Experiments will investigate the structure and formation of the collagen fibrils of hyaline cartilage from mammalian and avian species. The fibrils are assembled from type II, type IX and type XI collagen and are associated with several non-collagenous proteins including decorin, fibromodulin and cartilage matrix protein (CMP). Extensive use will be made to high affinity monoclonal antibodies (mAbs) specific for each collagen type for which the location of different epitopes along the collagen molecule is known. Monoclonal antibodies will be prepared using different approaches including i) use of synthetic peptides coupled to keyhole limpet hemocyanin (KLH), ii) adjuvants that heighten the immunogenicity of synthetic peptides, iii) expression of non-collagenous domains from cDNA constructs in E. Coli and use of the expressed protein as an immunogen. for type II collagen, monoclonal antibodies are already available that recognize epitopes at three different locations along the length of the molecule. One of these antibodies is of special interest since its epitope is located in a short amino acid sequence of the amino- teleopeptide. Both the amino- and carboxyl-telopeptides of type II collagen will be synthesized and coupled to KLH for the preparation of additional monoclonal antibodies. These antibodies will be used i) to analyze cross-links between type II and type IX collagen by rotary shadowing to determine if type IX collagen is parallel or antiparallel to type II collagen. ii) to look for non-collagenous proteins that are cross-linked to the collagen fibril and are released from the surface of the fibril by bacterial collagenase digestion. iii) to develop an antibody to the cross-linking peptides such that the epitope is lost when cross-linking occurs. iv) to develop in vitro model systems of collagen fibril formation by chondrocytes and to investigate which monoclonal antibodies inhibit fibril assembly. For type XI collagen the amino- terminal non-collagenous domains will be cloned from bovine cartilage cDNA, expressed in E. coli, and used as an immunogen to prepare monoclonal antibodies. These monoclonal antibodies will be used to investigate the role of type XI collagen in fibril structure and during fibrillogenesis. Using synthetic peptides, monoclonal antibodies will be developed to the chains of mammalian type IX collagen. These antibodies will be used to block incorporation of type IX collagen onto the surface of the fibril and to investigate if wider collagen fibrils result from the absence of type IX collagen. Synthetic peptides for the carboxyl-termini of the three chains will be prepared in order to determine if sufficient information is present in these short sequences to promote the assembly of the triple helix. The proposed experiments will provide new information on the structure and organization of cartilage matrix and will provide new reagents to analyze the degenerative diseases of cartilage and genetic diseases involving abnormal cartilage development in man.
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