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The bisecting N-acetylglucosamine of N-glycans appear dispensable for development and reproduction

University of British Columbia

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:β-D-mannoside β-4 N-acetylglucosaminyltransferase HI (GlcNAc-TIII, E.C. 2.4.1.144) catalyzes the addition of GlcNAc to the mannose that is itself β-4 linked to underlying Nacetylglucosamine. GlcNAc-TIII thereby produces what is known as a "bisecting" GlcNAc linkage which can be 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 a-mannosidase-II, GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and core αl-6-fucosyltransferase to act. 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 brain and kidney tissue. 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, Mgaf3-deficient mice were produced that lacked GlcNAc-TIII activity and E₄-PHA visualized GlcNAc-bisected N-linked oligosaccharides. Such GlcNAc-TIII null 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, red cells or in serum metabolite levels that reflect kidney function. Although we find that GlcNAc-TIII and the bisecting GlcNAc of N-glycans appear dispensable for normal development and reproduction in the mouse, it may have a role in physiologic responses to pathogenic and environmental stimuli. Moreover, it may be important for some function still to be determined.

Thesis/Dissertation

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2009-04-02

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http://hdl.handle.net/2429/6744

Genetics

University of British Columbia

UBCV

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