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Objectives: Anticentromere antibody (ACA) is generally considered to be a serological marker for systemic sclerosis (SSc). ACA-positive patients with primary Sjögren's syndrome (pSS) have also been reported. ACA often recognizes centromere proteins (CENPs): CENP-A, CENP-B, and CENP-C, and sometimes reacts to heterochromatin protein 1 (HP1)α. We compared the reactivity against six different epitopes for three ACA-positive clinical subgroups: 29 patients with pSS, 36 SSc patients with sicca symptoms, and 28 SSc patients without sicca symptoms., Methods: We utilized enzyme-linked immunosorbent assays (ELISAs) with recombinant proteins covering six different epitope regions of ACA (the amino terminus (Nt) of CENP-A, CENP-B, and CENP-C, the carboxyl terminus (Ct) of CENP-B and CENP-C, and HP1α)., Results: The patients with pSS were found to have IgG-class autoantibodies against CENP-C-Nt and HP1α, and IgA-class autoantibodies against CENP-C-Ct with significantly higher frequencies than the SSc patients with or without sicca symptoms. The positive predictive value and the negative predictive value of the combination of these three autoantibodies for pSS were 73% and 82%, respectively, for pSS., Conclusions: Based on the result that reactivities against CENP-C and HP1α in patients with pSS differ from those in patients with SSc, we propose ACA-positive pSS as a clinical subset of SS that is independent of SSc.

Objective: Myositis-specific autoantibodies (MSAs), which characterize certain forms of inflammatory myopathy, are useful in the diagnosis and prediction of prognosis in DM/PM. Anti-transcriptional intermediary factor 1-α (TIF1-α) antibodies were recently reported to be associated with cancer-associated DM in conjunction with anti-TIF1-γ antibodies. This study aimed to identify a subset of DM patients who have anti-TIF1-α antibodies by using biotinylated recombinant proteins and to clarify the clinical and other serological features of DM patients with these antibodies., Methods: Sera from 202 Japanese patients with CTDs, including 108 with DM and 20 healthy controls, were screened for anti-TIF1-α antibodies by our novel ELISAs. Positive sera were further examined by immunoprecipitation and also investigated for the detection of anti-TIF1-γ and anti-Mi-2 antibodies., Results: Sera from 12 patients with DM were confirmed to be positive for anti-TIF1-α antibodies. None of the patients with other CTDs and none of the healthy controls had the antibodies. Seven anti-TIF1-α-positive patients simultaneously had anti-TIF1-γ antibodies and the other five had anti-Mi-2 antibodies, both of which are well known to be MSAs. These double-positive patients with anti-TIF1-α and anti-γ antibodies included three JDM and two cancer-associated adult DM patients, whereas all the double-positive patients with anti-TIF1-α and anti-Mi-2 antibodies were classical adult DM., Conclusion: Although MSAs have been regarded as mutually exclusive, anti-Mi-2 antibody-positive patients simultaneously have anti-TIF1-α antibodies. Anti-Mi-2 antibody-positive patients are associated with classical DM without cancer even with the simultaneous presence of anti-TIF1-α antibodies.

Lens epithelium-derived growth factor/dense fine speckles 70 kDa protein (LEDGF/DFS70) is a transcriptional cofactor, a transcriptional activator, survival factor, and HIV-1 transporter. It is also a major autoantigen in patients with atopic dermatitis (AD), because autoantibodies to this protein are found in approximately 30% of AD patients. To better understand the role of autoantibodies and autoantigens in the pathogenesis of AD, we examined the distribution of LEDGF/DFS70 in the epidermis of normal human skin by light and electron microscopic immunocytochemistry. Increased amounts of LEDGF/DFS70 were located in the nuclei of cells in the basal layer, whereas the cytoplasm of cells in the granular layer stained for LEDGF/DFS70 by light microscopy. Using immunoelectron microscopy, we observed the accumulation of LEDGF/DFS70 in keratohyalin granules (KGs) in the cytoplasm of cells in the granular layer. In addition, Ig heavy chain-binding protein/glucose-regulated protein, 78-kDa (Bip/GRP78), a stress sensing protein in the endoplasmic reticulum, colocalized with LEDGF/DFS70 in the KGs. These results suggest that LEDGF/DFS70 is predominantly located in the nucleus of the basal epidermal cells and translocates into the cytoplasm during differentiation. Once in the cytoplasm, LEDGF/DFS70 accumulates in the KGs in the granular layer. Finally, LEDGF/DFS70, a "nuclear" autoantigen in AD, may play a functional role in the KGs.

