IgG N- Glycosylation from Patients with Pemphigus Treated with Rituximab

Abstract

Pemphigus is a life-threatening auto-immune blistering disease of the skin and mucous membrane that is caused by the production of auto-antibodies (auto-Abs) directed against adhesion proteins: desmoglein 1 and 3. We demonstrated in the "Ritux3" trial, the high efficacy of rituximab, an anti-CD20 recombinant monoclonal antibody, as the first-line treatment for pemphigus. However, 25% of patients relapsed during the six-month period after rituximab treatment. These early relapses were associated with a lower decrease in anti-desmoglein auto-Abs after the initial cycle of rituximab. The N-glycosylation of immunoglobulin-G (IgG) can affect their affinity for Fc receptors and their serum half-life. We hypothesized that the extended half-life of Abs could be related to modifications of IgG N-glycans. The IgG N-glycome from pemphigus patients and its evolution under rituximab treatment were analyzed. Pemphigus patients presented a different IgG N-glycome than healthy donors, with less galactosylated, sialylated N-glycans, as well as a lower level of N-glycans bearing an additional N-acetylglucosamine. IgG N-glycome from patients who achieved clinical remission was not different to the one observed at baseline. Moreover, our study did not identify the N-glycans profile as discriminating between relapsing and non-relapsing patients. We report that pemphigus patients present a specific IgG N-glycome. The changes observed in these patients could be a biomarker of autoimmunity susceptibility rather than a sign of inflammation.

Keywords: IgG; N-glycans; N-glycome; glycosylation; pemphigus; rituximab; sialic acid.

Figure 1

MALDI-TOF mass spectrum of total IgG N-glycans from healthy donors (A) with the proportion for each N-glycans subtype in the healthy donor population (n = 13) (B). Each N-glycan identified has been drawn according to the international nomenclature [25]. N-glycans were cleaved off from IgG using peptide-N-glycosidase F, then purified and permethylated before MALDI-TOF mass spectrometry. Mass spectra and data were obtained from FlexControl 3.3 and FlexAnalysis 3.3 software. The relationship between the corresponding ion and the N-glycan structure was confirmed based on MS-MS (Figure S1). The x-axis represents the mass-to-charge (m/z) ratio. The y-axis represents the relative percentage of the detected N-glycans.

Figure 2

The N-glycan profile of pemphigus patients before rituximab treatment (at baseline) compared to healthy donors. MALDI-TOF mass spectra of total IgG N-glycans from pemphigus patients (A). The N-glycan structures were drawn according to the international nomenclature [25]. Blue square: N-acetylglucosamine; green circle: mannose; yellow circle: galactose; purple diamond: N-acetylneuraminic acid; red triangle: fucose. The percentage based on the relative quantification of galactosylated (B), mono-galactosylated (C), bi-galactosylated (D), sialylated (E), mono-sialylated (F), bi-sialylated (G), N-acetylglucosamine (GlcNAc) (H), fucosylated (I) N-glycans from IgG. Differences were considered significant when p cor. < 0.006 with Bonferroni adjustment.

Figure 3

N-glycome evolution of pemphigus patients over time and comparison between before (baseline) and 6 months (M6) and 12 months (M12) after rituximab treatment. Comparison of percentages based on relative quantification of galactosylated (A), mono-galactosylated (B), bi-galactosylated (C), sialylated (D), mono-sialylated (E), bi-sialylated (F), N-acetylglucosamine (GlcNAc) (G), fucosylated (H) subgroups of N-glycans from IgG. Analyses were performed at baseline and M6 for all patients (n = 16) and at M12 only for non-relapsing patients (n = 8). Means ± standard deviations were compared using the Wilcoxon paired test. Differences were considered significant when p cor. < 0.006 with Bonferroni adjustment.

Figure 4

The N-glycome of relapsing patients during relapse occurrence compared to the N-glycome of non-relapsing patients at 6 months (M6). The percentage based on the relative quantification of galactosylated (A), mono-galactosylated (B), bi-galactosylated (C), sialylated (D), mono-sialylated (E), bi-sialylated (F), N-acetylglucosamine (GlcNAc) (G), fucosylated (H) subgroup of N-glycans from IgG of non-relapsing and relapsing patients for M6 and relapse occurrence, respectively. Means ± standard deviations were compared using a Mann–Whitney test. Differences were considered significant when p cor. < 0.006 with Bonferroni adjustment.

Figure 5

Pathogenicity evaluation of purified IgG from relapsing and non-relapsing patients at baseline and relapse time or M6, respectively. Keratinocyte dissociation assay was used to assess the pathogenicity of purified IgG from healthy donors (negative control) (n = 6), relapsing patients (at baseline and relapse time) (n = 3) and non-relapsing patients (at baseline and Month 6) (n = 3). In brief, HaCaT cells pre-incubated for 24 h with purified IgG were dissociated from the plate with dispase, and the monolayers were mechanically disrupted. The number of cell fragments was proportional to the IgG pathogenicity. The p-value was calculated in comparison to the healthy donor. A representative image of three independent experiments performed with serum from three patients is shown.