Simple Screening Method for Autoantigen Proteins Using the N-Terminal Biotinylated Protein Library Produced by Wheat Cell-Free Synthesis

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,*,†,|,⊥,# and Tatsuya Sawasaki*,†,|,⊥,# Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan, Department of Pathogenomics, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan, Proteo-Medicine Research Center, Ehime University, Toon, Ehime 791-0295, Japan, The Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan, and RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan Received November 18, 2009 Abstract: Autoimmune diseases are a heterogeneous with sera antibodies. Cross-referencing with the Gene group of diseases characterized by immune reactions Ontology Database, 26 and 38 of autoantigen proteins against either a major or a limited number of the bodies were predicted to have nuclear localization and identified own autoantigens, causing inflammation and damage to as membrane and/or extracellular proteins. The immune tissues and organs. Thus, identification of autoantigens reaction of six randomly selected proteins was confirmed is an important first step to understanding autoimmune by immunoprecipitation and/or immunoblot analyses. diseases. Here we demonstrate a simple screening method Interestingly, three autoantigen proteins were recognized for identification of autoantigens reacting with patient by immunoprecipitation but not by immunoblot analysis. serum antibodies by combination of an N-terminal bioti- These results suggest that the BPL-based method could nylated protein library (BPL), produced using a wheat cell- provide a simple system for screening of autoantigen free protein production system, and a commercially proteins and would help with identification of autoantigen proteins reacting with antibodies that recognize available luminescence system. Optimization studies us- folded proteins, rather than denatured or unfolded forms. ing well-characterized autoantigens showed specific in- teractions between N-terminal biotinylated proteins and Keywords: autoantigen • autoimmunity • biomarker • cell- antibody that were sensitively detected under homoge- free protein production • Gene Ontology • high-throughput neous reaction conditions. In this optimized assay, 1 µL screening • MRL/lpr mouse • proteomics of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by Introduction addition of diluted serum between 1:500 and 1:10 000 in Autoimmune diseases are generally characterized by the 25 µL of reaction volume. For the BPL construction, 214 body’s immune responses being directed against its own mouse genes, consisting of 103 well-known autoantigens tissues, causing prolonged inflammation and subsequent tissue 1 and 111 genes in the mouse autoimmune susceptibility destruction. A hallmark of autoimmune diseases is the production of autoantibodies such as antinuclear, anti-Sm and loci, and the sera of MRL/lpr mouse were used as an anti-dsDNA in systemic lupus erythematosus (SLE),2 and the autoimmune model. By this screening method, 25 well- presence of RF, hnRNP A2 and calpastatin in rheumatoid known autoantigens and 71 proteins in the loci were arthritis (RA).3 However, there are still a lot of autoimmune identified as autoantigen proteins specifically reacting diseases for which antibodies have not been identified.2 To understand the molecular mechanisms in autoimmune dis- * To whom correspondence should be addressed. Yaeta Endo, Cell-Free eases, it is important to identify the relevant autoantigens, and Science and Technology Research Center, Ehime University, Bunkyo-cho, 3-ban, Matsuyama 790-8577, Japan. Tel. +81-89-927-9936. Fax +81-89-927- moreover, they could be pathogenic in these diseases. It is 9941. E-mail [email protected]. Tatsuya Sawasaki, Cell-Free Science widely hypothesized that proteins are the major antigenic and Technology Research Center, Ehime University, Bunkyo-cho, 3-ban, targets associated with autoimmune diseases.2 Therefore, + + Matsuyama 790-8577, Japan. Tel. 81-89-927-8530. Fax 81-89-927-9941. development of methods that allow large-scale screening of E-mail [email protected]. † Cell-Free Science and Technology Research Center, Ehime University. autoantigen proteins is indispensable for elucidation and ‡ Ehime University Graduate School of Medicine. diagnosis of the autoimmune diseases. § Deceased March 7, 2009. To date, autoantigen proteins have been detected as anti- | Proteo-Medicine Research Center, Ehime University. ⊥ The Venture Business Laboratory, Ehime University. genic molecules that are recognized by humoral antibodies, # RIKEN Systems and Structural Biology Center. including those in serum.2 The