Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I, alpha3,6-mannosidase II and a specific membrane-bound beta-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 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.
Zhang
Wenli
W
Department of Structural Biology and Biochemistry, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
Cao
Pinjiang
P
Chen
Shihao
S
Spence
Andrew M
AM
Zhu
Shaoxian
S
Staudacher
Erika
E
Schachter
Harry
H
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D008361:Mannosidases / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis*
D011134:Polysaccharides; D017351:N-Acetylglucosaminyltransferases; C446926:UDP-GlcNAc alpha-D-mannoside beta-1,2-N-acetylglucosaminyltransferase; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D000118:Acetylglucosaminidase