Renal disease is a major health problem in the world. Prominent causes are diabetes, hypertension and various forms of glomerular disease, such as Goodpasture syndrome and Alport syndrome. These affect the glomerular filtration barrier and lead to end stage renal disease. Goodpasture syndrome, Alport syndrome, and diabetic nephropathy directly affect the glomerular basement membrane(GBM), a major component of the filtration barrier. The quest for the molecular bases of these GBM abnormalities had led to the discovery of six alpha(IV) chains ( alpha 1-alpha 6) of type IV collagen. These are distributed in distinct networks about the basement membranes and mesangiel matrix of glomerulus. The GBM network formed by alpha 3, alpha 4 and alpha 5 chains is involved in the pathogenesis of Goodpasture and Alport syndromes. In this renewal application, five specific aims are proposed that address structure/function relationships of human type IV collagen and its role in Goodpasture syndrome and Alport syndrome. The central research strategy is to express full length alpha(IV) chains and NC1 domains and chimeras in eukaryotic cells for elucidation of :1) structure and assembly mechanisms of human type IV collagen networks; 2) pathogenic mechanisms of how mutations in alpha5(IV)) chain cause defective assembly in Alport syndrome; and 3) location and pathogenicity of the epitopes for Goodpasture autoantibodies.
The specific aims are: 1. To express and characterize full-length triple-helical protomers comprised of alpha3, alpha 4, and alpha5 (IV) collagen chains. 2. To identify the molecular recognition sequences of NC1 domain that confer chain specificity of network assembly 3. To determine how mutations in alpha5(IV) chain cause defective assembly of alpha3, alpha4 and alpha5 network in Alport syndrome. 4. To elucidate the structural basis by which GP epitopes are inaccessible to antibodies (cryptic) in the NC1 hexamer. 5. To identify the pathogenic epitopes of the alpha3(IV) NC1 domain in the rat model of Goodpasture syndrome. The achievement of these aims requires the application of techniques in molecular biology, protein engineering and physical biochemistry. These techniques include construction of cDNA expression vectors, expression of proteins in cell culture, protein fractionations, and physical characterization of proteins by techniques such as Fourier-transform infrared spectroscopy, circular dichroism spectroscopy, analytical ultra-centrifugation, and electron microscopy.
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