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OP0036 Molecular cloning and characterisation of autoantigen recognised by anti-wa antibodies in patients with systemic sclerosis
  1. Y Shirai1,
  2. T Nojima1,
  3. M Hirakata1,
  4. H Yamagata2,
  5. K Miyachi3,
  6. T Mimori4
  1. 1Internal Medicine, Keio University
  2. 2Internal Medicine, Natl. Murayama. Hospital, Tokyo
  3. 3Internal Medicine, Keigu Clinic, Yokohama
  4. 4Clinical Immunology, Kyoto University, Kyoto, Japan

Abstract

Background Anti-Wa antibodies were first reported in patients with systemic sclerosis (SSc) as autoantibodies reactive with a 48kDa transfer RNA-associated protein.1 However, the nature of the Wa antigen has not been clarified so far.

Objectives To identify and characterise the Wa antigen by molecular cloning using anti-Wa serum as a probe.

Methods Molecular cloning of the target Wa antigen was performed using lambda gt11 cDNA library constructed from human liver mRNA and anti-Wa protoype serum as a probe. Bound antibodies were eluted from the fusion proteins of the isolated clones expressed in E.coli and were used for RNA-immunoprecipitation assay. The reactivity of fusion proteins prepared from lysogenic E.coli Y1089 carrying the recombinant phages were examined using five sera that were proved to have anti-Wa antibodies in double immunodiffusion assay. The cDNA inserts of the isolated clones were sequenced, and the detailed homologies of nucleotide and amino acid sequences were analysed through the BLAST server at NCBI DNA databank. Anti-Wa antibodies in sera from patients with various rheumatic diseases were detected by immunoblot analysis using the fusion protein from the positive clone.

Results 10 positive clones were recognised by the probe serum. In RNA-immunoprecipitation, the affinity-purified antibodies from only one clone (termed Wa-1) immunoprecipitated transfer RNA from HeLa cell extracts. In immunoblot study, the Wa-1 fusion protein was recognised by all five sera that were proved to have anti-Wa antibodies. These data confirmed that the Wa-1 clone encoded for the Wa antigen. The nucleotide sequence and deduced amino acid sequence of the 0.7-kb insert cDNA of Wa-1 were completely identical with the C-terminal part of NEFA (named after DNA-binding domain, two EF-hands, Acidic amino acid rich region)/nucleobindin-2.2 In immunoblots using the Wa-1 fusion protein, autoantibodies to Wa (NEFA/nucleobindin-2) were detected in 3 of 21 SSc (21%), one of 10 RA (10%) and one of 16 SLE sera (6%). However, neither RA nor SLE sera immunoprecipitated transfer RNA from HeLa cells, while all 3 SSc sera immunopricipitated transfer RNA, suggesting the diversity of autoepitopes among patient sera.

Conclusion These results demonstrated that the autoantigen recognised by anti-Wa antibodies were proved to be identical with NEFA/nucleobindin-2.

References

  1. Miyachi K, et al. J Rheumatol. 1991;18:373–8

  2. Barnikol-Watanabe S, et al. Biol Chem Hoppe Seyler 1994;375:497–512

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