We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its gly- Collagen IX is a heterotrimer of ␣1(IX), ␣2(IX), and ␣3(IX) polypeptide chains that fold into the triple helix characteristic of the members of the collagen family of extracellular matrix proteins (1). This helix consists in the case of collagen IX of COL1, COL2, and COL3 domains, numbered from the carboxyl terminus, which are flanked by short noncollagenous segments, domains NC1-NC4. The domain NC4 is formed by the 245 extreme N-terminal amino acid residues of the ␣1(IX) chain, since a corresponding region is absent from the ␣2(IX) and ␣3(IX) polypeptides (2).The function of collagen IX remains elusive. It is a minor component of the collagen fibrils of cartilage extracellular matrix and is also found in several other tissues. Collagen IX molecules are not present within the fibril body in cartilage but are instead associated with the surface of the collagen fibril and become covalently cross-linked to other collagen IX molecules and to collagen II, the main constituent of the fibril (2, 3). Collagen IX is not required for the assembly of the heterotypic collagen fibrils, but it is important for preservation of the long term stability of the cartilage extracellular matrix (4, 5). The molecular mechanism involved is not understood, however. The NC4 domain of collagen IX is seen in electron microscopy as a compact globulus projecting away from the fibril body, with the COL3 domain acting as a spacer arm (6, 7). This location and the high theoretical pI of the NC4 domain implicate collagen IX as a potential docking molecule, possibly connecting the host fibril to adjacent collagen fibrils or to other macromolecules of the extracellular matrix (8). Proteoglycans of the cartilage extracellular matrix may serve an intermediary purpose in these processes. A proteolytic fragment of collagen IX, lacking the NC4 domain and some other parts of the molecule, is indeed known to bind heparin with high affinity in vitro (9). The NC4 domain reportedly shows homology to the heparin-binding Nterminal domain of thrombospondin, but the residues believed to be crucial for heparin-binding potential of thrombospondin are not conserved in the NC4 domain (10). No research has yet been reported, however, on the glycosaminoglycan binding properties of the NC4 domain or full-length collagen IX.Studies in vitro have demonstrated that cartilage oligomeric