Glycobiology vol 7 no 1 pp. 45-56, 1997
© 1997
research-article |
Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction
Howard Hughes Medical Institute, Department of Medicine, and the Division of Cellular and Molecular Medicine 9500 Gilman Drive 0625, CMM-W 321-B University of California at San Diego, La Jolla, CA 92093, USA
1Department of Biochemistry, The Hospital for Sick Children and the University of Toronto 555 University Avenue, Toronto, Ontario, MSG 1X8, Canada
2To whom correspondence should be addressed: Howard Hughes Medical Institute, 9500 Gilman Drive 0625, University of California at San Diego, La Jolla, CA 92093, USA
Received on April 22, 1996; revised on July 23, 1996; accepted on July 23, 1996
The biosynthesis of complex asparagine (N)-linked oligosaccharides in vertebrates proceeds with the linkage of N-acetylglucosamine (GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:ß-D-mannoside ß1–4 N-acetylglucosaminyltransferase III (GlcNAc-TIII, EC2.4.1.144) catalyzes the addition of GlcNAc to the mannose that is itself ß1–4 linked to underlying N-acetylglucosamine. GlcNAc-TIII thereby produces what is known as a ‘bisecting’ GlcNAc linkage which is found on various hybrid and complex N-glycans. GlcNAc-TIII can also play a regulatory role in N-glycan biosynthesis as addition of the bisecting GlcNAc eliminates the potential for 
-mannosidase-II, GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and core 1–6-fucosyltransferase to act subsequently. To investigate the physiologic relevance of GlcNAc-TIII function and bisected N-glycans, the mouse gene encoding GlcNAc-TIII (Mgat3) was cloned, characterized, and inactivated using Cre/loxP site-directed recombination. The Mgat3 gene is highly conserved in comparison to the rat and human homologs and is normally expressed at high levels in mammalian brain and kidney tissues. Using fluorescence in situ hybridization (FISH), the Mgat3 gene was regionally mapped to chromosome 15E11, near the Scn8a sodium channel gene at 15F1. Following homologous recombination in embryonic stem cells and Cre mediated gene deletion, Mgat3-deficient mice were produced that lacked GlcNAc-TIII activity and were deficient in E4-PHA visualized GlcNAc-bisected N-linked oligosaccharides. Nevertheless, GlcNAc-TIII deficient mice were found to be viable and reproduced normally. Moreover, such mice exhibited normal cellularity and morphology among organs including brain and kidney. No alterations were apparent in circulating leukocytes, erythrocytes or in serum metabolite levels that reflect kidney function. We thus find that GlcNAc-TII and the bisecting GlcNAc in N-glycans appear dispensable for normal development, homeostasis and reproduction in the mouse.
GlcNAc brain kidney biosynthesis
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