Studies on the Structure of Basement Membranes

Vanderbilt University, Nashville TN

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Abstract

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 nephropathydirectly affect the glomerular basement membrane (GBM), a major component of the filtration barrier. The quest for the molecular bases of these GBM abnormalities has led to the discovery of six a(FV) chains (al-oc6) of type IV collagen. These are distributed in distinct networks about the basement membranes and mesangial matrix of the glomerulus. The GBM network formed by oc3,o4, and cx5 chains is involved in the pathogenesis of Goodpasture and Alport syndromes. In this renewal application, five specific aims are proposed that address structure/functionrelationships ofhuman type IV collagen and its role in Goodpasture syndrome and Alport syndrome. The central research strategy is to express full-length a(IV) chains and NCI 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 cc5(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 o3, a4 and o5(TV) collagen chains. 2. To identify the molecular recognition sequences of NCI domain that confer chain specificity for network assembly. 3. To determine how mutations in o5(IV) chain cause defective assembly of oc3- a4- o5 network in Alport syndrome. 4. To elucidate the structural basis by which GP epitopes are inaccessible to antibodies (cryptic) in the NCI hexamer. 5. To identify the pathogenic epitopes of the a3(IV) NCI 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 ultracentrifugation, and electron microscopy.