Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Abstract

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.

The Cell Adhesion Domain of Type XVII Collagen Promotes Integrin-mediated Cell Spreading by a Novel Mechanism

“…Both transmembrane collagens XIII and XVII are proteolytically released from cell surface to the extracellular matrix (7,8,31), which allows them to function as matrix proteins, contributing to cell adhesion and migration. We have recently shown that the ␣ 1 ␤ 1 integrin, rather than ␣ 2 ␤ 1 , is a cellular receptor for type XIII collagen (17).…”

“…Type XVII collagen is shed from the cell surface (7,8), which 2 ) with heat-denatured (dCOL15) human recombinant COL15 domain of collagen type XVII overnight. HaCaT cells (10,000 cells/well) were allowed to attach and spread for 120 min in the presence of synthetic COL15-derived peptides in the concentration of 0.01 or 0.1 mM (A).…”

“…Type XVII collagen (BP180) is hemidesmosomal transmembrane protein that is mutated in junctional epidermolysis bullosa and is targeted by autoantibodies in blistering skin diseases (4 -6). It occurs in two forms: as a full-length transmembrane protein and as a distinct, soluble ectodomain that is shed from the cell surface by a furin-mediated, proteolytic process (7,8). This collagen possesses intracellular and transmembrane domains that are of 560 and 23 amino acids in length, respectively.…”

Collagenous Transmembrane Proteins: Recent Insights into Biology and Pathology*

“…Collagen XVII Is Shed by ADAMs-A landmark feature of collagen XVII is its constitutive shedding yielding a shorter and soluble form of the molecule (17,25) that essentially spans the entire ectodomain (see Fig. 2S in supplemental material).…”

Context-Dependent Regulation of Collagen XVII Ectodomain Shedding in Skin

“…To address these issues, we produced rabbit antibodies specifically reacting with N-terminal cleavage sites of the shed COL17 ectodomain. COL17 is a type II-oriented transmembrane glycoprotein whose N-terminus and C-terminus are in the cytoplasm and the extracellular matrix (ECM), respectively 10,11 . As is true for other transmembrane collagen family members, the extracellular domain of COL17 can be cleaved from the cell surface in vitro by a disintegrin and metalloproteases (ADAMs) 9, 10 and 17 12, 13 , and interestingly, the ectodomain shedding occurs within the NC16A domain which contains major epitopes for BP autoAbs 14, 15 .…”