Quantitative Profiling of N-linked Glycosylation Machinery in Yeast Saccharomyces cerevisiae
2017; Elsevier BV; Volume: 17; Issue: 1 Linguagem: Inglês
10.1074/mcp.ra117.000096
ISSN1535-9484
AutoresKristina Poljak, Nathalie Selevsek, Elsy M. Ngwa, Jonas Grossmann, Marie E. Losfeld, Markus Aebi,
Tópico(s)Biofuel production and bioconversion
ResumoAsparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N-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 Saccharomyces cerevisiae 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. Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N-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 Saccharomyces cerevisiae 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. Glycosylation is a fundamental part of life whose impact and intricacy increase with the complexity of the organism (1.Zielinska D.F. Gnad F. Schropp K. Wiśniewski J.R. Mann M. Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery.Mol. Cell. 2012; 46: 542-548Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Glycans affect protein folding, stability and degradation, they mediate interactions of all cells and regulate essential biological, chemical and physical processes (2.Varki A. Biological roles of oligosaccharides: all of the theories are correct.Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4981) Google Scholar). Asparagine linked N-glycosylation is an essential modification of proteins conserved among eukaryotes, archaea and some bacteria (3.Szymanski C.M. Wren B.W. Protein glycosylation in bacterial mucosal pathogens.Nat. Rev. Microbiol. 2005; 3: 225-237Crossref PubMed Scopus (338) Google Scholar). In eukaryotes, the reaction is catalyzed by oligosaccharyltransferase (OST) 1The abbreviations used are: OST, Oligosaccharyltransferase; N, Asparagine; ER, Endoplasmatic Reticulum; GlcNAc, N-acetyl-glucosamine; LLO, Lipid-linked oligosaccharide; PRM, Parallel reaction monitoring; SILAC, Stable isotope labelling with amino acids in cell culture. 1The abbreviations used are: OST, Oligosaccharyltransferase; N, Asparagine; ER, Endoplasmatic Reticulum; GlcNAc, N-acetyl-glucosamine; LLO, Lipid-linked oligosaccharide; PRM, Parallel reaction monitoring; SILAC, Stable isotope labelling with amino acids in cell culture. in the lumen of the endoplasmic reticulum (ER) (4.Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47-62Crossref PubMed Scopus (416) Google Scholar). On arrival of protein substrate to the lumen of the ER, the preassembled oligosaccharide is transferred en bloc from the lipid carrier dolichol pyrophosphate (Dol-PP) to asparagines in selected glycosylation sequons (N-X-S/T; X≠P) (4.Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47-62Crossref PubMed Scopus (416) Google Scholar, 5.Kelleher D.J. Banerjee S. Cura A.J. Samuelson J. Gilmore R. Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms.J. Cell Biol. 2007; 177: 29-37Crossref PubMed Scopus (39) Google Scholar). 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Hypoglycosylation of proteins will lead to the accumulation of unfolded proteins in the ER (ER stress) and to the activation of an unfolded protein response (UPR) pathway, in which transcriptional induction of UPR target genes allows the cell to adjust the capacity of protein folding in the ER (8.Chapman R. Sidrauski C. Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus.Annu. Rev. Cell Dev. Biol. 1998; 14: 459-485Crossref PubMed Scopus (204) Google Scholar, 9.Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation.Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar). N-linked glycosylation starts with the assembly of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide (LLO) on a Dol-PP carrier on the ER membrane (10.Helenius A. Aebi M. 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The LLO is completed by the action of three glucosyltransferases Alg6, Alg8 and Alg10. Unlike the cytoplasmic glycosyltransferases, lumenal ALG enzymes use Dol-P-bound sugars as donors (16.Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Glycosyltransferases: Structures, Functions, and Mechanisms.Annu. Rev. Biochem. 