Selected Reaction Monitoring to Differentiate and Relatively Quantitate Isomers of Sulfated and Unsulfated Core 1 O-Glycans from Salivary MUC7 Protein in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis is a common and debilitating systemic inflammatory condition affecting up to 1% of the world's population. This study aimed to investigate the immunological significance of O-glycans in chronic arthritis at a local and systemic level. O-Glycans released from synovial glycoproteins during acute and chronic arthritic conditions were compared and immune-reactive glycans identified. The sulfated core 1 O-glycan (Gal␤1-3GalNAcol) was immune reactive, showing a different isomeric profile in the two conditions. From acute reactive arthritis, three isomers could be sequenced, but in patients with chronic rheumatoid arthritis, only a single 3-Gal sulfate-linked isomer could be identified. The systemic significance of this glycan epitope was investigated using the salivary mucin MUC7 in patients with rheumatoid arthritis and normal controls. To analyze this low abundance glycan, a selected reaction monitoring (SRM) method was developed to differentiate and relatively quantitate the core 1 O-glycan and the sulfated core 1 O-glycan Gal-and GalNAc-linked isomers. The acquisition of highly sensitive full scan linear ion trap MS/MS spectra in addition to quantitative SRM data allowed the 3-and 6-linked Gal isomers to be differentiated. The method was used to relatively quantitate the core 1 glycans from MUC7 to identify any systemic changes in this carbohydrate epitope. A statistically significant increase in sulfation was identified in salivary MUC7 from rheumatoid arthritis patients. This suggests a potential role for this epitope in chronic inflammation. This study was able to develop an SRM approach to specifically identify and relatively quantitate sulfated core 1 isomers and the unsulfated structure. The expansion of this method may afford an avenue for the high throughput investigation of O-glycans. Molecular & Cellular

Reduced Mucin-7 (Muc7) Sialylation and Altered Saliva Rheology in Sjögren's Syndrome Associated Oral Dryness

“…This same core 1 3-linked gal sulfation has been shown to be increased in another systemic inflammatory disease, rheumatoid arthritis (65). Although sulfate, like sialic acid, carries a negative charge, given the low abundance of the sulfated core 1 structure compared with the highly abundant sialic acid, it is unlikely that they play a major role in lubrication but, instead, may have immunological and/or binding functions.…”

Salivary Mucin 19 Glycoproteins

“…Moreover, chronic inflammatory conditions (i.e. ocular rosacea and rheumatoid arthritis) are associated with increased sulfation of salivary oligosaccharides (112,113), likely due to expression of sulfotransferases in response to systemic inflammatory mediators (114). Mucin sulfation has been shown to promote colonic barrier function (115), adhesion of leukocytes and Helicobacter pylori (116), and resistance to bacterial mucin-degrading enzymes (117).…”

Salivary mucin MUC7 oligosaccharides in patients with recurrent aphthous stomatitis

“…Differential changes in sulfation are possible as two different enzymes are responsible for sulfate attachment to these two residues [22]. The core 1-sulfated glycan has been shown to be upregulated on MUC7 in patients with the chronic systemic inflammatory disease rheumatoid arthritis, indicating that this glycan is reactive to the immune environment [12]. Here, the results show that patients with RAS also have changes in this epitope.…”

“…Further specific investigation of these important epitopes in a clinical setting will provide a greater insight into the pathogenesis of this disease and ultimately lead to the development of much needed preventatives. The recent improvements in sensitive quantitative large-scaletargeted O-glycomics make this type of large-scale study now a realistic possibility [12].…”

“…Changes in O-glycans have been observed in different disease states, particularly those involving an inflammatory response, where the changes in glycosylation can be observed in mucins also indirectly associated with the particular disease [10][11][12]. Given that mucins and their Oglycosylation affect a range of aspects of oral health and their changes in response to infection and inflammation, mucin glycosylation in RAS was investigated.…”