Biochemical and morphological analyses will be performed of the proteins and proteoglycans that form the structure of the vitreous humor and zonular apparatus. Collagen fibrils will be isolated from bovine and chicken vitreous and the organization of the three collagen types (type II, IX and V/XI collagen) present in these fibrils will be investigated. Analyses will be performed of the relative amounts of type V collagen [alpha1(V) and alpha2(V) chains] and type XI collagen [alpha1(XI) and alpha2(XI) and alpha3(XI) chains] that are present in the chicken and bovine vitreous. Using specific monoclonal antibodies, we will investigate if these type V/XI molecules form a core """"""""microfibril"""""""" around which the fibril of type II collagen is assembled. Crosslinked peptides (containing both hydroxypyridinium and reducible crosslinks) of the collagen fibrils of bovine and chicken vitreous will be isolated and the location of crosslinks will be determined by N-terminal amino acid sequencing. The location of potential crosslinks between type II and type V/XI collagen or type IX collagen will be determined. Proteins or proteoglycans that are associated with collagen fibrils and are released after extensive digestion with bacterial collagenase will be investigated. Such proteins will be identified by N-terminal amino acid sequencing after SDS-PAGE. Specific analyses will be made for cartilage matrix protein (CMP) in the vitreous and its association with collagen fibrils. Other experiments with bovine vitreous will investigate the binding of novel proteins to hyaluronan and to investigate such proteins biochemically. Collagen fibrils of bovine vitreous will be digested with collagenase, stromelysin, gelatinase, plasmin, etc. using recombinant forms of the enzymes and the mixture of enzymes required for the complete dissolution of a collagen fibril will be determined. Studies will also investigate the release of type IX collagen from the surface of the collagen fibril by limited pepsin digestion and to examine such preparations by rotary shadowing for crosslinking to type II collagen. Type IX collagen will be isolated from chicken vitreous and the N-terminal amino acid sequence of the alpha1(IX) chain will be obtained in order to demonstrate that the downstream promotor is used during the formation of vitreous type IX collagen. The formation of the vitreous humor and its associated collagen fibrils will be investigated during eye development in chicken embryos. Zonular fibrils will be dissected from adult bovine eyes and experiments performed to determine the structure of these fibrils. Zonular fibrils will be digested with a variety of proteases and unique fragments identified after SDS PAGE and N-terminal amino acid sequencing. By use of monoclonal antibodies previously obtained to fibrillin, or monoclonal antibodies to other and potentially novel proteins of the zonule, our present model for the structure of these fibrils will be further investigated.
