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Cloned β1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species

Abstract

Cloned human β1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyses the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains ofN-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activityin vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 productin vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.

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Abbreviations

GSL:

glycosphingolipid(s)

CHO:

Chinese hamster ovary

GSL structures: GM2:

GalNAcβ1,4(NeuAcα2,3)Galβ1,4GlcCer

GD2:

GalNacβ1,4(NeuAcα2,8NeuAcα2,3)Galβ1,4GlcCer

GM3:

NeuAcα2,3Galβ1,4GlcCer

Gg3 :

GalNAcβ1,4Galβ1,4GlcCer

LacCer:

Galβ1,4GlcCer

GlcCer:

glucosylceramide

PBS-BSA:

phosphate buffered saline pH 7.4 containing 1% bovine serum albumin

GalNAcT:

N-acetylgalactosaminyltransferase

GNT:

N-acetylglucosaminyltransferase I

Iip33:

p33 form of human invariant chain

HPTLC:

high performance thin layer chromatography

PCR:

polymerase chain reaction

BFA:

Brefeldin A

References

  1. 1.

    Machamer CE (1993)Curr Opin Cell Biol 5: 606–12.

  2. 2.

    Nilsson T, Slusarewicz P, Hoe MH, Warren G (1993)FEBS Lett 330: 1–4.

  3. 3.

    Nagata Y, Yamashiro S, Yodoi J, Lloyd KO, Furukawa K (1992)J Biol Chem 267: 12082–89.

  4. 4.

    Lutz MS, Jaskiewicz E, Darling DS, Furukawa K, Young WW, Jr (1994)J Biol Chem 269: 29227–31.

  5. 5.

    Hidari KI-PJ, Ichikawa S, Furukawa K, Yamasaki M, Hirabayashi Y (1994)Biochem J 303: 957–65.

  6. 6.

    Yamashiro S, Haraguchi M, Furukawa K, Takamiya K, Yamamoto A, Nagata Y, Lloyd KO, Shiku H (1995)J Biol Chem 270: 6149–55.

  7. 7.

    Kumar R, Yang J, Eddy RL, Byers MG, Shows TB, Stanley P (1992)Glycobiology 2: 383–93.

  8. 8.

    Lotteau V, Teyton L, Peleraux A, Nilsson T, Karlsson L, Schmid S, Quaranta V, Peterson PA (1990)Nature 348: 600–5.

  9. 9.

    Evans GI, Lewis GK, Ramsay G, Bishop JM (1985)Mol Cell Biol 5: 3610–16.

  10. 10.

    Tang BL, Wong SH, Low SH, Hong W (1992)J Biol Chem 267: 10122–26.

  11. 11.

    Kozak M (1983)Microbiol Rev 47: 1–45.

  12. 12.

    Natoli EJ Jr, Livingston PO, Pukel CS, Lloyd KO, Wiegandt H, Szalas J, Oettgen HF, Old LJ (1986)Cancer Res 46: 4116–20.

  13. 13.

    Young WW Jr, MacDonald EMS, Nowinski RC, Hakomori S (1979)J Exp Med 150: 1008–19.

  14. 14.

    Dippold WG, Lloyd KO, Li LTC, Ikeda H, Oettgen HF, Old LJ (1980)Proc Natl Acad Sci USA 77: 6114–18.

  15. 15.

    White T, Mandel U, Orntoft TF, Dabelsteen E, Karkov J, Kubeja M, Hakomori S, Clausen H (1990)Biochemistry 29: 2740–47.

  16. 16.

    Sambrook J, Fritsch EF, Maniatis T (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed, Vol. 1–3, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

  17. 17.

    Mount SL, Taatjes DJ, von Turkovich M, Tindle BH, Trainer TD (1993)Ultrastruct Pathol 17: 547–57.

  18. 18.

    Taatjes DJ, Roth J, Shaper NL, Shaper JH (1992)Glycobiology 2: 579–89.

  19. 19.

    Slot JW, Geuze HJ (1985)Eur J Cell Biol 38: 87–93.

  20. 20.

