A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

Abstract

High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (approximately 5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS-agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive beta-elimination and analyzed by nanoLC/mass spectrometry and MS(n). The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Gal beta 1-3/4-GlcNAc beta 1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were -Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.

Mucin-bacterial interactions in the human oral cavity and digestive tract

“…For example human colonic MUC2 contains more than 100 different O-linked glycans, which range in size from to 2 to 12 monosaccharides, most of which are based on the core 3 structure. 28 Strikingly, the spectrum of these O-glycans was very uniform among human individuals. 28 In the oral cavity, it was demonstrated that the glycosylation of the salivary mucins MUC5B and MUC7 is heterogeneous and can differ between individuals even with the same blood group.…”

“…28 Strikingly, the spectrum of these O-glycans was very uniform among human individuals. 28 In the oral cavity, it was demonstrated that the glycosylation of the salivary mucins MUC5B and MUC7 is heterogeneous and can differ between individuals even with the same blood group. These different mucin glycoforms are secreted in the oral cavity by physically separated salivary glands.…”

“…For this type of study, the proper procedure would be to isolate the genuine mucin(s) from the organ of interest. As indicated above, even pure mucins (e.g., human colonic MUC2 28 ) are very heterogeneous in nature due to their extensive and variable O-glycosylation, and the glycosylation varies from organ to organ even for one specific MUC-type mucin. In other words, only mucins from the organ of interest will do as appropriate targets for bacterial adhesion and/or degradation studies.…”

Glycosulfatase-Encoding Gene Cluster in Bifidobacterium breve UCC2003

“…However, the ability to utilize mucin, the glycoprotein component of the mucous layer that covers the epithelial cells of the gastrointestinal tract, is limited to members of the B. bifidum species (21,24). Approximately 60% of the predicted glycosyl hydrolases encoded by B. bifidum PRL2010 are predicted to be involved in mucin degradation, most of which are conserved exclusively within the B. bifidum species (21).Host-derived glycoproteins such as mucin and proteoglycans (e.g., chondroitin sulfate and heparan sulfate), which are found in the colonic mucosa and/or human milk, are often highly sulfated (25)(26)(27)(28)(29). Human colonic mucin is heavily sulfated, which is in contrast to mucin from the stomach or small intestine, the presumed purpose of which is to protect mucin against degradation by bacterial glycosidases (30)(31)(32).…”

Dynamic Interactions of a Conserved Enterotoxigenic Escherichia coli Adhesin with Intestinal Mucins Govern Epithelium Engagement and Toxin Delivery

“…Mucins in the intestine are O glycosylated by the addition of GalNAc to Ser and Thr moieties of the respective proteins, and further substitution of these GalNAc residues yields a diverse repertoire of glycan structures (29) that may be differentially glyco- sylated, dependent on their relative location within the intestine (30). Expression of blood group-related antigens that terminate in GalNAc are among glycans that exhibit varied expression in the gastrointestinal tract (31).…”