Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency

Markus Aebi,Benjamin L. Schulz

doi:10.1074/mcp.m800219-mcp200

Abstract

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.

Highlights

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase
As we suspected that Ost3/6p affect glycosylation of a defined but unknown subset of glycosylation sites, we developed an MS-based analytical method that could concurrently measure the glycan occupancy of many sites in different proteins
This method involved enrichment of glycoproteins covalently linked to the yeast polysaccharide cell wall, release of glycans from these glycoproteins with endoglycosidase H leaving previously glycosylated asparagines residues “tagged” with a single N-acetylglucosamine (GlcNAc), and LC-ESI-MS/MS detection of peptides and glycopeptides after protease digestion

Summary

EXPERIMENTAL PROCEDURES

Chemicals were obtained from Sigma-Aldrich unless specified otherwise. Yeast Strains—Yeast strains used were YG889 (MATa ade101 ura his3⌬200 tyr1 ⌬ost3::HIS3 ⌬ost6::kanMX4) [12] and SS330 (MATa ade101 ura his3⌬200 tyr1) [18]. Based on previously reported methods [19], covalently linked cell wall material was pelleted by centrifugation at 16,000 rcf for 1 min; washed three times with 50 mM Tris-HCl, pH 7.5; and resuspended in 50 mM Tris-HCl, pH 8, 2% SDS, 7 M urea, and 2 M thiourea. Peptide identities based on MS/MS data were assigned using an in-house installation of Mascot (Version 2.1., Matrix Science) searching the Saccharomyces cerevisiae protein database (downloaded from European Molecular Biology Laboratory-European Bioinformatics Institute: fgcz_4921 4932_yeast_contaminants_20070811; 6068 sequences; 2,968,192 residues) with the following parameters: fixed modification of carbamidomethylated cysteines; variable modification of oxidized methionines, deamidated asparagines, and HexNAc-asparagines; no enzyme specified; 0.01-Da peptide tolerance; 0.6-Da fragment ion tolerance; and detected ion-specific charge state. Multiple sequence alignment based on amino acid sequence was performed with MUSCLE (multiple sequence comparison by log-expectation) [21], and a phylogenetic tree was built from this alignment with SCI-PHY (subfamily classification in phylogenomics) [22]

RESULTS

DISCUSSION

Wild type