Differences in specificities of anti-centromere sera for the monomeric and dimeric C-terminal peptides of human centoromere protein C.
Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus. In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked. Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA(+) sera. The reactivities of the dimers were obviously higher in both IgG and IgM responses. The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera. Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined. The former two mutants decreased the dimerization activity. The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half. Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E. coli, GroEL, reacted to the centromere in culture cells. In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C.
Hayashi
Y
Y
Division of Connective Tissue Disease & Autoimmunity, Department of Dermatology, Nagoya University School of Medicine, Nagoya, Japan.
Muro
Y
Y
Kuriyama
K
K
Tomita
Y
Y
Sugimoto
K
K
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000276:immunology*; D002503:Centromere / Q000276:immunology*; D018834:Chaperonin 60 / Q000494:pharmacology; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000276:immunology*; D019281:Dimerization; D000939:Epitopes; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*
D001324:Autoantigens; D018834:Chaperonin 60; D002868:Chromosomal Proteins, Non-Histone; D000939:Epitopes; D007106:Immune Sera; D010446:Peptide Fragments; C060905:centromere protein C