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. 2002 Jan 1;361(Pt 1):153–162. doi: 10.1042/0264-6021:3610153

Cloning and expression of a novel UDP-GlcNAc:alpha-D-mannoside beta1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

Wenli Zhang 1, Doron Betel 1, Harry Schachter 1
PMCID: PMC1222290  PMID: 11742540

Abstract

A TBLASTN search with human UDP-GlcNAc:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(alpha1-6)[Man(alpha1-3)]Man(beta1-)O-octyl to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl (the substrate for beta-1,2-N-acetylglucosaminyltransferase II), Man(alpha1-)O-benzyl [with K(m) values of approximately 0.3 and >30 mM for UDP-GlcNAc and Man(alpha1-)O-benzyl respectively] and the glycopeptide CYA[Man(alpha1-)O-T]AV (K(m) approximately 12 mM). The product formed with Man(alpha1-)O-benzyl was identified as GlcNAc(beta1-2)Man(alpha1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:alpha-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3 kb message with a wide tissue distribution. The cDNA has a 1980 bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(beta1-)O-octyl, Man(beta1-)O-p-nitrophenyl and GlcNAc(beta1-2)Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-4)GlcNAc(beta1-4)GlcNAc(beta1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for alpha-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(alpha1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(beta1-2)Man(alpha1-)O-Ser/Thr moiety on alpha-dystroglycan and other O-mannosylated proteins.

