Basement membrane function is altered in three notable human diseases that affect the kidneys, Goodpasture syndrome, Alport syndrome and diabetes mellitus. Specifically, the ultrsfiltration function of the glomerular basement membrane (GBM) is altered which often leads to end- stage renal disease. Our investigations on the biochemistry and pathology of GBM have revealed the existence of two new chains (alpha3 & alpha4) of the type IV collagen constitutent, and that the alpha3 chain is the major auto-antigen of Goodpasture syndrome. Based on our work and that of others, a molecular commonality between the pathogenesis of Goodpasture and Alport syndromes is emerging wich involves the alpha3, alpha4, and the newly discovered alpha5 chain of type IV collagen. Potentially, these chains are important elements of the pathogenesis of diabetic nephropathy. The overall goal of this renewal project is: to further the understanding of the molecular structure and assembly of GBM type IV collagen and its role in the pathogenesis of Goodpasture and Alport syndromes. The objectives are: Objective I. To elucidate new information about critical structural features of GBM type IV collagen with respect to: 1) the primary structure of the alpha3 and alpha4 chains; 2) the structure and organization of the NC1 hexamer; and 3) the organization of promoters. Objective II. To delineate the role of GBM type IV collagen in the pathogenesis of Goodpasture syndrome with respect to: 1) the specificity of GP-antibodies reacting with NC1 dimers; 2) the pathogenicity of NC1 dimers in eliciting GP-like syndrome; and 3) the critical residues in the GP-epitope within NC1 domain. Objective III. To delinate the role of GBM type IV collagen on the pathogenesis of Alport syndrome with respect to; 1) the specificity of Alport alloantibodies reacting with NC1 dimers; and 2) the subunit composition of NC1 hexamers from Alport GBM. Objective IV. To elucidate new information about the sequestration of the GP-eptiope and the molecular assembly of type IV collagen with respect to: 1) the specificity of NC1 subunits in forming trimers and hexamers; and 2) the role of NC1 domain on triple helix formation and protomer lateral associations. The methodology involves a broad spectrum of technologies. These include, light and electron microscopy, immunochemical and protein chemistry techniques (electrophoresis, HPLC, peptide sequencing, ELISAs, and circular dichroism spectroscopy), molecular biology techniques, and animal model studies.
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