Determination of muc5b sulfated glycans in Sjögren’s syndrome patients

LANDONI M, ; VAZQUEZ TJ,; CASTRO I,; GONZÁLEZ MJ,; COUTO AS

virtual

Congreso; Reunion Conjunta SAIB-SAMIGE; 2021

SAIB

Sjögren’s syndrome (SS) is a systemic chronic autoimmune disease affecting mainly the exocrine glands. Secretory activity of salivary and lacrimal glands is highly compromised leading to a high number of patients that suffers of mouth and eye dryness. Salivary hypofunction and xerostomia caused by Sjögren’s disease seriously affect the quality of life of SS patients. MUC5B is the predominant mucin in saliva and it is known to be highly O-glycosylated. The associated carbohydrates are heterogeneous and include neutral, sulfated and sialylated oligosaccharides. Sulfated and sialylated oligosaccharides add negative charges to mucins, thereby conferring the ability to retain high amounts of water and contributing thereby to generate the hydrophilic gel essential for lubrication of the oral epithelium. Mucin oligosaccharides sulfation may occur on Gal and/or GlcNAc. These reactions are catalyzed by Gal3-O-sulfotransferases (Gal3STs) and GlcNAc-6-sulfotransferases (GlcNAc6ST), respectively. Previous studies from Dr. MJ González laboratory showed a decrease in the sulfo-Lewisa (SO3-3Galβ1-3(Fuc1-4)GlcNAc-R) levels present in patients in contrast with controls thus resulting in a concomitant decrease of the number of sulfo-Lewisa -positive mucous acini. In labial salivary glands (LSG), this sulfated glycan structure is associated exclusively with MUC5B. However, the levels of mRNA and protein didn´t present significant differences. On the other hand, it has been detected a decrease in sulfotransferase activity that may provide an explanation for mucin hyposulfation observed in the LSGs from SS patients.In order to determine if the lower activity of sulfotranferases is reflected in the glycan structure of MUC5B in control and patients we started a characterization of the oligosaccharide moiety. MUC5B isolated from salivary were analyzed by SDS-PAGE and transfered to a PVDF membrane. The band corresponding to this high molecular weight protein was excised and subjected to different treatments to get some insight on the glycan structure. In-membrane reductive -elimination was performed in order to get the O-glycans structures. The oligosaccharide mixtures obtained were analysis by HPAEC-PAD revealing a different profile for the samples. The oligosaccharides were also analyzed by UV-MALDI-TOF mass spectrometry that allowed to identified the more abundant species. In both samples, structures of oligosaccharides with and without sulfate were determined, probably not in the same proportion.The in-membrane digestion of the MUC5B band with trypsin followed by HILIC enrichment of glycopeptides and further analysis by nHPLC-ESI-Orbitrap was performed. The analysis of the reporter ions corresponding to the sulfated glycopeptides showed the prevalence of sulfated structures in the control samples over the patients’ ones. Therefore, post-translational modifications of MUC5B, rather than changes in mucin levels seems to contribute significantly to xerostomia.