Identification of Novel Autoantigen in the Synovial Fluid of Rheumatoid Arthritis Patients Using an Immunoproteomics Approach

Sagarika Biswas,Michael Oellerich,Saurabh Sharma,Abdul R Asif,Rajesh Malhotra,Muzna Zahur,Hasi R Das,Ashish Saroha,D S Bhakuni,Paul Proost

doi:10.1371/journal.pone.0056246

Abstract

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Highlights

During the last decade Rheumatoid arthritis (RA) has evolved rapidly, affecting about 0.5–1.0% of the general population
Diagnosis of RA was based on established criteria by American College of Rheumatology/European league (Table 1), that include a health assessment questionnaire (HAQ) scoring, general health (GH), tender-joint count (TJC), swollen-joint count (SJC), and disease activity score (DAS) calculated from online DAS28 tool based on 28 joint count
Antigens having molecular weight less than 75 kDa were designated as low molecular weight (LMW) and more than 75 kDa as high molecular weight (HMW) antigens

Summary

Introduction

During the last decade Rheumatoid arthritis (RA) has evolved rapidly, affecting about 0.5–1.0% of the general population. The early clinical presentation may not be specific since RA is initially indistinguishable from other forms of arthritis. The characteristic feature of this disorder is the presence of autoantibodies in the patient serum that distinguishes it from nonautoimmune joint pathogenesis like reactive arthritis or osteoarthritis (OA) [3]. Rheumatoid factor is the best-known autoantibody present, one third of RA patients have no rheumatoid factors. These antibodies are reported in other disorders and even in up to 15% of the healthy population [4]. The overall sensitivity of all these anti-citrullinated protein antibodies has very little additional diagnostic value over rheumatoid factor alone [4,5,6]

Methods

Results

Conclusion