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Inhibition of hybrid-and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

Inhibition of hybrid-and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway
Inhibition of hybrid-and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway
A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the α-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of α-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal α1–2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi α-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.
electrospray ionization mass spectrometry, fucosyltransferase, matrix-assisted laser desorption, ionization (MALDI)/N-linked glycosylation
0959-6658
748-756
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Chang, Veronica T.
e83571e9-6b2f-4f8e-adfc-3fd539e9102e
Yu, Chao
12304bbc-1182-4b35-a3e5-d50719156e2f
Aricescu, A. Radu
8ee3d2bd-7ef7-4e85-862a-e13f48ae0fa6
Jones, E. Yvonne
6b9004c9-137b-4f43-8a14-c7c83bcaa4be
Davis, Simon J.
77c9e91c-a2c6-498a-9128-bf164a8da8de
Dwek, Raymond A.
5fb1a0ce-1ac6-4825-8ff0-f62c7164ff0c
Rudd, Pauline M.
4bbd1e70-98ae-4c28-84e6-c6a18d98e7ee
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Harvey, David J.
8bb24417-3852-4b1f-827b-0d5d2c176744
Chang, Veronica T.
e83571e9-6b2f-4f8e-adfc-3fd539e9102e
Yu, Chao
12304bbc-1182-4b35-a3e5-d50719156e2f
Aricescu, A. Radu
8ee3d2bd-7ef7-4e85-862a-e13f48ae0fa6
Jones, E. Yvonne
6b9004c9-137b-4f43-8a14-c7c83bcaa4be
Davis, Simon J.
77c9e91c-a2c6-498a-9128-bf164a8da8de
Dwek, Raymond A.
5fb1a0ce-1ac6-4825-8ff0-f62c7164ff0c
Rudd, Pauline M.
4bbd1e70-98ae-4c28-84e6-c6a18d98e7ee

Crispin, Max, Harvey, David J., Chang, Veronica T., Yu, Chao, Aricescu, A. Radu, Jones, E. Yvonne, Davis, Simon J., Dwek, Raymond A. and Rudd, Pauline M. (2006) Inhibition of hybrid-and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. Glycobiology, 16 (8), 748-756. (doi:10.1093/glycob/cwj119).

Record type: Article

Abstract

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the α-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of α-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal α1–2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi α-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.

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e-pub ahead of print date: 3 May 2006
Published date: 1 August 2006
Keywords: electrospray ionization mass spectrometry, fucosyltransferase, matrix-assisted laser desorption, ionization (MALDI)/N-linked glycosylation

Identifiers

Local EPrints ID: 414126
URI: http://eprints.soton.ac.uk/id/eprint/414126
ISSN: 0959-6658
PURE UUID: 8b025be2-b4c4-48ef-8d9a-4de4878fb1fa
ORCID for Max Crispin: ORCID iD orcid.org/0000-0002-1072-2694

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Date deposited: 15 Sep 2017 16:30
Last modified: 24 Sep 2019 00:26

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Contributors

Author: Max Crispin ORCID iD
Author: David J. Harvey
Author: Veronica T. Chang
Author: Chao Yu
Author: A. Radu Aricescu
Author: E. Yvonne Jones
Author: Simon J. Davis
Author: Raymond A. Dwek
Author: Pauline M. Rudd

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