Glomerular basement membrane -: Identification of a novel disulfide-cross-linked network of α3, α4, and α5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome

Glomerular basement membrane (GEM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GEM was investigated. This was accomplished by using pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 degrees C and the other at 25 degrees C, and their chain composition was determined by use of monoclonal antibodies. The 4 degrees C protomers comprise the alpha 1(IV) and alpha 2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains along with some alpha 1(IV) and alpha 2(TV) chains. The structure of the 25 degrees C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between alpha 3(TV), alpha 4(IV), and alpha 5(IV) chains. These results establish a conceptual framework to explain several features of the GEM abnormalities of Alport syndrome. In particular, the alpha 3(IV).alpha 4(IV).alpha 5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha 5(IV) chain cause defective assembly of not only alpha 5(IV) chain but also the alpha 3(IV) and alpha 4(TV) chains in the GEM of patients with Alport syndrome.