Covalent Cross-linking of the NC1 Domain of Collagen Type IX to Collagen Type II in Cartilage

From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the α1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the α1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of α1(IX) and the known cross-linking lysine at residue 930 of the α1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network. From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the α1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the α1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of α1(IX) and the known cross-linking lysine at residue 930 of the α1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network. Collagen type IX is a member of the fibril-associated collagen with interrupted triple helix (FACIT) 1The abbreviations used are: FACIT, fibril-associated collagen with interrupted triple helix; COL1, COL2, etc., triple helical collagen domains; NC1, NC2, etc., non-triple helical domains; CB9,7, cyanogen bromide peptide; HPLC, high performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; mAb, monoclonal antibody. family of collagen molecules, which share homologous domains and are all believed to function in the extracellular matrix in association with collagen fibril surfaces (1.Shaw L.M. Olsen B.R. Trends Biochem. Sci. 1991; 16: 191-194Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 2.Olsen B.R. Int. J. Biochem. Cell Biol. 1997; 29: 555-558Crossref PubMed Scopus (83) Google Scholar). Collagen IX is unique among the FACIT molecules in binding covalently to fibril surfaces. The cross-links are formed through the lysyl oxidase mechanism (3.Wu J.J. Eyre D.R. Biochem. Biophys. Res. Commun. 1984; 123: 1033-1039Crossref PubMed Scopus (46) Google Scholar, 4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 5.van der Rest M. Mayne R. J. Biol. Chem. 1988; 263: 1615-1618Abstract Full Text PDF PubMed Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). Collagen IX is found mostly in cartilages, but it also occurs in the eye (vitreum (8.Fitch J.M. Mentzer A. Mayne R. Linsenmayer T.F. Dev. Biol. 1988; 128: 396-405Crossref PubMed Scopus (57) Google Scholar, 9.Bishop P.N. Crossman M.V. McLeod D. Ayad S. Biochem. J. 1994; 299: 497-505Crossref PubMed Scopus (82) Google Scholar) and avian cornea (8.Fitch J.M. Mentzer A. Mayne R. Linsenmayer T.F. Dev. Biol. 1988; 128: 396-405Crossref PubMed Scopus (57) Google Scholar)), ear (tectorial membrane (10.Richardson G.P. Russell I.J. Duance V.C. Bailey A.J. Hear. Res. 1987; 25: 45-60Crossref PubMed Scopus (114) Google Scholar)), and intervertebral disc (11.Newall J.F. Ayad S. Biochem. Soc. Trans. 1995; 23: 517SCrossref PubMed Scopus (5) Google Scholar, 12.Wu J.J. Eyre D.R. J. Biol. Chem. 2003; 278: 24521-24525Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), always in co-existence with type II collagen. A special role for collagen IX in the organization of type II collagen fibril networks has apparently evolved, which for articular cartilage seems to be essential for the long term normal functioning of joints. The evidence for this comes from the study of genetic defects. Mutations in all three collagen IX genes have been linked to a chondrodysplasia syndrome (multiple epiphyseal dysplasia), which features early onset osteoarthritis (13.Chapman K.L. Briggs M.D. Mortier G.R. Pediatr. Pathol. Mol. Med. 2003; 22: 53-75Crossref PubMed Scopus (21) Google Scholar). All three chains of the vertebrate collagen IX molecule, α1(IX), α2(IX), and α3(IX), contain intermolecular cross-linking sites, each with chain-specific properties (3.Wu J.J. Eyre D.R. Biochem. Biophys. Res. Commun. 1984; 123: 1033-1039Crossref PubMed Scopus (46) Google Scholar, 4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 5.van der Rest M. Mayne R. J. Biol. Chem. 1988; 263: 1615-1618Abstract Full Text PDF PubMed Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). All the cross-links are of the lysyl oxidase-mediated type, formed from modified lysine residues between type IX and type II collagen molecules or between type IX collagen molecules. The first evidence for this was an observation that type IX collagen triple helical domains purified from pepsin digests of bovine articular cartilage contained pyridinoline cross-linking residues (3.Wu J.J. Eyre D.R. Biochem. Biophys. Res. Commun. 