Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

<div><p>Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (<em>p</em><0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.</p> </div

Prediction of O-glycosylation sites using Net-O-Glyc3.1 program.

<p>Prediction of O-glycosylation sites using Net-O-Glyc3.1 program.</p

Glycosylation studies in A2HSG by lectin blotting.

<p>The binding of sialic acid specific lectin (MAA, <i>Maackia amurensis</i>) to A2HSG in (A) untreated (B) PNGase F treated plasma. Same pooled samples were used for lectin blot (C) and Western blot using anti-A2HSG antibody (D) to locate the protein before and after enzyme treatment. C1, C2 are pool 1 and 2 of control group 1 respectively while C3 represents pool 1 of control group 2. R1, R2 are pool 1 and 2 of patient group 1 respectively while R3 represents pool 1 of patient group 2.</p

List of differentially expressed proteins, as identified by MALDI-TOF MS, in plasma of RA patients with respect to normal control.

*<p>M.W and p<i>I</i> were estimated on the basis of the sequence submitted in swiss-prot database.</p><p>↑ indicates up regulation and ↓ indicates down regulation of proteins in RA with respect to control.</p

Two dimensional gel electrophoresis (2-DE) of jacalin bound plasma.

<p>A representative silver stained 2-DE gel image of jacalin agarose bound plasma proteins from four sets of pooled samples from controls and RA patients. The samples were pooled as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046374#s4" target="_blank">materials and methods</a> section. The gels were analyzed for differential expression of proteins using PD-Quest software and the differentially expressed proteins were labelled by arrows. The lower panel showed zoomed image and 3D view of A2HSG (spot no 9) and ITIH4 (spot no 15).</p

Glycosylation studies in A2HSG.

<p>2-DE Western blot of A2HSG in untreated plasma showed the higher p<i>I</i> of A2HSG in RA patients compared to control. The treatment with sialidase shifted the p<i>I</i> of control towards higher p<i>I</i> in comparison to that of RA, while the shift in p<i>I</i> was found to be equal in both the samples after PNGase F treatment. The experiment was repeated thrice with different pooled samples.</p

Western blot analysis of A2HSG and ITIH4.

<p>(A) In RA (n = 35), pool 1 and pool 2 of group 1 consisted of 7 and 8 samples respectively while each pool of group 2 contained 10 samples. Similarly, in control (n = 30) each pool of group 1 and group 2 consisted of 10 and 5 samples respectively.(B) Mean density of each protein band in the Western blot followed by statistical analysis using One way ANOVA (<i>p</i><0.05) showed the change in the expression of protein. (C) Representative Western blot image of A2HSG and ITIH4 from individual Control and RA samples. (D) Mean percentage relative density of each protein band in control and RA were plotted on Y-axis. Statistical analysis was done using Mann-Whitney U test (<i>p</i><0.05).</p

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

<div><p>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.</p> </div

Clinical and demographic characteristics of the study subjects.

<p>M-male, F-female, SD-standard deviation, CRP-C-reactive protein, mg-milligram, RF-rheumatoid factor, IU-international units, ml-milliliter, anti CCP- Anti-cyclic citrullinated peptide, EU- ELISA units, ESR-erythrocyte sedimentation rate, mm-millimeter, hr-hour, DAS28- disease activity score (28 joints maximum).</p

Identified high and low molecular weight RA antigens.

<p>Identification of high molecular weight (HMW) and low molecular weight (LMW) antigens using Q-TOF mass spectrometric (MS) analysis followed by online MASCOT search against the SwissProt and NCBInr protein databases (kDa-kilo Dalton, MW-molecular weight, Obs-observed, Thr- theoretical, pI- isoelectric point, MOWSE- Molecular Weight Search).</p