Quantitative Profiling of N-linked Glycosylation Machinery in Yeast Saccharomyces cerevisiae

Abstract

Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The -linked glycosylation machinery involves enzymes responsible for the assembly of the lipid-linked oligosaccharide (LLO), which is then transferred to the asparagine residues on the polypeptides by the enzyme oligosaccharyltransferase (OST). A major goal in the study of protein glycosylation is to establish quantitative methods for the analysis of site-specific extent of glycosylation. We developed a sensitive approach to examine glycosylation site occupancy in by coupling stable isotope labeling (SILAC) approach to parallel reaction monitoring (PRM) mass spectrometry (MS). We combined the method with genetic tools and validated the approach with the identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of OST. Based on the observations that alternations in LLO substrate structure and OST subunits activity differentially alter the systemic output of OST, we conclude that sequon recognition is a direct property of the catalytic subunit Stt3p, auxiliary subunits such as Ost3p and Ost6p extend the OST substrate range by modulating interfering pathways such as protein folding. In addition, our proteomics approach revealed a novel regulatory network that connects isoprenoid lipid biosynthesis and LLO substrate assembly.

Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae

“…Recently, N-glycoproteomics studies have been performed in the tissues of different organisms, including yeast [47][48], filamentous fungi [49][50], nematode [51], Drosophila [52], the plant Arabidopsis thaliana [53] and mammals [54][55][56]. Quantifying the N-glycoproteomes in the plant pathogenic fungus F. graminearum have revealed intensive glycosylation changes during exposure to fungicide: 774 sites in 406 proteins were identified, and the glycosylation level was found to be largely down-regulated upon fungicide treatment [50].…”

“…To generate GFP-fused constructs, the promoter and coding regions of Gls1 was amplified and fused with GFP, then cloned into pKN(S6 Table). In the resulting vectors, the GFP fusion constructs were expressed under the control of the native promoter [48]. These vectors were transformed to protoplasts of P131, the Δalg3 mutant or the Δgls1 mutant to obtain the GFP-tagged strains.…”

The consensus Nglyco-X-S/T motif and a previously unknown Nglyco-N -linked glycosylation are necessary for growth and pathogenicity ofPhytophthora

“…To identify sequence motifs that were generally associated with N-glycosylation sites, we examined a 21-amino acid sequence window surrounding the central Nglyco residues of each site using the web-based Motif-X program with a significance threshold of P < 0.000001. Previous research showed that the consensus sequence for N-glycosylation motif is Nglyco-X-S/T (X P) (10)(11)(12)(13). In this study, we found that most of the 248 N-glycosylation sites matched the Nglyco-X-S/T motif ( Fig.…”

“…Then, the oligosaccharyltransferase (OST) catalyzes the en bloc transfer of the LLO to the asparagine side chain of the acceptor polypeptides in the ER lumen (9,10). The OST selects polypeptides carrying an Nglyco-X-S/T (X P) motif and generates the N-glycosidic linkage between the side chain amide of asparagine and the LLO (9)(10)(11)(12). Moreover, other conserved N-glycosylation motifs have been identified through liquid chromatography -tandem mass spectrometry (LC-MS/MS).…”

Glycopeptide variable window SWATH for improved Data Independent Acquisition glycoprotein analysis

“…Oligosaccharyltransferase (OTase) catalyzes the attachment of the precursor oligosaccharide to selected Asn residues in glycosylation sequons (N-X-S/T; X¹P) in nascent polypeptides [14]. Mutations in ALG genes can cause altered glycan structures due to the accumulation and transfer of truncated glycans, and altered site-specific glycan occupancy due to inefficient transfer of these truncated glycans by OTase [15,16]. Further trimming and maturation of glycans take place in the ER and Golgi with various enzymes involved, leading to diverse N-glycan structures on mature glycoproteins [17,18].…”