1984; 123: 1033-1039Crossref PubMed Scopus (46) Google Scholar). The concentration was higher on a mole/mole basis than in type II collagen isolated from the same tissue. Further analysis showed that the most abundant cross-links in fetal cartilage were divalent keto-amines, the same intermediates found on the pathway to pyridinoline formation within collagen fibrils (6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar). Analysis of the structures of cross-linked peptides isolated from type IX collagen showed one class that had derived from the reaction of a hydroxylysine aldehyde in the N-telopeptide of type II collagen to a hydroxylysine at the N terminus of the triple helical COL2 domain (4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 5.van der Rest M. Mayne R. J. Biol. Chem. 1988; 263: 1615-1618Abstract Full Text PDF PubMed Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar). All three chains, α1(IX), α2(IX), and α3(IX), were able to cross-link to the α1(II) N-telopeptide. In addition, the α1(II) C-telopeptide was similarly cross-linked to another site in the middle of the COL2 domain but only to α3(IX) (4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). Studies on fetal human cartilage showed this same pattern of collagen IX cross-linking as in bovine cartilage (7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). From both human and bovine tissues, cross-linked peptides were also identified that had originated from an interaction between two type IX collagen molecules and in yields that indicated about an equal prominence as for IX-to-II linkages (6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). The IX-to-IX cross-links were from the NC1 domain (C terminus) of α3(IX) to the same COL2 site (in α1(IX) or α3(IX)) to which the α1(II) N-telopeptide could link. Of all the cross-linked peptides isolated from collagen IX, only two had properties that showed an origin from a lysyl oxidase-generated aldehyde in the type IX collagen molecule itself. These peptides had linked α3(IX)NC1 to a site in α1(IX) or α3(IX)COL2. All others were apparently based on precursor hydroxylysine aldehydes in α1(II)N- or C-telopeptides. From transmission electron microscopy we know that type IX collagen molecules physically decorate the surface of type II collagen fibrils in developing cartilage (14.Vaughan L. Mendler M. Huber S. Bruckner P. Winterhalter K.H. Irwin M.I. Mayne R. J. Cell Biol. 1988; 106: 991-997Crossref PubMed Scopus (259) Google Scholar) and are most concentrated on the thinnest fibrils (15.Hagg R. Bruckner P. Hedbom E. J. Cell Biol. 1998; 142: 285-294Crossref PubMed Scopus (100) Google Scholar) that form the network or basket around chondrocytes (16.Poole C.A. Gilbert R.T. Herbage D. Hartmann D.J. Osteoarthritis Cartilage. 1997; 5: 191-204Abstract Full Text PDF PubMed Scopus (52) Google Scholar). From the yield of cross-linked peptides we know that most of the collagen IX molecules in cartilage, even in fetal tissue, occur in covalent linkage to type II collagen fibrils (6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). The lack of extractability of collagen IX in protein denaturants confirms this. The relative yields of the different cross-linked peptides also predict that most collagen IX molecules are also linked to each other. These various observations make it challenging to present a molecular model that can explain how, in a network of fine collagen fibrils coated with collagen IX molecules, IX-to-IX molecular links can be so abundant when the opportunity for fibril-to-fibril surface interactions would seem to be relatively limited. Any viable molecular model of heterofibril assembly needs, however, to accommodate the cross-linking stoichiometry. Here we provide evidence for an additional, major site of interaction between types II and IX collagens that further constrains how the molecules can be organized but from its position suggests how the collagen IX molecules are folded on the surface of the collagen II polymer to accommodate all the cross-links. Preparation of Human Collagen Samples—Ribs, knees, and hip were harvested and minced from 94- to 98-day-old human fetuses (Central Laboratory for Human Embryology, University of Washington). The minced tissue samples were extracted in 4 m guanidine HCl, 0.05 m Tris, pH 7.4, containing protease inhibitors (2 mm EDTA, 5 mm benzamidine, 2 mm phenylmethylsulfonyl fluoride, and 5 mm phenanthroline) at 4 for to and The tissue residue was with in and with pepsin a of for at 4 was to the and the was to tissue. The collagen was with m to most of the the collagen was in and with in for at The was antibody was used to an This antibody was to a proteolytic in the C-telopeptide domain of type II collagen as a L.M. P. Eyre D.R. Trans. Res. Soc. 1998; Scholar). The recognized is at the C terminus of the sequence in which a C-terminal is essential for antibody of collagen II is at this and the used in the present study and this from type II collagen L.M. P. Eyre D.R. Trans. Res. Soc. 1998; Scholar). The C terminus of the human α1(IX) chain in a similar for to which also R. M. A. and monoclonal antibody was to and a digests of fetal human cartilage were in mm pH and through the The was with mm mm pH and mm mm pH the the and the were by The peptides were with mm mm pH peptides were and by mm a of in The were by and and and were by the of PubMed Scopus Google Scholar). N-terminal were to membrane a Western blot analysis, were to another membrane Western were with monoclonal and a residues of the α1(II) helical domain) in J. J. A. S. P. L. Eyre D.R. Osteoarthritis Cartilage. Full Text PDF PubMed Scopus Google Scholar). A. and D. R. with the antibody was by with The Western were and m human fetal cartilage collagen was in mm with