Autoantibodies against DFS70 (Dense Fine Speckles 70) are found in 30% of Japanese atopic dermatitis patients, and less frequently in patients with other diseases. We have recently reported that they are also seen in 11% of hospital workers, but in only approximately 2% of patients with systemic rheumatic disease. In this study, in order to investigate the possible pathological role of anti-DFS70 antibodies, fine epitope mapping was carried out using 93 anti-DFS70 autoantibody-positive sera. Immunoblotting using overlapping peptides failed to reveal major linear epitopes. Western blotting using various truncated proteins showed a strikingly uniform epitope distribution on a suspected tertiary structure expressed by DFS70(349-435). Some sera showed reactivity only in an immunoprecipitation assay using an in vitro translated DFS70. Circular dichroism analysis revealed that DFS(349-435) contains an approximately 40% alpha-helical conformation, while an overlapping, non-antigenic peptide is composed of random coiled structures. The skewed single major epitope enabled us to establish a highly quantitative ELISA for the epitope region. Antibody titers showed no significant differences between the diseased group and healthy individuals. We propose that anti-DFS70 antibody may be a natural autoantibody, which might modify or reflect the inflammatory process of various disorders.

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.

Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases. CENP-A is one of the major antigens against ACA and a histone H3-like protein. To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera. Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3. Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3-17 (peptide A) and 25-38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA.

We examined the clinical features of patients presenting antinuclear autoantibodies against p80-coilin and the IgG subclass distribution of anti- p80-coilin antibodies. Sera from 365 Japanese patients were analysed. Immunoblotting and indirect immunofluorescence microscopy techniques were used with a polyclonal rabbit antiserum against p80-coilin. Eleven patients with anti-p80-coilin antibodies were found. All the patients were female and nine were in their twenties. None could be diagnosed with differentiated rheumatic disease except for one case of systemic scleroderma and another of Sjögren's syndrome. Most patients had general fatigue, arthralgia, headaches, dysmenorrhea, lymph node swelling and/or low grade fever such as chronic fatigue syndrome (CFS), and showed low complement. One patient fulfilled the criteria for CFS. All were younger females than those often diagnosed with rheumatic disease in previous reports. Patients' sera had a predominant distribution of subclass IgG(1)anti-p80-coilin antibodies and five sera had concomitant subclass IgG(2). Two rheumatic disease patients had a relatively high titer of IgG(2)anti-p80-coilin antibodies. The IgG(2)subclass of anti-p80-coilin antibodies may be a specific marker for systemic autoimmune disease., (Copyright 1999 Academic Press.)

Using autoimmune serum from a patient with anti-centromere antibodies, we have identified and partially characterized a novel protein with a mol. wt of approximately 27 kD (hereafter referred to as p27). A cDNA expression library was screened with this serum, and two overlapping inserts were isolated among three positive clones other than clones corresponding to centromere protein (CENP)-B and CENP-C. Analysis of the sequence showed an open reading frame of approximately 0.6 kb encoding 199 amino acids with a predicted mol. wt of 21.5 kD. Immunoblotting analysis with bacterial recombinant p27 showed that approximately 2% of anti-centromere antibody-positive patients had autoantibodies to p27, whereas only one of 215 autoimmune patients without anti-centromere antibodies reacted with the recombinant. All five cases with anti-p27 antibodies, who were diagnosed as having scleroderma and/or Sjögren's syndrome, showed internal organ involvement. Although affinity-purified anti-p27 human or mouse polyclonal antibodies failed to stain any cellular structures in an immunofluorescence study, the potential association of anti-p27 with anti-centromere antibodies suggests that this novel autoantigen might play a role in mitosis.

