Kahsai T Z, Enders G C, Gunwar S, Brunmark C, Wieslander J, Kalluri R, Zhou J, Noelken M E, Hudson B G
Departments of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
J Biol Chem. 1997 Jul 4;272(27):17023-32. doi: 10.1074/jbc.272.27.17023.
Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six alpha-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of alpha3(IV) chain (40%) and the alpha4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six alpha(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of alpha(IV) chains, were characterized for their alpha(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with alpha(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the alpha1(IV) and alpha2(IV) chains and two novel networks, one composed of the alpha1(IV)-alpha6(IV) chains and the other composed of the alpha3(IV)-alpha6(IV) chains. The results establish a structural linkage between the alpha3(IV) and alpha5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of the alpha3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.
生精小管基底膜（STBM）在精子发生过程中起着重要作用。在本研究中，对牛STBM中IV型胶原蛋白的组成和结构组织进行了研究。基于免疫细胞化学和生化分析，发现STBM由IV型胶原蛋白的所有六条α链组成。α3（IV）链（40%）和α4（IV）链（18%）的含量明显高于任何其他基底膜胶原蛋白。使用假溶素（EC 3.4.24.26）消化以切除三螺旋分子，随后进行胶原酶消化以产生NC1六聚体，并通过抗体亲和色谱法解析NC1六聚体群体，从而研究了六条α（IV）链的超分子结构。利用高效液相色谱、二维电泳以及使用α（IV）链特异性抗体进行免疫印迹，对反映α（IV）链特定排列的六聚体的α（IV）链组成进行了表征。发现了三个主要的六聚体群体，它们代表了α1（IV）和α2（IV）链的经典网络以及两个新的网络，一个由α1（IV）-α6（IV）链组成，另一个由α3（IV）-α6（IV）链组成。这些结果在α3（IV）链和α5（IV）链之间建立了结构联系，为一个难题提供了分子基础，即在患有Alport综合征的患者肾小球基底膜中，编码α5（IV）链的基因突变会导致α3（IV）链组装缺陷。
