Complex asparagine (N)‐linked oligosaccharides appear late in phylogeny and are highly regulated in vertebrates. Variations in these structures are found on the majority of cell‐surface and secreted proteins. Complex N‐linked oligosaccharide biosynthesis is initiated in the Golgi apparatus by the action of Mgat‐1‐encoded UDP‐N‐acetylglucosamine:alpha‐3‐D‐ mannoside beta‐1,2‐N‐acetylglucosaminyltransferase I (GlcNAc‐TI). To determine if these structures govern ontogenic processes in mammals, mouse embryos were generated that lacked a functional Mgat‐1 gene. Inactivation of both Mgat‐1 alleles produced deficiencies in GlcNAc‐TI activity and complex N‐linked oligosaccharides. Embryonic lethality occurred by day 10.5, thus establishing that complex N‐linked oligosaccharides are required during post‐implantation development. Remarkably, embryonic development proceeded into day 9 with the differentiation of multiple cell types. Complex N‐linked oligosaccharides are important for morphogenic processes as neural tube formation, vascularization and the determination of left‐right body plan asymmetry were impaired in the absence of a functional Mgat‐1 gene.

N-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N-glycans and plays a critical role in mammalian development and possibly all metazoans. We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn 2+ at 1.5 and 1.8 A Ê resolution, respectively. The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining b-glycosidases. The structuring of a 13 residue loop, resulting from UDPGlcNAc/Mn 2+ binding, provides an explanation for the ordered sequential`Bi Bi' kinetics shown by GnT I. Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine b-1,4-galactosyltransferase 1 and Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase. The low sequence identity, conserved fold and related functional features shown by this domain de®ne a superfamily whose members probably share a common ancestor. Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.

SummaryCharacterization of a b1,2-xylosyltransferase from Arabidopsis thaliana (AtXylT) was carried out by expression in Sf9 insect cells using a baculovirus vector system. Serial deletions at both the N-and C-terminal ends proved that integrity of a large domain located between amino acid 31 and the C-terminal lumenal region is required for AtXylT activity expression. The influence of N-glycosylation on AtXylT activity has been evaluated using either tunicamycin or mutagenesis of potential N-glycosylation sites. AtXylT is glycosylated on two of its three potential N-glycosylation sites (Asn51, Asn301, Asn478) and the occupancy of at least one of these two sites (Asn51 and Asn301) is necessary for AtXylT stability and activity. Contribution of the N-terminal part of AtXylT in targeting and intracellular distribution of this protein was studied by expression of variably truncated, GFP-tagged AtXylT forms in tobacco cells using confocal and electron microscopy. These studies have shown that the transmembrane domain of AtXylT and its short flanking amino acid sequences are sufficient to specifically localize a reporter protein to the medial Golgi cisternae in tobacco cells. This study is the first detailed characterization of a plant glycosyltransferase at the molecular level.

UDP-GlcNAc:␣3-D-mannoside ␤1,2-N-acetylglucosaminyltransferase I (encoded by Mgat1) controls the synthesis of hybrid, complex, and paucimannose N-glycans. Mice make hybrid and complex N-glycans but little or no paucimannose N-glycans. In contrast, Drosophila melanogaster and Caenorhabditis elegans make paucimannose N-glycans but little or no hybrid or complex N-glycans. To determine the functional requirement for ␤1,2-N-acetylglucosaminyltransferase I in Drosophila, we generated null mutations by imprecise excision of a nearby transposable element. Extracts from Mgat1 1 /Mgat11 null mutants showed no ␤1,2-N-acetylglucosaminyltransferase I enzyme activity. Moreover, mass spectrometric analysis of these extracts showed dramatic changes in N-glycans compatible with lack of ␤1,2-N-acetylglucosaminyltransferase I activity. Interestingly, Mgat1 1 / Mgat11 null mutants are viable but exhibit pronounced defects in adult locomotory activity when compared with Mgat1 1 /CyO-GFP heterozygotes or wild type flies. In addition, in null mutants males are sterile and have a severely reduced mean and maximum life span. Microscopic examination of mutant adult fly brains showed the presence of fused ␤ lobes. The removal of both maternal and zygotic Mgat1 also gave rise to embryos that no longer express the horseradish peroxidase antigen within the central nervous system. Taken together, the data indicate that ␤1,2-N-acetylglucosaminyltransferase I-dependent N-glycans are required for locomotory activity, life span, and brain development in Drosophila.

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:beta-D-mannoside beta 1-4 N-acetylglucosaminyltransferase III (GlcNAc-TIII, EC2.4.1.144) catalyzes the addition of GlcNAc to the mannose that is itself beta 1-4 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 alpha-mannosidase-II, GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and core alpha 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-TIII and the bisecting GlcNAc in N-glycans appear dispensable for normal development, homeostasis and reproduction in the mouse.