The Ki-67 antigen, which is recognized by the monoclonal antibody Ki-67, is a marker of cell proliferation. During cDNA cloning using sera from a patient with systemic lupus erythematosus, we obtained a positive clone encoding part of Ki-67. We determined the frequency of autoantibodies against Ki-67 in patients with systemic autoimmune diseases. Serum samples from 252 patients with rheumatic diseases were examined by immunoblotting with HeLa nuclear extract and the recombinant N-terminus of the Ki-67 antigen. Autoantibodies against Ki-67 antigen were detected in two out of 76 patients with systemic lupus erythematosus and one out of 90 patients with scleroderma. While, in a previous report, anti-Ki-67 antibodies were frequently targeted by a certain strain of autoimmune mice, our results indicated that Ki-67 was a minor target of autoantibodies among Japanese patients with systemic autoimmune diseases., (Copyright 1997 Academic Press Limited.)

Autoantibodies against centromere proteins are commonly found in the serum of patients with scleroderma and other systemic autoimmune diseases. The reactivity of anticentromere autoantibodies (ACA) from 78 patients was investigated by ELISA using two kinds of a 15-amino-acid peptide corresponding to the N- and C-termini of CENP-A, one of the target molecules of ACA. The N-terminal peptide (residues 3-17) was recognized by 85% of ACA, while the C- terminal peptide (residues 126-140) was not. The ELISA result for the N-terminal peptide correlated with the immunoreactivity of CENP-A observed in immunoblotting. Moreover, the binding between autoantibodies and CENP-A was inhibited by the N-terminal peptide in 98.5% of anti-CENP-A- positive sera in immunoblotting. The sequence of peptide, PRRRSRKPEAPRRRS, is highly charged and has two repeats of PRRRS. These results indicate that the N-terminal-charged region forms a major epitope of CENP-A. This area may be involved in the induction of specific autoantibodies against centromere in autoimmune patients.

Heterochromatin protein 1 (HP1) is one of the nonhistone chromosomal components tightly associated with the pericentromeric heterochromatic region in Drosophila. The human homologue of HP1 is recognized by a subpopulation of anti-centromere antibodies (ACA). Such autoantibodies recognize a group of several nuclear proteins with Mr of 23-25 kDa and have been termed 'anti-chromo antibodies (AChA)' because an evolutionarily conserved N-terminal half called the 'chromo domain' of HP1 is the epitope. In this study, 84 ACA sera were examined by immunoblotting with recombinant 25-kDa chromo protein (p25). The p25 antigen was expressed as a glutathione S-transferase-fusion protein in E. coli and purified with glutathione-sepharose. Except for one serum specimen, AChA-positive sera reacted with the N-terminus (a.a 16-106) and/or the C-terminus (a.a. 83-191) of p25. Autoimmune response against the N-terminus of p25 in 33 patients was significantly associated with systemic lupus erythematosus and significantly related to leukopenia, thrombocytopenia and elevated erythrocyte sedimentation rate; C-terminal reactivity in 30 patients was significantly associated with primary Sjogren's syndrome and related to leukopenia. The internal 64-amino acid stretch (a.a 43-106) with DNA-binding activity was not autoantigenic. p25 has two separate homologous regions to Drosophila HP1 at the N- and C-termini; the chromo domain and the chromo shadow domain. Patients with autoimmune response against these conserved domains might form a clinical subset of patients positive for ACA.

