Abberant Immunoglobulin G Glycosylation in Rheumatoid Arthritis by LTQ-ESI-MS

Abstract

Aberrant glycosylation has been observed in many autoimmune diseases. For example, aberrant glycosylation of immunoglobulin G (IgG) has been implicated in rheumatoid arthritis (RA) pathogenesis. The aim of this study is to investigate IgG glycosylation and whether there is an association with rheumatoid factor levels in the serum of RA patients. We detected permethylated N-glycans of the IgG obtained in serum from 44 RA patients and 30 healthy controls using linear ion-trap electrospray ionization mass spectrometry (LTQ-ESI-MS), a highly sensitive and efficient approach in the detection and identification of N-glycans profiles. IgG N-glycosylation and rheumatoid factor levels were compared in healthy controls and RA patients. Our results suggested that total IgG purified from serum of RA patients shows significantly lower galactosylation (p = 0.0012), lower sialylation (p < 0.0001) and higher fucosylation (p = 0.0063) levels compared with healthy controls. We observed a positive correlation between aberrant N-glycosylation and rheumatoid factor level in the RA patients. In conclusion, we identified aberrant glycosylation of IgG in the serum of RA patients and its association with elevated levels of rheumatoid factor.

Keywords: IgG; glycosylation; mass spectrometry; rheumatoid arthritis.

Figure 1

Purity assessment of immunoglobulin G (IgG) from human serum. IgG was purified by protein G affinity chromatography in the serum of rheumatoid arthritis patients (A) and healthy controls (B), and the heavy chain (black dot) and light chain (arrow) of IgG were separated. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis was performed to determine the purity.

Figure 2

Representative MS¹ spectrums of N-glycans from healthy control Immunoglobulin G (IgG) (A) and rheumatoid arthritis patient IgG (B). The representative structure of IgG Fc N-glycan is shown. Based on the core structure (GlcNAc2-Man3-GlcNAc2), galactoses (H, lighter circle), Mannose (H, solid circle), bisecting GlcNAc (N, diamond), sialic acids (S, rhombus), or fucose (F, triangle) can be attached.

Figure 3

Multistage MS (MSⁿ) pathway of the doubly changed ion at m/z 937 in serum from a rheumatoid arthritis patient. The pathway consists of MS² m/z 937²⁺ (A), MS³ m/z 808²⁺ (B), MS⁴ m/z 678²⁺ (C) and MS⁵ m/z 567²⁺ (D), along with their respective chemical structures.

Figure 4

Aberrance of immunoglobulin G (IgG) glycosylation in serum of rheumatoid arthritis (RA) patients compared with healthy controls. IgG glycosylation in serum of patients with RA is different compared with healthy controls. Changes were also observed between patients with three various rheumatoid factor (RF) levels and healthy controls for total galactosylation (A), sialylation (B), core fucosylation (C), and bisecting GlcNAc (D). A two-tailed Student’s t-test comparison was performed between the healthy controls and patients, and patients with three RF levels. Error bar indicates standard deviation (ns, no significance; p < 0.05 indicated statistical significance).

Figure 5

Association between aberrant immunoglobulin G (IgG) glycosylation and rheumatoid factor (RF) titer in rheumatoid arthritis. The IgG galactosylation (A), sialylation (B), core fucosylation (C), bisection (D), G1F/G0F (E), G2F/G0F (F), G1F0/G0F0 (G), and G2F0/G0F0 (H) for all patients, as well as patients with different RF levels, are aberrant compared with healthy controls. Statistical significance was evaluated using two-tailed Student’s t-test. Error bar indicates standard deviation (ns, no significance; p < 0.05 indicated statistical significance).