Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly

K. Miyachi, Y. Hirano, T. Horigome, T. Mimori, H. Miyakawa, Y. Onozuka, M. Shibata, Michito Hirakata, A. Suwa, H. Hosaka, S. Matsushima, T. Komatsu, H. Matsushima, R. W. Hankins, M. J. Fritzler

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

We have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [35S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

Original languageEnglish
Pages (from-to)568-573
Number of pages6
JournalClinical and Experimental Immunology
Volume136
Issue number3
DOIs
Publication statusPublished - 2004 Jun

Fingerprint

Biliary Liver Cirrhosis
Nuclear Envelope
Autoantibodies
Anti-Idiotypic Antibodies
Serum
Immunodiffusion
Precipitins
Antibodies
Autoantigens
Fluorescence Microscopy
Immunoprecipitation
Recombinant Proteins
Confocal Microscopy
Methionine
Autoimmune Diseases
CDC48 protein
In Vitro Techniques
Proteins
Complementary DNA
Liver

Keywords

  • Autoantibody
  • Conformational epitope
  • Nuclear envelope assembly
  • p95c p97/VCP
  • Primary biliary cirrhosis

ASJC Scopus subject areas

  • Immunology

Cite this

Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly. / Miyachi, K.; Hirano, Y.; Horigome, T.; Mimori, T.; Miyakawa, H.; Onozuka, Y.; Shibata, M.; Hirakata, Michito; Suwa, A.; Hosaka, H.; Matsushima, S.; Komatsu, T.; Matsushima, H.; Hankins, R. W.; Fritzler, M. J.

In: Clinical and Experimental Immunology, Vol. 136, No. 3, 06.2004, p. 568-573.

Research output: Contribution to journalArticle

Miyachi, K, Hirano, Y, Horigome, T, Mimori, T, Miyakawa, H, Onozuka, Y, Shibata, M, Hirakata, M, Suwa, A, Hosaka, H, Matsushima, S, Komatsu, T, Matsushima, H, Hankins, RW & Fritzler, MJ 2004, 'Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly', Clinical and Experimental Immunology, vol. 136, no. 3, pp. 568-573. https://doi.org/10.1111/j.1365-2249.2004.02456.x
Miyachi, K. ; Hirano, Y. ; Horigome, T. ; Mimori, T. ; Miyakawa, H. ; Onozuka, Y. ; Shibata, M. ; Hirakata, Michito ; Suwa, A. ; Hosaka, H. ; Matsushima, S. ; Komatsu, T. ; Matsushima, H. ; Hankins, R. W. ; Fritzler, M. J. / Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly. In: Clinical and Experimental Immunology. 2004 ; Vol. 136, No. 3. pp. 568-573.
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AU - Miyachi, K.

AU - Hirano, Y.

AU - Horigome, T.

AU - Mimori, T.

AU - Miyakawa, H.

AU - Onozuka, Y.

AU - Shibata, M.

AU - Hirakata, Michito

AU - Suwa, A.

AU - Hosaka, H.

AU - Matsushima, S.

AU - Komatsu, T.

AU - Matsushima, H.

AU - Hankins, R. W.

AU - Fritzler, M. J.

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AB - We have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [35S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

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