Skip to main content
SpringerLink
Log in

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues

  • Glycoconjagate Journal
  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

UDP-GlcNAc:GlcNAc β1-2Manα1-6R (GlcNAc to Man) β1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAcβ1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAcβ1-2Manα1-6Glc/Manβ-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Manα1-3 residue and the 4-hydroxyl of the Manβ- residue of the Manα1-6(Manα1-3)Manβ-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

all:

allyl

AML:

acute myeloid leukaemia

BSA:

bovine serum albumin

CML:

chronic myelogenous leukaemia

Gal:

G,d-galactose

Glc:

d-glucose

GlcNAc:

Gn,N-acetyl-d-glucosamine

HPLC:

high performance liquid chromatography

Man:

M,d-mannose

mco:

8-methoxycarbonyl-octyl, (CH2)8COOCH3

Me:

methyl

MES:

2-(N-morpholino)ethanesulfonate

oct:

octyl

pnp:

p-nitrophenyl

T:

transferase

References

  1. Varki A (1993)Glycobiology 3:97–30.

    Google Scholar 

  2. Varki A (1992)Curr Opin Cell Biol 4:257–66.

    Google Scholar 

  3. Feizi T (1993)Curr Opin Struct Biol 3:701–10.

    Google Scholar 

  4. Dennis JW (1992) InCell Surface Carbohydrates and Cell Development, (Fukuda M, ed.) pp 161–94, Boca Raton, Florida: CRC Press, Inc.

    Google Scholar 

  5. Dennis JW, Laferte S (1988) InAltered Glycosylation in Tumor Cells (Reading CL, Hakomori S-I, Marcus DM, eds) pp. 257–67, New York, N.Y.: Alan R. Liss Inc.

    Google Scholar 

  6. Smets LA, Van Beek WP (1984)Biochim Biophys Acta 738:237–49.

    Google Scholar 

  7. Santer UV, DeSantis R, Hard KJ, Van-Kuik JA, Vliegenthart JFG, Won B, Glick MC (1989)Eur J Biochem 181:249–60.

    Google Scholar 

  8. Dennis JW, Laferte S, Waghorne C, Breitman ML, Kerbel RS (1987)Science 236:582–85.

    Google Scholar 

  9. Pierce M, Arango J (1986)J Biol Chem 261:10772–77.

    Google Scholar 

  10. Arango J, Pierce M (1988)J Cell Biochem 37:225–31.

    Google Scholar 

  11. Yamashita K, Tachibana Y, Ohkura T, Kobata A (1985)J Biol Chem 260:3963–69.

    Google Scholar 

  12. Palcic MM, Ripka J, Kaur KJ, Shoreibah M, Hindsgaul O, Pierce M (1990)J Biol Chem 265:6759–69.

    Google Scholar 

  13. Yousefi S, Higgins E, Daoling Z, Pollex-Krüger A, Hindsgaul O, Dennis JW (1991)J Biol Chem 266:1772–82.

    Google Scholar 

  14. Easton EW, Bolscher JGM, Van den Eijnden DH (1991)J Biol Chem 266:21674–80.

    Google Scholar 

  15. Dennis JW, Laferte S (1989)Cancer Res 49:945–50.

    Google Scholar 

  16. Easton EW, Blokland I, Geldof AA, Rao BR, Van den Eijnden DH (1992)Febs Lett 308:46–49.

    Google Scholar 

  17. Dennis JW, Kosh K, Bryce D-M, Breitman ML (1989)Oncogene 4:853–60.

    Google Scholar 

  18. Cummings RD, Kornfeld S (1984)J Biol Chem 259:6253–60.

    Google Scholar 

  19. van den Eijnden DH, Koenderman AHL, Schiphorst WECM (1988)J Biol Chem 263:12461–71.

    Google Scholar 

  20. Brockhausen I, Romero PA, Herscovics A (1991)Cancer Res 51:3136–42.

    Google Scholar 

  21. Heffernan M, Lotan R, Amos B, Palcic M, Takano R, Dennis JW (1993)J Biol Chem 268:1242–51.

    Google Scholar 

  22. Nakao H, Nishikawa A, Karasuno T, Nishiura T, Iida M, Kanayama Y, Yonezawa T, Tarui S, Taniguchi N (1990)Biochem Biophys Res Commun 172:1260–66.

