articular cartilage and bovine cornea, articular cartilage KS is modified with large amounts of sialic acid and fucose. The most interesting results
190 Chapter VIII: Final Discussion shown in chapter VI were those using N-glycosidase. These results show that rG1-G2 is substituted only with N-linked keratan sulphate.
In chapter VII, rG1-G2 was examined for aggrecanase cleavage. Cleavage was monitored by Western blot using an anti-NITEGE373 neoepitope antibody. By treating rG1-G2 with or without different glycosidase digests and then comparing each substrate’s response to aggrecanase digestion, I showed that the presence of N-linked keratan sulphate in the IGD enhanced cleavage at the EGE373-374ARG site.
Overview
One of the key results of this research project was discovering that the keratan sulphate substituted on rG1-G2 was entirely N-linked, despite the presence of a number O-linkage sites (table 12). This suggests that both tissue type and cell type determines the kind of linkage structures substituted on proteoglycans. In this case, the keratan sulphate was N-linked because corneas are not known to synthesise O-linked keratan sulphate proteoglycans (Funderburgh 2000; Funderburgh 2002).
The results presented in chapter VII show that the partially glycosylated form of rG1-G2, substituted with N-linked keratan sulphate, is better able to promote aggrecanase cleavage at the EGE373 site than the enzymatically deglycosylated rG1-G2. These observations fit those reported by Pratta et al. (Pratta et al. 2000) – increased glycosylation within the IGD appears to impart an aggrecanase-enhancing quality to the EGE373 site. Although there is still not enough data to formulate a definitive mechanism for this phenomenon, there are a number of possibilities to explain it. Previous experiments by Munteanu et al. (Munteanu et al. 2002) showed that exogenous keratan sulphate does not inhibit aggrecanase cleavage of the IGD, suggesting that either keratan sulphate does not directly bind to aggrecanase, or if it does, binding to aggrecanase has no inhibiting effect on the activity of aggrecanase. However it is important to note that aggrecanase enhancement was not studied by Munteanu et al., so it is possible that exogenous keratan sulphate
191 Chapter VIII: Final Discussion may enhance aggrecanase activity, perhaps via a conformational change in the enzyme. An alternative explanation was introduced in the previous chapter; this is discussed further below.
The charge on glycosaminoglycans gives them their hydrophilic property and is the reason why cartilage is able to maintain such a high hydroscopic pressure. I propose that the keratan sulphate of the IGD enhances aggrecanase cleavage at the EGE373 site, not by directly interacting with aggrecanase, but by introducing a hydroscopic pressure within the IGD, thereby extending the IGD and exposing it to aggrecanases. This extension of the interglobular domain has been demonstrated in figure 43 where the completely unglycosylated G1-G2 had an average length of 19 nm, whereas the ~40 kDa of keratan sulphate found in the native glycosylated G1-G2 extends the G1-G2 length to almost double, to an average of 37 nm. Since young animals of all species have very little keratan sulphate, these observations may help explain why the underglycosylated calf aggrecan is not cleaved by aggrecanase as well as the fully glycosylated steer aggrecan. It may be a matter of accessibility of the aggrecanase to the IGD. We must also not forget the work of Tortorella et al. (Tortorella et al. 2000) who determined that the thrombospondin domain of aggrecanase plays a critical role in binding to aggrecan via the glycosaminoglycan chains. This domain is presumably involved in initiating cleavage because aggrecanase lacking this domain was not very effective at cleaving aggrecan. With this in mind, keratan sulphate may have dual roles: the first, to modify the tertiary structure of the G1-G2 domain in such a way as to present a more effective substrate for aggrecanase cleavage, perhaps by making the 368NITEGEARGSV378 sequence more accessible to aggrecanase. The second role may be to serve as a general docking structure for aggrecanase-binding. However the mere presence of keratan sulphate in the IGD by no means guarantees increased cleavage. Occasionally during the course of characterisation of rG1-G2, more NITEGE373 neoepitope was detected in the digest containing enzymatically deglycosylated rG1-G2. It seems that the presence of N-linked KS only gives the IGD a greater likelihood of cleavage. When substrates completely devoid
192 Chapter VIII: Final Discussion of keratan sulphate are exposed to aggrecanase, cleavage still occurs, however it is not very efficient (Hughes et al. 1997; Mercuri et al. 1999).
Future directions
According to Barry et al. (Barry et al. 1995) there is a possibility for up to three N-linked keratan sulphate chains, two in the B-loops of the G1 domain, and another in the interglobular domain in the NITEGE373 sequence. While we know that rG1-G2 only contains N-linked keratan sulphate, it is still uncertain whether all of these sites are substituted. It would be useful to find out which of these sites are substituted in rG1-G2.
In addition to the N-linked keratan sulphate site on Asn368 there is also an additional O-linked keratan sulphate site on Thr370 (Barry et al. 1995). Although the rG1-G2 expressed in keratocytes allows for N-linked keratan sulphate only, its expression in another type of cell, perhaps a chondrocyte- related cell, may allow for O-linked keratan sulphate substitution on rG1-G2. If this is an authentic O-linkage site, it too may influence aggrecanase cleavage. An entirely O-linked G1-G2 substrate would allow us to investigate the effect of O-linked keratan sulphate on cleavage.
Examination of keratan sulphate substitution on rG1-G2 over time would be interesting. An experiment I had in mind was to infect cells in the usual manner, and collect and replace the culture medium each day for an extended period beyond the established 4 days that I had used throughout my project, perhaps until the cells had completely lysed. Using a combination of keratanase digestion and SDS-PAGE, the amount of secreted rG1-G2 could be determined, and more interesting, the degree of glycosylation at each stage of the timecourse could also be determined. It was always my contention that keratan sulphate synthesis continually decreases so that the keratan sulphate of rG1-G2 collected later in the timecourse would be less developed than the keratan sulphate substituted on rG1-G2 collected earlier. However, if rG1-G2 continues to be secreted beyond 4 days while still being
193 Chapter VIII: Final Discussion glycosylated, then it would be possible to collect enough rG1-G2 to properly analyse the sialic acid capping as well as the fucosylation of the substituted N-linked keratan sulphate. Although the keratan sulphate analysis I performed on the rG1-G2 KS generated useful data on the disaccharide composition of the glycosaminoglycan chains, the limited sialic acid capping and fucosylation was such that I was unable to accurately quantitate these residues. By using a significantly larger amount of keratan sulphate, analysis of these two structures may be possible.
From multiple rG1-G2 preparations, the ratio of keratan sulphate to core protein was approximately 1 : 10, based on approximations of mass using dye-binding and absorbance at 278nm respectively. Therefore to collect enough keratan sulphate to accurately analyse the less abundant products observed in figure 47, a minimum of 50 μg is required which in turn, requires approximately 500 μg of purified rG1-G2. The 500 μg of purified rG1-G2, which typically represents an entire large scale preparation, would be consumed for a single FACE experiment. Given more time, I would like to have made a new preparation of rG1-G2, this time on a scale much larger than all of my previous ones. An average yield of 500 μg of rG1-G2 requires approximately 2 x 108 primary keratocytes to be infected. The large scale preparation that I have in mind would involve up to 10 x the number of cells and perhaps the use of a bioreactor. The expected yield from such a preparation is expected to be as high as 5 mg of rG1-G2 with a total of 500 μg of keratan sulphate. This would allow for more than enough material for accurate analysis of the less abundant products. However an infection of this magnitude does pose one major problem and that is in keratocyte preparation. The logistics of handling and preparing so many fresh corneas is truly mind-boggling; isolating 2 x 109 cells requires up to 800 fresh bovine corneas. A major task such as this would have to be divided and staggered over ten separate experiments, extending the rG1-G2 production time considerably. However it is possible the number of cells required to produce this much rG1-G2 may be dramatically reduced if infected keratocytes continue to secrete glycosylated rG1-G2 beyond 4 days. Further experiments will confirm this.
194 Chapter VIII: Final Discussion
It may be useful to determine whether the thrombospondin domain binds chondroitin sulphate or keratan sulphate better. We know from Tortorella’s work (Tortorella et al. 2000) that this domain is necessary for aggrecan recognition and cleavage, and that it binds glycosaminoglycans. If we were to properly define the preferred ligand for this domain, whether it is keratan sulphate or chondroitin sulphate, we may be able to determine why the CS- rich domain is cleaved prior to cleavage in the IGD.
As a part of ongoing efforts to identify the factors involved in the progression of arthritis, the presence of keratan sulphate in serum has been examined as a possible biomarker of the early stages of osteoarthritis. It was thought that the majority of keratan sulphate present in serum is attributable to the catabolism of cartilage proteoglycans. Dramatic increases in serum keratan sulphate were hypothesised to be a biomarker of arthritis, or at least a sign of increased aggrecan proteolysis. Much of this work was done by Thonar et al. (Thonar et al. 1987; Thonar et al. 1988; Thonar 1990; Thonar et al. 1992; Thonar et al. 1993; Thonar et al. 1994; Thonar et al. 1995). However, the validity of keratan sulphate as a biomarker of cartilage catabolism remains controversial, for several reasons, not least of which is the fact that the keratan sulphate antigen is polyvalent and exhibits properties of co-operative binding in ELISA assays (Seibel et al. 1992). Experiments designed to measure the levels of keratan sulphate in the serum of arthritis patients have varied considerably (Thonar et al. 1987; Ratcliffe et al. 1988; Thonar et al. 1988; Pavelka et al. 1989; Pavelka et al. 1989; Thonar 1990; Campion et al. 1991; Thonar et al. 1992; Sarkozi et al. 1993; Thonar et al. 1993; Thonar et al. 1994; Thonar et al. 1995).
