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• W2336846619 abstract "Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro- and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3Δ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation. Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro- and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3Δ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation. Protein glycosylation is a highly conserved co- and post-translocational modification of proteins that influences protein folding, stability, solubility, and function (1Schulz B.L. Beyond the sequon: sites of N-glycosylation.in: Petrescu S. Glycosylation. Intech, Rijeka, Croatia2012: 21-40Google Scholar, 2Schwarz F. Aebi M. Mechanisms and principles of N-linked protein glycosylation.Curr. Opin. Struct. Biol. 2011; 21: 576-582Crossref PubMed Scopus (476) Google Scholar). N-glycosylation of Asparagine (Asn) 1The abbreviations used are:AsnAsparagineEREndoplasmic ReticulumGlcGlucoseGlcNAcN-acetyl-glucosamineLLOLipid-linked oligosaccharideManMannoseOTaseOligosaccharyltransferaseSerSerineSWATHSequential window acquisition of all theoretical fragment ion spectraThrThreonine. residues occurs in eukaryota, archaea, and some bacteria, although the biosynthetic pathways and glycan structures are diverse in these organisms (3Spiro R.G. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.Glycobiology. 2002; 12: 43R-56RCrossref PubMed Scopus (1066) Google Scholar). In eukaryotes, nascent polypeptides in the Endoplasmic Reticulum (ER) are the protein acceptor substrates for N-glycosylation, as folded proteins cannot be efficiently N-glycosylated and N-glycosylation is critical for efficient protein folding. After protein folding, glycan structures can be further truncated or extended by Golgi-resident glycosyltransferases. Glycan biosynthesis is inherently inefficient, resulting in structural diversity of mature glycoproteins. Diversity in the presence or absence of glycans on glycoproteins is termed macroheterogeneity, whereas diversity in the structures of glycans at a specific site is termed microheterogeneity. This structural diversity is key for the regulation of many biological functions of glycoproteins. Asparagine Endoplasmic Reticulum Glucose N-acetyl-glucosamine Lipid-linked oligosaccharide Mannose Oligosaccharyltransferase Serine Sequential window acquisition of all theoretical fragment ion spectra Threonine. Eukaryotic N-glycosylation is catalyzed in the ER lumen, where the enzyme oligosaccharyltransferase (OTase) transfers donor glycans en bloc from a dolichol pyrophosphate (DolP) carrier (Lipid-linked oligosaccharide; LLO) to selected Asn in nascent polypeptides. Approximately 80% of secretory proteins are N-glycosylated, making this modification of high fundamental, medical, and biotechnological relevance (4Aebi M. N-linked protein glycosylation in the ER.Biochim. Biophys. Acta. 2013; 1833: 2430-2437Crossref PubMed Scopus (455) Google Scholar, 5Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim. Biophys. Acta. 1999; 1473: 4-8Crossref PubMed Scopus (1492) Google Scholar). In Bakers' yeast Saccharomyces cerevisiae, the LLO is synthesized in a defined, sequential, stepwise process by a series of enzymes integral or attached to the ER membrane: Alg1–14 (Fig. 1A) (6Burda P. Aebi M. The dolichol pathway of N-linked glycosylation.Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (497) Google Scholar). LLO biosynthesis begins on the cytosolic face of the ER membrane, where Alg7 and the Alg13-Alg14 complex transfer the first and second N-acetylglucosamine (GlcNAc) residues to DolP. This is followed by the sequential addition of Mannose (Man) residues by Alg1, Alg2, and Alg11 to synthesize the Man5GlcNAc2 structure. This LLO is then flipped into the ER lumen by a process requiring Rft1 (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein.Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar). Man residues are then successively added by Alg3, Alg9, Alg12, and again Alg9 to construct the Man9GlcNAc2 structure (Fig. 1A). The LLO structure is completed by addition of three Glucose (Glc) residues to branch A through the sequential action of Alg6, Alg8, and Die2/Alg10 (Fig. 1A). This final Glc3Man9GlcNAc2 structure is the preferred LLO donor transferred to proteins by OTase, compared with biosynthetic intermediates. Because of the sequential nature of LLO biosynthesis, defects in LLO biosynthesis because of deficiency of an Alg enzyme result in accumulation of the precursor LLO structure (8Huffaker T.C. Robbins P.W. Yeast mutants deficient in protein glycosylation.Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar, 9Burda P. Aebi M. The dolichol pathway of N-linked glycosylation.Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar, 10Cipollo J.F. Trimble R.B. The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus.Glycobiology. 2002; 12: 749-762Crossref PubMed Scopus (19) Google Scholar). For instance, in alg6Δ yeast the unglucosylated Man9GlcNAc2 LLO structure accumulates and is transferred to protein (11Reiss G. te, Heesen S. Zimmerman J. Robbins P.W. Aebi M. Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway.Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar). Importantly, OTase transfers these truncated LLOs to proteins with reduced efficiency, resulting in hypoglycosylation of diverse proteins (11Reiss G. te, Heesen S. Zimmerman J. Robbins P.W. Aebi M. Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway.Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar). In particular, the terminal α1,2 Glc on the A branch of the LLO is critical for efficient in vivo glycosylation (12Burda P. Aebi M. The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation.Glycobiology. 1998; 8: 455-462Crossref PubMed Scopus (93) Google Scholar). The Man content of the B and C branches also influences OTase function (13Izquierdo L. Mehlert A. Ferguson M.A. The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases.Glycobiology. 2012; 22: 696-703Crossref PubMed Scopus (18) Google Scholar). Thus, although the glycan normally transferred by OTase has a canonical structure, OTase can transfer a variety of truncated glycans to polypeptides. This capability of OTase can increase the glycan heterogeneity of mature proteins and ensures that the essential process of protein glycosylation can continue even with perturbation of the glycan biosynthetic pathway. OTase catalyzes the transfer of glycans from LLO to selected Asn residues in nascent polypeptides. Asn are glycosylated with high efficiency if they are located in glycosylation sequons (NxS/T; x≠P), as this is the peptide acceptor-binding motif of Stt3, the catalytic subunit of OTase (14Lizak C. Gerber S. Numao S. Aebi M. Locher K.P. X-ray structure of a bacterial oligosaccharyltransferase.Nature. 2011; 474: 350-355Crossref PubMed Scopus (277) Google Scholar). 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 (15Schulz B.L. Aebi M. Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 16Schwarz M. Knauer R. Lehle L. Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit.FEBS Lett. 2005; 579: 6564-6568Crossref PubMed Scopus (44) Google Scholar, 17Knauer R. Lehle L. The oligosaccharyltransferase complex from Saccharomyces cerevisiae. Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex.J. Biol. Chem. 1999; 274: 17249-17256Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 18Jamaluddin M.F. Bailey U.M. Schulz B.L. Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.Mol. Cell Proteomics. 2014; 13: 3286-3293Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The function of the other OTase subunits is less well defined. However, lack of any of the nonessential subunits or mutations in essential subunits leads to inefficient N-glycosylation of diverse glycoproteins (15Schulz B.L. Aebi M. Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 19te Heesen S. Knauer R. Lehle L. Aebi M. Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.EMBO J. 1993; 12: 279-284Crossref PubMed Scopus (106) Google Scholar, 20Reiss G. te Heesen S. Gilmore R. Zufferey R. Aebi M. A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro.EMBO J. 1997; 16: 1164-1172Crossref PubMed Scopus (63) Google Scholar). The structures of N-glycans are substantially processed after transfer to protein. Immediately after the glycan is transferred to nascent polypeptides by OTase, the terminal α1,2 Glc is trimmed by Glucosidase I, whereas the remaining Glc residues and some Man residues are removed during protein folding (21Zacchi L.F. Caramelo J.J. McCracken A.A. Brodsky J.L. Endoplasmic Reticulum Associated Degradation and Protein Quality Control.in: Stahl R. B. a. P. The Encyclopedia of Cell Biol. 1st Ed. Academic Press, Waltham, MA2016: 596-611Crossref Scopus (6) Google Scholar). N-glycans are further modified in a protein-, cell-, and species-specific manner as glycoproteins progress through the Golgi Complex. In yeast this results in high mannose N-glycans with extended polymannose structures (22Fabre E. Hurtaux T. Fradin C. Mannosylation of fungal glycoconjugates in the Golgi apparatus.Curr. Opin. Microbiology. 