2008; 77: 521-555Crossref PubMed Scopus (1322) Google Scholar) and have a high LLO substrate specificity that ensures an assembly of an optimal OST LLO substrate that exposes the terminal α-1,2-linked glucose (17.Burda P. Aebi M. The ALG10 locus of Saccharomycescerevisiae 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). Because of sequential nature of LLO biosynthesis, deficiencies in ALG enzymes result in accumulation of LLO intermediates. Truncated LLO structures will still be transferred by the OST though with a lower efficiency, resulting in the appearance of hypoglycosylated protein substrates (12.Burda P. Aebi M. The dolichol pathway of N-linked glycosylation.Biochim. Biophys. Acta - Gen. Subj. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar). In higher eukaryotes, OST is a multiprotein complex consisting of different subunits (Stt3p, Ost1p, Ost5p, Wbp1p, Ost2p, Swp1p, Ost3p/Ost6p, and Ost4p in S. cerevisiae. Some protozoan, archaeal, and bacterial species have a single protein OST, homologous to Stt3p, the catalytic protein subunit of the eukaryotic OST (4.Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47-62Crossref PubMed Scopus (416) Google Scholar). Based on phylogenetic analysis, yeast STT3 is proposed to be a B type STT3 that is not associated with the translocon (18.Ruiz-Canada C. Kelleher D.J. Gilmore R. 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Several MS techniques for measuring N-linked glycosylation site occupancy in yeast have been described using different modes of operation for targeted glycopeptide analysis, such as selected reaction monitoring (SRM), sequential window acquisition of targeted fragment ions (SWAT), and data-independent techniques, such as sequential window acquisition of all theoretical fragment ion spectra (SWATH) (32.Yeo K.Y.B. Chrysanthopoulos P.K. Nouwens A.S. Marcellin E. Schulz B.L. High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity.Anal. Biochem. 2016; 510: 106-113Crossref PubMed Scopus (14) Google Scholar, 33.Zacchi L.F. Schulz B.L. SWATH-MS glycoproteomics reveals consequences of defects in the glycosylation machinery.Mol. Cell. Proteomics. 2016; 15: 2435-2447Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 34.Xu Y. Bailey U.M. Schulz B.L. 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In this paper, we describe a complementary method for the identification and relative quantification of N-linked glycosylation occupancy and protein abundance of yeast membrane and lumenal proteins using SILAC (stable isotope labeling by amino acids in cell culture) strategy combined with a PRM (parallel reaction monitoring) based MS method. The PRM technique is currently the most sensitive mode of targeted proteomics analyses for quantitative measurements in biological samples and is now being exploited for quantitative analysis of N-linked glycosylation (35.de Godoy L. Olsen J. de Souza G. Li G. Mortensen P. Mann M. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. - PubMed - NCBI.Genome Biol. 2006; 7: 50Crossref PubMed Scopus (231) Google Scholar, 36.Peterson A.C. Russell J.D. Bailey D.J. Westphall M.S. Coon J.J. Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics.Mol. Cell. Proteomics. 2012; 11: 1475-1488Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). The study aimed to define N-linked glycosylation occupancy of yeast proteins in distinct yeast mutants defective in the N-linked glycosylation pathway. SILAC coupled to PRM analysis was used to analyze quantitative changes in N-linked glycosylation occupancy. Each sample for MS analysis was generated by mixing two individual SILAC-labeled cell populations. Ribosomal proteins Rpl5 and Rsp1 were used as internal control to evaluate variability during mixing of light and heavy cells. All quantitative PRM experiments were performed in biological triplicates resulting in a total of 21 samples. Reproducibility of results was assessed by calculating coefficient of variation for each set of biological triplicates in Excel where > 83% (n = 62) of the data analyzed had CV value of < 20%. Statistical tests used to analyze significant differences indicated in the respective figure legends were performed using t test in Microsoft Excel. All yeast strains and plasmids used in this study are listed in supplemental Table S1 and S2. Standard yeast genetic techniques were used (37.Güldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. A new efficient gene disruption cassette for repeated use in budding yeast.Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1361) Google Scholar, 38.Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.Yeast. 1999; 15: 963-972Crossref PubMed Scopus (813) Google Scholar). All yeast strains were grown in an orbital shaker at 30 °C and 180 rpm to exponential phase (OD600 nm 1.0). For MS assay, cells were grown in appropriate synthetic drop-out (S.D.) medium (0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose with appropriate amino acid supplements) containing either 20 mg/L of light or heavy isotopes of arginine (13C6) and lysine (13C6-15N2) (Cambridge Isotope Laboratories). Cells were collected, frozen in liquid nitrogen and stored at −80 °C. For sterol inhibition assay, mid-log phase cultures of the wild-type yeast cells in light S.D. medium were exposed to miconazole (0.3 μg/ml, dissolved in DMSO; Janseen Geel) whereas the controls received an equivalent amount of DMSO. After 2.5 h, cells were washed once with ice cold 50 mm sodium acetate, 10 mm EDTA buffer (pH 4.5) before they were collected, frozen in liquid nitrogen and stored at −80 °C. Membrane proteins were prepared as described (39.Mueller S. Wahlander A. Selevsek N. Otto C. Ngwa E.M. Poljak K. Frey A.D. Aebi M. Gauss R. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.Mol. Biol. Cell. 2015; 26: 2596-2608Crossref PubMed Scopus (37) Google Scholar). In brief, yeast cells were lysed at 4 °C using glass beads, and the microsomal fraction was pelleted by centrifugation (16000 × g; 20 min), resuspended in 0.1 m Na2CO3, 1 mm EDTA, pH 11.3 buffer and pelleted again. Samples were resuspended in SDS buffer (2% SDS (w/v), 50 mm DTT, 0.1 m Tris HCl pH 7.6) and processed using the filter assisted sample preparation protocol (40.Wiœniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5097) Google Scholar). Membrane proteins were digested first with endopeptidase LysC (20 μg/ml; Wako Pure Chemical, Richmond, VA) at room temperature for 16 h followed by digestion with trypsin (20 μg/ml; Promega) at 37 °C for 4 h. For data-dependent acquisition (DDA)-based experiments in the discovery phase of PRM assay development, sample was enriched for glycopeptides using SPE ZIC-HILIC (SeQuant) column and collected in a single fraction as described (41.Neue K. Mormann M. Peter-Katalinic J. Pohlentz G. Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides.J. Proteome Res. 2011; 10: 2248-2260Crossref PubMed Scopus (73) Google Scholar). For PRM experiments protein digestion was directly followed by digestion of N-glycans with endo-β-N-acetylglucosaminidase H (EndoH; 500 U; New England Biolabs) in sodium citrate buffer (50 mm, pH 5.5) at 37 °C with agitation for 40 h. Peptides were desalted using Sep Pak C18 classic (Waters) and dried using speed vacuum. Desalted peptides were resuspended in ACN/H2O (3:97 (v/v)) with formic acid (FA; 0.1% (v/v)) and analyzed by LC-ESI-MS/MS. Samples were subjected to a Q Exactive HF mass spectrometer (Thermo Scientific) coupled to a nano EasyLC 1000 (Thermo Fisher Scientific) system and to an Acquity UPLC M-class System (Waters). Samples were loaded onto a self-made column (75 μm × 150 mm) packed with reverse-phase C18 material (ReproSil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch GmbH) when the EasyLC 1000 was coupled. ACQUITY UPLC M-Class column (75 μm × 150 mm) packed with reverse-phase C18 material (Waters HSS T3 100 C18, 1.8 μm) was used when the Acquity UPLC M-class system was coupled. Peptides were separated at a flow rate of 300 nL/min using a linear gradient of 1% to 35% solvent B (0.1% formic acid in acetonitrile) over 90 min, followed by an increase to 98% B over 2 min and held at 98% B for 5 min before returning to initial conditions of 1% B. In DDA-MS, the mass spectrometer was set to acquire full-scan MS spectra (300–1700 m/z) at a resolution of 60,000 after accumulation to an automated gain control (AGC) target value of 3e6 and a maximum injection time of 15 ms. Charge state screening was enabled and unassigned charge states and singly charged precursors were excluded. Ions were isolated using a quadrupole mass filter with a 1.2 m/z isolation window. A maximum injection time of 45 ms was set. HCD fragmentation