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.

Figure 1

Sensitivity and specificity for detection of biotinylated p53 protein−antibody complex. (A) Biotinylated p53 (lane 1) and dihydrofolate reductase (DHFR) (lane 2) was detected by immunoblotting analysis using Alexa488-STA. M indicates protein molecular weight marker. (B) Schematic diagram of detection of biotinylated protein−antibody interaction by luminescence analysis. When a biotinylated protein and antibody interact (upper panel), Protein A-conjugated acceptor beads bound to antibody and streptavidin (STA)-coated donor beads bound to biotinylated protein are in close proximity. Upon excitation at 680 nm, a singlet oxygen is generated by the donor beads, transferred to the acceptor beads within 200 nm, and the resultant reaction emits light at 520−620 nm. This emission is measured using an EnVision. (C) Detection sensitivity of the antibody concentration measured by luminescence analysis. Translation mixture (1 μL) expressing biotinylated or nonbiotinylated p53 protein and biotinylated DHFR were incubated with various concentration of monoclonal antibody from 5 × 10⁻³ to 5 × 10² pg/μL. (D) Biotinylated protein−antibody complex by interaction between biotinylated p53 protein and the monoclonal antibody in the presence of mouse serum was detected by luminescence analysis. (E) Minimum IgG amount in the presence of mouse serum to detect biotinylated p53 proteins. The relative luminescence signals between the specific luminescence and background signals indicated in the y-axis.

Figure 2

Detection of autoantibodies against Hars and LmnB2 proteins. (A) Biotinylated histidyl-tRNA synthetase (Hars) (lane 1), and lamin B2 (LmnB2) (lane 2) and DHFR (lane 3) proteins were detected by immunoblotting analysis using Alexa488-STA. M indicates protein molecular weight marker. (B) Various volumes (0.003 to 4 μL: representing 2.5−3280 nM Hars, 1.8−2400 nM LmnB2 and 6.4−8520 nM DHFR) of translation mixture expressing biotinylated Hars, LmnB2 or DHFR proteins were incubated with serum of MRL/lpr mouse sera (final 1:1000 dilution) in 25 μL of reaction volume. (C) Serum dilution between 1:100 and 1:10 000 was incubated with 1 μL of the translation mixtures in 25 μL of reaction volume.

Figure 3

BPL-based screening of autoantigen proteins using the MRL/lpr mouse sera. (A) Schematic of the BPL-based screening method. (B) Thirty-two randomly selected biotinylated proteins of the BPL were detected by immunoblotting analysis using Alexa488-STA. (C) Scatter plot showing the luminescent signals in each well of two independent screening data sets using MRL/lpr mouse sera. The x-axis indicates luminescence signals in MRL/lpr plate 1 whereas the y-axis represents those in MRL/lpr plate 2. (D) Each data point represents luminescence signals using MRL/lpr mouse sera or normal mouse sera (NMS). The x-axis indicates luminescence signals in NMS whereas the y-axis represents those in MRL/lpr mice. (E, F) Ninety-six proteins identified as autoantigen proteins were grouped by protein localization in cells (E) Membrane (GO:0016020), Nucleus (GO:0005634), Cytoplasm (GO:0005737), Extracellular region (GO:0005576) and Mitochondrion (GO:0005739) and biological function/process (F) Kinase activity (GO:0016301), Peptidase activity (GO:0008233), Ubiquitin (GO:0005551), Translation (GO:0006412) and Transcription (GO:0006350) according to Gene Ontology Database. Minor groups less than 3 proteins were belonged to “Other” group. More detailed information on individual proteins was indicated in the Table 1.

Figure 4

Detection of identified autoantigen proteins by immunoblotting and immunoprecipitation. (A) Immunoblotting analysis by using recombinant proteins. Purified recombinant proteins were separated by SDS-PAGE and stained with CBB (Upper). Purified recombinant proteins were reacted with serum from MRL/lpr mouse (Lower). (B) Immunoprecipitation analysis using recombinant proteins. Translation mixtures expressing biotinylated proteins were incubated with 1 μL of undiluted serum overnight at 4 °C. Immobilized Protein A sepharose was added to each sample, and incubated for 60 min at 4 °C. After washing, proteins were separated by SDS-PAGE, followed by immunoblotting with Alexa488-STA. M indicates a 45 kDa protein molecular weight marker. (C) Whole data of randomly selected proteins. IB, Immunoblotting; IP, Immunoprecipitation. Relative luminescence signal, 10² ≤ ++; 5 ≤ + < 10²; − < 5. (A, B, C) Lane 1, Hhex; Lane 2, Tdg; Lane 3, Sgta; Lane 4, Slc1a6; Lane 5, Lmnb2; Lane 6, Top2a; Lane 7, Cs; Lane 8, Car4. Lane 7 and 8 were negative controls. Detailed information on individual proteins was indicated in Supplementary Table 1 (Supporting Information).