Cleavage of BP180, a 180-kDa bullous pemphigoid antigen, yields a 120-kDa collagenous extracellular polypeptide.
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.
Hirako
Y
Y
Unit of Biosystems, Graduate School of Human Informatics, Nagoya University, Nagoya 464-01.
Usukura
J
J
Uematsu
J
J
Hashimoto
T
T
Kitajima
Y
Y
Owaribe
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D001324:Autoantigens / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D001485:Basement Membrane / Q000166:cytology / Q000378:metabolism; D002352:Carrier Proteins; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D017364:Collagenases / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D004847:Epithelial Cells; D019573:Epithelium, Corneal / Q000276:immunology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008321:Mammary Glands, Animal; D051379:Mice; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous; D010446:Peptide Fragments / Q000032:analysis; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000097805:Collagen Type XVII
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D017364:Collagenases; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010446:Peptide Fragments; D000097805:Collagen Type XVII; C486268:DST protein, human