We have previously reported, from the nematode worm Caenor-habditis elegans, three genes (gly-12, gly-13 and gly-14) encoding enzymically active UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid, paucimannose and complex N-glycan synthesis. We now describe a worm with null mutations in all three GnT I genes, gly-14 (III);gly-12 gly-13 (X) (III and X refer to the chromosome number). The triple-knock-out (TKO) worms have a normal phenotype, although they do not express GnT I activity and do not synthesize 31 paucimannose, complex and fucosylated oligomannose N-glycans present in the wild-type worm. The TKO worm has increased amounts of non-fucosylated oligomannose N-glycan structures, a finding consistent with the site of GnT I action. Five fucosylated oligomannose N-glycan structures were observed in TKO, but not wild-type, worms, indicating the presence of unusual GnT I-independent fucosyltransferases. It is concluded that wild-type C. elegans makes a large number of GnT I-dependent N-glycans that are not essential for normal worm development under laboratory conditions. The TKO worm may be more susceptible to mutations in other genes, thereby providing an approach for the identification of genes that interact with GnT I.

We have previously reported three Caenorhabditis elegans genes ( gly-12, gly-13 and gly-14 ) encoding UDP- N -acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2- N -acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manalpha1,6(Manalpha1,3)Manalpha1,6](Manalpha1,3)Manbeta1, 4GlcNAcbeta1,4GlcNAc-R, but not on Manalpha1,6(Manalpha1,3)Manbeta1- O -R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn(2+) concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man(6-9)GlcNAc(2)) and 'paucimannose' Man(3-5)GlcNAc(2)(+/-Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man(3)GlcNAc(2) requires the prior actions of GnT I, alpha3,6-mannosidase II and a membrane-bound beta- N -acetylglucosaminidase similar to an enzyme previously reported in insects. The beta- N -acetylglucosaminidase removes terminal N -acetyl-D-glucosamine from the GlcNAcbeta1, 2Manalpha1,3Manbeta- arm of Manalpha1,6(GlcNAcbeta1,2Manalpha1,3) Manbeta1,4GlcNAcbeta1,4GlcNAc-R to produce paucimannose Man(3)GlcNAc(2) N-glycan. N -acetyl-D-glucosamine removal was inhibited by two N -acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manalpha1,6(Manalpha1,3)Manalpha 1,6] (GlcNAcbeta1,2Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-R nor from the GlcNAcbeta1,2Manalpha1,6Manbeta- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development.