Glycosylation of intestinal mucins in health and disease

Abstract

The highly glycosylated proteins found on mucosal surfaces, the mucins, are heterogeneous glycosylated molecules. The mucin oligosaccharides are mainly of the O-linked type, that is linked to the oxygen of serine or threonine, even though also N-linked oligosaccharides (linked to aspargine) may be found. The mucins glycosylation is believed to be important for the protection of mucosal surfaces, and perhaps to modify the physical properties of the mucus. The fundamental knowledge of how mucins are constructed should increase the understanding both of the physiological function and its macroscopic and biochemical properties. This knowledge should give new insights into mucosal related diseases, like chronic bronchitis and cystic fibrosis.In order to characterize intestinal mucins with respect to their glycosylation, oligosaccharides were released and studied mainly by mass spectrometry. A method was developed for further sub-fractionation of released oligosaccharides into neutral, sialylated and sulphated species, based upon their chemical properties. Gas chromatography-mass spectrometry (GC-MS) was adopted for the neutral and sialylated species, while tandem mass spectrometry was used for the sulphated species. Chemical derivatization preceding the mass spectrometric characterization increased the volatility for analysing oligosaccharides with GC-MS, and increased the ionization efficiency for analysing with tandem mass spectrometry. Proton-NMR was used to characterize individual oligosaccharides isolated by high performance-liquid chromatography (HPLC). Monosaccharide composition was analysed by HPLC or GC. Immunological as well as lectin assays were used for the characterization of both mucin oligosaccharides and mucin protein backbone. The expression of mucins and glycosyltransferases were detected using Northern blotting, and the latter was also identified by in vitro enzyme assays.The main impression from the presented data was the complexity of the intestinal mucin glycosylation. Comparison of pig and rat small intestinal mucin oligosaccharides illustrated a species dependent glycosylation. The most obvious difference was among the sialylated oligosaccharides, showing a sialylation pathway in the pig small intestine (sialylation of the 6-position of the polypeptide linked N-acetylgalactosamine), almost absent in the rat small intestine.When analysing the glycosylation of a single intestinal mucin (Muc2) from different rat strains and tissues (small and large intestine), a tissue and individual dependent glycosylation could be found. These differences were due to the presence or absence of blood group related oligosaccharide antigens (blood group A- and Sda/Cad-like antigens). The modification of oligosaccharides by sulphation were found to be similar both between species (rats and pigs) and tissues (small and large intestine).Investigation of how a parasitic infection (Nippostrongylus brasiliensis) influenced the oligosaccharide expression on the rat small intestinal Muc2 mucin showed that the glycosylation was sensitive to this infection, indicating a mechanism for the gastrointestinal protection from microbes. Using the fast GC-MS technique for easy detection and characterization of mucin oligosaccharides, the appearance and disappearance of the glycosylation alterations during different stages of the infection were followed. The results suggested that the glycosylation modifications were not co-regulated, reaching maximal expression on different stages of the infection.