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Selected References

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  1. Amado M., Almeida R., Schwientek T., Clausen H. Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim Biophys Acta. 1999 Dec 6;1473(1):35–53. doi: 10.1016/s0304-4165(99)00168-3. [DOI] [PubMed] [Google Scholar]
  2. Chen S., Zhou S., Sarkar M., Spence A. M., Schachter H. Expression of three Caenorhabditis elegans N-acetylglucosaminyltransferase I genes during development. J Biol Chem. 1999 Jan 1;274(1):288–297. doi: 10.1074/jbc.274.1.288. [DOI] [PubMed] [Google Scholar]
  3. Chiba A., Matsumura K., Yamada H., Inazu T., Shimizu T., Kusunoki S., Kanazawa I., Kobata A., Endo T. Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin. J Biol Chem. 1997 Jan 24;272(4):2156–2162. doi: 10.1074/jbc.272.4.2156. [DOI] [PubMed] [Google Scholar]
  4. Endo T. O-mannosyl glycans in mammals. Biochim Biophys Acta. 1999 Dec 6;1473(1):237–246. doi: 10.1016/s0304-4165(99)00182-8. [DOI] [PubMed] [Google Scholar]
  5. Henry M. D., Campbell K. P. A role for dystroglycan in basement membrane assembly. Cell. 1998 Dec 11;95(6):859–870. doi: 10.1016/s0092-8674(00)81708-0. [DOI] [PubMed] [Google Scholar]
  6. Holt K. H., Crosbie R. H., Venzke D. P., Campbell K. P. Biosynthesis of dystroglycan: processing of a precursor propeptide. FEBS Lett. 2000 Feb 18;468(1):79–83. doi: 10.1016/s0014-5793(00)01195-9. [DOI] [PubMed] [Google Scholar]
  7. Ibraghimov-Beskrovnaya O., Ervasti J. M., Leveille C. J., Slaughter C. A., Sernett S. W., Campbell K. P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 1992 Feb 20;355(6362):696–702. doi: 10.1038/355696a0. [DOI] [PubMed] [Google Scholar]
  8. Ioffe E., Liu Y., Stanley P. Complex N-glycans in Mgat1 null preimplantation embryos arise from maternal Mgat1 RNA. Glycobiology. 1997 Oct;7(7):913–919. doi: 10.1093/glycob/7.7.913. [DOI] [PubMed] [Google Scholar]
  9. Ioffe E., Stanley P. Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):728–732. doi: 10.1073/pnas.91.2.728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kumar R., Yang J., Larsen R. D., Stanley P. Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9948–9952. doi: 10.1073/pnas.87.24.9948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Matsumura K., Chiba A., Yamada H., Fukuta-Ohi H., Fujita S., Endo T., Kobata A., Anderson L. V., Kanazawa I., Campbell K. P. A role of dystroglycan in schwannoma cell adhesion to laminin. J Biol Chem. 1997 May 23;272(21):13904–13910. doi: 10.1074/jbc.272.21.13904. [DOI] [PubMed] [Google Scholar]
  14. Metzler M., Gertz A., Sarkar M., Schachter H., Schrader J. W., Marth J. D. Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development. EMBO J. 1994 May 1;13(9):2056–2065. doi: 10.1002/j.1460-2075.1994.tb06480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Montanaro F., Lindenbaum M., Carbonetto S. alpha-Dystroglycan is a laminin receptor involved in extracellular matrix assembly on myotubes and muscle cell viability. J Cell Biol. 1999 Jun 14;145(6):1325–1340. doi: 10.1083/jcb.145.6.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mulder H., Dideberg F., Schachter H., Spronk B. A., De Jong-Brink M., Kamerling J. P., Vliegenthart J. F. In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis of incorporation GlcNAc by beta 2GlcNAc-transferase I is an essential prerequisite for the action of beta 2GlcNAc-transferase II and beta 2Xyl-transferase. Eur J Biochem. 1995 Aug 15;232(1):272–283. doi: 10.1111/j.1432-1033.1995.tb20809.x. [DOI] [PubMed] [Google Scholar]
  17. Möller G., Reck F., Paulsen H., Kaur K. J., Sarkar M., Schachter H., Brockhausen I. Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues. Glycoconj J. 1992 Aug;9(4):180–190. doi: 10.1007/BF00731163. [DOI] [PubMed] [Google Scholar]
  18. Nishikawa Y., Pegg W., Paulsen H., Schachter H. Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I. J Biol Chem. 1988 Jun 15;263(17):8270–8281. [PubMed] [Google Scholar]
  19. Oriol R., Mollicone R., Cailleau A., Balanzino L., Breton C. Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria. Glycobiology. 1999 Apr;9(4):323–334. doi: 10.1093/glycob/9.4.323. [DOI] [PubMed] [Google Scholar]
