Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p

Yusuf, Siti N. H. Mohd, Bailey, Ulla-Maja, Tan, Nikki Y., Jamaluddin, Muhammad Fairuz and Schulz, Benjamin L. (2013) Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p. Biochemical and Biophysical Research Communications, 432 3: 438-443. doi:10.1016/j.bbrc.2013.01.128

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Author Yusuf, Siti N. H. Mohd
Bailey, Ulla-Maja
Tan, Nikki Y.
Jamaluddin, Muhammad Fairuz
Schulz, Benjamin L.
Title Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p
Journal name Biochemical and Biophysical Research Communications   Check publisher's open access policy
ISSN 0006-291X
1090-2104
Publication date 2013-03-15
Year available 2013
Sub-type Article (original research)
DOI 10.1016/j.bbrc.2013.01.128
Open Access Status File (Author Post-print)
Volume 432
Issue 3
Start page 438
End page 443
Total pages 6
Place of publication Maryland Heights, MO, United States
Publisher Academic Press
Collection year 2014
Language eng
Formatted abstract
Oligosaccharyltransferase (OTase) glycosylates selected asparagine residues in secreted and membrane proteins in eukaryotes, and asparagine (N)-glycosylation affects the folding, stability and function of diverse glycoproteins. The range of acceptor protein substrates that are efficiently glycosylated depends on the action of several accessory subunits of OTase, including in yeast the homologous proteins Ost3p and Ost6p. A model of Ost3p and Ost6p function has been proposed in which their thioredoxin-like active site cysteines form transient mixed disulfide bonds with cysteines in substrate proteins to enhance the glycosylation of nearby asparagine residues. We tested aspects of this model with a series of in vitro assays. We developed a whole protein mixed disulfide interaction assay that showed that Ost6p could form mixed disulfide bonds with selected cysteines in pre-reduced yeast Gas1p, a model glycoprotein substrate of Ost3p and Ost6p. A complementary peptide affinity chromatography assay for mixed disulfide bond formation showed that Ost3p could also form mixed disulfide bonds with cysteines in selected reduced tryptic peptides from Gas1p. Together, these assays showed that the thioredoxin-like active sites of Ost3p and Ost6p could form transient mixed disulfide bonds with cysteines in a model substrate glycoprotein, consistent with the function of Ost3p and Ost6p in modulating N-glycosylation substrate selection by OTase in vivo.
Keyword N-glycosylation
Saccharomyces cerevisiae
Thioredoxin
Disulfide
Mass spectrometry
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Chromatography B. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochemical and Biophysical Research Communications, 432 (3) 2013 DOI:10.1016/j.bbrc.2013.01.128

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
School of Chemistry and Molecular Biosciences
 
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