    Roth J, Taatjes DJ, Warhol MJ (1989)Histochemistry 92: 47–56.

  21. 21.

    Kasahara K, Guo L, Nagai Y, Sanai Y (1994)Anal Biochem 218: 224–26.

  22. 22.

    Schnaar RL (1994)Methods Enzymol 230: 348–70.

  23. 23.

    Smith PK, Krohn RI, Hermanson GT, Mallic AK, Gartner FH, Provenzano MD, Fujimoto EK, Gocke NM, Olson BJ, Klenk DC (1985)Anal Biochem 150: 76–85.

  24. 24.

    Saito T, Hakomori S (1971)J Lipid Res 12: 257–59.

  25. 25.

    Dunphy WG, Rothman JE (1985)Cell 42: 13–21.

  26. 26.

    Roth J (1987)Biochim Biophys Acta 906: 405–36.

  27. 27.

    Nilsson T, Hoe MH, Slusarewicz P, Rabouille C, Watson R, Hunte F, Watzele G, Berger EG, Warren G (1994)EMBO J 13: 562–74.

  28. 28.

    Young WW Jr, Lutz MS, Mills SE, Lechler-Osborn S (1990)Proc Natl Acad Sci USA 87: 6838–42.

  29. 29.

    Van Echten G, Sandhoff K (1993)J Biol Chem 268: 5341–44.

  30. 30.

    Palleros DR, Welch WJ, Fink AL (1991)Proc Natl Acad Sci USA 88: 5719–23.

  31. 31.

    Trinchera M, Ghidoni R (1989)J Biol Chem 264: 15766–69.

  32. 32.

    Trinchera M, Pirovano B, Ghidoni R (1990)J Biol Chem 265: 18242–47.

  33. 33.

    Wattenberg BW (1990)J Cell Biol 111: 421–28.

  34. 34.

    Young WW Jr, Lutz MS, Blackburn WA (1992)J Biol Chem 267: 12011–15.

  35. 35.

    Van Echten G, Iber H, Stotz H, Takatsuki A, Sandhoff K (1990)Eur J Cell Biol 51: 135–39.

  36. 36.

    Kleene R, Berger EG (1993)Biochim Biophys Acta Rev Biomembr 1154: 283–325.

  37. 37.

    Roth J, Taatjes DJ, Weinstein J, Paulson JC, Greenwell P, Watkins WM (1986)J Biol Chem 261: 14307–12.

  38. 38.

    Nilsson T, Pypaert M, Hoe MH, Slusarewicz P, Berger EG, Warren G (1993)J Cell Biol 120: 5–13.

  39. 39.

    Colley KJ, Lee EU, Paulson JC (1992)J Biol Chem 267: 7784–93.

  40. 40.

    Rabouille C, Hui N, Hunte F, Kieckbusch R, Berger EG, Warren G, Nilsson T (1995)J Cell Sci 108: 1617–27.

  41. 41.

    Schutze M-P, Peterson PA, Jackson MR (1994)EMBO J 13: 1696–705.

  42. 42.

    Jackson MR, Nilsson T, Peterson PA (1993)J Cell Biol 121: 317–33.

  43. 43.

    Mandon E, Kempner ES, Ishihara M, Hirschberg CB (1994)J Biol Chem 269: 11729–33.

  44. 44.

    Yamaguchi N, Fukuda MN (1995)J Biol Chem 270: 12170–76.

  45. 45.

    Claesson L, Larhammar D, Rask L, Peterson PA (1983)Proc Natl Acad Sci USA 80: 7395–99.

  46. 46.

    Strubin M, Long EO, Mach B (1986)Cell 47: 619–25.

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Author information

Correspondence to William W. Young Jr.

Additional information

This paper is dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.

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Jaskiewicz, E., Zhu, G., Taatjes, D.J. et al. Cloned β1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species. Glycoconjugate J 13, 213–223 (1996). https://doi.org/10.1007/BF00731496

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Keywords

  • glycosyltransferase
  • Golgi
  • epitope tagging
  • glycosphingolipid