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Emerging Paradigms for the Initiation of Mucin-type Protein O-Glycosylation by the Polypeptide GalNAc Transferase Family of Glycosyltransferases*

  • Thomas A. Gerken
    Correspondence
    To whom correspondence should be addressed: Dept. of Pediatrics, Case Western Reserve University, School of Medicine, BRB 823, 2109 Adelbert Rd., Cleveland, OH 44104-4948. Tel.: 216-368-4556; Fax: 216-368-4223;
    Affiliations
    From the Departments of Pediatrics (W. A. Bernbaum Center for Cystic Fibrosis Research),

    Departments of Biochemistry,

    Chemistry, and
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  • Oliver Jamison
    Affiliations
    From the Departments of Pediatrics (W. A. Bernbaum Center for Cystic Fibrosis Research),
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  • Author Footnotes
    2 Present address: Meridian Life Science Inc., 5171 Wilfong Rd., Memphis, TN 38134.
    Cynthia L. Perrine
    Footnotes
    2 Present address: Meridian Life Science Inc., 5171 Wilfong Rd., Memphis, TN 38134.
    Affiliations
    Chemistry, and
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  • Jeremy C. Collette
    Affiliations
    From the Departments of Pediatrics (W. A. Bernbaum Center for Cystic Fibrosis Research),
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  • Helen Moinova
    Affiliations
    Departments of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106 and
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  • Lakshmeswari Ravi
    Affiliations
    Departments of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106 and
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  • Sanford D. Markowitz
    Affiliations
    Departments of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106 and
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  • Author Footnotes
    3 Present address: Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, NCI, National Institutes of Health, Frederick, MD 21702.
    Wei Shen
    Footnotes
    3 Present address: Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, NCI, National Institutes of Health, Frederick, MD 21702.
    Affiliations
    the Section on Biological Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892
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  • Author Footnotes
    4 Present address: Dept. of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287.
    Himatkumar Patel
    Footnotes
    4 Present address: Dept. of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287.
    Affiliations
    the Section on Biological Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892
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  • Lawrence A. Tabak
    Affiliations
    the Section on Biological Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants CA78834 (to T. A. G.), P30-CA43703, and CA130901 (to S. D. M.) from the NCI and NIDDK intramural program grant (to L. A. T.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental “Experimental Procedures,” Tables S1–S4, Figs. S1–S3, and additional references.
    3 Present address: Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, NCI, National Institutes of Health, Frederick, MD 21702.
    4 Present address: Dept. of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287.
    2 Present address: Meridian Life Science Inc., 5171 Wilfong Rd., Memphis, TN 38134.
Open AccessPublished:February 24, 2011DOI:https://doi.org/10.1074/jbc.M111.218701
      Mammalian mucin-type O-glycosylation is initiated by a large family of ∼20 UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer α-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the −1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.

      Introduction

      Mucin-type O-glycosylation is one of the most common post-translational modifications of secreted and membrane-associated proteins. Glycoproteins containing O-glycosylated mucin domains serve many important biological roles chiefly because of their unique biophysical and structural properties that include an extended peptide conformation and robust resistance to proteases. Consequently, glycoproteins containing O-glycosylated mucin domains function in the protection of the cell surface, the modulation of cell-cell interactions, in the inflammatory and immune response, in metastasis and tumorigenesis, and in protein sorting, targeting, and turnover (for examples see Refs.
      • Tian E.
      • Ten Hagen K.G.
      ,
      • Tabak L.A.
      ,
      • Kato K.
      • Takeuchi H.
      • Kanoh A.
      • Miyahara N.
      • Nemoto-Sasaki Y.
      • Morimoto-Tomita M.
      • Matsubara A.
      • Ohashi Y.
      • Waki M.
      • Usami K.
      • Mandel U.
      • Clausen H.
      • Higashi N.
      • Irimura T.
      ,
      • Zhang L.
      • Ten Hagen K.G.
      ,
      • Lee S.H.
      • Yu S.Y.
      • Nakayama J.
      • Khoo K.H.
      • Stone E.L.
      • Fukuda M.N.
      • Marth J.D.
      • Fukuda M.
