Boutaud A, Borza D B, Bondar O, Gunwar S, Netzer K O, Singh N, Ninomiya Y, Sado Y, Noelken M E, Hudson B G
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
J Biol Chem. 2000 Sep 29;275(39):30716-24. doi: 10.1074/jbc.M004569200.
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 GBM is composed of five (alpha1 to alpha5) of the six chains of type IV collagen, organized into an alpha1.alpha2(IV) and an alpha3.alpha4.alpha5(IV) network. In Alport syndrome, mutations in any of the genes encoding the alpha3(IV), alpha4(IV), and alpha5(IV) chains cause the absence of the alpha3. alpha4.alpha5 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 alpha5 chain was shown to be linked to the alpha3 and alpha4 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 alpha1-alpha5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an alpha1.alpha2 and alpha3.alpha4.alpha5 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 alpha1.alpha2 and alpha3.alpha4.alpha5 networks of GBM. Moreover, hexamer formation from the alpha3, alpha4, and alpha5 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 alpha3.alpha4.alpha5 network by interfering with the assembly of the alpha3.alpha4.alpha5 NC1 hexamer.
在影响IV型胶原的遗传性和获得性疾病中，肾脏肾小球基底膜（GBM）的超滤功能受损。GBM由IV型胶原六条链中的五条（α1至α5）组成，组织成一个α1.α2（IV）和一个α3.α4.α5（IV）网络。在Alport综合征中，编码α3（IV）、α4（IV）和α5（IV）链的任何基因发生突变都会导致α3.α4.α5网络缺失，进而导致进行性肾衰竭。在本研究中，通过进一步表征链的组织和阐明控制网络组装的鉴别性相互作用，探索了网络缺陷背后的分子机制。通过对非胶原（NC1）结构域的六聚体复合物进行分析，进一步证实了这两个网络的存在，并且显示α5链通过其各自的NC1结构域之间的相互作用与α3和α4链相连。通过比较天然NC1六聚体与体外解离和重组的六聚体以及由纯化的α1-α5（IV）NC1单体体外组装的六聚体的组成，研究了NC1结构域在网络组装中的潜在识别功能。结果表明，NC1单体缔合形成具有两种不同群体特征的天然样六聚体，即α1.α2和α3.α4.α5异源六聚体。这些发现表明，NC1单体包含用于选择链和原聚体的识别序列，这些序列足以编码GBM的α1.α2和α3.α4.α5网络的组装。此外，由α3、α4和α5 NC1单体形成六聚体需要所有三种单体共同组装，这表明Alport综合征中NC1结构域的突变可能通过干扰α3.α4.α5 NC1六聚体的组装而破坏α3.α4.α5网络的组装。
