Major antigenic epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein. Evidence using a 55 kDa recombinant protein.
In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M(r) (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.
Gaucherand
M
M
Inserm 346, Clinique Dermatologique, Université Claude Bernard Lyon I, Hopital Edouard Y Herriot, France.
Nicolas
J F
JF
Paranhos Baccala
G
G
Rouault
J P
JP
Réano
A
A
Magaud
J P
JP
Thivolet
J
J
Jolivet
M
M
Schmitt
D
D
D000818:Animals; D001323:Autoantibodies / Q000032:analysis; D001483:Base Sequence; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010391:Pemphigoid, Bullous / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D001323:Autoantibodies; D000939:Epitopes; D011994:Recombinant Proteins