  20. Reck F., Springer M., Paulsen H., Brockhausen I., Sarkar M., Schachter H. Synthesis of tetrasaccharide analogues of the N-glycan substrate of beta-(1-->2)-N-acetylglucosaminyltransferase II using trisaccharide precursors and recombinant beta-(1-->2)-N-acetylglucosaminyltransferase I. Carbohydr Res. 1994 Jun 2;259(1):93–101. doi: 10.1016/0008-6215(94)84200-0. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sarkar M. Expression of recombinant rabbit UDP-GlcNAc: alpha 3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells. Glycoconj J. 1994 Jun;11(3):204–209. doi: 10.1007/BF00731219. [DOI] [PubMed] [Google Scholar]
  23. Sarkar M., Hull E., Nishikawa Y., Simpson R. J., Moritz R. L., Dunn R., Schachter H. Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):234–238. doi: 10.1073/pnas.88.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sarkar M., Pagny S., Unligil U., Joziasse D., Mucha J., Glössl J., Schachter H. Removal of 106 amino acids from the N-terminus of UDP-GlcNAc: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I does not inactivate the enzyme. Glycoconj J. 1998 Feb;15(2):193–197. doi: 10.1023/a:1006928624913. [DOI] [PubMed] [Google Scholar]
  25. Sarkar M., Schachter H. Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I. Biol Chem. 2001 Feb;382(2):209–217. doi: 10.1515/BC.2001.028. [DOI] [PubMed] [Google Scholar]
  26. Sasaki T., Yamada H., Matsumura K., Shimizu T., Kobata A., Endo T. Detection of O-mannosyl glycans in rabbit skeletal muscle alpha-dystroglycan. Biochim Biophys Acta. 1998 Nov 27;1425(3):599–606. doi: 10.1016/s0304-4165(98)00114-7. [DOI] [PubMed] [Google Scholar]
  27. Schachter H. Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem Cell Biol. 1986 Mar;64(3):163–181. doi: 10.1139/o86-026. [DOI] [PubMed] [Google Scholar]
  28. Schachter H. The 'yellow brick road' to branched complex N-glycans. Glycobiology. 1991 Nov;1(5):453–461. doi: 10.1093/glycob/1.5.453. [DOI] [PubMed] [Google Scholar]
  29. Silberstein S., Gilmore R. Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J. 1996 Jun;10(8):849–858. [PubMed] [Google Scholar]
  30. Stanley P. Glycosylation engineering. Glycobiology. 1992 Apr;2(2):99–107. doi: 10.1093/glycob/2.2.99. [DOI] [PubMed] [Google Scholar]
  31. Takahashi S., Sasaki T., Manya H., Chiba Y., Yoshida A., Mizuno M., Ishida H., Ito F., Inazu T., Kotani N. A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans. Glycobiology. 2001 Jan;11(1):37–45. doi: 10.1093/glycob/11.1.37. [DOI] [PubMed] [Google Scholar]
  32. Tan J., D'Agostaro A. F., Bendiak B., Reck F., Sarkar M., Squire J. A., Leong P., Schachter H. The human UDP-N-acetylglucosamine: alpha-6-D-mannoside-beta-1,2- N-acetylglucosaminyltransferase II gene (MGAT2). Cloning of genomic DNA, localization to chromosome 14q21, expression in insect cells and purification of the recombinant protein. Eur J Biochem. 1995 Jul 15;231(2):317–328. doi: 10.1111/j.1432-1033.1995.tb20703.x. [DOI] [PubMed] [Google Scholar]
  33. Tessier D. C., Thomas D. Y., Khouri H. E., Laliberté F., Vernet T. Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene. 1991 Feb 15;98(2):177–183. doi: 10.1016/0378-1119(91)90171-7. [DOI] [PubMed] [Google Scholar]
  34. Unligil U. M., Zhou S., Yuwaraj S., Sarkar M., Schachter H., Rini J. M. X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J. 2000 Oct 16;19(20):5269–5280. doi: 10.1093/emboj/19.20.5269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Williamson R. A., Henry M. D., Daniels K. J., Hrstka R. F., Lee J. C., Sunada Y., Ibraghimov-Beskrovnaya O., Campbell K. P. Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1-null mice. Hum Mol Genet. 1997 Jun;6(6):831–841. doi: 10.1093/hmg/6.6.831. [DOI] [PubMed] [Google Scholar]
  36. Yamada H., Chiba A., Endo T., Kobata A., Anderson L. V., Hori H., Fukuta-Ohi H., Kanazawa I., Campbell K. P., Shimizu T. Characterization of dp6troglycan-laminin interaction in peripheral nerve. J Neurochem. 1996 Apr;66(4):1518–1524. doi: 10.1046/j.1471-4159.1996.66041518.x. [DOI] [PubMed] [Google Scholar]
  37. Yip B., Chen S. H., Mulder H., Höppener J. W., Schachter H. Organization of the human beta-1,2-N-acetylglucosaminyltransferase I gene (MGAT1), which controls complex and hybrid N-glycan synthesis. Biochem J. 1997 Jan 15;321(Pt 2):465–474. doi: 10.1042/bj3210465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yoshida A., Kobayashi K., Manya H., Taniguchi K., Kano H., Mizuno M., Inazu T., Mitsuhashi H., Takahashi S., Takeuchi M. Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell. 2001 Nov;1(5):717–724. doi: 10.1016/s1534-5807(01)00070-3. [DOI] [PubMed] [Google Scholar]

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