Type IV collagen of the glomerular basement membrane - Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GEM is composed of five (alpha 1 to alpha 5) of the six chains of type IV collagen, organized into an alpha 1.alpha 2(IV) and an alpha 3.alpha 4.alpha 5(IV) network. In Alport syndrome, mutations in any of the genes encoding the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains cause the absence of the alpha 3.alpha 4.alpha 5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the alpha 5 chain was shown to be linked to the alpha 3 and alpha 4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified alpha 1-alpha 5(IV) NC1 monomers. The results showed that NC1 monomers associate to form nativelike hexamers characterized by two distinct populations, an alpha 1.alpha 2 and alpha 3.alpha 4.alpha 5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the alpha 1.alpha 2 and alpha 3.alpha 4.alpha 5 networks of GEM. Moreover, hexamer formation from the alpha 3, alpha 4, and alpha 5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the alpha 3.alpha 4.alpha 5 network by interfering with the assembly of the alpha 3.alpha 4.alpha 5 NC1 hexamer.