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Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells.

Pult, Frauke and Fallah, Ghada and Braam, Ursula and Detro-Dassen, Silvia and Niculescu, Cristina and Laube, Bodo and Schmalzing, Günther (2011):
Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells.
In: Glycobiology, pp. 1147-1160, 21, (9), ISSN 1460-2423, [Article]

Abstract

N-glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged approximately 12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilisation of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein, and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in HEK293 cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.

Item Type: Article
Erschienen: 2011
Creators: Pult, Frauke and Fallah, Ghada and Braam, Ursula and Detro-Dassen, Silvia and Niculescu, Cristina and Laube, Bodo and Schmalzing, Günther
Title: Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells.
Language: English
Abstract:

N-glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged approximately 12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilisation of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein, and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in HEK293 cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.

Journal or Publication Title: Glycobiology
Volume: 21
Number: 9
Divisions: 10 Department of Biology
10 Department of Biology > Neurophysiology and Neurosensory Systems
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Date Deposited: 11 Apr 2011 09:08
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