The biosynthesis of collagen is fundamental to the production of new connective tissue matrix, which is an integral component of all tissues. New collagen formation requires enzymatic cleavage of the terminal propeptides from its precursor procollagen. Recent evidence suggests that the enzyme which removes the carboxyl propeptide from procollagen may be Cathepsin D, a lysosomal enzyme. This proposal will test the hypothesis that Cathepsin D can correctly process procollagen under certain conditions, and that cleavage may occur during intracellular transport and secretion. Preliminary in vitro studies show that purified Cathepsin D will specifically cleave procollagen. Also, organ culture experiments show that agents which alter lysosomal enzyme biosynthesis inhibit COOH-propeptide removal from procollagen. The in vitro studies will be extended to determine the cleavage site specificity. The collagen portion of the cleavage product will be examined for the size and COOH-terminal sequence of its COOH-telopeptide, using a combination of CNBr- and carboxypeptidase-digestions of the cleavage product. The sequence of the amino terminus of the carboxyl propeptide released by Cathepsin D action will be determined manually. This combination of sequence studies should show that Cathepsin D can cleave native procollagen at the authentic cleavage site. The organ culture observations will also be extended. Using subcellular fractionation and pulse chase studies the distribution of procollagen and Cathepsin D will be determined. The emphasis will be on determining the site of divergence of procollagen and Cathepsin D during their biosynthesis, and whether any proteolytic processing of procollagen occurs subcellularly. Electron microscopy, using both antibodies to procollagen and Cathepsin D and a bioaffinity probe for Cathepsin D, will be used to determine their intracellular and extracellular distribution. These studies will clearly define the role of Cathepsin D in procollagen processing, and elucidate the intracellular pathways by which procollagen and lysosomal enzymes are processed. This will provide a better understanding of the proteolytic steps which regulate the biosynthesis of the collagen matrix, and aid in clarification of the molecular defect in procollagen processing in certain forms of lysosomal storage diseases.