      ). It is also likely that such O-glycosylated domains may further present a molecular code for the specific recognition of additional binding partners, enzymes, or even other glycosyltransferases. In some instances, specific mucin-type O-glycosylation may modulate receptor activity (
      • Herr P.
      • Korniychuk G.
      • Yamamoto Y.
      • Grubisic K.
      • Oelgeschläger M.
      ,
      • Wagner K.W.
      • Punnoose E.A.
      • Januario T.
      • Lawrence D.A.
      • Pitti R.M.
      • Lancaster K.
      • Lee D.
      • von Goetz M.
      • Yee S.F.
      • Totpal K.
      • Huw L.
      • Katta V.
      • Cavet G.
      • Hymowitz S.G.
      • Amler L.
      • Ashkenazi A.
      ) and protein hormone processing (
      • Kato K.
      • Jeanneau C.
      • Tarp M.A.
      • Benet-Pagès A.
      • Lorenz-Depiereux B.
      • Bennett E.P.
      • Mandel U.
      • Strom T.M.
      • Clausen H.
      ); hence, mucin-type O-glycosylation may be sufficiently regulated to actively serve as a modulator of complex biological processes and even signaling. Recent studies clearly indicate the critical role of mucin-type O-glycosylation in vertebrate and nonvertebrate development (
      • Tabak L.A.
      ,
      • Zhang L.
      • Ten Hagen K.G.
      ,
      • Xia L.
      • Ju T.
      • Westmuckett A.
      • An G.
      • Ivanciu L.
      • McDaniel J.M.
      • Lupu F.
      • Cummings R.D.
      • McEver R.P.
      ,
      • Fu J.
      • Gerhardt H.
      • McDaniel J.M.
      • Xia B.
      • Liu X.
      • Ivanciu L.
      • Ny A.
      • Hermans K.
      • Silasi-Mansat R.
      • McGee S.
      • Nye E.
      • Ju T.
      • Ramirez M.I.
      • Carmeliet P.
      • Cummings R.D.
      • Lupu F.
      • Xia L.
      ,
      • ten Hagen K.G.
      • Zhang L.
      • Tian E.
      • Zhang Y.
      ,
      • Zhang L.
      • Tran D.T.
      • Ten Hagen K.G.
      ,
      • Lin Y.R.
      • Reddy B.V.
      • Irvine K.D.
      ).
      Mucin-type protein O-glycosylation is initiated in the Golgi compartment by the transfer of α-GalNAc, from UDP-GalNAc,
      The abbreviations used are: UDP-GalNAc, uridine diphosphate N-α-acetylgalactosamine; ppGalNAc T, UDP-α-GalNAc, polypeptide N-α-acetylgalactosaminyltransferase; T-synthase, UDP-Gal, glycoprotein-α-GalNAc β3 galactosyltransferase; Core 2-O-sLex, NeuNAcα2–3Galβ1–4 (Fucα1-3)GlcNAcβ1–6 (Galβ1–3)GalNAcα1-O-Thr/Ser; h, human.
      to Ser and Thr residues of polypeptide acceptors by the large family (∼20) of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts). By subsequent action of a series of specific glycosyltransferases, the O-linked glycan can be further elongated to produce a vast array of glycan structures (
      • Brockhausen I.
      ). Unlike the N-glycosylation of Asn residues and the O-xylosylation of Ser residues of proteoglycans, there are no highly specific sequence motifs (i.e. Asn-Xaa (not Pro)-(Ser/Thr) and acidic-acidic-Xaa-Ser-Gly-Xaa-Gly, respectively (
      • Walmsley A.R.
      • Hooper N.M.
      ,
      • Kearns A.E.
      • Campbell S.C.
      • Westley J.
      • Schwartz N.B.
      )) that enable the facile prediction or recognition of sites of mucin-type O-glycosylation based on peptide sequence. Nevertheless, data base analysis of known mucin-type O-glycosylation sites have resulted in a number of algorithms (
      • Elhammer A.P.
      • Poorman R.A.
      • Brown E.
      • Maggiora L.L.
      • Hoogerheide J.G.
      • Kézdy F.J.
      ,
      • Gupta R.
      • Birch H.
      • Rapacki K.
      • Brunak S.
      • Hansen J.E.
      ,
      • Chen Y.Z.
      • Tang Y.R.
      • Sheng Z.Y.
      • Zhang Z.
