The long-term objectives of this grant application are: 1) to elucidate the normal structure and function of type VI collagen and 2) to understand the role of type VI collagen in pathological conditions. Defects in structure and expression of collagen have been shown to be directly involved in several heritable diseases of connective tissue. Type VI collagen is a component of the extracellular matrix of almost all tissues. Because of the limitations of biochemical approaches, very little is known about its possible role in pathological conditions. As a prerequisite to a detailed understanding of the involvement of type VI collagen in diseases, we propose to develop new approaches to analyze this protein and its genes in detail. Specifically, the aims are (1) to isolate cDNA clones corresponding to mRNAs for the three chains of type VI collagen; (2) to determine the whole amino acid sequence of collagen VI from the base sequences of overlapping cDNA clones and to confirm the sequencing by Edman degradation of selected peptides obtained from pepsin-solubilized human placenta collagen VI; (3) to prepare monospecific antibodies against synthetic peptides selected from the non-collagenous domains of collagen VI and to use these antibodies in analysis of the topology and assembly of the protein and in the development of sensitive radioimmunoassays for clinical studies; (4) to determine the chromosomal localization of type VI collagen genes by Southern blot analysis of hybrid cells; (5) to isolate cDNA probes corresponding to murine collagen VI and to use these and human probes for tissue and cell analysis of collagen VI expression under normal conditions; (6) to characterize collagen VI genes and to study their expression in cultured fibroblasts from patients with inherited connective diseases.
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