This is a study of structural and metabolic defects in procollagen in genetic diseases of connective tissues. Each simple inherited defect in a procollagen gene is a unique opportunity to study one or more steps in the metabolism of procollagen. Abnormalities in the structure and metabolism of procollagen will be identified and characterized in ascorbate stimulated post-confluent cultures of skin and tendon fibroblasts, and in collagens and procollagens extracted from affected tissues. We will study the effects of each mutation on collagen synthesis, post-translational modification, secretion, extracellular processing of procollagen to collagen, and covalent cross-linking of collagen within fibrils. The initial studies will utilize pulse chase experiments with cultured fibroblasts from humans and domestic animals with various forms of the Ehlers-Danlos syndrome (EDS), Marfans syndrome, and osteogenesis imperfect (OI) (J. Biol> Chem. 261:10006, 1986). The radiolabeled and extracted proteins will be separated and characterized with liquid chromatography, SDS-PAGE, peptide mapping, amino acid analysis, CD analysis and by partial enzymatic degradation at different temperatures. These studies will serve to identify primary and secondary effects of each mutation of different types of procollagen, and identify those mutations that result in regulatory defects that affect the transcription of procollagen genes. The date on both the primary structural and metabolic defects in procollagen chains will be used by laboratories of molecular genetics to study the mutation in procollagen genes. At the same time these data will be correlated with analyses of the effects of the structural and metabolic abnormalities on the thermal stability, secretion, and processing of procollagens, and on the deposition and cross-linking of collagens within fibrils.
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