    Google Scholar 

  23. Cummings RD, Trowbridge IS, Kornfeld S (1982)J Biol Chem 257:13421–27.

    Google Scholar 

  24. Hindsgaul O, Kaur KJ, Srivastava G, Blaszczyk-Thurin M, Crawley SC, Heerze LD, Palcic MM (1991)J Biol Chem 266:17858–62.

    Google Scholar 

  25. Shoreibah MG, Hindsgaul O, Pierce M (1992)J Biol Chem 267:2920–27.

    Google Scholar 

  26. Gu J, Nishikawa A, Tsuruoka N, Ohno M, Yamaguchi N, Kanagawa K, Taniguchi N (1993)J Biochem 113:614–19.

    Google Scholar 

  27. Shoreibah M, Perng GS, Adler B, Weinstein J, Basu R, Cupples R, Wen D, Browne JK, Buckhaults P, Fregien N, Pierce M (1993)J Biol Chem 268:15381–85.

    Google Scholar 

  28. Saito H, Nishikawa A, Gu JG, Ihara Y, Soejima H, Wada Y, Sekiya C, Niikawa N, Taniguchi N (1994)Biochem Biophys Res Commun 198:318–27.

    Google Scholar 

  29. Saito H, Nishikawa A, Gu JG, Ihara Y, Soejima H, Wada Y, Sekiya C, Niikawa N, Taniguchi N (1994)Biochem Biophys Res Commun 200:668–69.

    Google Scholar 

  30. Hindsgaul O, Tahir SH, Srivastava OP, Pierce M (1988)Carbohydrate Res 173:263–72.

    Google Scholar 

  31. Srivastava OP, Hindsgaul O, Shoreibah M, Pierce M (1988)Carbohydrate Res 179:137–61.

    Google Scholar 

  32. Crawley SC, Hindsgaul O, Alton G, Pierce M, Palcic MM (1990)Anal Biochem 185:112–17.

    Google Scholar 

  33. Brockhausen I, Grey AA, Pang H, Schachter H, Carver JP (1988)Glycoconjugate J 5:419–48.

    Google Scholar 

  34. Brockhausen I, Carver J, Schachter H (1988)Biochem Cell Biol 66:1134–51.

    Google Scholar 

  35. Kanie O, Crawley SC, Palcic MM, Hindsgaul O (1993)Carbohydr Res 243:139–64.

    Google Scholar 

  36. Khan SH, Crawley SC, Kanie O, Hindsgaul O (1993)J Biol Chem 268:2468–73.

    Google Scholar 

  37. Brockhausen I, Matta KL, Orr J, Schachter H (1985)Biochemistry 24:1866–74.

    Google Scholar 

  38. Khan SH, Abbas SA, Matta KL (1989)Carbohydr Res 193:125–39.

    Google Scholar 

  39. Brockhausen I, Möller G, Yang JM, Khan SH, Matta KL, Paulsen H, Grey AA, Shah RN, Schachter H (1992)Carbohydr Res 236:281–99.

    Google Scholar 

  40. Khan SH, Matta KL (1993)J Carbohydr Chem 12:335–48.

    Google Scholar 

  41. Khan SH, Abbas SA, Matta KL (1990)Carbohydr Res 205:385–97.

    Google Scholar 

  42. Khan SH, Matta KL (1993)Carbohydr Res 243:29–42.

    Google Scholar 

  43. Paulsen H, Meinjohanns E, Reck F, Brockhausen I (1993)Liebigs Ann Chem 737–750.

  44. Brockhausen I, Kuhns W, Schachter H, Matta KL, Sutherland DR, Baker MA (1991)Cancer Res 51:1257–63.

    Google Scholar 

  45. Toki D, Granovsky MA, Reck F, Kuhns W, Baker MA, Matta KL, Brockhausen I (1994)Biochem Biophys Res Commun 198:417–23.

