Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

Jillianne Eyring,Chia-Wei Lin,Elsy Mankah Ngwa,Jérémy Boilevin,Giorgio Pesciullesi,Kaspar P Locher,Tamis Darbre,Jean-Louis Reymond,Markus Aebi

doi:10.1016/j.jbc.2021.100809

Abstract

Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.

Highlights

Nitrogen of the asparagine side chain and the C1 carbon of the reducing end N-acetylglucosamine (GlcNAc) residue of the oligosaccharide
Silver staining of purified OST complexes showed that all eight subunits were present and either Ost3p or Ost6p was solely incorporated into each respective complex (Fig. 1A)
Analysis of the glycosylation site occupancy of the OST complex glycoproteins Stt3p, Wbp1p, and Ost1p by mass spectrometry confirmed that the OST6 complex was generally less efficiently glycosylated than the OST3 complex, but the Stt3p N539 glycosite was fully glycosylated in both OST complexes (Fig. 1C)

Summary

Introduction

Nitrogen of the asparagine side chain and the C1 carbon of the reducing end N-acetylglucosamine (GlcNAc) residue of the oligosaccharide. Reactions were performed with 0.1 μM purified OST3 complex, 100 μM LLO C20, and varying concentrations of the peptide.

Results

Conclusion