Basement membranes are specialized extracellular matrices which serve three major biological functions: physical support of tissues, cell attachment sites, and ultrafiltration. In a number of human diseases, these matrices are affected which lead to serious and often fatal clinical consequences. Such diseases include Goodpasture syndrome and diabetes mellitus which affect the kidney glomerular basement membrane (GBM) and often lead to end-stage renal failure. Great advances have been made in the last decade in understanding the molecular structure and role of basement membranes in health and disease. Several constituents have been identified which are apparently common to all basement membranes. These are: collagen IV, laminin, entactin, and heparan sulfate proteoglycan. Specific knowledge of the molecular structure of collagen IV of kidney GBM led to the discovery that, the Goodpasture epitope was localized in the globular domain of collagen IV, a finding which advanced the understanding of the molecular basis of Goodpasture syndrome, an autoimmune disorder. The long-term goals of the proposed research are to define the detailed molecular structure of collagen IV of kidney GBM to correlate its structure with its function in glomerular ultrafiltration, and to determine its role in Goodpasture syndrome and diabetic nephropathy. In the present application, efforts are focused on elucidation of the detailed properties of collagen IV of bovine GBM with an amphasis on the molecular properties of the Goodpasture epitope.
The specific aim i s to characterize collagen IV with respect to: collagen chain origin of Goodpasture epitope; location and pairing of disulfide bonds and subunit organization in the globular domain; molecular structure of Goodpasture epitope; structure and location of oligosaccharides of the 7S and globular domains; chemical nature of aldehyde-derived crosslinks; stability, assembly, and reactivity of globular domain with Goodpasture antibodies; and pathogenicity of the Goodpasture epitope. The methodology includes a broad spectrum of techniques, such as rotary-shadowing electron microscopy, circular dichroism spectroscopy, analytical ultracentrifugation, 500 MHz nuclear magnetic resonance spectroscopy, amino acid sequence analysis, immunochemical procedures, gas-liquid chromatography, high-pressure liquid chromatography, liquid scintillation counting, electrophoresis, and amino acid and carbohydrate analyses.
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