      )
      See also OGEPT version 1.0, a program for predicting mucin-type O-glycosylation sites (Torres, Jr., R., Almeida, I. C., Dayal, Y., and Leung, M.-Y., O-Glycosylation Prediction Electronic Tool, University of Texas, El Paso).
      for the approximate prediction of mucin-type O-glycosylation. None of these approaches, however, readily account for the wide range and remarkable reproducibility of the O-glycan site-to-site occupancy observed in the mucins that have been characterized to date (
      • Gerken T.A.
      • Gilmore M.
      • Zhang J.
      ,
      • Gerken T.A.
      • Tep C.
      • Rarick J.
      ). Importantly, the predictive approaches do not take into account the different peptide substrate specificities of the various ppGalNAc T isoforms.
      Structurally the ppGalNAc Ts consist of an N-terminal catalytic domain tethered by a short linker to a C-terminal ricin-like lectin domain containing three recognizable carbohydrate-binding sites (
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      ). Some members of the ppGalNAc T family prefer substrates that have been previously modified with O-linked GalNAc on nearby Ser/Thr residues, hence having so-called glycopeptide or filling-in activities, i.e. ppGalNAc T7 and T10 (
      • Bennett E.P.
      • Hassan H.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Cheng L.
      • Tachibana K.
      • Zhang Y.
      • Guo J.
      • Kahori Tachibana K.
      • Kameyama A.
      • Wang H.
      • Hiruma T.
      • Iwasaki H.
      • Togayachi A.
      • Kudo T.
      • Narimatsu H.
      ,
      • Pratt M.R.
      • Hang H.C.
      • Ten Hagen K.G.
      • Rarick J.
      • Gerken T.A.
      • Tabak L.A.
      • Bertozzi C.R.
      ). Others simply possess altered preferences against glycopeptide substrates, i.e. ppGalNAc T2 and T4 (
      • Hassan H.
      • Reis C.A.
      • Bennett E.P.
      • Mirgorodskaya E.
      • Roepstorff P.
      • Hollingsworth M.A.
      • Burchell J.
      • Taylor-Papadimitriou J.
      • Clausen H.
      ,
      • Hanisch F.G.
      • Reis C.A.
      • Clausen H.
      • Paulsen H.
      ,
      • Wandall H.H.
      • Irazoqui F.
      • Tarp M.A.
      • Bennett E.P.
      • Mandel U.
      • Takeuchi H.
      • Kato K.
      • Irimura T.
      • Suryanarayanan G.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      ), or may be inhibited by neighboring glycosylation, i.e. ppGalNAc T1 and T2 (
      • Gerken T.A.
      • Gilmore M.
      • Zhang J.
      ,
      • Gerken T.A.
      • Tep C.
      • Rarick J.
      ,
      • Pratt M.R.
      • Hang H.C.
      • Ten Hagen K.G.
      • Rarick J.
      • Gerken T.A.
      • Tabak L.A.
      • Bertozzi C.R.
      ). These latter transferases have been called early or initiating transferases, preferring nonglycosylated over glycosylated substrates. The roles of the catalytic and lectin domains on modulating ppGalNAc T peptide and glycopeptide specificity are not fully understood. It has been shown that the presence of the lectin domain of ppGalNAc T2 significantly shifts the preferred sites of glycosylation on glycopeptide substrates (
      • Bennett E.P.
      • Hassan H.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Cheng L.
      • Tachibana K.
      • Zhang Y.
      • Guo J.
      • Kahori Tachibana K.
      • Kameyama A.
      • Wang H.
      • Hiruma T.
      • Iwasaki H.
      • Togayachi A.
      • Kudo T.
      • Narimatsu H.
      ,
      • Wandall H.H.
      • Irazoqui F.
      • Tarp M.A.
      • Bennett E.P.
      • Mandel U.
      • Takeuchi H.
      • Kato K.
      • Irimura T.
      • Suryanarayanan G.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      ), although other studies have demonstrated that the catalytic domain of ppGalNAc T10 is responsible for its near absolute glycopeptide specificity (
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      ,
      • Perrine C.L.
      • Ganguli A.
      • Wu P.
      • Bertozzi C.R.
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      • Gerken T.A.