Another complication with keratan sulphate as a biomarker of articular cartilage pathology is that there are many other tissues that can contribute to the pool of serum keratan sulphate, such as blood vessels and non-articular cartilage sources such as the sternum and surrounding ribcage. The differences in serum keratan sulphate levels may have been due to variations in antigen content arising from the various sources of keratan sulphate in the
195 Chapter VIII: Final Discussion body, or they may reflect the problems with polyvalency and co-operative binding in ELISA assays, as described.
Recently, our group has determined that the keratan sulphate of the IGD has a microstructure that is distinct from the keratan sulphate found elsewhere on aggrecan (Fosang et al. 2004). While the keratan sulphate in the KS-rich domain has a high proportion of disulphated disaccharides (55%) and a low proportion of unsulphated disaccharides (11%), the keratan sulphate of the IGD has a lower proportion of disulphated disaccharides (33%) and a higher proportion of unsulphated disaccharides (33%). It is possible that this configuration of keratan sulphate is unique to the IGD of aggrecan and if this proves to be true, then it may be possible to develop an antibody to recognise the IGD with its unique keratan sulphate microstructure. An antibody which recognises the IGD could potentially make a useful biomarker of cartilage damage. MMPs are involved primarily in late stage severe, cartilage damage whereas aggrecanases are involved primarily in the early stages (van Meurs et al. 1999; Caterson et al. 2000). During early stages of arthritic joint damage, aggrecanases cleave aggrecan in the CS-rich domain as well as in the IGD at EGE373 - 374ARG, however the G1-NITEGE fragment remains in the cartilage. When MMPs are active and available in the tissue, they can process the G1-NITEGE fragment further by cleaving at the PEN341 - 342FFG site to generate a 32mer fragment bearing the majority of the keratan sulphate in the IGD. This 32mer fragment, or unique keratan sulphate epitope that may be present in the IGD, could be a suitable biomarker of late stage cartilage damage by aggrecanases and MMPs. Although the use of biomarkers may not be particularly useful in diagnosing arthritis patients in the late stage of their disease, it may still be useful in analysing the proteolytic events that contribute to the disease.
Conclusion
This thesis describes the production of recombinant G1-G2 fragments substituted with 5 kDa of N-linked keratan sulphate, as well as the many steps required to optimise levels of keratan sulphate substitution in a tissue culture
196 Chapter VIII: Final Discussion environment. To my knowledge, this is the first account of a recombinant protein substituted with keratan sulphate. Using this recombinant protein as a substrate for aggrecanase digests, I have shown that the N-linked keratan sulphate of the interglobular domain enhances cleavage at the NITEGE373 ↓ 374ARGSV site. The results contained in this thesis add to the data already established in the literature, giving us new insights into the mechanisms of aggrecanase-mediated cartilage destruction. These results also introduce a potential target for new drugs that may be useful in reducing aggrecanase cleavage and prolonging the health of joint cartilage.
197 References
References
Abbaszade, I., R. Q. Liu, F. Yang, S. A. Rosenfeld, O. H. Ross, J. R. Link, D. M. Ellis, M. D. Tortorella, M. A. Pratta, J. M. Hollis, R. Wynn, J. L. Duke, H. J. George, M. C. Hillman, Jr., K. Murphy, B. H. Wiswall, R. A. Copeland, C. P. Decicco, R. Bruckner, H. Nagase, Y. Itoh, R. C. Newton, R. L. Magolda, J. M. Trzaskos, T. C. Burn and et al. (1999). "Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family." J Biol Chem 274(33): 23443-50.
Ahrens, D., A. E. Koch, R. M. Pope, M. Stein-Picarella and M. J. Niedbala (1996). "Expression of matrix metalloproteinase 9 (96-kd gelatinase B) in human rheumatoid arthritis." Arthritis Rheum 39(9): 1576-87.
Akama, T. O., A. K. Misra, O. Hindsgaul and M. N. Fukuda (2002). "Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratan sulfate." J Biol Chem 277(45): 42505-13.
Akama, T. O., J. Nakayama, K. Nishida, N. Hiraoka, M. Suzuki, J. McAuliffe, O. Hindsgaul, M. Fukuda and M. N. Fukuda (2001). "Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate." J Biol Chem 276(19): 16271-8.
Angata, T. and A. Varki (2002). "Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective." Chem Rev 102(2): 439-69.
Antonsson, P., D. Heinegard and A. Oldberg (1989). "The keratan sulfate- enriched region of bovine cartilage proteoglycan consists of a consecutively repeated hexapeptide motif." J Biol Chem 264(27): 16170-3.
198 References
Arciniegas, E., C. Y. Neves, D. Candelle and D. Parada (2004). "Differential versican isoforms and aggrecan expression in the chicken embryo aorta." Anat Rec A Discov Mol Cell Evol Biol 279(1): 592-600.
Aspberg, A., S. Adam, G. Kostka, R. Timpl and D. Heinegard (1999). "Fibulin- 1 is a ligand for the C-type lectin domains of aggrecan and versican." J Biol Chem 274(29): 20444-9.
Aspberg, A., C. Binkert and E. Ruoslahti (1995). "The versican C-type lectin domain recognizes the adhesion protein tenascin-R." Proc Natl Acad Sci U S A 92(23): 10590-4.
Aspberg, A., R. Miura, S. Bourdoulous, M. Shimonaka, D. Heinegard, M. Schachner, E. Ruoslahti and Y. Yamaguchi (1997). "The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety." Proc Natl Acad Sci U S A 94(19): 10116-21.
Balsamo, J., H. Ernst, M. K. Zanin, S. Hoffman and J. Lilien (1995). "The interaction of the retina cell surface N- acetylgalactosaminylphosphotransferase with an endogenous proteoglycan ligand results in inhibition of cadherin-mediated adhesion." J Cell Biol 129(5): 1391-401.
Barry, F. P., J. U. Gaw, C. N. Young and P. J. Neame (1992). "Hyaluronan- binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate." Biochem J 286 ( Pt 3): 761-9.
Barry, F. P., P. J. Neame, J. Sasse and D. Pearson (1994). "Length variation in the keratan sulfate domain of mammalian aggrecan." Matrix Biol 14(4): 323- 8.
199 References
Barry, F. P., L. C. Rosenberg, J. U. Gaw, T. J. Koob and P. J. Neame (1995). "N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature and immature bovine cartilage." J Biol Chem 270(35): 20516-24.
Barta, E., F. Deak and I. Kiss (1993). "Evolution of the hyaluronan-binding module of link protein." Biochem J 292 ( Pt 3): 947-9.
Beales, M. P., J. L. Funderburgh, J. V. Jester and J. R. Hassell (1999). "Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture." Invest Ophthalmol Vis Sci 40(8): 1658-63.
Bethea, C. L. and S. L. Kozak (1984). "Effect of extracellular matrix on PC 12 cell shape and dopamine processing." Mol Cell Endocrinol 37(3): 319-29.
Biedler, J. L., L. Helson and B. A. Spengler (1973). "Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture." Cancer Res 33(11): 2643-52.
Birkedal-Hansen, H. (1995). "Proteolytic remodeling of extracellular matrix." Curr Opin Cell Biol 7(5): 728-35.
Bleckmann, H. and H. Kresse (1979). "[Influence of glycosaminoglycan synthesis of cultured cornea stroma cells by variation of culture condition]." Albrecht Von Graefes Arch Klin Exp Ophthalmol 210(4): 291-300.
Boone, R. F. and B. Moss (1977). "Methylated 5'-terminal sequences of vaccinia virus mRNA species made in vivo at early and late times after infection." Virology 79(1): 67-80.
Brown, G. M., T. N. Huckerby, B. L. Abram and I. A. Nieduszynski (1996). "Characterization of a non-reducing terminal fragment from bovine articular cartilage keratan sulphates containing alpha(2-3)-linked sialic acid and
200 References alpha(1-3)-linked fucose. A sulphated variant of the VIM-2 epitope." Biochem J 319 ( Pt 1): 137-41.
Brown, G. M., T. N. Huckerby, M. T. Bayliss and I. A. Nieduszynski (1998). "Human aggrecan keratan sulfate undergoes structural changes during adolescent development." J Biol Chem 273(41): 26408-14.
Brummendorf, T., S. Kenwrick and F. G. Rathjen (1998). "Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations." Curr Opin Neurobiol 8(1): 87-97.
Butler, C. D., S. A. Schnetz, E. Y. Yu, J. B. Davis, K. Temple, J. Silver and A. T. Malouf (2004). "Keratan sulfate proteoglycan phosphacan regulates mossy fiber outgrowth and regeneration." J Neurosci 24(2): 462-73.
Cai, C. X., E. Gibney, M. K. Gordon, J. K. Marchant, D. E. Birk and T. F. Linsenmayer (1996). "Characterization and developmental regulation of avian corneal beta-1,4-galactosyltransferase mRNA." Exp Eye Res 63(2): 193-200.
Calabro, A., M. Benavides, M. Tammi, V. C. Hascall and R. J. Midura (2000). "Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)." Glycobiology 10(3): 273-81.
Calabro, A., V. C. Hascall and R. J. Midura (2000). "Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage." Glycobiology 10(3): 283-93.
Calabro, A., R. Midura, A. Wang, L. West, A. Plaas and V. C. Hascall (2001). "Fluorophore-assisted carbohydrate electrophoresis (FACE) of glycosaminoglycans." Osteoarthritis Cartilage 9 Suppl A: S16-22.
Calvete, J. J., W. Schafer, T. Soszka, W. Q. Lu, J. J. Cook, B. A. Jameson and S. Niewiarowski (1991). "Identification of the disulfide bond pattern in
201 References albolabrin, an RGD-containing peptide from the venom of Trimeresurus albolabris: significance for the expression of platelet aggregation inhibitory activity." Biochemistry 30(21): 5225-9.
