Abstract
BackgroundAntibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). Anti-Sm autosera have been reported to specifically recognize Sm D1 and D3 with symmetric di-methylarginines (sDMA). We investigated if anti-Sm sera from local SLE patients can differentially recognize Sm proteins or any other proteins due to their methylation states.ResultsWe prepared HeLa cell proteins at normal or hypomethylation states (treated with an indirect methyltransferase inhibitor adenosine dialdehyde, AdOx). A few signals detected by the anti-Sm positive sera from typical SLE patients decreased consistently in the immunoblots of hypomethylated cell extracts. The differentially detected signals by one serum (Sm1) were pinpointed by two-dimensional electrophoresis and identified by mass spectrometry. Three identified proteins: splicing factor, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein D-like (hnRNP DL) and cellular nucleic acid binding protein (CNBP) are known to contain methylarginines in their glycine and arginine rich (GAR) sequences. We showed that recombinant hnRNP DL and CNBP expressed in Escherichia coli can be detected by all anti-Sm positive sera we tested. As CNBP appeared to be differentially detected by the SLE sera in the pilot study, differential recognition of arginine methylated CNBP protein by the anti-Sm positive sera were further examined. Hypomethylated FLAG-CNBP protein immunopurified from AdOx-treated HeLa cells was less recognized by Sm1 compared to the CNBP protein expressed in untreated cells. Two of 20 other anti-Sm positive sera specifically differentiated the FLAG-CNBP protein expressed in HeLa cells due to the methylation. We also observed deferential recognition of methylated recombinant CNBP proteins expressed from E. coli by some of the autosera.ConclusionOur study showed that hnRNP DL and CNBP are novel antigens for SLE patients and the recognition of CNBP might be differentiated dependent on the level of arginine methylation.
Highlights
Antibodies against spliceosome Sm proteins are specific to the autoimmune disease systemic lupus erythematosus (SLE)
We showed that recombinant heterogeneous nuclear ribonucleoprotein D-like and cellular nucleic acid binding protein (CNBP) expressed in Escherichia coli can be detected by anti-Sm positive sera
The results indicated that the CNBP protein is arginine methylated and the methylation level of the protein can be reduced by Adenosine dialdehyde (AdOx) treatment
Summary
Antibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). A common feature of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and mixed connective tissue disease (MCTD) is the breakdown of tolerance to self antigens that leads to the production of antibodies reactive with multiple self. The methylation is catalyzed by the type I protein arginine methyltransferase (PRMT) [5, 6] Another type II PRMT modifies other methyl-accepting proteins such as myelin basic protein [7], core small nuclear ribonucleoprotein (SnRNP) Sm B/B′, D1, D3 [8], and the Sm-like proteins LSm4 [9] to form MMA and symmetric NG, N′G– dimethylarginine (sDMA). Peptides with aDMA modification were identified as natural MHC class I ligands, indicating that specific cytotoxic T-cell response against cells presenting aDMA modified peptides can be elicited [13]
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