Screening And Identification Of Molecular Markers In Serum Circulating Immune Complexes In Patients With Rheumatoid Arthritis

Objective: To screen the new autoantigen spectrum from serum circulating immune complexes of patients with early rheumatoid arthritis(RA)and analyze its functions and signal pathways.To further explore the clinical significance of antibody to peptidoglycan recognition protein(Anti-PGLYRP)-2 in RA and analyze the possible B cell linear antigen epitopes in order to provide a new treatment basis.Methods: 10 serum samples of early RA and 10 serum samples of healthy controls were fully mixed in equal proportion respectively,and Protein G beads was used to precipitate the circulating immune complex in the mixed serum.After the samples were boiled,the proteins with different molecular weight were separated by SDS-PAGE electrophoresis,the 25 KD immunoglobulin light chain and 75 KD immunoglobulin heavy chain in the colloidal particles were removed,and the proteins in the remaining colloidal particles were hydrolyzed by trypsin.The antigenic substance expression profile of circulating immune complex in patients with RA was identified by Orbitrap electric field orbital well cyclotron resonance mass spectrometer and its functions and pathways were analyzed by biological information methods.Furthermore,indirect enzyme-linked immunosorbent assay(ELISA)was used to detect Anti-PGLYRP-2 levels in RA sera and control groups(rheumatic immune diseases and healthy controls).The Anti-PGLYRP-2 expression levels in serum of RA patients and other control groups were analyzed and compared,meantime its diagnostic value for RA was judged.Further data analysis was conducted to clarify its relationship with clinical manifestations and activity indicators of RA.The PGLYRP-2 B cell linear epitopes were comprehensively predicted by the biological information analysis.Result:(1)By using immunoprecipitation and Orbitrap high resolution mass spectrometry,it was found that there were 33 antigenic substances in circulating immune complexes of RA,which were involved in the pathophysiological processes such as immune response,coagulation cascade,oxidative stress and cell differentiation.(2)Further verification by ELISA showed that the Anti-PGLYRP-2 levels in RA patients were significantly higher than that in disease and healthy controls(P < 0.05).(3)The positive rate of Anti-PGLYRP-2 in RA patients was 42.70%,which was significantly higher than that in osteoarthritis(18.64%),systemic lupus erythematosus(16.95%),ankylosing spondylitis(14.55%),primary Sjogren’s syndrome(17.89%),and healthy controls(2%)(P < 0.05).(4)The sensitivity of AntiPGLYRP-2 in RA was 42.70%,and the specificity was 85.22%.The positive rate of AntiPGLYRP-2 was also higher in RA subgroups,with 34.72% in early RA group,35.29% in rheumatoid factor negative group,and 42.86% in anti-cyclic citrullinated peptide negative group.Anti-PGLYRP-2 combined with rheumatoid factor and anti-cyclic citrullinated peptide antibody could improve the diagnostic value.(5)Further analysis showed that there was a positive correlation between Anti-PGLYRP-2 and rheumatoid factor,hidden rheumatoid factor-immunoglobulin G,immunoglobulin A,immunoglobulin M and immunoglobulin G(P < 0.05).(6)In addition,higher levels of rheumatoid factor,immunoglobulin G,immunoglobulin M and anti-neutrophil cytoplasmic autoantibodyproteinase 3 were associated with Anti-PGLYRP-2 titer(P < 0.05).(7)The possible B cell epitopes in PGLYRP-2 were(IGANTPDATKGCPD)at position 185-198,(FLRGSQTQSHPDLGTEGCWD)at position 231-250 and(TVKPRPARSVSKRSRREPPPRTLPATD)at position 548-574,respectively.In addition,there was 80% identity between PGLYRP-2563-567 and Epstein-Barr virus nuclear antigen(EBNA)1396-400.Conclusion:(1)33 autoantigens in serum circulating immune complexes of RA patients were screened by Orbitrap mass spectrometry,and their functions were related to immune response,coagulation cascade,oxidative stress and so on.(2)Anti-PGLYRP-2 was a promising biomarker of RA,especially in early and serologically negative patients.PGLYRP-2548-574 may be the best epitope to induce antibody production,and there was a possible cross-reaction with EBNA1.