Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.
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abstract
Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.
