Simple screening method for autoantigen proteins using the n-terminal biotinylated protein library produced by wheat cell-free synthesis

Kazuhiro Matsuoka, Hiroaki Komori, Masato Nose, Yaeta Endo, Tatsuya Sawasaki

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)

Abstract

Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 μL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10000 in 25 μL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with identification of autoantigen proteins reacting with antibodies that recognize folded proteins, rather than denatured or unfolded forms.

Original languageEnglish
Pages (from-to)4264-4273
Number of pages10
JournalJournal of Proteome Research
Volume9
Issue number8
DOIs
Publication statusPublished - 2010 Aug 6

Keywords

  • Gene Ontology
  • MRL/lpr mouse
  • autoantigen
  • autoimmunity
  • biomarker
  • cellfree protein production
  • high-throughput screening
  • proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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