2014; 20: 103-110Crossref PubMed Scopus (15) Google Scholar, 23Nakayama K. Nagasu T. Shimma Y. Kuromitsu J. Jigami Y. OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides.EMBO J. 1992; 11: 2511-2519Crossref PubMed Scopus (244) Google Scholar). Despite the critical function of the presence and structure of N-glycans on key aspects of protein maturation, N-glycosylation by the OTase can proceed even with immature LLO structures. Remarkably, yeast is viable in the absence of genes encoding several Alg enzymes and OTase subunits (9Burda P. Aebi M. The dolichol pathway of N-linked glycosylation.Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar). However, many reports have shown that these mutations result in inefficient glycosylation of diverse proteins (8Huffaker T.C. Robbins P.W. Yeast mutants deficient in protein glycosylation.Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar, 11Reiss G. te, Heesen S. Zimmerman J. Robbins P.W. Aebi M. Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway.Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar, 12Burda P. Aebi M. The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation.Glycobiology. 1998; 8: 455-462Crossref PubMed Scopus (93) Google Scholar, 20Reiss G. te Heesen S. Gilmore R. Zufferey R. Aebi M. A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro.EMBO J. 1997; 16: 1164-1172Crossref PubMed Scopus (63) Google Scholar, 24Bailey U.M. Jamaluddin M.F. Schulz B.L. Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins.J. Proteome Res. 2012; 11: 5376-5383Crossref PubMed Scopus (39) Google Scholar, 25Frank C.G. Aebi M. ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis.Glycobiology. 2005; 15: 1156-1163Crossref PubMed Scopus (51) Google Scholar, 26Runge K.W. Robbins P.W. A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues.J. Biol. Chem. 1986; 261: 15582-15590Abstract Full Text PDF PubMed Google Scholar). Here, we describe a simple, semi-quantitative, automated, and sensitive SWATH-mass spectrometry method to simultaneously measure macro- and microheterogeneity of diverse glycoproteins. We use this method to analyze the cell wall glycoproteome of the complete set of viable yeast deletion mutants in N-glycan biosynthesis to understand how OTase activity is affected by sub-optimal glycan donor structure at a systems level. All yeast strains used in this study derive from the BY4741 wild-type strain and were obtained from the genome deletion collection (Open Biosystems) (Table I). Yeast strains were grown in YPAD medium (1% yeast extract, 2% dextrose, 2% Bactopeptone) at 30 °C.Table IStrains used in this studyNameGenotypeSourceBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Open Biosystemsalg3ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 alg3Δ∷KANMXOpen Biosystemsalg6ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 alg6Δ∷KANMXOpen Biosystemsalg8ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 alg8Δ∷KANMXOpen Biosystemsalg9ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 alg9Δ∷KANMXOpen Biosystemsdie2ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 alg10Δ∷KANMXOpen Biosystemsalg12ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 alg12Δ∷KANMXOpen Biosystemsost3ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ost3Δ∷KANMXOpen Biosystemsost5ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ost5Δ∷KANMXOpen Biosystemsost6ΔMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ost6Δ∷KANMXOpen Biosystems Open table in a new tab Yeast were grown in 50 ml YPAD in an orbital shaker at 30 °C and 200 rpm, harvested at mid-log phase (OD600 nm 1.0) by centrifugation, and frozen at −20 °C. Proteins covalently linked to the polysaccharide cell wall were prepared following previously published protocols (15Schulz B.L. Aebi M. Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), but excluding endoglycosidase treatment. Briefly, cells were completely lysed, cysteines were reduced and alkylated with acrylamide, the insoluble cell wall polysaccharide with covalently attached proteins was thoroughly washed in strongly denaturing conditions, and proteins were digested with trypsin. Peptides and glycopeptides were desalted using C18 ZipTips (Millipore) prior to analysis by LC-ESI-MS/MS. Desalted peptides were analyzed by LC-ESI-MS/MS using a Prominence nanoLC system (Shimadzu) and TripleTof 5600 mass spectrometer with a Nanospray III interface (SCIEX) as previously described (27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Crossref PubMed Scopus (61) Google Scholar). Peptides were separated with buffer A (1% acetonitrile and 0.1% formic acid) and buffer B (80% acetonitrile with 0.1% formic acid) with a gradient of 10–60% buffer B over 45 min. Gas and voltage setting were adjusted as required. For information-dependent acquisition (IDA), an MS TOF scan from m/z of 350–1800 was performed for 0.5 s followed by IDA of MS/MS in high sensitivity mode with automated CE selection of the top 20 peptides from m/z of 40–1800 for 0.05 s per spectrum and dynamic exclusion of peptides for 5 s after 2 selections. Identical LC conditions were used for SWATH-MS, with an MS-TOF scan from an m/z of 350–1800 for 0.05 s followed by high-sensitivity information- independent acquisition with 26 m/z isolation windows with 1 m/z window overlap each for 0.1 s across an m/z range of 400–1250. Collision energy was automatically assigned by the Analyst software (SCIEX) based on m/z window ranges. Peptide identification was performed essentially as previously described (27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Crossref PubMed Scopus (61) Google Scholar) using ProteinPilot 4.1 (SCIEX), searching the UniProt database (downloaded from http://uniprot.org on 15 January 2015; 16,818,973 sequences) with standard settings: sample type, identification; cysteine alkylation, acrylamide; instrument, TripleTof 5600; species yeast; ID focus, biological modifications; enzyme, trypsin; Search effort, thorough ID. False discovery rate analysis using ProteinPilot was performed on all searches with limits of 99% identification confidence and 1% local false discovery rate. The ProteinPilot search results were used as ion libraries for SWATH analyses. For glycopeptide analysis, ion libraries were manually created for each possible glycopeptide using fragment ions from the nonglycosylated version of each peptide and parent masses corresponding to various glycan structures ranging from GlcNAc2 to Man15GlcNAc2. Ion libraries used are included as Supporting Information (supplemental Table S1). The abundance of peptides was measured using PeakView Software with standard settings, summing the integrated areas of up to six fragment ions per peptide. Protein abundance was measured using the sum of the abundances of up to six peptides per protein. The accuracy of peak selection by PeakView was manually confirmed in each sample for all sequon-containing peptides. When detected, different charge states of sequon-containing peptides were considered independently. Glycopeptides were typically detected at +1 charge state relative to their corresponding unglycosylated version. Macroheterogeneity was measured by the fraction of the summed abundance of all glycosylated forms of a peptide to the summed abundance of all glycosylated and unglycosylated forms of that peptide. Microheterogeneity was measured by the fraction of a given glycosylated form of a peptide to the summed abundance of all glycosylated forms of that peptide. This allowed comparison of microheterogeneity between yeast strains independently of macroheterogeneity. All analyses were performed in biological triplicate. Statistical analyses for SWATH proteomics were performed using MSStats in R (27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Crossref PubMed Scopus (61) Google Scholar). Mass spectrometry data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD003091. All experiments using the wild-type strain as control and the 9 mutants (Table I) were performed in biological triplicates, as in previously published studies (15Schulz B.L. Aebi M. Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24Bailey U.M. Jamaluddin M.F. Schulz B.L. Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins.J. Proteome Res. 2012; 11: 5376-5383Crossref PubMed Scopus (39) Google Scholar, 27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Crossref PubMed Scopus (61) Google Scholar). Statistical analyses for SWATH-MS cell wall proteomics were performed using MSStats in R (27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Crossref PubMed Scopus (61) Google Scholar, 28Choi M. Chang C.Y. Clough T. Broudy D. Killeen T. MacLean B. Vitek O. MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments.Bioinformatics. 