Hirako Y, Usukura J, Uematsu J, Hashimoto T, Kitajima Y, Owaribe K
Unit of Biosystems, Graduate School of Human Informatics, Nagoya University, Nagoya 464-01.
J Biol Chem. 1998 Apr 17;273(16):9711-7. doi: 10.1074/jbc.273.16.9711.
The hemidesmosome (HD) is a cell-to-substrate adhesion apparatus found in stratified and complex epithelia. One of the putative cell-matrix adhesion molecules present in the HD is the 180-kDa bullous pemphigoid antigen (BP180), also termed type XVII collagen. In our previous study, using a monoclonal antibody (mAb) 1337, we have detected a 120-kDa collagenase-sensitive polypeptide in the HD fraction (Uematsu, J. and Owaribe, K. (1993) Cell Struct. Funct. 18, 588 (abstr.)). The present study was undertaken to assess the relation of the 120-kDa polypeptide to this BP180. Immunofluorescence microscopy of bovine skin revealed the basement membrane zone of skin to be stained clearly with mAb 1337, whereas the lateral surfaces of basal cells, which were decorated by typical antibodies against BP180, were not. The antibody did not detect HDs in cultured cells but rather in the culture medium. These results indicate a localization of mAb 1337 antigen distinct from BP180. However, the same polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Therefore, the polypeptide was identified as an extracellular fragment of BP180. mAb 1337 immunoprecipitated the 120-kDa fragment from the medium, but not the 180-kDa molecule of BP180 extracted from cultured cells, indicating that the antibody specifically recognizes the fragment. The mAb 1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Hence, the staining pattern observed for bovine skin demonstrated the presence of the 120-kDa extracellular fragment. Rotary shadow electron microscopy of affinity-purified 120-kDa fragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb 1337 shows unique epitope specificity, recognizing only the 120-kDa extracellular fragment of BP180, which is constitutively cleaved on the cell surface as a 120-kDa fragment both in in vivo and in vitro.
半桥粒（HD）是一种存在于复层和复杂上皮组织中的细胞与基底附着结构。半桥粒中存在的一种假定的细胞 - 基质黏附分子是180kDa的大疱性类天疱疮抗原（BP180），也称为XVII型胶原蛋白。在我们之前的研究中，使用单克隆抗体（mAb）1337，我们在HD组分中检测到一种120kDa的胶原酶敏感多肽（上松，J.和小针，K.（1993年）细胞结构与功能18，588（摘要））。本研究旨在评估该120kDa多肽与这种BP180的关系。牛皮肤的免疫荧光显微镜检查显示，皮肤的基底膜区被mAb 1337清晰染色，而由针对BP180的典型抗体标记的基底细胞侧面则未被染色。该抗体在培养细胞中未检测到半桥粒，而是在培养基中检测到。这些结果表明mAb 1337抗原的定位与BP180不同。然而，同一种多肽也被针对BP180细胞外而非细胞质部分的单克隆抗体识别，并发现与针对BP180非胶原16A结构域的多克隆抗体发生反应。因此，该多肽被鉴定为BP180的细胞外片段。mAb 1337从培养基中免疫沉淀出120kDa片段，但未从培养细胞中提取的180kDa的BP180分子中免疫沉淀，这表明该抗体特异性识别该片段。mAb 1337显然识别一个独特的表位，该表位由切割暴露或形成。因此，在牛皮肤上观察到的染色模式证明了120kDa细胞外片段的存在。对亲和纯化的120kDa片段进行旋转阴影电子显微镜检查表明，它们具有独特的分子形状，由一个中心杆和一条柔性尾巴组成，没有BP180分子中存在的球状头部。从这些结果中，我们得出结论，mAb 1337显示出独特的表位特异性，仅识别BP180的120kDa细胞外片段，该片段在体内和体外均在细胞表面以120kDa片段的形式组成性切割。