large-scale screening of au- 4264 Journal of Proteome Research 2010, 9, 4264–4273 10.1021/pr9010553 2010 American Chemical Society Published on Web 06/24/2010 Autoantigen Screening Method Using Biotinylated Protein Library technical notes toantigen proteins reacting with patient serum antibodies has or by autoradiography. The wheat germ extract was purchased been carried out by mainly three technologies: serological from Cell-Free Science Co. (Yokohama, Japan). Anti-p53 mono- proteome analysis (SERPA), serological expression cloning clonal antibody (D01) was purchased from Santa Cruz Bio- (SEREX) and protein microarray.4 The utility of SERPA and technology (Santa Cruz, CA). Mouse serum for mouse immu- SEREX for this screening is limited because particular cells and noglobulin in Figure 1 was purchased from Calbiochem tissues are generally used as antigen resources in these systems (Darmstadt, Germany). Other reagents used in this study were and they are dependent on artificial membranes for immuno- described previously.10,15 blotting which do not maintain native protein structure.5 Serum Samples. MRL/lpr mice were originally purchased Recent advances in protein microarray technology have allowed from the Jackson Laboratory (Bar Harbor, ME). All of the mice large-scale screening of autoantigens reacting with the sera of used in this paper were maintained in clean rooms at the - patients suffering from autoimmune disorders and cancer.5 7 Animal Research Institute, School of Medicine, Ehime Univer- However, protein microarray is not yet a commonly used sity. Sera of female mice were collected from 15 mice and 8 biochemical tool for screening. One issue with protein mi- pooled and stocked in -20 °C until use. All experiments were croarrays is that purified recombinant proteins are required, done according to the Guidelines for the Care and Use of which demonstrate batch-to-batch variation and limited stabil- Laboratory Animals at Ehime University. 5 ity and shelf life. Additionally, it is difficult to maintain the DNA Template Construction for the BPL. Functional an- functional form of a protein after their immobilization on a notation of mouse (FANTOM) as a mouse full-length cDNA microplate. Many proteins needed to be appropriately oriented resource is purchased from a company (Danaform, Tokyo, 9 for proper functioning. In fact, a number of spotted autoan- Japan). The DNA templates for transcription were constructed tigens were not always detectable with planar arrays, presum- by “split-primer” PCR technique described previous reports.10,17 ably due to loss of three-dimensional structures, steric inter- The first PCR was amplified with 10 nM of each of the following 6 ference or electrostatic repulsion. primers: a gene specific primer, 5′- CCACCCACCACCACCAAT- In this work, we developed a novel autoantigen protein Gnnnnnnnnnnnnnnnnnnn (n denotes the coding region of the screening method that overcame the following issues high- target gene), and AODA2303 (5′-GTCAGACCCCGTAGAAAAGA) lighted above: (1) utilization of a high-throughput and genome- or AODS (5′-TTTCTACGGGGTCTGACGCT). The second PCR wide protein expression system, (2) specific protein labeling products for protein synthesis were constructed with 100 nM for assay using unpurified protein samples and (3) high- SPu 5′-GCGTAGCATTTAGGTGACACT, 1 nM deSP6E02bls-S1 throughput detection system of properly folded antigen. To- (5′-GGTGACACTATAGAACTCACCTATCTCTCTACACAAAACA- ward the first, we recently developed an automated protein TTTCCCTACATACAACTTTCAACTTCCTATTATGGGCCTGAAC- production robot utilizing a high-throughput wheat embryo GACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAACTCCA- derived cell-free protein production system.10,11 The combina- CCCACCACCACCAATG) and 100 nM AODA2303 or AODS. By tion of an automatic cell-free protein production system and this “split-primer” PCR, the bls was fused onto the N-terminals the full-length cDNA allowed for facile construction of a robust of all the genes for protein biotinylation.13 protein library.12 To enhance the utility of the library, per the second issue above, specific labeling of each protein is required Construction of the BPL by the Cell-Free Protein Synthesis for efficient detection. We selected biotin as the labeling System. Cell-free construction of the BPL is based on the compound because it is readily available and demonstrates previously described bilayer diffusion system in which 1 µL (50 high specificity for streptavidin binding. The biotinylated ng) crude cell-free expressed BirA was added to the translation protein library (BPL) was constructed using target proteins

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