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

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 microL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 microL 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.

SUBMITTER: Matsuoka K

PROVIDER: S-EPMC2917173 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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

Matsuoka Kazuhiro K Komori Hiroaki H Nose Masato M Endo Yaeta Y Sawasaki Tatsuya T

Journal of proteome research 20100801 8

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 lib ...[more]

PMID: 20575507

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Project description:BACKGROUND: Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production. RESULTS: The histidine tag-based self-cleavage method for purifying proteins produced by the wheat cell-free protein synthesis system showed high background, low recovery, and unexpected cleavage between the N-terminally fused sortase and target protein during the protein synthesis. Addition of calcium chelator BAPTA to the cell-free reaction inhibited the cleavage. In order to adapt the sortase-based purification method to the cell-free system, we next used biotin as the affinity tag. The biotinylated sortase self-cleavage purification (BISOP) method provided tag-free, highly purified proteins due to improved recovery of proteins from the resin. The N-terminal sequence analysis of the GFP produced by the BISOP method revealed that the cleavage indeed occurred at the right cleavage site. Using this method, we also successfully purified the E2 heterocomplex of USE2N and USE2v1. The c-terminal src kinase (CSK) obtained by the BISOP method showed high activity in phosphorylating the Src protein. Furthermore, we demonstrated that this method is suitable for automatically synthesizing and purifying proteins using robots. CONCLUSION: We demonstrated that the newly developed BISOP method is very useful for obtaining high quality, tag-free recombinant proteins, produced using the cell-free system, for biochemical and structural analyses.

Project description:Anti-endothelial-cell antibodies are associated with psychiatric manifestations in systemic lupus erythematosus (SLE). Our primary aim in this study was to seek and characterize molecules that behave as endothelial autoantigens in SLE patients with psychiatric manifestations. By screening a cDNA library from human umbilical artery endothelial cells with serum from an SLE patient with psychosis, we identified one positive strongly reactive clone encoding the C-terminal region (C-ter) of Nedd5, an intracytoplasmatic protein of the septin family. To evaluate anti-Nedd5 serum immunoreactivity, we analyzed by ELISA specific IgG responses in 17 patients with SLE and psychiatric manifestations (group A), 34 patients with SLE without psychiatric manifestations (group B), 20 patients with systemic sclerosis, 20 patients with infectious mononucleosis, and 35 healthy subjects. IgG specific to Nedd5 C-ter was present in 14 (27%) of the 51 SLE patients. The mean optical density value for IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients of group A than in those of group B, those with infectious mononucleosis, or healthy subjects (0.17 +/- 0.14 vs, respectively, 0.11 +/- 0.07, P = 0.04; 0.11 +/- 0.06, P = 0.034; and 0.09 +/- 0.045, P = 0.003, on Student's t-test). Moreover, IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients with systemic sclerosis than in patients of group B or healthy subjects (0.18 +/- 0.18 vs, respectively, 0.11 +/- 0.07, P = 0.046; and 0.09 +/- 0.045, P = 0.003). The percentage of patients with anti-Nedd5 C-ter serum IgG was higher in group A than in group B (8 (47%) of 17, vs 6 (17%) of 34, P = 0.045, on Fisher's exact test). In order to clarify a possible mechanism by which Nedd5 might be autoantigenic, we observed that Nedd5 relocated from cytoplasm to the plasma membrane of EAhy926 endothelial cells after apoptotic stimuli. In conclusion, Nedd5 is a novel autoantigen of potential clinical importance that could be successfully used for a more thorough investigation of the pathogenesis of psychiatric manifestations in SLE. Although anti-Nedd5 autoantibodies are not specific to SLE, they are significantly associated with neuropsychiatric SLE and may represent immunological markers of psychiatric manifestations in this pathology.

Dataset Information

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

Publications

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

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