Intracellular Folding of Tissue-type Plasminogen Activator
EFFECTS OF DISULFIDE BOND FORMATION ON N-LINKED GLYCOSYLATION AND SECRETION (*)
- From the (1)University of Manchester, School of Biological Sciences, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom and the
- (2)Institute für Mikrobiologie, Heinrich Heine Universität Düsseldorf, Universitätsstrasse 1, Gebaude 26.12, D-4000 Düsseldorf, Federal Republic of Germany
- §To whom reprint requests should be addressed. Tel.: 44-61-275-5103; Fax: 44-61-275-5082; neil.bulleid{at}man.ac.uk
Abstract
The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded, and was secreted. We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.
Footnotes
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↵* This work was supported by the Wellcome Trust (Grant 35853) and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- ER
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endoplasmic reticulum
- t-PA
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tissue-type plasminogen activator
- DTT
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dithiothreitol
- PAGE
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polyacrylamide gel electrophoresis
- CHO
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Chinese hamster ovary
- NEM
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N-ethylmaleimide.
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↵2S. Allen and N. J. Bulleid, unpublished results.
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- Received August 26, 1994.
- Revision received December 5, 1994.