    Google Scholar 

  46. Brockhausen I, Hull E, Hindsgaul O, Schachter H, Shah RN, Michnick SW, Carver JP (1989)J Biol Chem. 264:11211–21.

    Google Scholar 

  47. Kobata A (1988)Biochimie 70:1575–85.

    Google Scholar 

  48. Yamashita K, Hitoi A, Taniguchi N, Yokosawa N, Tsukada Y, Kobata A (1983)Cancer Res 43:5059–63.

    Google Scholar 

  49. Baker MA, Taub RN, Kanani A, Brockhausen I, Hindenburg A (1985)Blood 66:1068–71.

    Google Scholar 

  50. Baker MA, Kanani A, Brockhausen I, Schachter H, Hindenburg A, Taub RN (1987)Cancer Res 47:2763–66.

    Google Scholar 

  51. Kanani A, Sutherland DR, Fibach E, Matta KL, Hindenburg A, Brockhausen I, Kuhns W, Taub RN, Van den Eijnden DH, Baker MA (1990)Cancer Res 50:5003–7.

    Google Scholar 

  52. Linker T, Crawley SC, Hindsgaul O (1993)Carbohydr Res 245:323–31.

    Google Scholar 

  53. Vella GJ, Paulsen H, Schachter H (1984)Can J Biochem Cell Biol 62:409–17.

    Google Scholar 

  54. Möller G, Reck F, Paulsen H, Kaur KJ, Sarkar M, Schachter H, Brockhausen I (1992)Glycoconjugate J 9:180–90.

    Google Scholar 

  55. Bendiak B, Schachter H (1987)J Biol Chem 262:5775–83.

    Google Scholar 

  56. Bendiak B, Schachter H (1987)J Biol Chem 262:5784–90.

    Google Scholar 

  57. Reck F, Meinjohanns E, Springer M, Wilkens R, Van Dorst JALM, Paulsen H, Möller G, Brockhausen I, Schachter H (1994)Glycoconjugate J 11:210–16.

    Google Scholar 

  58. Nishikawa Y, Pegg W, Paulsen H, Schachter H (1988)J Biol Chem 263:8270–81.

    Google Scholar 

  59. Kanie O, Crawley SC, Palcic M, Hindsgaul O (1994)Bioorg. Medicinal Chem 2:1231–41.

    Google Scholar 

  60. Gleeson PA, Schachter H (1983)J Biol Chem 258:6162–73.

    Google Scholar 

  61. Narasimhan S (1982)J Biol Chem 257:10235–42.

    Google Scholar 

  62. Khan SH, Duus JØ, Crawley SC, Palcic MM, Hindsgaul O (1994)Tetrahedron Asymmetry 5:2415–35.

    Google Scholar 

Download references

Author information

Author notes

  1. Shaheer Khan

    Present address: Perkin-Elmer, Applied Biosystems Division, 850 Lincoln Center Drive, 94404, Foster City, CA, USA

Authors and Affiliations

  1. Research Institute, The Hospital for Sick Children, 555 University Avenue, M5G 1X8, Toronto, Ontario, Canada

    Inka Brockhausen, Folkert Reck, William Kuhns & Harry Schachter

  2. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Inka Brockhausen & Harry Schachter

  3. Department of Gynecologic Oncology, Roswell Park Memorial Institute, Buffalo, NY, USA

    Shaheer Khan & Khushi L. Matta

  4. Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany

    Ernst Meinjohanns & Hans Paulsen

  5. Department of Medical Genetics and Biophysics, University of Toronto, Toronto, Ontario, Canada

    Rajan N. Shah

  6. Department of Medicine, Toronto Hospital, Toronto, Ontario, Canada

    Michael A. Baker

Authors
  1. Inka Brockhausen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Folkert Reck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William Kuhns
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shaheer Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Khushi L. Matta
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ernst Meinjohanns
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hans Paulsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rajan N. Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael A. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Harry Schachter
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brockhausen, I., Reck, F., Kuhns, W. et al. Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues. Glycoconjugate J 12, 371–379 (1995). https://doi.org/10.1007/BF00731340

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00731340

Keywords

Search

Navigation