was performed at a normalized collision energy (NCE) of 28%. Selected ions were dynamically excluded for 20 s. For PRM measurements, the Q Exactive HF performed MS1 scans (400–1200 m/z) followed by 12 MS/MS acquisitions in PRM mode. The full scan event was collected at a resolution of 60,000 (at m/z 400) and a AGC value of 3e6 and a maximum injection time of 120 ms. The PRM scan events used an Orbitrap resolution of 30,000 or 60,000, maximum fill time of 55 ms or 110 ms respectively, with an isolation width of 2 m/z and an AGC value of 2e5. HCD fragmentation was performed at a normalized collision energy (NCE) of 28% and MS/MS scans were acquired with a starting mass of m/z 120. Scan windows were set to 10 min for each peptide in the final PRM method to ensure the measurement of 6–10 points per LC peak per transition. All samples were analyzed using two PRM methods based on scheduled inclusion lists containing the 175 target precursor ions, including Biognosys iRT standard peptides, at an Orbitrap resolution of 30 000 and two PRM methods based on scheduled inclusion lists containing the 128 target precursor ions, including Biognosys iRT standard peptides (Biognosys AG, Zurich), at an Orbitrap resolution of 60,000 (supplemental Table S3). MS and MS/MS spectra generated from enriched and nonenriched glycopeptides extracts were converted to Mascot generic format (MGF) using Proteome Discoverer, v1.4 (Thermo Fisher Scientific, Bremen, Germany) using the automated rule based converter control (42.Barkow-Oesterreicher S. Türker C. Panse C. FCC – An automated rule-based processing tool for life science data.Source Code Biol. Med. 2013; 8: 3Crossref PubMed Scopus (12) Google Scholar). The MGFs were searched with Mascot Server v.2.5.1.3 (www.matrixscience.com) using the following parameters: a precursor ion mass tolerance of 15 ppm, product ion mass tolerance of 0.05 Dalton, enzyme specificity to trypsin and LysC and up to two missed cleavages were allowed, as variable modifications methionine oxidation and asparagine N-linked HexNAc (CID/HCD) glycosylation, and as fixed modification carbamidomethylation of cysteine, for the glycoenriched samples. Searches were made against the Saccharomyces cerevisiae reference proteome database (containing 6649 entries, downloaded 24/04/2012), concatenated to a reversed decoyed FASTA database and 261 common mass spectrometry protein contaminants. A Mascot score >20 and an expectation value 20 corresponding to the localization probability of > 99% were considered to identify correctly assigned glycosylation sites (43.Searle B.C. Scaffold: A bioinformatic tool for validating MS/MS-based proteomic studies.Proteomics. 2010; 10: 1265-1269Crossref PubMed Scopus (396) Google Scholar, 44.Vincent-Maloney N. Searle B.C. Turner M. Probabilistically Assigning Sites of Protein Modification with Scaffold PTM.J. Biomol. Tech. 2011; 22: S36Google Scholar). Majority of software used to visualize and examine MS/MS data, including Scaffold PTM software, do not discriminate among asparagines located in consensus glycosylation sequons, thus the localization score reported for some glycopeptides is 0. Such sites were accepted given that they only contain one asparagine as possible glycosylation site (corresponding to known glycosylation motif Asn-X-Ser/Thr) (supplemental Table S4). The Mascot search results (dat. files) were imported into the Skyline software (v2.6.0) (45.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: An open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2983) Google Scholar) and spectral libraries were built using the BiblioSpec algorithm (46.Frewen, B., and MacCoss, M. J., (2007) Using BiblioSpec for creating and searching tandem MS peptide libraries. Curr. Protoc. Bioinformatics Chapter 13, Unit 13.7,Google Scholar). Additionally, published spectral libraries (47.Selevsek N. Chang C.-Y. Gillet L.C. Navarro P. Bernhardt O.M. Reiter L. Cheng L.-Y. Vitek O. Aebersold R. Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH-MS.Mol. Cell. Proteomics. 2015; (10.1074/jbc.M114.604033)Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) were used to populate the final PRM assays. For glycopeptides in which ion libraries could
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