2014; 30: 2524-2526Crossref PubMed Scopus (518) Google Scholar). Statistical analysis of differences in micro- and macroheterogeneity were performed using t test in Microsoft excel. Comparisons with p < 0.05 were considered significant. N-glycosylation is an enzymatic process catalyzed by the multiprotein subunit OTase enzyme. OTase displays preferences both for the acceptor protein to be glycosylated and the LLO glycan donor to be transferred to the protein (15Schulz B.L. Aebi M. Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 29Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (416) Google Scholar). Our goal was to quantify how differences in LLO structure affect the ability of OTase to glycosylate substrates in vivo and identify the repercussions of these changes on glycan macro- and microheterogeneity in mature cell wall proteins. To generate different LLO structures in vivo we utilized the full set of viable mutants in Alg enzymes responsible for the ER lumenal portion of the LLO biosynthetic pathway (Fig. 1A). The strains selected carry single mutations in genes encoding the ER lumenal mannosyltransferases (Alg3, Alg9, and Alg12) and glucosyltransferases (Alg6, Alg8, and Die2/Alg10) (Fig. 1A). Lack of these enzymes causes accumulation of the LLO precursor substrate of the enzyme, which is inefficiently transferred to proteins by OTase (8Huffaker T.C. Robbins P.W. Yeast mutants deficient in protein glycosylation.Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar, 11Reiss G. te, Heesen S. Zimmerman J. Robbins P.W. Aebi M. Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway.Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar, 12Burda P. Aebi M. The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation.Glycobiology. 1998; 8: 455-462Crossref PubMed Scopus (93) Google Scholar, 25Frank C.G. Aebi M. ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis.Glycobiology. 2005; 15: 1156-1163Crossref PubMed Scopus (51) Google Scholar, 26Runge K.W. Robbins P.W. A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues.J. Biol. Chem. 1986; 261: 15582-15590Abstract Full Text PDF PubMed Google Scholar, 30Burda P. Jakob C.A. Beinhauer J. Hegemann J.H. Aebi M. Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.Glycobiology. 1999; 9: 617-625Crossref PubMed Scopus (80) Google Scholar). We focused our analysis on proteins covalently linked to the yeast cell wall, a well-defined subcellular fraction enriched in glycoproteins that has been previously extensively characterized by MS (glyco)proteomics (15Schulz B.L. Aebi M. Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24Bailey U.M. Jamaluddin M.F. Schulz B.L. Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins.J. Proteome Res. 2012; 11: 5376-5383Crossref PubMed Scopus (39) Google Scholar, 27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Crossref PubMed Scopus (61) Google Scholar, 31Yin Q.Y. de Groot P.W. de Jong L. Klis F.M. de Koster C.G. Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae.FEMS Yeast Res. 2007; 7: 887-896Crossref PubMed Scopus (27) Google Scholar). To measure glycan macro- and microheterogeneity we designed and used a novel SWATH-MS glycoproteomic workflow to measure cell wall protein site-specific glycan structure and occupancy, and cell wall proteome abundance. The structure of glycans on mature proteins can have profound impacts on glycoproteins' activity and stability. To be able to directly measure site-specific glycan occupancy and structure on peptides in a complex mixture we employed a variation of our previously published SWATH-MS glycoproteomic protocol (27Xu Y. Bailey U.M. Schulz B.L. Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS.Proteomics. 2015; 15: 2177-2186Cr" @default.
• W2336846619 created "2016-06-24" @default.
• W2336846619 creator A5062804481 @default.
• W2336846619 creator A5080285308 @default.
• W2336846619 date "2016-07-01" @default.
• W2336846619 modified "2023-10-13" @default.
• W2336846619 title "SWATH-MS Glycoproteomics Reveals Consequences of Defects in the Glycosylation Machinery" @default.
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