Campion, G. V., F. McCrae, T. J. Schnitzer, M. E. Lenz, P. A. Dieppe and E. J. Thonar (1991). "Levels of keratan sulfate in the serum and synovial fluid of patients with osteoarthritis of the knee." Arthritis Rheum 34(10): 1254-9.
Carlson, E. C., I. J. Wang, C. Y. Liu, P. Brannan, C. W. Kao and W. W. Kao (2003). "Altered KSPG expression by keratocytes following corneal injury." Mol Vis 9: 615-23.
Carpino, L. A. and G. Y. Han (1972). "9-Fluorenylmethoxycarbonyl amino- protecting group." J Org Chem 37(22): 3404-3409.
Caterson, B., A. Calabro, P. J. Donohue and M. R. Jahnke (1986). Articular Cartilage Biochemistry. Articular Cartilage Biochemistry. K. E. Kuettner, R. Schleyerbach and V. C. Hascall. New York, Raven Press: 59-73.
Caterson, B., C. R. Flannery, C. E. Hughes and C. B. Little (2000). "Mechanisms involved in cartilage proteoglycan catabolism." Matrix Biol 19(4): 333-44.
Chakravarti, S., T. Magnuson, J. H. Lass, K. J. Jepsen, C. LaMantia and H. Carroll (1998). "Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican." J Cell Biol 141(5): 1277-86.
Chandrasekaran, L. and M. L. Tanzer (1992). "Molecular cloning of chicken aggrecan. Structural analyses." Biochem J 288 ( Pt 3): 903-10.
Chandrasekaran, L. and M. L. Tanzer (1993). "Molecular cloning of chicken aggrecan." Biochem J 296 ( Pt 3): 885-7.
202 References
Chang, A. and D. H. Metz (1976). "Further investigations on the mode of entry of vaccinia virus into cells." J Gen Virol 32(2): 275-82.
Chaplin, M. F. and J. F. Kennedy, Eds. (1986). Carbohydrate analysis: a practical approach, IRL Press Limited.
Chiu, C. P., A. G. Watts, L. L. Lairson, M. Gilbert, D. Lim, W. W. Wakarchuk, S. G. Withers and N. C. Strynadka (2004). "Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog." Nat Struct Mol Biol 11(2): 163-70.
Collins-Racie, L. A., C. R. Flannery, W. Zeng, C. Corcoran, B. Annis- Freeman, M. J. Agostino, M. Arai, E. DiBlasio-Smith, A. J. Dorner, K. E. Georgiadis, M. Jin, X. Y. Tan, E. A. Morris and E. R. LaVallie (2004). "ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage." Matrix Biol 23(4): 219-30.
Cyrklaff, M., C. Risco, J. J. Fernandez, M. V. Jimenez, M. Esteban, W. Baumeister and J. L. Carrascosa (2005). "Cryo-electron tomography of vaccinia virus." Proc Natl Acad Sci U S A 102(8): 2772-7.
Dahl, R. and J. R. Kates (1970). "Synthesis of vaccinia virus "early" and "late" messenger RNA in vitro with nucleoprotein structures isolated from infected cells." Virology 42(2): 463-72.
Dales, S. and B. G. T. Pogo (1981). "Biology of Poxviruses." Virol Monogr 1981: 1-156.
Dall'Olio, F., M. Chiricolo, A. D'Errico, E. Gruppioni, A. Altimari, M. Fiorentino and W. F. Grigioni (2004). "Expression of beta-galactoside alpha2,6 sialyltransferase and of alpha2,6-sialylated glycoconjugates in normal human liver, hepatocarcinoma, and cirrhosis." Glycobiology 14(1): 39-49.
203 References
Danielson, K. G., H. Baribault, D. F. Holmes, H. Graham, K. E. Kadler and R. V. Iozzo (1997). "Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility." J Cell Biol 136(3): 729-43.
Davies, Y., D. Lewis, N. J. Fullwood, I. A. Nieduszynski, B. Marcyniuk, J. Albon and A. Tullo (1999). "Proteoglycans on normal and migrating human corneal endothelium." Exp Eye Res 68(3): 303-11.
Day, A. J. and G. D. Prestwich (2002). "Hyaluronan-binding proteins: tying up the giant." J Biol Chem 277(7): 4585-8.
Day, J. M., A. D. Murdoch and T. E. Hardingham (1999). "The folded protein modules of the C-terminal G3 domain of aggrecan can each facilitate the translocation and secretion of the extended chondroitin sulfate attachment sequence." J Biol Chem 274(53): 38107-11.
Day, J. M., A. I. Olin, A. D. Murdoch, A. Canfield, T. Sasaki, R. Timpl, T. E. Hardingham and A. Aspberg (2004). "Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C and other extracellular matrix proteins." J Biol Chem 279(13): 12511-8.
Dennis, M. S., W. J. Henzel, R. M. Pitti, M. T. Lipari, M. A. Napier, T. A. Deisher, S. Bunting and R. A. Lazarus (1990). "Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet- aggregation inhibitors." Proc Natl Acad Sci U S A 87(7): 2471-5.
Dickenson, J. M., T. N. Huckerby and I. A. Nieduszynski (1991). "A non- reducing terminal fragment from tracheal cartilage keratan sulphate chains contains alpha (2-3)-linked N-acetylneuraminic acid." Biochem J 278 ( Pt 3): 779-85.
Doege, K. J., M. Sasaki, T. Kimura and Y. Yamada (1991). "Complete coding sequence and deduced primary structure of the human cartilage large
204 References aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms." J Biol Chem 266(2): 894-902.
Doolittle, R. F. (1995). "The multiplicity of domains in proteins." Annu Rev Biochem 64: 287-314.
D'Souza, S. E., M. H. Ginsberg and E. F. Plow (1991). "Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif." Trends Biochem Sci 16(7): 246-50.
Dunlevy, J. R., P. J. Neame, J. P. Vergnes and J. R. Hassell (1998). "Identification of the N-linked oligosaccharide sites in chick corneal lumican and keratocan that receive keratan sulfate." J Biol Chem 273(16): 9615-21.
Elroy-Stein, O., T. R. Fuerst and B. Moss (1989). "Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system." Proc Natl Acad Sci U S A 86(16): 6126-30.
Eriksson, I., D. Sandback, B. Ek, U. Lindahl and L. Kjellen (1994). "cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N- deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin." J Biol Chem 269(14): 10438-43.
Farndale, R. W., C. A. Sayers and A. J. Barrett (1982). "A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures." Connect Tissue Res 9(4): 247-8.
Farris, A. D., G. Koelsch, G. J. Pruijn, W. J. van Venrooij and J. B. Harley (1999). "Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis." Nucleic Acids Res 27(4): 1070-8.
Fini, M. E. and M. T. Girard (1990). "The pattern of metalloproteinase expression by corneal fibroblasts is altered by passage in cell culture." J Cell Sci 97 ( Pt 2): 373-83.
205 References
Firestein, G. S., G. S. Panayi and F. A. Wollheim, Eds. (2000). Rheumatoid Arthritis - Frontiers in pathogenesis and treatment, Oxford University Press.
Flannery, C. R., M. W. Lark and J. D. Sandy (1992). "Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage." J Biol Chem 267(2): 1008-14.
Flannery, C. R., C. B. Little, C. E. Hughes and B. Caterson (1999). "Expression of ADAMTS homologues in articular cartilage." Biochem Biophys Res Commun 260(2): 318-22.
Flannery, C. R. and J. D. Sandy (1993). "Aggrecan catabolism in cartilage: Studies on the structure of a novel proteinase (aggrecanase) which cleaves the Glu 373-Ala 374 bond of the interglobular domain." Trans Orthop Res Soc 18: 190-190.
Fosang, A. J. and T. E. Hardingham (1989). "Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein." Biochem J 261(3): 801-9.
Fosang, A. J., K. Last, Y. Fujii, M. Seiki and Y. Okada (1998). "Membrane- type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain." FEBS Lett 430(3): 186-90.
Fosang, A. J., K. Last, V. Knauper, G. Murphy and P. J. Neame (1996). "Degradation of cartilage aggrecan by collagenase-3 (MMP-13)." FEBS Lett 380(1-2): 17-20.
Fosang, A. J., K. Last, V. Knauper, P. J. Neame, G. Murphy, T. E. Hardingham, H. Tschesche and J. A. Hamilton (1993). "Fibroblast and
206 References neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain." Biochem J 295 ( Pt 1): 273-6.
Fosang, A. J., K. Last, A. Plaas and C. J. Poon (2004). "Keratan sulphate in the aggrecan interglobular domain has a microstructure that is distinct from keratan sulphate elsewhere on aggrecan." Trans Orthop Res Soc 29: #0232.
Fosang, A. J., K. Last, H. Stanton, D. B. Weeks, I. K. Campbell, T. E. Hardingham and R. M. Hembry (2000). "Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants." J Biol Chem 275(42): 33027-37.
Fosang, A. J., P. J. Neame, T. E. Hardingham, G. Murphy and J. A. Hamilton (1991). "Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins." J Biol Chem 266(24): 15579-82.
Fosang, A. J., P. J. Neame, K. Last, T. E. Hardingham, G. Murphy and J. A. Hamilton (1992). "The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B." J Biol Chem 267(27): 19470-4.
Friedlander, D. R., P. Milev, L. Karthikeyan, R. K. Margolis, R. U. Margolis and M. Grumet (1994). "The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth." J Cell Biol 125(3): 669-80.
Fuerst, T. R., E. G. Niles, F. W. Studier and B. Moss (1986). "Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase." Proc Natl Acad Sci U S A 83(21): 8122-6.
