Iwata M, Imamura Y, Sasaki T, Hayashi T
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo.
J Biochem. 1995 Jun;117(6):1298-304. doi: 10.1093/oxfordjournals.jbchem.a124858.
The extracts from bovine lens capsule with acetic acid contained, after reduction, three major collagenous polypeptides with M(r) = 180k, 175k, and 160k, which were specifically immuno-stained with anti-type IV collagen polyclonal antibody. The biochemical properties of 180k and 160k polypeptides were akin and were distinct from that of 175k polypeptide [J. Biochem. (1993) 114,358-362]. In the present study, evidence that the 160k and 180k polypeptides from bovine lens capsule both originated from alpha 1(IV) was obtained on the basis of reactivity with a monoclonal antibody that recognizes alpha 1(IV) chain at the collagenous sequence contained in [KGEPGLPGRGFPGFP]. The epitope-bearing sequence was identified from the following three experiments. Pepsin-solubilized polypeptides from human placenta were purified by affinity chromatography on the antibody-coupled column and sequenced. The restriction map of the clones positively reactive with the monoclonal antibody from human placenta cDNA library was superimposed on that of human alpha 1(IV) cDNA at a specific region. Synthetic peptides corresponding to the sequence were assayed for inhibitory activity against the reaction between epitope-bearing pepsin fragments and the antibody. The 180k and 160k polypeptides showed similar intensities in protein staining as well as in immuno-staining with the monoclonal antibody. In contrast, the 175k polypeptide did not react with the monoclonal antibody, indicating that it is a genetically distinct type IV collagen chain, presumably alpha 2(IV) from its abundance. The 160k, a major type IV collagen polypeptide, is a short form of alpha 1(IV) present as a tissue form in bovine lens capsule.
用乙酸从牛晶状体囊膜中提取的物质，经还原后含有三种主要的胶原多肽，其相对分子质量分别为180k、175k和160k，它们能被抗IV型胶原多克隆抗体特异性免疫染色。180k和160k多肽的生化特性相似，与175k多肽的特性不同[《生物化学杂志》（1993年）114卷，358 - 362页]。在本研究中，基于与一种单克隆抗体的反应性，获得了证据表明牛晶状体囊膜中的160k和180k多肽均源自α1(IV)，该单克隆抗体能识别[KGEPGLPGRGFPGFP]中所含胶原序列的α1(IV)链。通过以下三个实验确定了表位携带序列。用胃蛋白酶溶解的人胎盘多肽在抗体偶联柱上进行亲和层析纯化并测序。将来自人胎盘cDNA文库且与该单克隆抗体呈阳性反应的克隆的限制性图谱与人类α1(IV) cDNA在特定区域进行比对。测定了与该序列相对应的合成肽对表位携带胃蛋白酶片段与抗体之间反应的抑制活性。180k和160k多肽在蛋白质染色以及与单克隆抗体的免疫染色中显示出相似的强度。相比之下，175k多肽不与该单克隆抗体反应，表明它是一种基因上不同的IV型胶原链，根据其丰度推测可能是α2(IV)。160k，一种主要的IV型胶原多肽，是α1(IV)的一种短形式，以组织形式存在于牛晶状体囊膜中。