Recently a novel nuclear protein mainly expressed in S and G2 phase cells was characterized using autoantibodies from a cancer patient (Exp. Cell Res., 212, 255-261, 1994). cDNA clones were isolated by immunoscreening, and sequence analysis revealed that the cDNA clones encoded a 713-amino acid residue polypeptide which is provisionally named SG2NA (S/G2 nuclear antigen). In the carboxyl terminal half of SG2NA, there are 6 copies of WD-40 motifs characteristic of a large family of proteins of which the prototype is beta-transducin/enhancer of split (TLE). In addition, there are nuclear localization sequence and phosphorylation sites related to the TLE proteins. Autoantibodies occurring spontaneously in certain cancer patients have been useful reagents for identifying cellular proteins and this is the first report of a novel WD-40 protein which is the target of an autoimmune response.

Mammalian centromere proteins (CENPs) can be divided into those that translocate from centromere to midzone in the progress of mitosis, and those that remain at the centromere throughout the cell cycle. The latter including CENP-A, CENP-B, and CENP-C is the candidate for DNA-binding protein. CENP-B has been shown previously to possess the specific DNA-binding activity to 17-base pair sequences dispersed on human centromeric alphoid repeats. In this study, we examined DNA-binding property of CENP-C that is localized to inner kinetochore plate of the metaphase chromosome. We independently isolated a full-length cDNA encoding human CENP-C and expressed it as the polypeptide tagged with histidine oligomer in Escherichia coli. After affinity purification with Ni(2+)-chelated resin, DNA-binding activity of the recombinant CENP-C renatured on the membrane was demonstrated by using human genomic DNA and an alphoid subfamily in South-Western-type blotting analysis. By constructing a series of truncated products, the DNA-binding domain was located at an internal 101-amino-acid stretch with no apparent homology to any other DNA-binding proteins. This may suggest that CENP-C is directly involved in formation of kinetochore chromatin fibers.

Proliferating cell nuclear antigen (PCNA), also known as auxiliary protein of DNA polymerase delta, is involved in DNA replication and repair. Certain patients with systemic lupus erythematosus (lupus) produce autoantibodies to PCNA and the autoantibody-defined epitopes of PCNA have been inferred to be conformation-dependent. Based on antigenic properties of continuous primary structure peptides, compound peptides composed of covalently linked discontinuous sequences were synthesized and used as immunogens in rabbits. One compound peptide induced an antibody response to a nuclear antigen which demonstrated a cell cycle-related pattern of nuclear immunofluorescence with maximum expression in the DNA synthesis phase of the cell cycle associated with other features which simulated the conformation-dependent properties of lupus defined auto-epitopes. Other rabbit antibodies raised against different compound peptides did not show such properties and recognized epitopes which were continuous primary sequence regions. These results show that from analysis of antigenic sites on a protein, it might be possible to construct synthetic compound peptides which can mimic conformation-dependent epitopes recognized by spontaneously occurring autoantibodies.

The alphoid DNA-CENP-B (centromere protein B) complex is the first sequence-specific DNA/protein complex detected in the centromeric region of human chromosomes. In the reaction, CENP-B recognizes a 17-bp sequence (CENP-B box) and assembles two alphoid DNA molecules into a complex, which is designated complex A (Muro, Y., H. Masumoto, K. Yoda, N. Nozaki, M. Ohashi, and T. Okazaki. 1992. J. Cell Biol. 116:585-596). Since CENP-B gene is conserved in mammalian species and CENP-B boxes are found also in mouse centromere satellite DNA (minor satellite), this sequence-specific DNA-protein interaction may be important for some kind of common centromere function. In this study we have characterized the structure of CENP-B and CENP-B-alphoid DNA complex. We have shown by chemical cross-linking that CENP-B formed a dimer, and have estimated by molecular weight determination the composition of complex A to be a CENP-B dimer and two molecules of alphoid DNA. The DNA binding domain has been delimited within the NH2-terminal 125-amino acid region containing four potential alpha-helices using truncated CENP-B made in Escherichia coli cells. We have shown that CENP-B had sites highly sensitive to proteases and that the DNA binding domain was separable from the dimerizing activity by the proteolytic cleavage at 20 kD from the COOH terminus of the molecule. Thus, CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the alphoid DNA repeat through both the DNA-protein and protein-protein interactions.