Fukuda, M., N. Hiraoka, T. O. Akama and M. N. Fukuda (2001). "Carbohydrate-modifying sulfotransferases: structure, function, and pathophysiology." J Biol Chem 276(51): 47747-50.
207 References
Fukuta, M., J. Inazawa, T. Torii, K. Tsuzuki, E. Shimada and O. Habuchi (1997). "Molecular cloning and characterization of human keratan sulfate Gal- 6-sulfotransferase." J Biol Chem 272(51): 32321-8.
Fukuta, M., K. Uchimura, K. Nakashima, M. Kato, K. Kimata, T. Shinomura and O. Habuchi (1995). "Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase." J Biol Chem 270(31): 18575-80.
Funderburgh, J. L. (2000). "Keratan sulfate: structure, biosynthesis, and function." Glycobiology 10(10): 951-8.
Funderburgh, J. L. (2002). "Keratan sulfate biosynthesis." IUBMB Life 54(4): 187-94.
Funderburgh, J. L., B. Caterson and G. W. Conrad (1987). "Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan." J Biol Chem 262(24): 11634-40.
Funderburgh, J. L., M. L. Funderburgh, M. M. Mann and G. W. Conrad (1991). "Unique glycosylation of three keratan sulfate proteoglycan isoforms." J Biol Chem 266(22): 14226-31.
Funderburgh, J. L., M. L. Funderburgh, M. M. Mann, L. Corpuz and M. R. Roth (2001). "Proteoglycan expression during transforming growth factor beta -induced keratocyte-myofibroblast transdifferentiation." J Biol Chem 276(47): 44173-8.
Funderburgh, J. L., M. L. Funderburgh, M. M. Mann, S. Prakash and G. W. Conrad (1996). "Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro." J Biol Chem 271(49): 31431-6.
208 References
Funderburgh, J. L., M. M. Mann and M. L. Funderburgh (2003). "Keratocyte phenotype mediates proteoglycan structure: a role for fibroblasts in corneal fibrosis." J Biol Chem 278(46): 45629-37.
Funderburgh, J. L., P. R. Stenzel-Johnson and J. W. Chandler (1982). "Monoclonal antibodies to rabbit corneal keratan sulfate proteoglycan." Curr Eye Res 2(11): 769-76.
Gao, G., A. Plaas, V. P. Thompson, S. Jin, F. Zuo and J. D. Sandy (2004). "ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1." J Biol Chem 279(11): 10042-51.
Gao, G., J. Westling, V. P. Thompson, T. D. Howell, P. E. Gottschall and J. D. Sandy (2002). "Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation." J Biol Chem 277(13): 11034-41.
Geremia, R. A., A. Harduin-Lepers and P. Delannoy (1997). "Identification of two novel conserved amino acid residues in eukaryotic sialyltransferases: implications for their mechanism of action." Glycobiology 7(2): v-vii.
Gersten, K. M., S. Natsuka, M. Trinchera, B. Petryniak, R. J. Kelly, N. Hiraiwa, N. A. Jenkins, D. J. Gilbert, N. G. Copeland and J. B. Lowe (1995). "Molecular cloning, expression, chromosomal assignment, and tissue-specific expression of a murine alpha-(1,3)-fucosyltransferase locus corresponding to the human ELAM-1 ligand fucosyl transferase." J Biol Chem 270(42): 25047-56.
Giannelli, G., R. Erriquez, F. Iannone, F. Marinosci, G. Lapadula and S. Antonaci (2004). "MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in patients with rheumatoid arthritis and psoriatic arthritis." Clin Exp Rheumatol 22(3): 335-8.
Gomis-Ruth, F. X. (2003). "Structural aspects of the metzincin clan of metalloendopeptidases." Mol Biotechnol 24(2): 157-202.
209 References
Gospodarowicz, D., A. L. Mescher and C. R. Birdwell (1977). "Stimulation of corneal endothelial cell proliferations in vitro by fibroblast and epidermal growth factors." Exp Eye Res 25(1): 75-89.
Griffiths, G., R. Wepf, T. Wendt, J. K. Locker, M. Cyrklaff and N. Roos (2001). "Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis." J Virol 75(22): 11034-55.
Grodzinsky, A. J., M. E. Levenston, M. Jin and E. H. Frank (2000). "Cartilage tissue remodeling in response to mechanical forces." Annu Rev Biomed Eng 2: 691-713.
Grogan, M. J., M. R. Pratt, L. A. Marcaurelle and C. R. Bertozzi (2002). "Homogeneous glycopeptides and glycoproteins for biological investigation." Annu Rev Biochem 71: 593-634.
Grumet, M., A. Flaccus and R. U. Margolis (1993). "Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules." J Cell Biol 120(3): 815-24.
Grumet, M., P. Milev, T. Sakurai, L. Karthikeyan, M. Bourdon, R. K. Margolis and R. U. Margolis (1994). "Interactions with tenascin and differential effects on cell adhesion of neurocan and phosphacan, two major chondroitin sulfate proteoglycans of nervous tissue." J Biol Chem 269(16): 12142-6.
Hardingham, T. E. and H. Muir (1973). "Binding of oligosaccharides of hyaluronic acid to proteoglycan." Biochem J 135: 905-908.
Harduin-Lepers, A., V. Vallejo-Ruiz, M. A. Krzewinski-Recchi, B. Samyn-Petit, S. Julien and P. Delannoy (2001). "The human sialyltransferase family." Biochimie 83(8): 727-37.
210 References
Hascall, V. C. and D. Heinegard (1974). "Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction." J Biol Chem 249(13): 4242-9.
Hascall, V. C. and K. E. Kuettner, Eds. (2002). The Many Faces of Osteoarthritis. Basel - Boston - Berlin., Birkhäuser Verlag.
Hashimoto, Y., A. Orellana, G. Gil and C. B. Hirschberg (1992). "Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase." J Biol Chem 267(22): 15744-50.
Hassell, J. R., P. K. Schrecengost, J. A. Rada, N. SundarRaj, G. Sossi and R. A. Thoft (1992). "Biosynthesis of stromal matrix proteoglycans and basement membrane components by human corneal fibroblasts." Invest Ophthalmol Vis Sci 33(3): 547-57.
Heinegard, D. and V. C. Hascall (1974). "Aggregation of cartilage proteoglycans. 3. Characteristics of the proteins isolated from trypsin digests of aggregates." J Biol Chem 249(13): 4250-6.
Hering, T. M., T. Huynh, L. Duesler, N. Kazmi and D. Flory (2002). "Recombinant aggrecan as a substrate for adamts and mmp enzymes." Trans Orthop Res Soc 27: 437.
Hocking, A. M., R. A. Strugnell, P. Ramamurthy and D. J. McQuillan (1996). "Eukaryotic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity." J Biol Chem 271(32): 19571-7.
Hohenester, E. and J. Engel (2002). "Domain structure and organisation in extracellular matrix proteins." Matrix Biol 21(2): 115-28.
211 References
Holland, J. W., K. L. Meehan, S. L. Redmond and H. J. Dawkins (2004). "Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican." Prostate 59(3): 252-9.
Homeister, J. W., A. D. Thall, B. Petryniak, P. Maly, C. E. Rogers, P. L. Smith, R. J. Kelly, K. M. Gersten, S. W. Askari, G. Cheng, G. Smithson, R. M. Marks, A. K. Misra, O. Hindsgaul, U. H. von Andrian and J. B. Lowe (2001). "The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing." Immunity 15(1): 115-26.
Honke, K., M. Tsuda, Y. Hirahara, A. Ishii, A. Makita and Y. Wada (1997). "Molecular cloning and expression of cDNA encoding human 3'- phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase." J Biol Chem 272(8): 4864-8.
Horita, D. A., P. J. Hajduk, P. F. Goetinck and L. E. Lerner (1994). "NMR studies of peptides derived from the putative binding regions of cartilage proteins. No evidence for binding to hyaluronan." J Biol Chem 269(3): 1699- 704.
Huang, Y., H. Tanimukai, F. Liu, K. Iqbal, I. Grundke-Iqbal and C. X. Gong (2004). "Elevation of the level and activity of acid ceramidase in Alzheimer's disease brain." Eur J Neurosci 20(12): 3489-97.
Huckerby, T. N. and R. M. Lauder (2000). "Keratan sulfates from bovine tracheal cartilage structural studies of intact polymer chains using H and 13C NMR spectroscopy." Eur J Biochem 267(11): 3360-9.
Hughes, C. E., F. H. Buttner, B. Eidenmuller, B. Caterson and E. Bartnik (1997). "Utilization of a recombinant substrate rAgg1 to study the biochemical properties of aggrecanase in cell culture systems." J Biol Chem 272(32): 20269-74.
212 References
Ilic, M. Z., C. J. Handley, H. C. Robinson and M. T. Mok (1992). "Mechanism of catabolism of aggrecan by articular cartilage." Arch Biochem Biophys 294(1): 115-22.
Imber, M. J., L. R. Glasgow and S. V. Pizzo (1982). "Purification of an almond emulsin fucosidase on Cibacron blue-sepharose and demonstration of its activity toward fucose-containing glycoproteins." J Biol Chem 257(14): 8205- 10.
Inatani, M., H. Tanihara, A. Oohira, M. Honjo and Y. Honda (1999). "Identification of a nervous tissue-specific chondroitin sulfate proteoglycan, neurocan, in developing rat retina." Invest Ophthalmol Vis Sci 40(10): 2350-9.
Iozzo, R. V. (1998). "Matrix proteoglycans: from molecular design to cellular function." Annu Rev Biochem 67: 609-52.
Jackson, P. (1991). "Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device." Anal Biochem 196(2): 238-44.
