A 230-kD basic protein is the major bullous pemphigoid antigen.
Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of BP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP sera by both immunoprecipitation of extracts of radiolabeled cultured human keratinocytes and immunoblotting of extracts of normal human epidermis. Thirty-six of 37 BP sera immunoprecipitated the 230-kD polypeptide, and 11 of 13 BP sera identified the 230-kD polypeptide by immunoblotting. Eighty-six disease and normal control sera did not bind the 230-kD BP antigen. Immunoprecipitation was a more sensitive assay than was immunoblotting to detect the 230-kD antigen; BP sera that were weak or negative by immunoblotting were strongly positive by immunoprecipitation. To demonstrate that this 230-kD polypeptide shared epitopes, defined by BP sera, with the epidermal basement membrane zone, we showed that BP IgG affinity purified on this polypeptide bound the epidermal basement membrane zone by immunofluorescence. To further characterize the BP antigen and to compare the antigen synthesized by cultured human keratinocytes to the antigen extracted from epidermis, we analyzed the 230-kD BP antigen by two-dimensional gel electrophoresis. BP antigen immunoprecipitated from culture extracts or BP antigen identified by immunoblots of epidermal extracts was a single spot on two-dimensional gels, with a pI of about 8. These data indicate that, although other polypeptides are sometimes identified (e.g., a 166 kD band by immunoprecipitation or a 180 kD band on immunoblots), the major BP antigen identified in cultured human keratinocytes is similar to the antigen found in normal human epidermis and is a basic 230-kD polypeptide.
Mueller
S
S
Dermatology Branch, National Cancer Institute, Bethesda, Maryland 20892.
Klaus-Kovtun
V
V
Stanley
J R
JR
D001324:Autoantigens / Q000032:analysis*; D001485:Basement Membrane / Q000276:immunology; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D004586:Electrophoresis; D004817:Epidermis / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007633:Keratins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000276:immunology*; D010455:Peptides / Q000276:immunology*; D011233:Precipitin Tests; D012872:Skin Diseases, Vesiculobullous / Q000276:immunology*; D000097805:Collagen Type XVII
D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D007633:Keratins; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010455:Peptides; D000097805:Collagen Type XVII; C486268:DST protein, human