mm pH at a concentration of The was for at to the collagen and to was at a of and the was at for was to a concentration of 5 mm to the The cross-linked was on the as for the on the and for by containing were by were in and coated on with bovine in m m Tris, pH were with or by antibody to were with m containing between each was and was at a or a N-terminal were to N-terminal on a with analysis of protein from were to mass analysis with or an for protease M. and Using Scholar). peptides were by mm with an mass were by to peptides a for of collagen genes L. J. Soc. 1994; 5: PubMed Scopus Google Scholar) to and The cross-linked by from a of fetal cartilage collagen was by the from the for mass and from human fetal and human articular cartilage were in for 5 and with 2 in pH at for The were with of antibody and the and The pattern of the tissue was with a with a which was the type II collagen C-telopeptide the C terminus of the sequence L.M. P. Eyre D.R. Trans. Res. Soc. 1998; Scholar). also with a high to a similar sequence by for S. M. D.J. L. Biol. 1998; PubMed Scopus Google Scholar) that occurs at the C terminus of the chain for Western blot of collagen from human cartilage in type IX the binding to the α1(IX) chain and to cross-linked of it was binding to α1(IX) chains in a 4 m guanidine of human fetal cartilage and to of the collagen from the same tissue In the of the two the α1(II) chain, one and the had properties an interaction between the from type IX collagen and the α1(II) a another which is specific for an in the α1(II) chain the of α1(II) in the was 2 that fetal cartilage matrix with cartilage of fetal and human cartilage the antibody. of fetal cartilage showed a of extracellular matrix with articular cartilage with the known higher of type IX collagen in fetal Collagen IX in tissue is also to be most concentrated confirm this molecular by sequence analysis, a of collagen was from fetal bovine A similar which to molecular on was in a and a containing this was by molecular chromatography analysis of the N of the two one the N terminus of the α1(II) chain by pepsin J.J. Eyre D.R. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) and the a sequence at the N terminus of the domain of cross-linking between the domain of type IX collagen and an α1(II) chain, we the sites of used human cartilage for the bovine α1(IX) chain, which in sequence at the C terminus from human α1(IX) Wu and D. R. 4 Western of digests of cartilage matrix and of collagen on a The from the than This is with a cross-link between the domain the and The sequence a that would be by to a C-terminal containing the putative cross-linking lysine and the S. M. D.J. L. Biol. 1998; PubMed Scopus Google Scholar). A immunoaffinity was used to and the cross-linked NC1 to the for sequence analysis. the of the on by and pyridinoline and Western blot the helical sequence and The results are with a cross-link from to a site in confirm the of was on and to and the to the was to N-terminal sequence analysis. two were as is from the N terminus of The is from the NC1 with evidence of two the and a form from a of fetal cartilage collagen similar results that the NC1 domain had the N-terminal sequence the of than the two and from collagen. The were from and by mass or The results of this analysis the identity of CB9,7, but the cross-linked was identified in the of the of the two lysine residues in the divalent cross-link was by a of type II collagen from fetal human cartilage with and the to the The was by mass spectrometry. the results of this analysis, which the of the two peptides cross-linked through lysine The results showed the of two of the same cross-linked that in mass of the cross-linking residue by two molecules. form the mass of the chain The mass equal to two molecules and an would the but structures are to explain this mass The Western blot results in establish the of for the human α1(IX) chain and lack of of α1(II) chains from the same cartilage These and observations binding of to the C terminus of the α1(IX) chain but to the same cross-linking sequence in the α1(II) C-telopeptide The is recognized only it C-terminal through for through by matrix as has been L.M. P. Eyre D.R. Trans. Res. Soc. 1998; Scholar). This was to establish was to the proteolytic in of type II collagen L.M. P. Eyre D.R. Trans. Res. Soc. 1998; Scholar), the in that was for binding to from fetal human cartilage or of α1(II) chains when similarly The results of 2 further this with a lack of of cartilage when was used for on fetal cartilage the These that to a in type IX collagen but in type II collagen and are with the higher and of type IX collagen in fetal cartilage matrix of with or of collagen (6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, D.R. Wu and The of a similar of cross-linked chains from fetal bovine cartilage that this is to human fetal tissue, and the results the component from and Western The predicted molecular identity was of an domain with its NC1 sequence to which an α1(II) chain was of the NC1 domain by pepsin would explain the of