title: High-sensitivity O-glycomic analysis of mice deficient in core 2 {beta}1,6-N-acetylglucosaminyltransferases.
abstract: Core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1-3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500 Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids.
authors.affiliation: Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London, UK.
authors.forename: Mohd Nazri
authors.initials: MN
authors.lastname: Ismail
authors.forename: Erica L
authors.initials: EL
authors.lastname: Stone
authors.forename: Maria
authors.initials: M
authors.lastname: Panico
authors.forename: Seung Ho
authors.initials: SH
authors.lastname: Lee
authors.forename: Ying
authors.initials: Y
authors.lastname: Luu
authors.forename: Kevin
authors.initials: K
authors.lastname: Ramirez
authors.forename: Samuel B
authors.initials: SB
authors.lastname: Ho
authors.forename: Minoru
authors.initials: M
authors.lastname: Fukuda
authors.forename: Jamey D
authors.initials: JD
authors.lastname: Marth
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
mesh_terms: D000818:Animals; D003106:Colon / Q000378:metabolism; D054794:Glycomics; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics
chemical_list: D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase