Gunwar S, Ballester F, Noelken M E, Sado Y, Ninomiya Y, Hudson B G
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
J Biol Chem. 1998 Apr 10;273(15):8767-75. doi: 10.1074/jbc.273.15.8767.
Glomerular basement membrane (GBM) 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 GBM 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 alpha1(IV) and alpha2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha3(IV), alpha4(IV), and alpha5(IV) chains along with some alpha1(IV) and alpha2(IV) 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 alpha3(IV), alpha4(IV), and alpha5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha3(IV). alpha4(IV).alpha5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of not only alpha5(IV) chain but also the alpha3(IV) and alpha4(IV) chains in the GBM of patients with Alport syndrome.
肾小球基底膜（GBM）在肾脏对血浆的超滤过程中发挥着关键作用。在阿尔波特综合征中，这种功能会受到损害，该综合征是一种遗传性疾病，由编码IV型胶原的基因突变引起，但尚不清楚这些突变如何导致GBM出现缺陷。在本研究中，对GBM的IV型胶原超分子组织进行了研究。这是通过使用假溶素（EC 3.4.24.26）消化来切除截短的三螺旋原聚体以进行结构研究来实现的。溶解了两组不同的截短原聚体，一组在4℃，另一组在25℃，并使用单克隆抗体确定了它们的链组成。4℃的原聚体包含α1（IV）和α2（IV）链，而25℃的原聚体主要由α3（IV）、α4（IV）和α5（IV）链以及一些α1（IV）和α2（IV）链组成。通过电子显微镜检查了25℃原聚体的结构，发现其特征是一个包含环和超螺旋三螺旋的网络，这些由α3（IV）、α4（IV）和α5（IV）链之间的二硫键交联稳定。这些结果建立了一个概念框架来解释阿尔波特综合征GBM异常的几个特征。特别是，α3（IV）、α4（IV）、α5（IV）网络涉及这些链之间的共价连接，这为一个难题提供了分子基础，即编码α5（IV）链的基因突变不仅导致α5（IV）链组装缺陷，还导致阿尔波特综合征患者GBM中的α3（IV）和α4（IV）链组装缺陷。