both the molecular species chains cross-linked to NC1 with and α1(II) chains a NC1 domain cross-linked to The sequence would the of α1(II) on of similar to the α1(II) This was by the results of analysis of peptides and immunoaffinity 4 and The lack of in the cross-linked the N-terminal and the mass the site of cross-linking between two and the cross-link as a divalent with evidence of pyridinoline cross-link formation at this from The pyridinoline cross-links found in type IX collagen were all formed between collagen II and the COL2 domain (4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar). These results establish a previously unrecognized site of intermolecular cross-linking between types IX and II collagen molecules in with known cross-linking sites in type IX collagen (4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 5.van der Rest M. Mayne R. J. Biol. Chem. 1988; 263: 1615-1618Abstract Full Text PDF PubMed Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar), this one is unique in the molecule to the triple helix of type II collagen. The (4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 5.van der Rest M. Mayne R. J. Biol. Chem. 1988; 263: 1615-1618Abstract Full Text PDF PubMed Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar) defined sites of linkage from type II collagen to the triple helical COL2 domain of type IX collagen. on evidence that COL1, the triple helical domain most in sequence all FACIT collagen family J. J.F. FEBS Lett. PubMed Scopus Google Scholar, D.R. der Rest M. Olsen B.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), with the NC1 domain and its is physically for the interaction of collagen IX molecules with fibril surfaces D.R. der Rest M. Olsen B.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we that the identified covalent from to type II collagen is the first cross-link to form in the interaction pathway and in This is in a molecular model of the interaction and of collagen IX molecules on the collagen II fibril surface that the of all the sites of covalent cross-linking. is similar to one of a of by C.A. L. Bailey A.J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar) on and the NC1 link to type II collagen was In the domain through so that it to the COL2 This the and the between COL2 and the type II helix to accommodate both the cross-link and (4.Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (183) Google Scholar, 6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar). is the of collagen IX molecules on the fibril relative to each by the by which collagen molecules are in to IX-to-IX intermolecular cross-links to form between α3(IX)NC1 and the COL2 N The previously interaction sites for α3(IX)NC1 on COL2 are in α1(IX) and α3(IX) (6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 7.Diab M. Wu J.J. Eyre D.R. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (86) Google Scholar, S. Wu J.J. Eyre D.R. Biochem. Biophys. PubMed Scopus Google Scholar). The of IX-to-IX cross-links can be by an interaction of type IX collagen molecules the fibril surface. The of collagen II molecules as a for this the model also has the potential to interfibrillar links between collagen IX molecules when the opportunity between nascent fibrils coated with collagen IX molecules. the of interfibrillar be a but observations have previously been as evidence for of collagen IX at sites of fibril in the matrix of developing cartilage M. P. Winterhalter K.H. Bruckner P. J. Cell Biol. PubMed Scopus Google Scholar) as well as a fibrils (14.Vaughan L. Mendler M. Huber S. Bruckner P. Winterhalter K.H. Irwin M.I. Mayne R. J. Cell Biol. 1988; 106: 991-997Crossref PubMed Scopus (259) Google Scholar). chain of the vertebrate collagen IX molecule has its pattern of cross-linking interactions in the collagen The α1(IX) chain is the of this in a of collagen IX protein normal of and R. Hedbom E. A. R. Bruckner P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is also evidence for a of α1(IX) in a A. M. M. R. Biophys. PubMed Scopus Google Scholar). seems that and α3(IX) in higher are the of and that have and cross-linking to the cross-link to type II collagen based on identified it is that the α3(IX)NC1 sequence was to be cross-linked to the COL2 domain of a type IX molecule (6.Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar). this an function from that of but we have evidence that a lysine residue in also can link to type II collagen at the the NC1 domains of all three have a cross-linking Using it was previously that the NC1 domain and the C-terminal of contain all the for formation D.R. Mayne R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). collagen IX has to be a covalent of collagen type II networks in and of higher avian of collagen IX has been with matrix J. Linsenmayer T.F. Dev. 1998; PubMed Scopus Google Scholar). A similar mechanism for the of cartilage collagen is and a is known to at a specific site in all three chains of the domain of type IX collagen J.J. Eyre D.R. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar).