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STRUCTURE AND FUNCTION OF A NOVEL BASEMENT MEMBRANE-ASSOCIATED COLLAGEN

新型基底膜相关胶原蛋白的结构和功能

基本信息

批准号：
03454538
负责人：
HAYASHI Toshihiko
金额：
$ 0.64万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

The novel collagen in the present project was defined as a protein specifically recognized by a monoclonal antibody originally developed for type IV collagen fraction from human placenta by pepsinization. The epitope appeared to reside on a polypeptide which contained an unidetified amino acid sequence. The collagen was found to be associated with a restricted basement membrane in the immunohistochemical examination on a vascular basement membrane and at least a portion of the polypeptide was associated with alpha 1(IV) chain, since a sandwith ELISA system using the other monoclonal antibody which recognizes alpha 1(IV) showed a positive reaction to the type IV collagen fraction, while the reaction became negative by treatment with heat or/and SH reagent. Molecular structure and function of the novel collagen was characterized by comparison with other extracellular components in solubility, intact size of the polypeptide, susceptibility to proteases. The study indicated similarity to an alpha chain of type IV collagen; that is 180kD chain was recovered only after treatment with SH reagent and denaturants from human placenta. The monoclonal antibody crossed with a particular sequence of alpha 1(IV) chain weakly. An important observation was that human fetal lung fibroblasts produced and deposited the novel collagen as well as alpha 1(IV) on the cell layer by culture, although human dermal fibroblasts deposited the collagen to a much less quantity from the study using cell-layer ELISA. Immunohistochemical localization indicated that deposition of novel collagen appeared to increase by fibroses of liver or kidney. Serum levels of the complex of the novel collagen as determined by ELISA were increased in patients with liver cirrhosis.

本项目中的新型胶原蛋白被定义为一种由单克隆抗体特异性识别的蛋白质，该单克隆抗体最初是通过胃蛋白酶消化从人胎盘中提取IV型胶原蛋白组分而开发的。该表位似乎位于含有未确定的氨基酸序列的多肽上。在对血管基底膜的免疫组织化学检查中发现胶原蛋白与限制性基底膜结合，并且至少一部分多肽与α 1（IV）链结合，因为使用识别α 1（IV）的另一种单克隆抗体的Sandwith ELISA系统显示对IV型胶原蛋白组分的阳性反应，而加热或/和SH试剂处理则使反应变为阴性。通过与其他胞外组分在溶解性、多肽完整大小、对蛋白酶的敏感性等方面的比较，对新型胶原的分子结构和功能进行了表征。该研究表明与IV型胶原的α链相似;即仅在用SH试剂和来自人胎盘的变性剂处理后才回收180 kD链。单克隆抗体与α 1（IV）链的特定序列弱交叉。一个重要的观察结果是，人胎肺成纤维细胞通过培养产生并沉积新的胶原蛋白以及α 1（IV）在细胞层上，尽管使用细胞层ELISA的研究中人真皮成纤维细胞沉积胶原蛋白的量少得多。免疫组织化学定位显示，新胶原沉积似乎增加肝或肾纤维化。通过ELISA测定的新胶原蛋白复合物的血清水平在肝硬化患者中增加。