High-sensitivity O-glycomic analysis of mice deficient in core 2 {beta}1,6-N-acetylglucosaminyltransferases.
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.
Ismail
Mohd Nazri
MN
Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London, UK.
Stone
Erica L
EL
Panico
Maria
M
Lee
Seung Ho
SH
Luu
Ying
Y
Ramirez
Kevin
K
Ho
Samuel B
SB
Fukuda
Minoru
M
Marth
Jamey D
JD
Haslam
Stuart M
SM
Dell
Anne
A
D000818:Animals; D003106:Colon / Q000378:metabolism; D054794:Glycomics; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics
D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase