Maintenance of the cartilage matrix structure depends upon interactions among collagens, proteoglycans, cartilage matrix protein, chondrocalcin and other matrix components. Cartilage contains four distinct collagen types, II, IX, X, and XI. Type IX collagen is an intriguing member of this group since it is a hybrid molecule containing triple-helical domains as well as one or two covalently attached chondroitin sulfate side chains Preliminary evidence presented in support of this application suggests that type IX is intimately associated with type II collagen fibers, but may also form its own limited network which then interacts with other cartilage components.
The specific aims of this proposal are to (1) identify and describe the network formed by type IX collagen in vivo, (2) establish conditions to study self assembly of type IX collagen in vitor, (3) identify the geometry of initial aggregates which leads to the formation of the network, and (4) characterize the participation of subdomains of subdomains of type IX collagen in the intermolecular interactions involved in aggregation. Type IX collagen isolated from 17 day day chick sterna cartilage treated with beta-aminopropionitrile to prevent covalent crosslinking will be used for this study. Both static and kinetic analysis of the association of type XI coillagen in vitro will be monitored by a combination of biphysical techniques, including turbidity, laser light scattering, velocity sedimentation, and electron microscopy. Subdomains will be isolated following fragmentation of the molecule and their effects on the assembly process studied. A monoclonal antibody to the NC1 domain of type XI will be analyzed for its ability to interfere with assembly and so generate intermediates in the assembly process in sufficient quantities for further investigation. In vivo analysis of type IX will be limited to immunoelectron microscopy with the monoclonal antibody and conjugated colloidal gold. Many components have yet to be identified and the functions of most of those which have been identified are unknown. Knowledge of structure and interactions of these components is necessary before understanding of their function is possible. Such information will provide clues to the development of cartilage and endochondral bone as well as to the many pathological states of this tissue, since few human heritable disorders of cartilage have been identified at the molecular level.
| Thom, J R; Morris, N P (1991) Biosynthesis and proteolytic processing of type XI collagen in embryonic chick sterna. J Biol Chem 266:7262-9 |
