Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics
2019; Elsevier BV; Volume: 18; Issue: 7 Linguagem: Inglês
10.1074/mcp.ra118.001121
ISSN1535-9484
AutoresYoshiki Narimatsu, Hiren J. Joshi, Katrine T. Schjoldager, John Hintze, Adnan Halim, Catharina Steentoft, Rebecca Nason, Ulla Mandel, Eric Bennett, Henrik Clausen, Sergey Y. Vakhrushev,
Tópico(s)Galectins and Cancer Biology
ResumoMost proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats. Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats. O-glycosylation of the N-acetylgalactosamine (GalNAc)-type is one of the most abundant and diverse forms of protein glycosylation, and it is uniquely positioned in the secretory pathway to fine-tune protein function (1Bennett E.P. Mandel U. Clausen H. Gerken T.A. Fritz T.A. Tabak L.A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family.Glycobiology. 2012; 22: 736-756Crossref PubMed Scopus (531) Google Scholar, 2Schjoldager K.T. Clausen H. Site-specific protein O-glycosylation modulates proprotein processing - deciphering specific functions of the large polypeptide GalNAc-transferase gene family.Biochim. Biophys. Acta. 2012; 1820: 2079-2094Crossref PubMed Scopus (154) Google Scholar). GalNAc-type O-glycosylation (hereafter simply O-glycosylation) is controlled by many polypeptide GalNAc-transferase (GalNAc-T) 1The abbreviations used are:GalNAcN-acetylgalactosamineGalNAc-TGalNAc-transferaseKOknockoutSCSimpleCellWTwildtypeLDLRlow-density lipoprotein receptorLRFlipoprotein related proteinZFNzinc finger nuclease. isoenzymes encoded for by up to 20 distinct GALNT genes in mammals that catalyze the addition of GalNAc residues to select serine and threonine (and possibly tyrosine) residues. GalNAc-T isoenzymes have distinct, albeit partly overlapping, peptide substrate specificities and kinetic properties, they are differentially expressed in cells and tissues, and their expression patterns change during cellular maturation, differentiation, and malignant transformation (1Bennett E.P. Mandel U. Clausen H. Gerken T.A. Fritz T.A. Tabak L.A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family.Glycobiology. 2012; 22: 736-756Crossref PubMed Scopus (531) Google Scholar, 3Gill D.J. Tham K.M. Chia J. Wang S.C. Steentoft C. Clausen H. Bard-Chapeau E.A. Bard F.A. Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E3152-E3161Crossref PubMed Scopus (139) Google Scholar, 4Tran D.T. Ten Hagen K.G. Mucin-type O-glycosylation during development.J. Biol. Chem. 2013; 288: 6921-6929Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The repertoire of isoenzymes expressed in cells direct the positions and patterns of O-glycans found on proteins (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar), but our insight into the specific contributions of individual isoenzymes and the seemingly coordinated process leading to proficient O-glycosylation of both isolated and clustered glycosites is still highly limited. GalNAc-Ts are unique among metazoan glycosyltransferases in that they employ a C-terminal GalNAc-binding lectin domain to modulate the substrate specificity and properties of the catalytic domain, which is predicted to enable follow-up glycosylation in high-density O-glycosylation regions (6Hassan H. Reis C.A. Bennett E.P. Mirgorodskaya E. Roepstorff P. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities.J. Biol. Chem. 2000; 275: 38197-38205Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 7Revoredo L. Wang S. Bennett E.P. Clausen H. Moremen K.W. Jarvis D.L. Ten Hagen K.G. Tabak L.A. Gerken T.A. Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family.Glycobiology. 2016; 26: 360-376Crossref PubMed Scopus (54) Google Scholar). The lectin domains coordinate glycosylation of distant glycosites (± 8–10 residues of initial O-glycosites) with isoform-specific orientation (7Revoredo L. Wang S. Bennett E.P. Clausen H. Moremen K.W. Jarvis D.L. Ten Hagen K.G. Tabak L.A. Gerken T.A. Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family.Glycobiology. 