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

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

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

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