Altered O-Glycans in stimulated whole saliva from patients with primary Sjögren's syndrome and non-pSS sicca

Abstract

To investigate if salivary O-linked glycans are altered in primary Sjögren's syndrome (pSS), and thus contributing to explain symptoms of oral dryness, and an impaired oral mucosal barrier function leading to changes in microbial metabolism and colonization by both pathogenic and commensal microorganisms and increased prevalence of oral diseases. O-linked oligosaccharides from stimulated whole saliva (SWS) samples from 24 patients with pSS, 38 patients with non-pSS sicca, and 23 healthy controls were analyzed using liquid chromatography mass spectrometer (LC-MS). Non-fractionated reduced and alkylated saliva was dot-blotted to PVDF-membrane and O-linked oligosaccharides were released using reductive beta-elimination. The 50 most abundant glycans were identified and their intensity compared for each sample, reflecting the relative abundance of individual monosaccharide residues. Comparison of the compositions of O-glycans in SWS samples revealed higher relative levels of sialic acid (NeuAc) and lower levels of neutral amino-monosaccharides (HexNAc) in pSS and non-pSS sicca patients than in the healthy controls. MS² fragmentation analysis of salivary O-glycans suggests that altered sulfation, fucosylation, sialylation and distribution of core types may all contribute to the observed alteration, directly or indirectly. Additionally, the short disaccharide sialyl-Tn was most abundant in the saliva samples from patients with pSS. Our findings indicate that the salivary mucin-type O-glycan profile is altered in pSS, reflecting a dysfunction of the post-translational modification of salivary mucins leading to rheological changes of saliva, oral dryness symptoms, and impaired oral mucosal barrier function. The pathophysiological significance of the aberrant O-glycosylation needs further elucidation.

Keywords: Hyposalivation; O-glycans; Primary Sjögren’s syndrome; Sialyl-Tn.

Fig. 1

MSAC metrics of O-glycans in stimulated whole saliva samples by LC-MS. A, B: Summary of all analyzed samples (A), and with non-secretors (determined by MS) excluded (B). Two-way ANOVA with Tukey’s multiple comparisons correction. Mean with standard deviation is shown. **p < 0.01, *p < 0.05.C, D: Average glycan size (sum of MSAC metrics for the individual sample (C) and acidity across the three groups (D). Non-parametric Kruskal Wallis test with Dunn’s multiple comparisons test. Mean with standard deviation is shown.

Fig. 2

MSAC metric ratios between patients with pSS and non-pSS sicca and healthy controls. Non-parametric Kruskal Wallis test with Dunn’s multiple comparisons test. **** p < 0.0005; ***, p < 0.001; **p < 0.01; *p < 0.05. Mean with standard deviation is shown.

Fig. 3

Relative abundance of Sialyl Tn (NeuAcα2-6GalNAc) in saliva, identified among the 50 most abundant glycans, measured with LC-MS. Numbers in parenthesis indicate number of positive identifications in each group (i.e., 3 individuals of total 23 analyzed). Non-parametric Kruskal Wallis test with Dunn’s multiple comparisons test. **p < 0.01. Mean with standard deviation is shown.