The structure and function of the minor cartilage collagens called type IX collagen and 1 Alpha, 2 Alpha, 3 Alpha collagen will be investigated. Previously, two pepsin-resistant fragments of type IX collagen were isolated from chicken sterna and called the high molecular weight and low molecular weight components or HMW and LMW. During the past year, three genetically distinct chains were fractionated from both HMW and LMW after reduction and alkylation of interchain disulfide bridges. A cDNA was prepared which encodes for one of the three chains of both HMW and LMW and, by converting the nucleotide sequence of this cDNA to an amino acid sequence, it was possible to detrmine the general structure of the intact type IX collagen molecule and the location of HMW and LMW. Future experiments will involve the isolation and identification of cDNA clones for the other two chains of type IX collagen, and the location of interchain disulfide bridges after the preparation of collagenase-resistant fragments of the molecule. Intact type IX collagen will be isolated either from suspension cultures of chick chondrocytes or from lathyritic rat chondrosarcoma, and its structure investigated by rotary shadowing. Experiments will be performed to investigate the influence both of type IX collagen and 1 Alpha, 2 Alpha, 3 Alpha collagen on the formation of fibrils of type II collagen in vitro. Monoclonal antibodies of high affinity will be prepared against both type IX collagen and 1 Alpha, 2 Alpha, 3 Alpha collagen, and the location of the epitopes for these collagens determined by rotary shadowing and by biochemical analyses. The monoclonal antibodies will be used to localize these collagens in the cartilage matrix of developing long bones using both light and electron microscopy. For electron microscopy, each monoclonal antibody will be coupled to peroxidase or to biotin. For high resolution EM immunolocalization, avidin-colloidal gold will be used with biotinylated antibodies. These experiments will test the hypothesis that 1 Alpha, 2 Alpha, 3 Alpha collagen and type IX collagen are involved in the organization of the three demensional network of type II collagen fibrils in cartilage matrix. The results will enhance our undertanding of the structure of cartilage matrix and the changes which occur during the degenerative diseases of cartilage.
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