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Partial characterization of the C-terminal non-collagenous domain (NC1) of collagen type X.

ABSTRACT: Collagen type X is composed of three identical alpha 1(X) chains of 59 kDa, each containing a triple-helical region of 45 kDa flanked by a short N-terminal sequence and a larger non-collagenous C-terminal (NC1) domain of approx. 15 kDa. Collagen type X molecules can associate via their C-termini to form a regular hexagonal lattice in vitro, which in vivo may provide a modified extracellular matrix for the events of endochondral ossification. The NC1 domain of chick collagen type X was isolated and purified from a highly purified bacterial collagenase digest of hypertrophic chondrocyte medium proteins. The structure and aggregation properties of the NC1 domain of collagen X were investigated, independently of the triple helix. A trimer, a dimer and a monomer of the individual alpha-chain NC1 polypeptides were identified from a bacterial collagenase digest of cartilage collagens using [14C]tyrosine labelling, N-chlorosuccinimide peptide mapping and N-terminal sequencing. The trimer (50 kDa) remained intact in Laemmli sample buffer unless boiled, upon which it dissociated into the dimer (38 kDa) and the monomer (20 kDa). The dimer persisted even after prolonged periods of heating or reduction with beta-mercaptoethanol, and in preparations obtained from chondrocyte cultures treated with beta-aminoproprionitrile, indicating the presence of non-reducible, non-lysine-derived, covalent cross-links. Hexamers of the individual C-termini were observed in rotary-shadowed preparations of purified NC1 domain, reflecting the ability of collagen type X to self-assemble via its C-termini under appropriate conditions.

SUBMITTER: Barber RE

PROVIDER: S-EPMC1217955 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Project description:Type X collagen is a short-chain non-fibrillar collagen that is deposited exclusively at sites of new bone formation. Although this collagen has been implicated in chondrocyte hypertrophy and endochondral ossification, its precise function remains unclear. One possible function could be to regulate the processes of chondrocyte hypertrophy through direct cell-type X collagen interactions. Adhesions of embryonic chick chondrocytes, and cell lines with known expression of collagen-binding integrins (MG63 and HOS), were assayed on chick type X collagen substrates, including the native, heat-denatured and pepsin-digested collagen, and the isolated C-terminal non-collagenous (NC1) domain. Type X collagen supported the greatest level of adhesion for all cell types tested. The involvement of the alpha2beta1 integrin in type X collagen-cell interaction was demonstrated by adhesion studies in the presence of Mg(2+) and Ca(2+) ions and integrin-function-blocking antibodies. Cells expressing alpha2beta1 integrin (chick chondrocytes and MG63 cells) also adhered to heat-denatured type X collagen and the isolated NC1 domain; however, removal of the non-collagenous domains by limited pepsinization of type X collagen resulted in very low levels of adhesion. Both focal contacts and actin stress-fibre formation were apparent in cells plated on type X collagen. The presence of alpha2 and beta1 integrin subunits in isolated chondrocytes and epiphyseal cartilage was also confirmed by immunolocalization. Our results demonstrate, for the first time, that type X collagen is capable of interacting directly with chondrocytes and other cells, primarily via alpha2beta1 integrin. These findings are atypical from the fibrillar collagen-cell interactions via collagen binding integrins in that: (1) the triple-helical conformation is not strictly required for cell adhesion; (2) the NC1 domain is also involved in the adhesion of alpha2beta1-expressing cells. These data form the basis for further studies into the mechanism and biological significance of type X collagen deposition in the growth plate.

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Partial characterization of the C-terminal non-collagenous domain (NC1) of collagen type X.

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