Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 , are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , or ␣ 10 ␤ 1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant ␣I domains in Escherichia coli. For each of the four ␣I domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both ␣ 1 I-and ␣ 2 Icollagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by ␣ 2 I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the ␣ 2 I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.Collagen IX was the first member to be discovered of a subgroup of collagens, now known as the fibril-associated collagens with interrupted triple helix (FACITs) 1 (1). At the present, FACITs also include collagens XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is composed of three different ␣ chains, termed ␣1(IX), ␣2(IX), and ␣3(IX). Structurally the collagen IX molecule can be divided into three triple helical domains (COL1, COL2, and COL3) separated and flanked by non-triple helical (NC) domains. Collagen IX is expressed in cartilage and in a limited number of other locations, including developing eye. Collagen IX can be covalently cross-linked to collagen II, and typically it covers the large fibrils formed by collagens II and XI (2-5). There is a kink between COL2 and COL3 domains making the COL3 domain project into the perifibrillar space, whereas the COL1 and COL2 domains are arranged on the surface of the fibril. Many but not all collagen IX molecules are proteoglycans because a glycosaminoglycan (GAG) chain may be attached to NC3 domain via a serine residue in the ␣2(IX) chain (6). Collagen IX is essential for the normal st...

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

Our data suggest that MMP-2 is associated with histological malignancy and poor survival in brain tumors.

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.