Basement membranes (BM) are specialized extracellular matrices which are composed of distinctive macromolecules, such as laminin, type IV (t.IV) collagen, proteopheparen sulfate and nidogen. In the microvasulature, endothelial cells and their BM form the wall across which nutrients and proteins exchange. In the kidney, the glomerular basement membrane (GBM) form the only barrier to the extravasation of plasma proteins and serves as a selective filter. This function of perselectively becomes impaired in nephrotic syndromes and excessive protein leakage occurs. The molecular mechanisms which lead to such defects of the GBM are not understood. In diabetes, t.IV collagen is chemically modified by non-enzymatic binding of glucose to the epsilon-amino groups of lysine residues. In Goodpasture nephritis, an auto-antibody forms to the main non-collagenous, NC1 domain of t.IV collagen. In Alport's nephritis there is a lack of recognition of the Goodpasture (NC1) antigen. However, it is not known whether structural or other changes of t.IV collagen play a causal role in the development of the above mentioned nephrotic syndromes. It is the purpose of this study to examine the structural and functional changes of t.IV collagen and the NCI domain in one of these pathological conditions. T.IV collagen has the ability to self-assemble by end-to-end and lateral interactions to a complex network. The NC1 domain is required for network formation because a) it binds to an adjacent NC1 and forms dimers b) it binds to the collagenous part of adjacent t.IV collagen molecules and initiates complex formation. In the proposed study the binding of NC1 to t.IV collagen and the formation of networks will be assessed in 1) control, 2) diabetic conditions. The effect of principally in vitro and to a lesser extent in vivo non-enzymatic glucosylation of t.IV collagen will be explore. Network formation by control and glucosylated t.IV collagen will be examined by rotary shadowing. The binding of control and glucosylated NC1 domain to diabetically modified t.IV collagen and to control or glucosylated triple-helical domain of type IV collagen will be evaluated, by rotary shadowing, zonal rate, velocity sedimentation, solid phase binding assays and turbidometry. Furthermore, chemically or proteolytically derived fragments from the NC1 and triple-helical domains as well as synthetic peptides will be used to identify active binding sites which mediate assembly of t.IV collagen. These studies will examine the possibility that supromolecular assembly of t.IV collagen in altered following non-enzymatic glucosylation.
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