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

• Published in: Glycobiology (Oxford) Vol. 19; no. 7; pp. 756 - 766
• Main Authors: Holmén Larsson, Jessica M, Karlsson, Hasse, Sjövall, Henrik, Hansson, Gunnar C
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.07.2009; Oxford Publishing Limited (England)
• Subjects: colon; glycan; oligosaccharide; mass spectrometry; large intestine
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwp048

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 (∼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 β-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 Galβ1-3 4-GlcNAcβ1-3(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.