We have previously reported three Caenorhabditis elegans genes (gly-12, gly-13 and gly-14) encoding UDP-N-acetyl-d-glucosamine:?-3-d-mannoside ?1,2-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 [Man?1,6(Man?1,3)Man?1,6](Man?1,3)Man?1, 4GlcNAc?1,4GlcNAc-R, but not on Man?1,6(Man?1,3)Man?1-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 (Man6�9GlcNAc2) and �paucimannose� Man3�5GlcNAc2(�Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man3GlcNAc2 requires the prior actions of GnT I, ?3,6-mannosidase II and a membrane-bound ?-N-acetylglucosaminidase similar to an enzyme previously reported in insects. The ?-N-acetylglucosaminidase removes terminal N-acetyl-d-glucosamine from the GlcNAc?1, 2Man?1,3Man?- arm of Man?1,6(GlcNAc?1,2Man?1,3) Man?1,4GlcNAc?1,4GlcNAc-R to produce paucimannose Man3GlcNAc2 N-glycan. N-acetyl-d-glucosamine removal was inhibited by two N-acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Man?1,6(Man?1,3)Man?1,6] (GlcNAc?1,2Man?1,3)Man?1,4GlcNAc?1,4GlcNAc-R nor from the GlcNAc?1,2Man?1,6Man?- 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.
Keywords
N-acetylglucosaminidase,
N-acetylglucosaminyl-transferase I,
Caenorhabditis elegans,
N-glycan biosynthesis,
mannosidase II,
paucimannose N-glycans