Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-D-glucosamine:α-3-D-mannoside β1,2-N-acetylglucosaminyltransferase 1, α3,6-mannosidase II and a specific membrane-bound β-N-acetylglucosaminidase

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 1,2-N-acetylglucosaminyltrans-ferase I (GnT 1), 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 Mana1,6(Manalpha1 1,3)Manbeta1-O-R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn2+ 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 1, alpha3,6-mannosidase 11 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 GlcNAc 1, 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)Manalpha1,6](GIcNAcbeta1,2Manalpha1,3)Man 1,4GlcNAcbeta1,4GlcNAc-R nor from the GlcNAc 1,2Manalpha1,6Manbeta- 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.