Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis

Abstract

Objective: To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA).

Methods: Analysis of N-glycan in serum samples from multiple cohorts was performed. The IgG N-glycan content and the timing of N-glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti-cyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N-glycan content was also compared between epitope affinity-purified autoantibodies and the remaining IgG repertoire in RA patients.

Results: Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean +/- SD 1.36 +/- 0.43 versus 1.01 +/- 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's rho = 0.37, P < 0.0001). This correlation was higher in women (Spearman's rho = 0.60, P < 0.0001) than in men (Spearman's rho = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction.

Conclusion: Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in autoantibodies.

Figure 1. Oligosaccharide structures attached to Asp-297 of IgG

Shown are fully galactosylated (G2, FA2BG2), monogalactosylated (G1, FA2[6]BG1) and agalactosylated (G0, FA2B) form of the biantennary oligosaccharide attached to the C_H2 Fc region of IgG (67). In this nomenclature, since all N-glycans have two core GlcNAc residues, a F at the start of the abbreviation indicates a core fucose in α1-6 linkage to the inner GlcNAc. A2 denotes a biantennary structure with both GlcNAcs β1-2 linked. B symbolizes a bisecting GlcNAc linked β1-4 to β1-3 mannose. Gx communicates the number of β1-4 linked galactose residues on each antenna. Here, [6]G1 indicates that the galactose is on the antenna of the α1-3 or α1-6 mannose.

Figure 2. IgG galactosylation (sG0/G1) ratios are aberrant in RA and this aberrancy precedes disease onset

A, sG0/G1 ratios for RA subjects (N = 232) and gender-matched controls (N = 232) were determined using the in-gel digest assay on whole serum (44). Individual values as well as averages for RA subjects and healthy donors are shown. (P < 0.0001). B, sG0/G1 ratios were determined in a longitudinal series of healthy and pre- and post-disease onset serum samples in the DoDSR cohort. Shown are mean and SEM values for subjects with RA (red and black) and age- and gender-matched healthy controls (blue and green). Values are reported longitudinally relative to onset of disease in the RA cohort. Note the sG0/G1 ratios between healthy and disease subjects diverge significantly ~3.5 years prior to disease onset.

Figure 3. sG0/G1 ratios in anti-citrulline autoantibodies vs. repertoire IgG

Shown are sG0/G1 ratios of IgG autoantobodies reactive to the citrullinated JED autoantigen and residual repertoire IgG after epitope affinity removal of anti-JED autoantibodies. Symbols portray individual subject values. Horizontal lines denote average values. (P=0.006).