title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients.
abstract: BACKGROUND
  Antibodies 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Our 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.
authors: Institute of Oral Biology, Chung Shan Medical University, Taichung, Taiwan, Republic of China.
authors: Hong-How
authors: HH
authors: Chang
authors: Huan-Hsuan
authors: HH
authors: Hu
authors: Yu-Jen
authors: YJ
authors: Lee
authors: Hung-Ming
authors: HM
authors: Wei
authors: Ming-Chun
authors: MC
authors: Fan-June
authors: Tsai-Ching
authors: TC
authors: Hsu
authors: Gregory J
authors: GJ
authors: Tsay
authors: Chuan
authors: C
authors: Li
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001323:Autoantibodies / Q000276:immunology*; D006367:HeLa Cells; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D008745:Methylation; D000072670:PTB-Associated Splicing Factor; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
chemical_list: D001323:Autoantibodies; C433227:CNBP protein, human; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D000072670:PTB-Associated Splicing Factor; D016601:RNA-Binding Proteins; D001120:Arginine
references: Mol Cell Proteomics. 2012 Nov;11(11):1489-99
references: 22865923
references: Arthritis Rheum. 2008 Feb;58(2):511-20
references: 18240226
references: Mol Cell Biol. 1995 May;15(5):2800-8
references: 7739561
references: Autoimmun Rev. 2007 Jan;6(3):176-82
references: 17289554
references: Autoimmunity. 2005 Feb;38(1):25-32
references: 15804702
references: Curr Opin Immunol. 2012 Feb;24(1):112-8
references: 22209691
references: J Biochem. 2004 Sep;136(3):371-6
references: 15598895
references: Protein Sci. 2000 Nov;9(11):2210-7
references: 11152131
references: Autoimmunity. 2008 Mar;41(2):123-32
references: 18324481
references: Science. 2001 Aug 3;293(5531):864-7
references: 11486088
references: Arthritis Rheum. 1982 Nov;25(11):1271-7
references: 7138600
references: Ann N Y Acad Sci. 2005 Jun;1050:1-9
references: 16014515
references: Curr Opin Immunol. 2004 Dec;16(6):753-8
references: 15511669
references: Autoimmunity. 2005 Feb;38(1):47-54
references: 15804705
references: Protein J. 2009 May;28(3-4):139-47
references: 19365714
references: Nat Methods. 2004 Nov;1(2):119-26
references: 15782174
references: J Biol Chem. 1994 Jan 14;269(2):1075-82
references: 8288564
references: Science. 1971 Feb 12;171(3971):579-81
references: 4924231
references: J Biol Chem. 2003 Sep 12;278(37):35145-51
references: 12840030
references: Mol Cell Proteomics. 2003 Dec;2(12):1319-30
references: 14534352
references: J Cell Biochem. 2012 Aug;113(8):2721-8
references: 22442049
references: Arthritis Rheum. 2000 Aug;43(8):1768-78
references: 10943867
references: J Biol Chem. 1985 Nov 15;260(26):14304-10
references: 2414294
references: RNA. 2001 Nov;7(11):1531-42
references: 11720283
references: Mol Cell. 2009 Jan 16;33(1):1-13
references: 19150423
references: Autoimmun Rev. 2003 Mar;2(2):57-62
references: 12848959
references: J Biol Chem. 2000 Jun 2;275(22):17122-9
references: 10747894
references: FEBS J. 2012 Mar;279(6):932-45
references: 22251447
references: J Biol Chem. 2002 Jan 25;277(4):2732-9
references: 11705999