Jackson, P. (1993). "Fluorophore-assisted carbohydrate electrophoresis: a new technology for the analysis of glycans." Biochem Soc Trans 21(1): 121-5.
Jackson, P. (1994). "The analysis of fluorophore-labeled glycans by high- resolution polyacrylamide gel electrophoresis." Anal Biochem 216(2): 243-52.
Jackson, P. (1994). "High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides." Methods Enzymol 230: 250-65.
Jackson, P., M. G. Pluskal and W. Skea (1994). "The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2-aminoacridone." Electrophoresis 15(7): 896-902.
213 References
Jaworski, D. M., G. M. Kelly and S. Hockfield (1994). "BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain." J Cell Biol 125(2): 495-509.
Jaworski, D. M., G. M. Kelly, J. M. Piepmeier and S. Hockfield (1996). "BEHAB (brain enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines." Cancer Res 56(10): 2293-8.
Jentoft, N. (1990). "Why are proteins O-glycosylated?" Trends Biochem Sci 15(8): 291-4.
Jester, J. V., P. A. Barry-Lane, H. D. Cavanagh and W. M. Petroll (1996). "Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes." Cornea 15(5): 505-16.
Joklik, W. K. (1964). "The Intracellular Uncoating of Poxvirus DNA. I. The Fate of Radioactively-Labeled Rabbitpox Virus." J Mol Biol 78: 263-76.
Jones, L. L. and M. H. Tuszynski (2002). "Spinal cord injury elicits expression of keratan sulfate proteoglycans by macrophages, reactive microglia, and oligodendrocyte progenitors." J Neurosci 22(11): 4611-24.
Kamei, A. (1998). "Partial properties of four glycosidases in normal human lens and variations in their enzyme activities during aging and with the advance of lens coloration." Biol Pharm Bull 21(9): 982-6.
Kaneko, M., T. Kudo, H. Iwasaki, Y. Ikehara, S. Nishihara, S. Nakagawa, K. Sasaki, T. Shiina, H. Inoko, N. Saitou and H. Narimatsu (1999). "Alpha1,3- fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX." FEBS Lett 452(3): 237-42.
214 References
Kao, W. W. and C. Y. Liu (2002). "Roles of lumican and keratocan on corneal transparency." Glycoconj J 19(4-5): 275-85.
Kashiwagi, M., J. J. Enghild, C. Gendron, C. Hughes, B. Caterson, Y. Itoh and H. Nagase (2004). "Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing." J Biol Chem 279(11): 10109-19.
Kates, J. R. and B. R. McAuslan (1967). "Poxvirus DNA-dependent RNA polymerase." Proc Natl Acad Sci U S A 58(1): 134-41.
Kavanagh, E., A. C. Osborne, D. E. Ashhurst and A. A. Pitsillides (2002). "Keratan sulfate epitopes exhibit a conserved distribution during joint development that remains undisclosed on the basis of glycosaminoglycan charge density." J Histochem Cytochem 50(8): 1039-47.
Kido, A., N. Komatsu, Y. Ose and M. Oya (1987). "alpha-L-fucosidase phenotyping in human tissues, dental pulps and hair roots." Forensic Sci Int 33(1): 53-9.
Kimata, K., H. J. Barrach, K. S. Brown and J. P. Pennypacker (1981). "Absence of proteoglycan core protein in cartilage from the cmd/cmd (cartilage matrix deficiency) mouse." J Biol Chem 256(13): 6961-8.
Kimura, H., N. Shinya, S. Nishihara, M. Kaneko, T. Irimura and H. Narimatsu (1997). "Distinct substrate specificities of five human alpha-1,3- fucosyltransferases for in vivo synthesis of the sialyl Lewis x and Lewis x epitopes." Biochem Biophys Res Commun 237(1): 131-7.
Kobayakawa, M., H. Iwata, K. S. Brown and K. Kimata (1985). "Abnormal collagen fibrillogenesis in epiphyseal cartilage of CMD (cartilage matrix deficiency) mouse." Coll Relat Res 5(2): 137-47.
Kohda, D., C. J. Morton, A. A. Parkar, H. Hatanaka, F. M. Inagaki, I. D. Campbell and A. J. Day (1996). "Solution structure of the link module: a
215 References hyaluronan-binding domain involved in extracellular matrix stability and cell migration." Cell 86(5): 767-75.
Krautstrunk, M., F. Scholtes, D. Martin, J. Schoenen, A. B. Schmitt, D. Plate, W. Nacimiento, J. Noth and G. A. Brook (2002). "Increased expression of the putative axon growth-repulsive extracellular matrix molecule, keratan sulphate proteoglycan, following traumatic injury of the adult rat spinal cord." Acta Neuropathol (Berl) 104(6): 592-600.
Krusius, T., J. Finne, R. K. Margolis and R. U. Margolis (1986). "Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain." J Biol Chem 261(18): 8237-42.
Kudo, T., Y. Ikehara, A. Togayachi, M. Kaneko, T. Hiraga, K. Sasaki and H. Narimatsu (1998). "Expression cloning and characterization of a novel murine alpha1, 3-fucosyltransferase, mFuc-TIX, that synthesizes the Lewis x (CD15) epitope in brain and kidney." J Biol Chem 273(41): 26729-38.
Kuno, K., N. Kanada, E. Nakashima, F. Fujiki, F. Ichimura and K. Matsushima (1997). "Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene." J Biol Chem 272(1): 556-62.
Kuno, K., Y. Okada, H. Kawashima, H. Nakamura, M. Miyasaka, H. Ohno and K. Matsushima (2000). "ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan." FEBS Lett 478(3): 241-5.
Kurpakus Wheater, M., K. A. Kernacki and L. D. Hazlett (1999). "Corneal cell proteins and ocular surface pathology." Biotech Histochem 74(3): 146-59.
Landolt, R. M., L. Vaughan, K. H. Winterhalter and D. R. Zimmermann (1995). "Versican is selectively expressed in embryonic tissues that act as barriers to
216 References neural crest cell migration and axon outgrowth." Development 121(8): 2303- 12.
Lechner, J. and F. Wieland (1989). "Structure and biosynthesis of prokaryotic glycoproteins." Annu Rev Biochem 58: 173-94.
Leerhoy, J. (1965). "Cytopathic Effect of Rubella Virus in a Rabbit-Cornea Cell Line." Science 149: 633-4.
Li, H., T. C. Leung, S. Hoffman, J. Balsamo and J. Lilien (2000). "Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan." J Cell Biol 149(6): 1275-88.
Li, S. Y., J. V. Holtje and K. D. Young (2004). "Comparison of high- performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli." Anal Biochem 326(1): 1-12.
Locker, J. K. and G. Griffiths (1999). "An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins." J Cell Biol 144(2): 267-79.
Lohmander, L. S., P. J. Neame and J. D. Sandy (1993). "The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis." Arthritis Rheum 36(9): 1214-22.
Long, C. J., M. R. Roth, E. S. Tasheva, M. Funderburgh, R. Smit, G. W. Conrad and J. L. Funderburgh (2000). "Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro." J Biol Chem 275(18): 13918-23.
Loulakis, P., A. Shrikhande, G. Davis and C. A. Maniglia (1992). "N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-
217 References treated articular-cartilage cultures. Putative site(s) of enzymic cleavage." Biochem J 284 ( Pt 2): 589-93.
Mace, K., R. Saxod, C. Feuerstein, R. Sadoul and F. J. Hemming (2002). "Chondroitin and keratan sulfates have opposing effects on attachment and outgrowth of ventral mesencephalic explants in culture." J Neurosci Res 70(1): 46-56.
Martin, S. A., E. Paoletti and B. Moss (1975). "Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions." J. Biol. Chem. 250(24): 9322-9329.
Mathew, E. C., C. M. Sanderson, R. Hollinshead and G. L. Smith (2001). "A mutational analysis of the vaccinia virus B5R protein." J Gen Virol 82(Pt 5): 1199-213.
Matthews, R. T., S. C. Gary, C. Zerillo, M. Pratta, K. Solomon, E. C. Arner and S. Hockfield (2000). "Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member." J Biol Chem 275(30): 22695-703.
McQuillan, D. J., S. Neung-Seon, A. M. Hocking and C. I. McQuillan (2001). Recombinant Expression of Proteoglycans in Mammalian Cells - Utility and Advantages of the Vaccinia Virus/T7 Bacteriophage Hybrid Expression System. Methods in Molecular Biology. R. V. Iozzo. Totowa, NJ, Humana Press Inc. 171: Proteoglycan Protocols: 201-219.
Mercuri, F. A., K. J. Doege, E. C. Arner, M. A. Pratta, K. Last and A. J. Fosang (1999). "Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase." J Biol Chem 274(45): 32387-95.
218 References
Mercuri, F. A., R. A. Maciewicz, J. Tart, K. Last and A. J. Fosang (2000). "Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity." J Biol Chem 275(42): 33038-45.
Merrifield, R. B. (1969). "Solid-phase peptide synthesis." Adv Enzymol Relat Areas Mol Biol 32: 221-96.
Michelacci, Y. M. (2003). "Collagens and proteoglycans of the corneal extracellular matrix." Braz J Med Biol Res 36(8): 1037-46.
Midura, R. J., O. M. Toledo, M. Yanagishita and V. C. Hascall (1989). "Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. I. Characterization of the culture system with emphasis on stromal proteoglycan biosynthesis." J Biol Chem 264(3): 1414-22.
Milev, P., A. Chiba, M. Haring, H. Rauvala, M. Schachner, B. Ranscht, R. K. Margolis and R. U. Margolis (1998). "High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase- zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth- associated molecule." J Biol Chem 273(12): 6998-7005.
