Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.
Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.
Stanley
J R
JR
Dermatology Branch, National Cancer Institute, Bethesda, Maryland 20892.
Tanaka
T
T
Mueller
S
S
Klaus-Kovtun
V
V
Roop
D
D
D000595:Amino Acid Sequence; D001323:Autoantibodies; D001324:Autoantigens / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D004247:DNA / Q000302:isolation & purification*; D000071821:Dystonin; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000235:genetics* / Q000276:immunology; D012313:RNA / Q000032:analysis; D012872:Skin Diseases, Vesiculobullous / Q000235:genetics*; D000097805:Collagen Type XVII
D001323:Autoantibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D004247:DNA; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D012313:RNA; D000097805:Collagen Type XVII; C486268:DST protein, human