Isolation and Comparison of Natural and Recombinant Human CENP-A Autoantigen

Martı́nez, Antı́gona; Sun, Dongxu; Billings, Peter B.; Swiderek, Kristine M.; Sullivan, Kevin F.; Hoch, Sallie O.

doi:10.1006/jaut.1998.0249

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…These facts reasonably suggest that centromere/kinetochore and telomere proteins are present only in small amounts compared with the overall chromosomal proteins. In fact, the quantitative purification of centromere protein A and telomeric repeat-binding factor-1 from human cells revealed markedly small molar amounts of ϳ1:70,000 and 1:100,000 histone H4, respectively (61,62).…”

Section: Discussionmentioning

confidence: 99%

Proteome Analysis of Human Metaphase Chromosomes

Uchiyama¹,

Kobayashi²,

Takata³

et al. 2005

Journal of Biological Chemistry

117

142

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DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.

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Section: Discussionmentioning

confidence: 99%

Proteome Analysis of Human Metaphase Chromosomes

Uchiyama¹,

Kobayashi²,

Takata³

et al. 2005

Journal of Biological Chemistry

117

142

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“…The ELISA system developed by Hoch's group was very sensitive for ACA [23]. It might be due to the usage of the baculovirus system, which can induce modifications in the protein [23,29]. In the N-terminal part of CENP-A, four copies of the peptide Ser/Thr-Pro, which are good candidates for phosphorylation, are present [16].…”

Section: Discussionmentioning

confidence: 99%

Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3

Muro

Azuma²,

Onouchi³

et al. 2000

Clinical and Experimental Immunology

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SUMMARYAnti-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 1 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.

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“…This protein, cloned in 1987 by Earnshaw et al, was eventually expressed as a eukaryotic recombinant protein and then adapted in an ELISA for autoantibody (aab) detection [18-22]. Similarly, the CENP-A protein was also cloned and a recombinant protein used for the detection of ACA by ELISA [23,24]. Despite these advances, only a few commercial diagnostic kits used the recombinant CENP-A protein because it has been assumed that CENP-B was the major autoantigen reactive with SSc sera [18].…”

Section: Introductionmentioning

confidence: 99%

Clinical and serological evaluation of a novel CENP-A peptide based ELISA

Mähler

Maes²,

Blockmans³

et al. 2010

Arthritis Res Ther

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IntroductionAnti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc). ACA are found in 20 to 40% of SSc patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. The objective of this study was to evaluate a novel CENP-A peptide ELISA.MethodsSera collected from SSc patients (n = 334) and various other diseases (n = 619) and from healthy controls (n = 175) were tested for anti-CENP-A antibodies by the novel CENP-A enzyme linked immunosorbent assay (ELISA). Furthermore, ACA were determined in the disease cohorts by IIF (ImmunoConcepts, Sacramento, CA, USA), CENP-B ELISA (Dr. Fooke), EliA® CENP (Phadia, Freiburg, Germany) and line-immunoassay (LIA, Mikrogen, Neuried, Germany). Serological and clinical associations of anti-CENP-A with other autoantibodies were conducted in one participating centre. Inhibition experiments with either the CENP-A peptide or recombinant CENP-B were carried out to analyse the specificity of anti-CENP-A and -B antibodies.ResultsThe CENP-A ELISA results were in good agreement with other ACA detection methods. According to the kappa method, the qualitative agreements were: 0.73 (vs. IIF), 0.81 (vs. LIA), 0.86 (vs. CENP-B ELISA) and 0.97 (vs. EliA® CENP). The quantitative comparison between CENP-A and CENP-B ELISA using 265 samples revealed a correlation value of rho = 0.5 (by Spearman equation). The receiver operating characteristic analysis indicated that the discrimination between SSc patients (n = 131) and various controls (n = 134) was significantly better using the CENP-A as compared to CENP-B ELISA (P < 0.0001). Modified Rodnan skin score was significantly lower in the CENP-A negative group compared to the positive patients (P = 0.013). Inhibition experiments revealed no significant cross reactivity of anti-CENP-A and anti-CENP-B antibodies. Statistically relevant differences for gender ratio (P = 0.0103), specific joint involvement (Jaccoud) (P = 0.0006) and anti-phospholipid syndrome (P = 0.0157) between ACA positive SLE patients and the entire SLE cohort were observed.ConclusionsAnti-CENP-A antibodies as determined by peptide ELISA represent a sensitive, specific and independent marker for the detection of ACA and are useful biomarkers for the diagnosis of SSc. Our data suggest that anti-CENP-A antibodies are a more specific biomarker for SSc than antibodies to CENP-B. Furthers studies are required to verify these findings.

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Isolation and Comparison of Natural and Recombinant Human CENP-A Autoantigen

Cited by 6 publications

References 35 publications

Proteome Analysis of Human Metaphase Chromosomes

Proteome Analysis of Human Metaphase Chromosomes

Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3

Clinical and serological evaluation of a novel CENP-A peptide based ELISA

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