Milev, P., P. Maurel, M. Haring, R. K. Margolis and R. U. Margolis (1996). "TAG-1/axonin-1 is a high-affinity ligand of neurocan, phosphacan/protein- tyrosine phosphatase-zeta/beta, and N-CAM." J Biol Chem 271(26): 15716- 23.
Mjaatvedt, C. H., H. Yamamura, A. A. Capehart, D. Turner and R. R. Markwald (1998). "The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation." Dev Biol 202(1): 56-66.
219 References
Morgunova, E., A. Tuuttila, U. Bergmann, M. Isupov, Y. Lindqvist, G. Schneider and K. Tryggvason (1999). "Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed." Science 284(5420): 1667-70.
Moss, B. (1990). Poxviridae and their replication. Fields Virology. B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath and B. Roizman. New York, N.Y., Raven Press.
Moss, B. (1990). "Regulation of vaccinia virus transcription." Annu Rev Biochem 59: 661-88.
Moulharat, N., C. Lesur, M. Thomas, G. Rolland-Valognes, P. Pastoureau, P. Anract, F. De Ceuninck and M. Sabatini (2004). "Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro." Osteoarthritis Cartilage 12(4): 296-305.
Munteanu, S. E., M. Z. Ilic and C. J. Handley (2002). "Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures." Matrix Biol 21(5): 429-40.
Munyon, W. H. and S. Kit (1966). "Induction of cytoplasmic ribonucleic acid (RNA) synthesis in vaccinia-infected LM cells during inhibition of protein synthesis." Virology 29(2): 303-9.
Muthukrishnan, S., B. Moss, J. A. Cooper and E. S. Maxwell (1978). "Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA." J Biol Chem 253(5): 1710-5.
Nagase, H. and K. Brew (2003). "Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors." Biochem Soc Symp(70): 201-12.
220 References
Nakayama, F., S. Nishihara, H. Iwasaki, T. Kudo, R. Okubo, M. Kaneko, M. Nakamura, M. Karube, K. Sasaki and H. Narimatsu (2001). "CD15 expression in mature granulocytes is determined by alpha 1,3-fucosyltransferase IX, but in promyelocytes and monocytes by alpha 1,3-fucosyltransferase IV." J Biol Chem 276(19): 16100-6.
Nakazawa, K., A. Morita, H. Nakano, C. Mano and N. Tozawa (1996). "Keratan sulfate synthesis by corneal stromal cells within three-dimensional collagen gel cultures." J Biochem (Tokyo) 120(1): 117-25.
Nakazawa, K., S. Suzuki and K. Wada (1995). "Proteoglycan synthesis by corneal explants from developing embryonic chicken." J Biochem (Tokyo) 117(4): 707-18.
Nakazawa, K., I. Takahashi, Y. Ohno and M. Sato (1997). "Modification of proteoglycan synthesis by corneal stromal cells on co-culture with either epithelial or endothelial cells." J Biochem (Tokyo) 122(4): 851-8.
Nakazawa, K., I. Takahashi and Y. Yamamoto (1998). "Glycosyltransferase and sulfotransferase activities in chick corneal stromal cells before and after in vitro culture." Arch Biochem Biophys 359(2): 269-82.
Narimatsu, H., H. Iwasaki, F. Nakayama, Y. Ikehara, T. Kudo, S. Nishihara, K. Sugano, H. Okura, S. Fujita and S. Hirohashi (1998). "Lewis and secretor gene dosages affect CA19-9 and DU-PAN-2 serum levels in normal individuals and colorectal cancer patients." Cancer Res 58(3): 512-8.
Narimatsu, H., H. Iwasaki, S. Nishihara, H. Kimura, T. Kudo, Y. Yamauchi and S. Hirohashi (1996). "Genetic evidence for the Lewis enzyme, which synthesizes type-1 Lewis antigens in colon tissue, and intracellular localization of the enzyme." Cancer Res 56(2): 330-8.
221 References
Nieduszynski, I. A., T. N. Huckerby, J. M. Dickenson, G. M. Brown, G. H. Tai, H. G. Morris and S. Eady (1990). "There are two major types of skeletal keratan sulphates." Biochem J 271(1): 243-5.
Nutt, C. L., R. T. Matthews and S. Hockfield (2001). "Glial tumor invasion: a role for the upregulation and cleavage of BEHAB/brevican." Neuroscientist 7(2): 113-22.
Oleszewski, M., S. Beer, S. Katich, C. Geiger, Y. Zeller, U. Rauch and P. Altevogt (1999). "Integrin and neurocan binding to L1 involves distinct Ig domains." J Biol Chem 274(35): 24602-10.
Olin, A. I., M. Morgelin, T. Sasaki, R. Timpl, D. Heinegard and A. Aspberg (2001). "The proteoglycans aggrecan and Versican form networks with fibulin- 2 through their lectin domain binding." J Biol Chem 276(2): 1253-61.
Ong, E., J. C. Yeh, Y. Ding, O. Hindsgaul and M. Fukuda (1998). "Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids." J Biol Chem 273(9): 5190-5.
Oohira, A., F. Matsui, E. Watanabe, Y. Kushima and N. Maeda (1994). "Developmentally regulated expression of a brain specific species of chondroitin sulfate proteoglycan, neurocan, identified with a monoclonal antibody IG2 in the rat cerebrum." Neuroscience 60(1): 145-57.
Overall, C. M. (2001). Matrix metalloproteinase substrate binding domains, modules and exosites. Matrix Metalloproteinase Protocols. I. Clark. Totowa, NJ, Humana: 79-120.
Paulsson, M., M. Morgelin, H. Wiedemann, M. Beardmore-Gray, D. Dunham, T. Hardingham, D. Heinegard, R. Timpl and J. Engel (1987). "Extended and globular protein domains in cartilage proteoglycans." Biochem J 245(3): 763- 72.
222 References
Pavelka, K. and M. J. Seibel (1989). "[Quantitative detection of keratan sulfate specific epitopes in synovial fluid in inflammatory and degenerative joint diseases]." Z Rheumatol 48(6): 294-300.
Pavelka, K., M. J. Seibel and W. Muller (1989). "[Measurement of keratan sulfate levels in synovial fluid in joint diseases]." Vnitr Lek 35(12): 1203-10.
Perkins, S. J., A. S. Nealis, J. Dudhia and T. E. Hardingham (1989). "Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein." J Mol Biol 206(4): 737-53.
Perkins, S. J., A. S. Nealis, D. G. Dunham, T. E. Hardingham and I. H. Muir (1991). "Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X- ray scattering." Biochemistry 30(44): 10708-16.
Phillips, C. A., J. L. Melnick and M. Burkhardt (1966). "Isolation, propagation and neutralization of rubella virus in cultures of rabbit cornea (SIRC) cells." Proc Soc Exp Biol Med 122(3): 783-6.
Plaas, A. H., L. West, R. J. Midura and V. C. Hascall (2001). "Disaccharide composition of hyaluronan and chondroitin/dermatan sulfate. Analysis with fluorophore-assisted carbohydrate electrophoresis." Methods Mol Biol 171: 117-28.
Plaas, A. H., L. A. West and R. J. Midura (2001). "Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-beta-galactosidase digestion products." Glycobiology 11(10): 779-90.
Plaas, A. H., L. A. West, E. J. Thonar, Z. A. Karcioglu, C. J. Smith, G. K. Klintworth and V. C. Hascall (2001). "Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy." J Biol Chem 276(43): 39788-96.
223 References
Poon, C. J., A. H. Plaas, D. R. Keene, D. J. McQuillan, K. Last and A. J. Fosang (2005). "N-linked keratan sulphate in the aggrecan interglobular domain potentiates aggrecanase activity." J Biol Chem.
Porter, S., I. M. Clark, L. Kevorkian and D. R. Edwards (2005). "The ADAMTS metalloproteinases." Biochem J 386(Pt 1): 15-27.
Pratta, M. and E. Arner (1997). "Effect of aggrecan glycosylation on digestion by "aggrecanase"." Trans Orthop Res Soc 22: 453.
Pratta, M. A., P. A. Scherle, G. Yang, R. Q. Liu and R. C. Newton (2003). "Induction of aggrecanase 1 (ADAM-TS4) by interleukin-1 occurs through activation of constitutively produced protein." Arthritis Rheum 48(1): 119-33.
Pratta, M. A., M. Tortorella and E. C. Arner (1999). "Aggrecan glycosylation affects cleavage by aggrecanase." Trans Orthop Res Soc 24: 407.
Pratta, M. A., M. D. Tortorella and E. C. Arner (2000). "Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase." J Biol Chem 275(50): 39096-102.
Primakoff, P. and D. G. Myles (2000). "The ADAM gene family: surface proteins with adhesion and protease activity." Trends Genet 16(2): 83-7.
Ramamurthy, P., A. M. Hocking and D. J. McQuillan (1996). "Recombinant decorin glycoforms. Purification and structure." J Biol Chem 271(32): 19578- 84.
Ratcliffe, A., M. Doherty, R. N. Maini and T. E. Hardingham (1988). "Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease." Ann Rheum Dis 47(10): 826-32.
224 References
Rauch, U., K. Feng and X. H. Zhou (2001). "Neurocan: a brain chondroitin sulfate proteoglycan." Cell Mol Life Sci 58(12-13): 1842-56.
Rauch, U., P. Gao, A. Janetzko, A. Flaccus, L. Hilgenberg, H. Tekotte, R. K. Margolis and R. U. Margolis (1991). "Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies." J Biol Chem 266(22): 14785-801.
Retzler, C., W. Gohring and U. Rauch (1996). "Analysis of neurocan structures interacting with the neural cell adhesion molecule N-CAM." J Biol Chem 271(44): 27304-10.