      ). Clearly, detailed studies of the catalytic and lectin domain specificities of these transferases are necessary to fully understand their properties.
      Several ppGalNAc T isoforms have been shown to be necessary for, or associated with, normal development, cellular processes, or specific disease states, presumably by possessing specific protein targets that other coexpressed ppGalNAc T isoforms fail to recognize.
      Note that there is also a possibility that the observation of the loss of a glycosylation site in vivo may simply be that no other ppGalNAc Ts are expressed and not necessarily because the mutant transferase is necessarily substrate-specific. The possibility of mis-trafficking/localization at the Golgi membrane could also result in the apparent loss of transferase activity.
      For example, inactive mutations in the fly PGANT35A (the ppGalNAc T11 orthologue in mammals) are lethal (
      • Ten Hagen K.G.
      • Tran D.T.
      ,
      • Schwientek T.
      • Bennett E.P.
      • Flores C.
      • Thacker J.
      • Hollmann M.
      • Reis C.A.
      • Behrens J.
      • Mandel U.
      • Keck B.
      • Schäfer M.A.
      • Haselmann K.
      • Zubarev R.
      • Roepstorff P.
      • Burchell J.M.
      • Taylor-Papadimitriou J.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Tian E.
      • Ten Hagen K.G.
      ), although mutations in PGANT3 (with no close mammalian homologue) result in wing blistering (
      • Zhang L.
      • Zhang Y.
      • Hagen K.G.
      ). Both of these transferases play significant roles modulating specific cell-cell interactions in the developing fly (
      • Zhang L.
      • Tran D.T.
      • Ten Hagen K.G.
      ,
      • Tian E.
      • Ten Hagen K.G.
      ). In humans, mutations in ppGalNAc T3 cause a form of familial tumoral calcinosis, due to abnormal cleavage and secretion of the phosphaturic factor FGF23 (
      • Kato K.
      • Jeanneau C.
      • Tarp M.A.
      • Benet-Pagès A.
      • Lorenz-Depiereux B.
      • Bennett E.P.
      • Mandel U.
      • Strom T.M.
      • Clausen H.
      ,
      • Topaz O.
      • Shurman D.L.
      • Bergman R.
      • Indelman M.
      • Ratajczak P.
      • Mizrachi M.
      • Khamaysi Z.
      • Behar D.
      • Petronius D.
      • Friedman V.
      • Zelikovic I.
      • Raimer S.
      • Metzker A.
      • Richard G.
      • Sprecher E.
      ,
      • Bennett E.P.
      • Chen Y.W.
      • Schwientek T.
      • Mandel U.
      • Schjoldager K.B.
      • Cohen S.M.
      • Clausen H.
      ). Human ppGalNAc T14 may modulate apoptotic signaling in tumor cells by glycosylating the proapoptotic receptors DLR4 and DLR5 (
      • Wagner K.W.
      • Punnoose E.A.
      • Januario T.
      • Lawrence D.A.
      • Pitti R.M.
      • Lancaster K.
      • Lee D.
      • von Goetz M.
      • Yee S.F.
      • Totpal K.
      • Huw L.
      • Katta V.
      • Cavet G.
      • Hymowitz S.G.
      • Amler L.
      • Ashkenazi A.
      ), although the specific O-glycosylation of the TGFB-II receptor (ActR-II) by GALNTL1 (ppGalNAc T16) modulates its signaling in Xenopus and mammalian development (
      • Herr P.
      • Korniychuk G.
      • Yamamoto Y.
      • Grubisic K.
      • Oelgeschläger M.
      ). Specific ppGalNAc Ts have also been linked to Williams-Beuren syndrome (WBSCR17, pt-GalNAc-T, or GALNTL3) (
      • Nakamura N.
      • Toba S.
      • Hirai M.
      • Morishita S.
      • Mikami T.
      • Konishi M.
      • Itoh N.
      • Kurosaka A.
      ,
      • Merla G.
      • Ucla C.
      • Guipponi M.
      • Reymond A.
      ) and hereditary multiple exostoses (ppGalNAc T5) (
      • Simmons A.D.
      • Musy M.M.
      • Lopes C.S.
      • Hwang L.Y.
      • Yang Y.P.
      • Lovett M.