2016; 26: 360-376Crossref PubMed Scopus (54) Google Scholar, 8Gerken T.A. Revoredo L. Thome J.J. Tabak L.A. Vester-Christensen M.B. Clausen H. Gahlay G.K. Jarvis D.L. Johnson R.W. Moniz H.A. Moremen K. The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation.J. Biol. Chem. 2013; 288: 19900-19914Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and recently the molecular basis for this has been shown to involve combined docking of the partially glycosylated substrate into the lectin and catalytic domain (9de Las Rivas M. Lira-Navarrete E. Daniel E.J.P. Companon I. Coelho H. Diniz A. Jimenez-Barbero J. Peregrina J.M. Clausen H. Corzana F. Marcelo F. Jimenez-Oses G. Gerken T.A. Hurtado-Guerrero R. The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences.Nat. Commun. 2017; 8: 1959Crossref PubMed Scopus (28) Google Scholar). Moreover, a subset of the GalNAc-T isoenzymes selectively or exclusively recognizes acceptor sites with GalNAc residues found immediately adjacent (10Kubota T. Shiba T. Sugioka S. Furukawa S. Sawaki H. Kato R. Wakatsuki S. Narimatsu H. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).J. Mol. Biol. 2006; 359: 708-727Crossref PubMed Scopus (98) Google Scholar), and structural studies demonstrate that the catalytic domain accommodates the first introduced GalNAc residue (10Kubota T. Shiba T. Sugioka S. Furukawa S. Sawaki H. Kato R. Wakatsuki S. Narimatsu H. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).J. Mol. Biol. 2006; 359: 708-727Crossref PubMed Scopus (98) Google Scholar, 11de Las Rivas M. Daniel E.J.P. Coelho H. Lira-Navarrete E. Raich L. Companon I. Diniz A. Lagartera L. Jimenez-Barbero J. Clausen H. Rovira C. Rovira C. Marcelo F. Corzana F. Gerken T.A. Hurtado-Guerrero R. Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.ACS Cent. Sci. 2018; 4: 1274-1290Crossref PubMed Scopus (28) Google Scholar). Most of our current understanding is, however, still based largely on in vitro analyses with synthetic peptide and glycopeptide substrates (12Gerken T.A. Jamison O. Perrine C.L. Collette J.C. Moinova H. Ravi L. Markowitz S.D. Shen W. Patel H. Tabak L.A. Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases.J. Biol. Chem. 2011; 286: 14493-14507Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Kong Y. Joshi H.J. Schjoldager K.T. Madsen T.D. Gerken T.A. Vester-Christensen M.B. Wandall H.H. Bennett E.P. Levery S.B. Vakhrushev S.Y. Clausen H. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.Glycobiology. 2015; 25: 55-65Crossref PubMed Scopus (77) Google Scholar). N-acetylgalactosamine GalNAc-transferase knockout SimpleCell wildtype low-density lipoprotein receptor lipoprotein related protein zinc finger nuclease. Recently, we developed a quantitative O-glycoproteomics strategy for sensitive mapping of contribution of individual GalNAc-Ts with identification of their nonredundant functions (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). This strategy is partly based on genetic simplification of the O-glycan structures produced in cells with the so-called "SimpleCell" strategy, where the second step in the biosynthesis of O-glycans is eliminated by knockout (KO) of the private chaperone Cosmc for the core1 synthase C1GalT1 (14Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (263) Google Scholar). This facilitates the lectin enrichment required for sensitive mass spectrometric identification and quantification of glycopeptides (15Levery S.B. Steentoft C. Halim A. Narimatsu Y. Clausen H. Vakhrushev S.Y. Advances in mass spectrometry driven O-glycoproteomics.Biochim. Biophys. Acta. 2015; 1850: 33-42Crossref PubMed Scopus (93) Google Scholar, 16Steentoft C. Bennett E.P. Clausen H. Glycoengineering of human cell lines using zinc finger nuclease gene targeting: SimpleCells with homogeneous GalNAc O-glycosylation allow isolation of the O-glycoproteome by one-step lectin affinity chromatography.Methods Mol. Biol. 2013; 1022: 387-402Crossref PubMed Scopus (24) Google Scholar). The strategy is further based on isogenic SimpleCells with KO of individual GALNT genes and comparative analysis of O-glycoproteomes using stable isotope dimethyl labeling of tryptic digests, lectin enrichment and sensitive mass spectrometry (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar, 17Boersema P.J. Foong L.Y. Ding V.M. Lemeer S. van Breukelen B. Philp R. Boekhorst J. Snel B. den Hertog J. Choo A.B. Heck A.J. In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.Mol. Cell Proteomics. 2010; 9: 84-99Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18Vakhrushev S.Y. Steentoft C. Vester-Christensen M.B. Bennett E.P. Clausen H. Levery S.B. Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells.Mol. Cell Proteomics. 2013; 12: 932-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In a previous study, we applied the strategy to human liver HepG2 cells targeting the two most abundantly expressed GALNT1 and T2 genes, and demonstrated that these two isoforms each serve a small unique subset of substrates, whereas the majority of the O-glycosylation capacity was predicted to be redundantly shared (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). Similarly, we found that the two close isoforms, GalNAc-T3 and T6, predicted to have highly similar functions selectively served a minor set of unique substrates (19Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy.J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Lavrsen K. Dabelsteen S. Vakhrushev S.Y. Levann A.M.R. Haue A.D. Dylander A. Mandel U. Hansen L. Frodin M. Bennett E.P. Wandall H.H. De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium.J. Biol. Chem. 2018; 293: 1298-1314Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These results are in good agreement with the past extensive in vitro studies performed (12Gerken T.A. Jamison O. Perrine C.L. Collette J.C. Moinova H. Ravi L. Markowitz S.D. Shen W. Patel H. Tabak L.A. Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases.J. Biol. Chem. 2011; 286: 14493-14507Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Kong Y. Joshi H.J. Schjoldager K.T. Madsen T.D. Gerken T.A. Vester-Christensen M.B. Wandall H.H. Bennett E.P. Levery S.B. Vakhrushev S.Y. Clausen H. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.Glycobiology. 2015; 25: 55-65Crossref PubMed Scopus (77) Google Scholar), indicating that the strategy can be used to explore the contribution of GalNAc-T isoenzymes to the O-glycoproteome. Here, we took the strategy a step further, and used precision gene engineering to develop a comprehensive panel of isogenic HEK293 SimpleCell (SC) and corresponding wildtype (WT) cell lines with different repertoires of the six major GALNT genes that are expressed, to explore regulation of the O-glycoproteome in detail in a human cell widely used for recombinant expression of glycoproteins. We confirm that a major part of the O-glycoproteome is covered by redundant functions of GalNAc-T isoenzymes, and we identify distinct O-glycosites that are regulated by specific GalNAc-T isoforms. We present the first evidence in cells that GalNAc-T7 and T10 serve to follow-up initial partial O-glycosylation in regions with clustered O-glycosites. Moreover, we find that the GalNAc-T11 isoform exhibits the most restricted nonredundant functions found so far for GalNAc-T isoforms, and T11 essentially only controls O-glycosylation of the low-density lipoprotein receptor (LDLR) and related proteins (LRPs) specifically in the C6XXXTC1 sequence motif in the linker regions of LDLR class A repeats. The panel of HEK293 WT isogenic cell lines with knockout of GALNTs presented here are useful for dissection and validation of biological functions of site-specific O-glycosylation. Gene targeting using Zinc finger nucleases (ZFNs) were performed in the HEK293SC cell line with COSMC KO established previously (14Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (263) Google Scholar) and in HEK293 WT. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 1% glutamax. ZFN GFP/Crimson fusion constructs were transfected by electroporation using Amaxa Nucleofector 2B system (Lonza, Switzerland), and GFP/Crimson double positive cells were enriched by FACS sorting. After 1–2 weeks of culture bulk sorted cell populations were further single cell sorted for GFP/Crimson negative population. KO clones with frameshift mutations were identified by Indel Detection Amplification Assay (IDAA), as previously described (21Yang Z. Steentoft C. Hauge C. Hansen L. Thomsen A.L. Niola F. Vester-Christensen M.B. Frodin M. Clausen H. Wandall H.H. Bennett E.P. Fast and sensitive detection of indels induced by precise gene targeting.Nucleic Acids Res. 2015; 43: e59Crossref PubMed Scopus (115) Google Scholar). All ZFN target sites and the primers used for IDAA are listed in supplemental Table S1. All selected clones were further verified by TOPO cloning and Sanger sequencing for characterization of mutations introduced. HEK293 cells were grown on sterile cover slides and fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 3% BSA. Fixed cells were incubated with monoclonal antibodies (MAbs) to GalNAc-T1 (4D8), T2 (4C4), T3 (2D10), T7 (8B8), or T11 (1B2) (22Mandel U. Hassan H. Therkildsen M.H. Rygaard J. Jakobsen M.H. Juhl B.R. Dabelsteen E. Clausen H. Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family.Glycobiology. 1999; 9: 43-52Crossref PubMed Scopus (105) Google Scholar, 23Schwientek T. Bennett E.P. Flores C. Thacker J. Hollmann M. Reis C.A. Behrens J. Mandel U. Keck B. Schafer M.A. Haselmann K. Zubarev R. Roepstorff P. Burchell J.M. Taylor-Papadimitriou J. Hollingsworth M.A. Clausen H. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila.J. Biol. Chem. 2002; 277: 22623-22638Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) overnight at 4 °C, followed by FITC-conjugated rabbit anti-mouse Ig (Dako) for 45 min, and mounted with ProLong Gold antifade reagent (DAPI) (Invitrogen, Carlsbad, CA). Fluorescence microscopy was performed using a Zeiss Axioskop 2 plus with an AxioCam MR3 (Axioskop, Zeiss, Oberkochen, Germany). The sample preparation for O-glycoproteomics was previously described (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). Briefly, packed cell pellets (0.5 ml) from HEK293SC ± GALNT KO were lysed with 0.1% RapiGest (Waters Corporation, Maliford, MA) or 200 ml cell culture supernatant were applied to a 1 ml Viscia Villosa lectin (VVA) lectin agarose (Vector Laboratories, Inc., Burlingame, CA) column and bound glycoproteins were eluted by heating a slurry of the agarose in 0.02% RapiGest. Cleared lysates and secretome samples were heated for 10 min at 80 °C, followed by reduction (5 mm DTT, 60 °C, 30 min), alkylation (10 mm iodoacetamide, RT, 30 min), and digestion with trypsin (25 μg/sample) (Roche, Hvidovre, Denmark) (37 °C, ON). Cleared acidified digests were loaded onto equilibrated SepPak C18 cartridges (Waters) followed by 0.1% TFA wash and labeled on-column by adding 30 mm NaBH3CN and 0.2% formaldehyde (light-labeling, L) or 30 mm NaBH3CN and 0.2% d-formaldehyde (medium-labeling, M). Columns were washed using 0.1% FA and eluted with 50% MeOH in 0.1% FA. Labeled GalNAc-glycopeptides were separated from nonglycosylated peptides using VVA-agarose LWAC as previously described (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). The glycopeptide quantification based on M/L isotope labeled doublet ratios was evaluated to estimate a meaningful cut-off ratio for substantial changes (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). The labeled glycopeptides produced doublets with varying ratios of the isotopic ions as well as a significant number of single precursor ions without evidence of ion pairs. Labeled samples from HEK293SC and HEK293SC with KO of individual GALNT genes were mixed 1:1 and subjected to LWAC separation. The distribution of labeled peptides from the LWAC flow-through showed that the quantitated peptide M/L ratios were normally distributed with 99% falling within ±1 (Log10). We selected doublet with less/more than −1/+1 (Log10) value and singlets as candidates for isoform-specific O-glycosylation events. LWAC fractions most enriched in glycopeptides were pooled together, dried by vacuum centrifugation, reconstituted in IPG rehydration buffer, and submitted to IEF fractionation as previously described (18Vakhrushev S.Y. Steentoft C. Vester-Christensen M.B. Bennett E.P. Clausen H. Levery S.B. Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells.Mol. Cell Proteomics. 2013; 12: 932-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Isoelectric focusing (IEF) was performed by a 3100 OFFGEL fractionator (Agilent Technologies, La Jolla, CA) using pH 3–10 strips (GE Healthcare, Hertfodshire, UK) 12 fractions were collected and desalted by custom Stage Tips (C18 sorbent from Empore 3 m) and submitted to LC-MS/MS analysis. EASY-nLC 1000 UHPLC (Thermo Scientific, Odense, DK) interfaced via nanoSpray Flex ion source to an LTQ-Orbitrap Velos Pro spectrometer (Thermo Scientific, San Jose, CA) was used for the glycoproteomic study. The nLC was operated in a single analytical column set up with PicoFrit Emitters (New Objectives, 75 μm inner diameter) packed with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 1.9-μm particle size, 19–21 cm column length). Each sample was injected onto the column and