Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

Abstract

Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptidebinding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. Molecular & Cellular Proteomics

SWATH-MS Glycoproteomics Reveals Consequences of Defects in the Glycosylation Machinery

“…Mutants in OTase subunits showed clear site-specific hypoglycosylation (Figs. 2 and 3), consistent with previous studies showing that Ost3-OTase and Ost6-OTase have distinct protein substrate preferences at the level of individual glycosylation sites (15,18,36). In contrast, alg mutations led to a consistent pattern of underglycosylation across different sites (Figs.…”

“…The yeast OTase is a hetero-oligomeric complex composed of essential (Ost1, Ost2, Wbp1, Stt3, and Swp1) and nonessential (Ost3, Ost4, Ost5, and Ost6) subunits. There are two OTase isoforms in yeast containing one of either of the paralogous Ost3 or Ost6 subunits, which have different protein substrate specificity at the level of individual glycosylation sites (15)(16)(17)(18). The function of the other OTase subunits is less well defined.…”

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

“…Saccharomyces cerevisiae strains used were wild-type BY4742 and the ∆ost3/∆ost6 derivative thereof, both carrying the empty YEp352 plasmid [22]. Yeast was grown on synthetic defined (SD) media (2% Yeast Nitrogen Base Without Amino Acids, 0.67% Yeast Synthetic Drop-out Medium Supplements [all Sigma-Aldrich, St. Louis, Missouri] and 2% D-glucose) lacking uracil.…”

“…Tethering of substrate protein may increase glycosylation efficiency by inhibiting substrate protein folding and by presenting substrate protein to the Stt3p active site of OTase. Yeast lacking either Ost3p or Ost6p have mild site-specific defects in glycosylation, while yeast lacking both Ost3p and Ost6p have widespread severe glycosylation defects [22][23][24]. So far only 20 glycosylation sites have been found to require either Ost3p or Ost6p for efficient glycosylation [10].…”

“…Each OTase complex incorporates only one Ost3p/Ost6p protein, and Ost3p-OTase and Ost6p-OTase have distinct protein substrate specificities [15,16]. Ost3p/Ost6p are proposed to increase the efficiency of N-glycosylation at specific asparagines by transiently binding nearby stretches of the nascent polypeptide [17] through non-covalent interactions [17][18][19] or a mixed disulphide bond [19][20][21]. Tethering of substrate protein may increase glycosylation efficiency by inhibiting substrate protein folding and by presenting substrate protein to the Stt3p active site of OTase.…”