Rhim, J. S., K. Schell and R. J. Huebner (1967). "Plaque assays of rubella virus in cultures of rabbit cornea (SIRC) cells." Proc Soc Exp Biol Med 125(4): 1271-4.
Rittenhouse, E., L. C. Dunn, J. Cookingham, C. Calo, M. Spiegelman, G. B. Dooher and D. Bennett (1978). "Cartilage matrix deficiency (cmd): a new autosomal recessive lethal mutation in the mouse." J Embryol Exp Morphol 43: 71-84.
Rodriguez-Manzaneque, J. C., J. Westling, S. N. Thai, A. Luque, V. Knauper, G. Murphy, J. D. Sandy and M. L. Iruela-Arispe (2002). "ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors." Biochem Biophys Res Commun 293(1): 501-8.
Roeb, E., K. Schleinkofer, T. Kernebeck, S. Potsch, B. Jansen, I. Behrmann, S. Matern and J. Grotzinger (2002). "The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist." J Biol Chem 277(52): 50326-32.
Roos, N., M. Cyrklaff, S. Cudmore, R. Blasco, J. Krijnse-Locker and G. Griffiths (1996). "A novel immunogold cryoelectron microscopic approach to
225 References investigate the structure of the intracellular and extracellular forms of vaccinia virus." Embo J 15(10): 2343-55.
Roughley, P. J. (2001). "Articular cartilage and changes in arthritis: noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage." Arthritis Res 3(6): 342-7.
Roughley, P. J., M. Alini and J. Antoniou (2002). "The role of proteoglycans in aging, degeneration and repair of the intervertebral disc." Biochem Soc Trans 30(Pt 6): 869-74.
Ruoslahti, E. and M. D. Pierschbacher (1986). "Arg-Gly-Asp: a versatile cell recognition signal." Cell 44(4): 517-8.
Sajdera, S. W. and V. C. Hascall (1969). "Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures." J Biol Chem 244(1): 77-87.
Salmons, T., A. Kuhn, F. Wylie, S. Schleich, J. R. Rodriguez, D. Rodriguez, M. Esteban, G. Griffiths and J. K. Locker (1997). "Vaccinia virus membrane proteins p8 and p16 are cotranslationally inserted into the rough endoplasmic reticulum and retained in the intermediate compartment." J Virol 71(10): 7404- 20.
Sandy, J. D., P. J. Neame, R. E. Boynton and C. R. Flannery (1991). "Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain." J Biol Chem 266(14): 8683-5.
Sandy, J. D., A. H. Plaas and T. J. Koob (1995). "Pathways of aggrecan processing in joint tissues. Implications for disease mechanism and monitoring." Acta Orthop Scand Suppl 266: 26-32.
Sandy, J. D. and C. Verscharen (2001). "Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is
226 References responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo." Biochem J 358(Pt 3): 615-26.
Sandy, J. D., J. Westling, R. D. Kenagy, M. L. Iruela-Arispe, C. Verscharen, J. C. Rodriguez-Mazaneque, D. R. Zimmermann, J. M. Lemire, J. W. Fischer, T. N. Wight and A. W. Clowes (2001). "Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4." J Biol Chem 276(16): 13372-8.
Sarkozi, A. M., G. Hamori, C. Fulop, E. Buzas, M. Brozik, K. Meretey and T. T. Glant (1993). "[Serum keratan sulfate studies and their significance in the evaluation of cartilage degradation in degenerative and inflammatory joint diseases]." Orv Hetil 134(9): 461-7.
Schmelz, M., B. Sodeik, M. Ericsson, E. J. Wolffe, H. Shida, G. Hiller and G. Griffiths (1994). "Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network." J Virol 68(1): 130-47.
Schrecengost, P. K., T. C. Blochberger and J. R. Hassell (1992). "Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts." Arch Biochem Biophys 292(1): 54-61.
Schwartz, N. B., M. Domowicz, R. C. Krueger, Jr., H. Li and D. Mangoura (1996). "Brain aggrecan." Perspect Dev Neurobiol 3(4): 291-306.
Seals, D. F. and S. A. Courtneidge (2003). "The ADAMs family of metalloproteases: multidomain proteins with multiple functions." Genes Dev 17(1): 7-30.
Seibel, M. J., W. Macaulay, R. Jelsma, F. Saed-Nejad and A. Ratcliffe (1992). "Antigenic properties of keratan sulfate: influence of antigen structure,
227 References monoclonal antibodies, and antibody valency." Arch Biochem Biophys 296(2): 410-8.
Seidenbecher, C. I., K. Richter, U. Rauch, R. Fassler, C. C. Garner and E. D. Gundelfinger (1995). "Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms." J Biol Chem 270(45): 27206-12.
Seyfried, N. T., G. F. McVey, A. Almond, D. J. Mahoney, J. Dudhia and A. J. Day (2005). "Expression and Purification of Functionally Active Hyaluronan- binding Domains from Human Cartilage Link Protein, Aggrecan and Versican: FORMATION OF TERNARY COMPLEXES WITH DEFINED HYALURONAN OLIGOSACCHARIDES." J Biol Chem 280(7): 5435-48.
Sheng, W., G. Wang, Y. Wang, J. Liang, J. Wen, P. S. Zheng, Y. Wu, V. Lee, J. Slingerland, D. Dumont and B. B. Yang (2005). "The roles of versican v1 and v2 isoforms in cell proliferation and apoptosis." Mol Biol Cell 16(3): 1330- 40.
Shindo, T., H. Kurihara, K. Kuno, H. Yokoyama, T. Wada, Y. Kurihara, T. Imai, Y. Wang, M. Ogata, H. Nishimatsu, N. Moriyama, Y. Oh-hashi, H. Morita, T. Ishikawa, R. Nagai, Y. Yazaki and K. Matsushima (2000). "ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function." J Clin Invest 105(10): 1345-52.
Shworak, N. W., J. Liu, L. M. Fritze, J. J. Schwartz, L. Zhang, D. Logeart and R. D. Rosenberg (1997). "Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate D-glucosaminyl 3-O- sulfotransferase." J Biol Chem 272(44): 28008-19.
Silbert, J. E. and G. Sugumaran (2002). "A starting place for the road to function." Glycoconj J 19(4-5): 227-37.
228 References
Snow, H. E., L. M. Riccio, C. H. Mjaatvedt, S. Hoffman and A. A. Capehart (2005). "Versican expression during skeletal/joint morphogenesis and patterning of muscle and nerve in the embryonic mouse limb." Anat Rec A Discov Mol Cell Evol Biol 282(2): 95-105.
Somerville, R. P., J. M. Longpre, K. A. Jungers, J. M. Engle, M. Ross, S. Evanko, T. N. Wight, R. Leduc and S. S. Apte (2003). "Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1." J Biol Chem 278(11): 9503-13.
Spicer, A. P., A. Joo and R. A. Bowling, Jr. (2003). "A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes: the missing links." J Biol Chem 278(23): 21083-91.
Stanton, H., F. M. Rogerson, C. J. East, S. B. Golub, K. E. Lawlor, C. T. Meeker, C. B. Little, K. Last, P. J. Farmer, I. K. Campbell, A. M. Fourie and A. J. Fosang (2005). "ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro." Nature 434(7033): 648-52.
Starr, C. M., R. I. Masada, C. Hague, E. Skop and J. C. Klock (1996). "Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates." J Chromatogr A 720(1-2): 295- 321.
Sternlicht, M. D. and Z. Werb (2001). "How matrix metalloproteinases regulate cell behavior." Annu Rev Cell Dev Biol 17: 463-516.
Sugimoto, K., M. Takahashi, Y. Yamamoto, K. Shimada and K. Tanzawa (1999). "Identification of aggrecanase activity in medium of cartilage culture." J Biochem (Tokyo) 126(2): 449-55.
Suzuki, M. (1939). "Prosthetic group of cornea mucoid." J Biochem (Tokyo) 30: 185-191.
229 References
Tai, G. H., T. N. Huckerby and I. A. Nieduszynski (1996). "Multiple non- reducing chain termini isolated from bovine corneal keratan sulfates." J Biol Chem 271(38): 23535-46.
Tai, G. H., I. A. Nieduszynski, N. J. Fullwood and T. N. Huckerby (1997). "Human corneal keratan sulfates." J Biol Chem 272(45): 28227-31.
Tchetverikov, I., H. K. Ronday, B. Van El, G. H. Kiers, N. Verzijl, J. M. TeKoppele, T. W. Huizinga, J. DeGroot and R. Hanemaaijer (2004). "MMP profile in paired serum and synovial fluid samples of patients with rheumatoid arthritis." Ann Rheum Dis 63(7): 881-3.
Thiele, H., M. Sakano, H. Kitagawa, K. Sugahara, A. Rajab, W. Hohne, H. Ritter, G. Leschik, P. Nurnberg and S. Mundlos (2004). "Loss of chondroitin 6- O-sulfotransferase-1 function results in severe human chondrodysplasia with progressive spinal involvement." Proc Natl Acad Sci U S A 101(27): 10155-60.
Thonar, E. J. (1990). "Serum keratan sulfate concentration as a measure of the catabolism of cartilage proteoglycans." Ryumachi 30(6): 461-8.
Thonar, E. J. and T. Glant (1992). "Serum keratan sulfate--a marker of predisposition to polyarticular osteoarthritis." Clin Biochem 25(3): 175-80.
Thonar, E. J. and D. H. Manicourt (1994). "[Biological markers of osteoarthritis]." Rev Rhum Ed Fr 61(9 Pt 2): 99S-102S.
Thonar, E. J., K. Masuda, M. E. Lenz, H. J. Hauselmann, K. E. Kuettner and D. H. Manicourt (1995). "Serum markers of systemic disease processes in osteoarthritis." J Rheumatol Suppl 43: 68-70.
