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

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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

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Authors and Affiliations

  1. Departments of Biological and Biophysical Sciences and Biochemistry, Health Sciences Center, University of Louisville, 501 S. Preston St., 40292, Louisville, KY, USA

    Ewa Jaskiewicz, Guofen Zhu, Douglas S. Darling, Glenn E. Zwanzig Jr & William W. Young Jr

  2. Department of Pathology, University of Vermont, 05405, Burlington, VT, USA

    Douglas J. Taatjes

Authors
  1. Ewa Jaskiewicz
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  2. Guofen Zhu
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  3. Douglas J. Taatjes
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  4. Douglas S. Darling
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  5. Glenn E. Zwanzig Jr
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  6. William W. Young Jr
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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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  • DOI: https://doi.org/10.1007/BF00731496

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