Antibodies recognising sulfated carbohydrates are prevalent in systemic sclerosis and associated with pulmonary vascular disease

Abstract

Background: Glycosylation represents an important modification that regulates biological processes in tissues relevant for disease pathogenesis in systemic sclerosis (SSc), including the endothelium and extracellular matrix. Whether patients with SSc develop antibodies to carbohydrates is not known.

Objectives: To determine the prevalence and clinical phenotype associated with serum IgG antibodies recognising distinct glycans in patients with SSc.

Methods: Pooled serum samples from patients with SSc and controls were screened for the presence of specific anticarbohydrate antibodies using a novel array containing over 300 glycans. Antibody titres to 4-sulfated N-acetyl-lactosamine (4S-LacNAc, (4OSO3)Galβ1-4GlcNAc) were determined in 181 individual serum samples from patients with SSc by ELISA and associated with disease phenotype.

Results: 4S-LacNAc was identified as a target in pooled SSc serum. Anti-4S-LAcNAc antibodies were detected in 27/181 patients with SSc (14.9%) compared with 1/40 healthy controls (2.5%). Sulfation at position C4 of galactose (4S-LacNAc) was found to be critical for immunogenicity. Anti-4S-LacNAc antibody-positive patients with SSc had a higher prevalence of pulmonary hypertension by echocardiography than anti-4S-LacNAc-negative patients (15/27 (55.7%) vs 49/154 (31.8%), p=0.02) with an OR of 2.6 (95% CI 1.1 to 6.3). Anti-4S-LacNAc-positive patients accounted for 23.4% of all patients with pulmonary hypertension.

Conclusion: Serum from patients with SSc contains IgG antibodies targeting distinct sulfated carbohydrates. The presence of anti-4S-LacNAc antibodies is associated with a high prevalence of pulmonary hypertension. These results suggest that specific post-translational carbohydrate modifications may act as important immunogens in SSc and may contribute to disease pathogenesis.

Figure 1. Recognition of distinct carbohydrates by serum antibodies in SSc

(A) Antibody binding ratio (ABR) of pooled SSc serum to 320 glycans determined by glycan array CFG 3.0 as outlined in Materials and Methods. (B) List of glycan structures that are highly recognized by SSc serum with an ABR > 2 SD above the mean (mean 1.57; SD 1.58). Dotted line represents cut-off for 2SD above mean. (C) Comparison of ABR between pooled serum from SSc and SLE. SSc serum recognizes a distinct set of glycans ( ABR > 2 SD from mean).

Figure 2. Antibodies to 4SLacNAc are frequent and of high titer in patients with SSc

(A) Antibodies to 4SLacNAc were determined by ELISA as outlined in Materials and Methods in individual sera from 181 patients with SSc, 40 disease patients (SLE) and 40 healthy controls. A.U. values are significantly higher in SSc patients compared to normal healthy controls (p=0.008). Dotted line represents cut-off for 2SD above mean (B) Correlation between fluorescence values obtained by glycan array and A.U. obtained by ELISA in 17 selected SSc patients, r²=0.89

Figure 3. Differential recognition of sulfated glycans by individual sera from SSc patients

Antibody binding to all sulfated glycans present in glycan array CFG version 3.1 from 17 preselected individual patient sera (9 anti-4S-LacNAc positive patients/8 antibody negative patients as determined by ELISA). ( ) low positive, ( ) medium positive, ( ) high positive (as defined in Materials and Methods). (*) denotes anti-4S-LacNAc positive patients as determined by ELISA.

Figure 4. Preferential recognition of 4S-LacNAc by individual sera from SSc patients

(A) ABR to sulfated LacNAc glycans present in glycan array CFG version 3.1 in 17 preselected sera from patients with SSc (9 anti-4S-LacNAc positive/8 antibody negative patients as determined by ELISA). (*) denotes anti-4S-LacNAc positive patients as determined by ELISA. (B) Confirmation of preferential recognition of 4S-LacNAc compared to 3S- or 6S-LacNAc by ELISA. Results from 26/181 previously identified anti-4S-LacNAc positive patients (figure 2A) are shown.(□) 4S-LacNAc, (■) 3S-LacNAc, ( ) 6S-LacNAc). 1 of the 27 anti-4S-LacNAc positive sera was not available for further testing.

Figure 5. Antibodies to 4S-LacNAc are not increased in patients with sicca or pulmonary hypertension independent from a diagnosis of systemic sclerosis

Antibodies to 4-sulfated LacNAc were determined by ELISA as outlined in Materials and Methods in individual sera from 181 patients with SSc, 40 patients with primary Sjogren’s, 16 patients with SLE and secondary Sjogren’s, 12 patients with RA and sicca and 25 patients with IPAH (* p=0.042).