Thonar, E. J., L. M. Pachman, M. E. Lenz, J. Hayford, P. Lynch and K. E. Kuettner (1988). "Age related changes in the concentration of serum keratan sulphate in children." J Clin Chem Clin Biochem 26(2): 57-63.
230 References
Thonar, E. J., T. J. Schnitzer and K. E. Kuettner (1987). "Quantification of keratan sulfate in blood as a marker of cartilage catabolism." J Rheumatol 14 Spec No: 23-4.
Thonar, E. J., M. Shinmei and L. S. Lohmander (1993). "Body fluid markers of cartilage changes in osteoarthritis." Rheum Dis Clin North Am 19(3): 635-57.
Tortorella, M., M. Pratta, R. Q. Liu, I. Abbaszade, H. Ross, T. Burn and E. Arner (2000). "The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage." J Biol Chem 275(33): 25791-7.
Tortorella, M. D., T. C. Burn, M. A. Pratta, I. Abbaszade, J. M. Hollis, R. Liu, S. A. Rosenfeld, R. A. Copeland, C. P. Decicco, R. Wynn, A. Rockwell, F. Yang, J. L. Duke, K. Solomon, H. George, R. Bruckner, H. Nagase, Y. Itoh, D. M. Ellis, H. Ross, B. H. Wiswall, K. Murphy, M. C. Hillman, Jr., G. F. Hollis, E. C. Arner and et al. (1999). "Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins." Science 284(5420): 1664-6.
Tortorella, M. D., R. Q. Liu, T. Burn, R. C. Newton and E. Arner (2002). "Characterization of human aggrecanase 2 (ADAM-TS5): substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4)." Matrix Biol 21(6): 499-511.
Tortorella, M. D., M. Pratta, R. Q. Liu, J. Austin, O. H. Ross, I. Abbaszade, T. Burn and E. Arner (2000). "Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4)." J Biol Chem 275(24): 18566-73.
Touab, M., J. Villena, C. Barranco, M. Arumi-Uria and A. Bassols (2002). "Versican is differentially expressed in human melanoma and may play a role in tumor development." Am J Pathol 160(2): 549-57.
231 References
Tsuji, S., A. K. Datta and J. C. Paulson (1996). "Systematic nomenclature for sialyltransferases." Glycobiology 6(7): v-vii.
Uchimura, K., K. Kadomatsu, H. Nishimura, H. Muramatsu, E. Nakamura, N. Kurosawa, O. Habuchi, F. M. El-Fasakhany, Y. Yoshikai and T. Muramatsu (2002). "Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates." J Biol Chem 277(2): 1443-50.
Unverzagt, C., S. Andre, J. Seifert, S. Kojima, C. Fink, G. Srikrishna, H. Freeze, K. Kayser and H. J. Gabius (2002). "Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake." J Med Chem 45(2): 478-91. van Meurs, J. B., P. L. van Lent, A. E. Holthuysen, Singer, II, E. K. Bayne and W. B. van den Berg (1999). "Kinetics of aggrecanase- and metalloproteinase- induced neoepitopes in various stages of cartilage destruction in murine arthritis." Arthritis Rheum 42(6): 1128-39.
Van Wart, H. E. and H. Birkedal-Hansen (1990). "The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family." Proc Natl Acad Sci U S A 87(14): 5578-82.
Vankemmelbeke, M. N., I. Holen, A. G. Wilson, M. Z. Ilic, C. J. Handley, G. S. Kelner, M. Clark, C. Liu, R. A. Maki, D. Burnett and D. J. Buttle (2001). "Expression and activity of ADAMTS-5 in synovium." Eur J Biochem 268(5): 1259-68.
Visco, D. M., B. Johnstone, M. A. Hill, G. A. Jolly and B. Caterson (1993). "Immunohistochemical analysis of 3-B-(-) and 7-D-4 epitope expression in canine osteoarthritis." Arthritis Rheum 36(12): 1718-25.
232 References
Visse, R. and H. Nagase (2003). "Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry." Circ Res 92(8): 827-39.
Vuillermoz, B., A. Khoruzhenko, M. F. D'Onofrio, L. Ramont, L. Venteo, C. Perreau, F. Antonicelli, F. X. Maquart and Y. Wegrowski (2004). "The small leucine-rich proteoglycan lumican inhibits melanoma progression." Exp Cell Res 296(2): 294-306.
Walcz, E., F. Deak, P. Erhardt, S. N. Coulter, C. Fulop, P. Horvath, K. J. Doege and T. T. Glant (1994). "Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan." Genomics 22(2): 364-71.
Wang, P., M. Tortorella, K. England, A. M. Malfait, G. Thomas, E. C. Arner and D. Pei (2004). "Proprotein convertase furin interacts with and cleaves pro- ADAMTS4 (Aggrecanase-1) in the trans-Golgi network." J Biol Chem 279(15): 15434-40.
Ward, B. M. and B. Moss (2001). "Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera." J Virol 75(10): 4802-13.
Ward, B. M., A. S. Weisberg and B. Moss (2003). "Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins." J Virol 77(7): 4113-26.
Watanabe, H. and Y. Yamada (1999). "Mice lacking link protein develop dwarfism and craniofacial abnormalities." Nat Genet 21(2): 225-9.
Watanabe, H. and Y. Yamada (2002). "Chondrodysplasia of gene knockout mice for aggrecan and link protein." Glycoconj J 19(4-5): 269-73.
233 References
Weikert, S., D. Papac, J. Briggs, D. Cowfer, S. Tom, M. Gawlitzek, J. Lofgren, S. Mehta, V. Chisholm, N. Modi, S. Eppler, K. Carroll, S. Chamow, D. Peers, P. Berman and L. Krummen (1999). "Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins." Nat Biotechnol 17(11): 1116-21.
White, J. M. (2003). "ADAMs: modulators of cell-cell and cell-matrix interactions." Curr Opin Cell Biol 15(5): 598-606.
Wight, T. N. (2002). "Versican: a versatile extracellular matrix proteoglycan in cell biology." Curr Opin Cell Biol 14(5): 617-23.
Williams, J., B. Rucinski, J. Holt and S. Niewiarowski (1990). "Elegantin and albolabrin purified peptides from viper venoms: homologies with the RGDS domain of fibrinogen and von Willebrand factor." Biochim Biophys Acta 1039(1): 81-9.
Wu, Y., W. Sheng, L. Chen, H. Dong, V. Lee, F. Lu, C. S. Wong, W. Y. Lu and B. B. Yang (2004). "Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth." Mol Biol Cell 15(5): 2093-104.
Yamada, H., K. Watanabe, M. Shimonaka and Y. Yamaguchi (1994). "Molecular cloning of brevican, a novel brain proteoglycan of the aggrecan/versican family." J Biol Chem 269(13): 10119-26.
Yamada, H., K. Watanabe, M. Shimonaka, M. Yamasaki and Y. Yamaguchi (1995). "cDNA cloning and the identification of an aggrecanase-like cleavage site in rat brevican." Biochem Biophys Res Commun 216(3): 957-63.
Yamaji, N., K. Nishimura, K. Abe, O. Ohara, T. Nagase and N. Nomura (2001). Novel metalloprotease having aggrecanase activity. Japan, Yamanouchi Pharmaceutical C. Ltd.
234 References
Yang, B. L., Y. Zhang, L. Cao and B. B. Yang (1999). "Cell adhesion and proliferation mediated through the G1 domain of versican." J Cell Biochem 72(2): 210-20.
Yoshihara, Y., H. Nakamura, K. Obata, H. Yamada, T. Hayakawa, K. Fujikawa and Y. Okada (2000). "Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis." Ann Rheum Dis 59(6): 455-61.
Yoshihara, Y., K. Obata, N. Fujimoto, K. Yamashita, T. Hayakawa and M. Shimmei (1995). "Increased levels of stromelysin-1 and tissue inhibitor of metalloproteinases-1 in sera from patients with rheumatoid arthritis." Arthritis Rheum 38(7): 969-75.
Yuan, W., R. T. Matthews, J. D. Sandy and P. E. Gottschall (2002). "Association between protease-specific proteolytic cleavage of brevican and synaptic loss in the dentate gyrus of kainate-treated rats." Neuroscience 114(4): 1091-101.
Zhou, X. H., C. Brakebusch, H. Matthies, T. Oohashi, E. Hirsch, M. Moser, M. Krug, C. I. Seidenbecher, T. M. Boeckers, U. Rauch, R. Buettner, E. D. Gundelfinger and R. Fassler (2001). "Neurocan is dispensable for brain development." Mol Cell Biol 21(17): 5970-8.
235 Appendix
Appendix The following article is as it appears in press. PMID: 15849197.
236 Appendix
237 Appendix
238 Appendix
239 Appendix
240 Appendix
241 Appendix
242
Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Poon, C. J.
Title: The role of keratan sulphate in the modulation of aggrecanase activity
Date: 2005
Citation: Poon, C. J. (2005). The role of keratan sulphate in the modulation of aggrecanase activity. PhD Thesis, Department of Paediatrics, Faculty of Medicine, Dentistry & Health Sciences, Paediatrics Royal Children's Hospital, The University of Melbourne.
Publication Status: Unpublished
Persistent Link: http://hdl.handle.net/11343/35154
File Description: The role of keratan sulphate in the modulation of aggrecanase activity
Terms and Conditions: Terms and Conditions: Copyright in works deposited in Minerva Access is retained by the copyright owner. The work may not be altered without permission from the copyright owner. Readers may only download, print and save electronic copies of whole works for their own personal non-commercial use. Any use that exceeds these limits requires permission from the copyright owner. Attribution is essential when quoting or paraphrasing from these works.