eluted with either a 2 h gradient (2–20% B in 95 min, 20–80% B in 10 min, and 80% B for 15 min) or a 4 h gradient (2–20% B in 215 min, 20–80% B in 10 min, and 80% B for 15 min) at 200 nL/min (Solvent A: 100% H2O; Solvent B: 100% acetonitrile; both containing 0.1% (v/v) formic acid). A precursor MS1 scan (m/z 350–1,700) of intact peptides was acquired in the Orbitrap at a nominal resolution setting of 30,000. The five most abundant multiply charged precursor ions in the MS1 spectrum at a minimum MS1 signal threshold of 50,000 was triggered for sequential Orbitrap HCD-MS2 and ETD-MS2 (m/z of 100–2,000). MS2 spectra were acquired at a resolution of 7,500 for HCD MS2 and 15,000 for ETD MS2. Activation times were 30 and 200 ms for HCD and ETD fragmentation, respectively; isolation width was 4 mass units, and 1 microscan was collected for each spectrum. Automatic gain control targets were 1,000,000 ions for Orbitrap MS1 and 100,000 for MS2 scans, and the automatic gain control for fluoranthene ion used for ETD was 300,000. Supplemental activation (20%) of the charge-reduced species was used in the ETD analysis to improve fragmentation. Dynamic exclusion for 60 s was used to prevent repeated analysis of the same components. Polysiloxane ions at m/z 445.12003 were used as a lock mass in all runs. The mass spectrometry glycoproteomics raw data and annotated spectra have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) (24Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2071) Google Scholar) via the PRIDE partner repository with the data set identifier PXD009955. The total number of samples (WT & KO pair) analyzed and described was 14. Each sample was represented by 12 IEF fractions. Each fraction was analyzed one time and no replicate analyses were performed because this is an exploratory study to identify targets for further validation. Median quantification ratios were calculated for each identified glycopeptide species. Data processing was performed using Proteome Discoverer 1.4 software (Thermo Scientific) using Sequest HT Node as previously described (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). Briefly, all spectra were initially searched with trypsin full cleavage specificity, filtered according to the confidence level (medium, low and unassigned) and further searched with the semi-specific enzymatic cleavage. The maximum number of missed cleavage sites was set to 2. In all cases the precursor mass tolerance was set to 6 ppm and fragment ion mass tolerance to 20 mmu. Carbamidomethylation on cysteine residues was used as a fixed modification. Methionine oxidation and HexNAc attachment to serine, threonine and tyrosine were used as variable modifications for ETD MS2. All HCD MS2 were preprocessed as described (25Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (909) Google Scholar) and searched under the same conditions mentioned above using only methionine oxidation as variable modification. All spectra were searched against a concatenated forward/reverse human-specific database (UniProt, January 2013, containing 20,232 canonical entries and another 251 common contaminants) using a target false discovery rate (FDR) of 1%. FDR was calculated using target decoy PSM validator node. The resulting list was filtered to include only peptides with glycosylation as a modification. Glycopeptide M/L ratios were determined using dimethyl 2plex method as previously described (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). The cellular repertoire of GalNAc-T isoforms determines the O-glycosylation capacity and the part of the cellular proteome that undergoes O-glycosylation (5Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO reports. 2015; 16: 1713-1722Crossref PubMed Scopus (80) Google Scholar). The repertoire of expressed GALNTs in HEK293 cells was analyzed by RNAseq as well as by immunocytochemisty (ICC) for isoforms for which validated monoclonal antibodies (mAbs) were available (Fig. 1A and supplemental Fig. S1). RNAseq analysis indicated that GALNT1, T2, T3, T7, T10, and T11 were the predominant isoforms expressed, whereas GALNT4, T6, T12, T13, T16, and T18 were barely detectable. Expression of the former isoforms were confirmed by ICC with specific mAbs, and for the latter group no detectable expres
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