GLOMERULAR-BASEMENT-MEMBRANE - IDENTIFICATION OF A NOVEL DISULFIDE-CROSS-LINKED NETWORK OF ALPHA-3, ALPHA-4, AND ALPHA-5 CHAINS OF TYPE-IV COLLAGEN AND ITS IMPLICATIONS FOR THE PATHOGENESIS OF ALPORT SYNDROME

Glomerular basement membrane (GEM) plays a crucial function in the ult rafiltration 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 mut ations lead to a defective GBM. In the present study, the supramolecul ar organization of type IV collagen of GEM was investigated. This was accomplished by using pseudolysin (EC 3.4.24.26) digestion to excise t runcated 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 th e alpha 1(IV) and alpha 2(IV) chains, whereas the 25 degrees C protome rs comprised mainly alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains a long with some alpha 1(IV) and alpha 2(TV) chains. The structure of th e 25 degrees C protomers was examined by electron microscopy and was f ound 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 estab lish a conceptual framework to explain several features of the GEM abn ormalities of Alport syndrome. In particular, the alpha 3(IV).alpha 4( IV).alpha 5(IV) network, involving a covalent linkage between these ch ains, 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) c hains in the GEM of patients with Alport syndrome.