O-glycosylation of intestinal and respiratory mucins in health and disease

Abstract

Mucins are large glycoproteins with a diverse O-glycosylation constituting up to 80% of the total mucin mass. The mucus layer covering the epithelial cells of the gastrointestinal and respiratory tract is largely made up of gel-forming mucins. MUC2 is the main gel-forming mucin in the intestinal tract and its O-glycosylation has been studied in health and disease in this thesis. Glycosylation alterations in relation to infection/inflammation in diseases affecting the mucosa as Cystic Fibrosis and Ulcerative colitis, have been identified. The glycosylation of mouse small intestinal mucins was studied during an infection cycle induced by the parasite Nippostrongylus brasiliensis. The O-linked oligosaccharides were released from the guanidinium chloride insoluble mucins and structurally characterized by gas chromatography/mass spectrometry. Two oligosaccharides containing blood group H-type epitopes (Fuc Ñ1-2Gal-) were transiently expressed with a peak at day 6. Northern blot analysis on total RNA showed a transient expression at day 4-6 of the Fut2 gene encoding the Fuc Ñ1-2 fucosyltransferase synthesizing the H-epitope. Additional oligosaccharides with the common structure HexNAc-Gal-3GalNAcol were transiently expressed with a peak at day 10. Secretor-negative women have an increased risk for recurrent vaginitis caused by C. albicans. A model of this disease is the Fut2-LacZ-null mice lacking the Fut2 enzyme. The aim of the study was to determine if the lack of Fut2 affected the glycosylation of mucins in the gastrointestinal tract. Mass spectrometry showed a complete loss of terminal fucosylation on the O-linked oligosaccharides of the colonic insoluble mucins in the mutant mice. Inoculation by gastric lavage with C. albicans showed no differences in colonization between mouse genotypes. The results suggest that the increased risk of recurrent vaginitis in secretor negative women, is not due to less intestinal colonization.There has been a long time controversy in Cystic Fibrosis (CF) research regarding the observed changes in mucin glycosylation. Are they due to an absent CFTR channel or secondary effects due to infection/ inflammation? We addressed this question by studying mucins secreted by non-infected second passage primary human bronchial epithelial (HBE) cell cultures. The O-linked oligosaccharides, released from purified non-CF and CF mucins, showed large inividual variations, but no significant differences between the two groups. To conclude, no differences in the mucin O-glycan repertoire was found, suggesting that observed CF glycosylation alterations are due to infection/inflammation. Novel proteomic and glycoproteomic methods were used to study sigmoid colon biopsies from active and inactive ulcerative colitis patients and compared to controls. In a total of 50 patients, the monomeric form of MUC2 was semiquantified and 5-10-fold individual differences in MUC2 amounts were observed. The O-glycosylation of colonic MUC2 was studied with a high sensitivity nanoLC/MS setup, developed in-house. More than 50 O-linked oligosaccharides were identified and quantified. Some of the glycan structures have not been characterized previously. A subpopulation of patients with ulcerative colitis showed an accumulation of some precursor glycans and a decrease of complex glycans. This glycan pattern was especially frequent among the active ulcerative colitis patients.