Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies

• Published in: The Journal of clinical investigation Vol. 82; no. 6; pp. 1864 - 1870
• Main Authors: STANLEY, J. R, TANAKA, T, MUELLER, S, KLAUS-KOVTUN, V, ROOP, D
• Format: Journal Article
• Language: English
• Published: Ann Arbor, MI American Society for Clinical Investigation 01.12.1988
• Subjects: Amino Acid Sequence; Autoantibodies; Autoantigens - genetics; Autoantigens - immunology; Base Sequence; Biological and medical sciences; Bullous diseases of the skin; Carrier Proteins; Collagen; Collagen Type XVII; Cytoskeletal Proteins; Dermatology; DNA - isolation & purification; Dystonin; Humans; Immunoenzyme Techniques; Medical sciences; Molecular Sequence Data; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous - genetics; Pemphigoid, Bullous - immunology; RNA - analysis; Skin Diseases, Vesiculobullous - genetics; Bullous dermatosis; Human; Immunopathology; Skin disease; Nucleotide sequence; Autoantibody; Autoimmune disease; Gene library; Open reading frame; Antigen; Complementary DNA; Bullous pemphigoid; Genetic engineering; Molecular cloning; Aminoacid sequence
• ISSN: 0021-9738; 1558-8238
• DOI: 10.1172/jci113803

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.