Centromere protein B (CENP-B), which is an alphoid DNA binding protein, is the target antigen in autoimmune disease patients (often with scleroderma). From our previous analysis of the reactivity of anticentromere sera, four independent epitopes were identified on recombinant CENP-B. The anticentromere sera displayed heterogeneity in their patterns of reactivity to the four epitopes. We have investigated to what extent this heterogeneity of the target autoepitope on CENP-B accounts for the clinical diversity of anticentromere antibody (ACA)-positive patients. A major autoepitope, epitope I, was recognized by all 40 ACA-positive sera; however, the other three epitopes were recognized differently from case to case. We could not find any significant correlation between the reactivity to CENP-B autoepitopes and the clinical presentation of ACA-positive patients. There was considerable clinical diversity, even among the nine patients showing specificity for the single major autoepitope. In conclusion, we found that, although ACA-positive patients were both clinically and immunologically heterogeneous, in most respects the clinical expression appeared to be independent of the reactivity to the CENP-B autoepitope, a finding which suggests that identification of the target epitope of CENP-B is unlikely to assist in the clinical classification of the disease in ACA-positive patients. The identification of multiple B cell epitopes on CENP-B is consistent with the concept that the self-antigen drives the antibody response. However, factors other than CENP-B autoepitope specificity must determine the clinical expression of ACA responses.

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromeric heterochromatin of human chromosomes. This protein was originally identified as the target antigen in autoimmune disease patients (often with scleroderma). In this study, we cloned a human CENP-B cDNA which was longer than the previously isolated one and expressed functional recombinant CENP-B in Escherichia coli. The DNA binding domain was finely located within the N-terminal 134-amino-acid residues covering a predicted helix-loop-helix (HLH) structure, by using a set of recombinant products with stepwise deletions from the C-terminus. From the analysis of their reactivity to anti-centromere sera from autoimmune disease patients, four epitopes were mapped on CENP-B antigen. In addition to two epitopes at the C-terminus, two were found on the HLH region at the N-terminus. In the analysis of the interaction between the antigen and autoantibodies, we found that the DNA binding activity of CENP-B was distorted by the attack of the anti-HLH domain antibodies in in vitro binding reactions. Our results suggest that the direct inhibition of the DNA binding activity by the autoantibodies might be involved in patients' autoimmune reactions in vivo.

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

Centromere protein B (CENP-B), which is an alphoid DNA binding protein, is the target antigen in autoimmune disease patients (often those with scleroderma). In this study, we analysed activities of anti-CENP-B-DNA complex in anticentromere antibody (ACA)-positive patients using DNA immunoprecipitation with purified CENP-B. The activities correlated with ACA titres and were closely associated with Raynaud's phenomenon. Patients with CREST symptoms (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) showed higher activities than those with no symptoms. Our results suggest that autoimmune responses to native CENP-B may have an important role in the pathogenesis of scleroderma.

Centromere protein-B (CENP-B) was purified from HeLa nuclear extract by using a combination of Q-sepharose ion change and oligonucleotide sepharose column chromatography. CENP-B was purified more than 10,000 times and was analyzed by immunoblotting and DNA immunoprecipitation. Purified CENP-B was used as an antigen to develop DNA immunoprecipitation for rapid and specific detection of anti-CENP-B antibody in human sera. In this analysis, none of the tested sera immunoprecipitated DNA alone. All of the 40 anticentromere antibody (ACA)-positive sera immunoprecipitated CENP-B-alphoid DNA fragment complex, whereas 10 healthy control sera and 10 other autoantibody positive sera did not. Five of the ACA-positive sera, which did not show reactivity of CENP-B in immunoblotting analysis, immunoprecipitated CENP-B-DNA complex. In some sera antigenicity to CENP-B may be weakened by denaturation.

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.