title: Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p.
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.
authors: School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
authors: Siti N H
authors: SN
authors: Mohd Yusuf
authors: Ulla-Maja
authors: UM
authors: Bailey
authors: Nikki Y
authors: NY
authors: Tan
authors: Muhammad Fairuz
authors: MF
authors: Jamaluddin
authors: Benjamin L
authors: BL
authors: Schulz
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D006602:Hexosyltransferases / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry
chemical_list: C066709:GAS1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D013879:Thioredoxins; D006602:Hexosyltransferases; C507060:OST6 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D003545:Cysteine