Fine-specificity of the anti-CENP-A B-cell autoimmune response.
The major targets recognized by anti-centromere autoantibodies are the three centromere-associated proteins (CENPs) A, B, and C, with apparent molecular masses of 19, 80, and 140 kDa, respectively. Previously a major epitope region on the 19-kDa CENP-A antigen was identified by synthesis of a soluble synthetic 15-mer peptide (amino acids 3-17) to be used in enzyme-linked immunosorbent assay and western blot competition assays. However, no systematic experimental scanning for epitope regions on the CENP-A autoantigen has yet been performed. In this study we scanned the complete CENP-A amino acid sequence for epitopes using 19 previously characterized autoimmune-sera. Overlapping peptides 15 amino acids in length and offset by three amino acids were synthesized on activated membranes, covering the whole CENP-A autoantigen. Probing of the membranes with various anti-centromere sera showed that all epitopes are clustered in the N-terminal 45 amino acids. For fine-mapping of this autoreactive region the N-terminus of CENP-A (amino acids 1-45) was scanned again by probing overlapping 15-mer, 12-mer, 10-mer, 8-mer, 7-mer, 6-mer, and 5-mer peptides, all offset by one amino acid, with anti-centromere sera. In this way we localized two epitope core regions within the N-terminal 45 amino acids, one covering amino acids 2-17, recognized by 17 sera, and the other covering amino acids 22-38, recognized by 18 sera. One serum did not react with CENP-A at all. Several sera seem to recognize overlapping individual epitopes within these two epitope core regions. All sera, however, recognize a sequence motif G/A-P-R/S-R-R.
Mahler
M
M
Institute of Molecular Genetics, University of Heidelberg, Germany.
Mierau
R
R
Blüthner
M
M
D000328:Adult; D000595:Amino Acid Sequence; D000917:Antibody Formation; D001172:Arthritis, Rheumatoid / Q000276:immunology; D001324:Autoantigens / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D006528:Carcinoma, Hepatocellular; D002503:Centromere / Q000276:immunology; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D003240:Connective Tissue Diseases / Q000276:immunology; D000939:Epitopes / Q000737:chemistry; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008113:Liver Neoplasms; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012595:Scleroderma, Systemic / Q000276:immunology; D014407:Tumor Cells, Cultured
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D000939:Epitopes