      ). Genome-wide sequencing studies have also revealed biochemically inactivating germ lines and somatic mutations in GALNT5 and GALNT12 (ppGalNAc T5 and T12) in individuals with breast and colon cancers (
      • Wood L.D.
      • Parsons D.W.
      • Jones S.
      • Lin J.
      • Sjöblom T.
      • Leary R.J.
      • Shen D.
      • Boca S.M.
      • Barber T.
      • Ptak J.
      • Silliman N.
      • Szabo S.
      • Dezso Z.
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      • Shipitsin M.
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      • Sukumar S.
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      • Guda K.
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      ). Other genome-wide association scans suggest that GALNT2 (ppGalNAc T2) variants may be associated with levels of HDL cholesterol and coronary artery disease (
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      • Waeber G.
      • Vollenweider P.
      • Voight B.F.
      • Vitart V.
      • Uitterlinden A.G.
      • Uda M.
      • Tuomilehto J.
      • Thompson J.R.
      • Tanaka T.
      • Surakka I.
      • Stringham H.M.
      • Spector T.D.
      • Soranzo N.
      • Smit J.H.
      • Sinisalo J.
      • Silander K.
      • Sijbrands E.J.
      • Scuteri A.
      • Scott J.
      • Schlessinger D.
      • Sanna S.
      • Salomaa V.
      • Saharinen J.
      • Sabatti C.
      • Ruokonen A.
      • Rudan I.
      • Rose L.M.
      • Roberts R.
      • Rieder M.
      • Psaty B.M.
      • Pramstaller P.P.
      • Pichler I.
      • Perola M.
      • Penninx B.W.
      • Pedersen N.L.
      • Pattaro C.
      • Parker A.N.
      • Pare G.
      • Oostra B.A.
      • O'Donnell C.J.
      • Nieminen M.S.
      • Nickerson D.A.
      • Montgomery G.W.
      • Meitinger T.
      • McPherson R.
      • McCarthy M.I.
      • McArdle W.
      • Masson D.
      • Martin N.G.
      • Marroni F.
      • Mangino M.
      • Magnusson P.K.
      • Lucas G.
      • Luben R.
      • Loos R.J.
      • Lokki M.L.
      • Lettre G.
      • Langenberg C.
      • Launer L.J.
      • Lakatta E.G.
      • Laaksonen R.
      • Kyvik K.O.
      • Kronenberg F.
      • König I.R.
      • Khaw K.T.
      • Kaprio J.
      • Kaplan L.M.
      • Johansson A.
      • Jarvelin M.R.
      • Janssens A.C.
      • Ingelsson E.
      • Igl W.
      • Kees Hovingh G.
      • Hottenga J.J.
      • Hofman A.
      • Hicks A.A.
      • Hengstenberg C.
      • Heid I.M.
      • Hayward C.
      • Havulinna A.S.
      • Hastie N.D.
      • Harris T.B.
      • Haritunians T.
      • Hall A.S.
      • Gyllensten U.
      • Guiducci C.
      • Groop L.C.
      • Gonzalez E.
      • Gieger C.
      • Freimer N.B.
      • Ferrucci L.
      • Erdmann J.
      • Elliott P.
      • Ejebe K.G.
      • Döring A.
      • Dominiczak A.F.
      • Demissie S.
      • Deloukas P.
      • de Geus E.J.
      • de Faire U.
      • Crawford G.
      • Collins F.S.
      • Chen Y.D.
      • Caulfield M.J.
      • Campbell H.
      • Burtt N.P.
      • Bonnycastle L.L.
      • Boomsma D.I.
      • Boekholdt S.M.
      • Bergman R.N.
      • Barroso I.
      • Bandinelli S.
      • Ballantyne C.M.
      • Assimes T.L.
      • Quertermous T.
      • Altshuler D.
      • Seielstad M.
      • Wong T.Y.
      • Tai E.S.
      • Feranil A.B.
      • Kuzawa C.W.
      • Adair L.S.
      • Taylor Jr., H.A.
      • Borecki I.B.
      • Gabriel S.B.
      • Wilson J.G.
      • Holm H.
      • Thorsteinsdottir U.
      • Gudnason V.
      • Krauss R.M.
      • Mohlke K.L.
      • Ordovas J.M.
      • Munroe P.B.
      • Kooner J.S.
      • Tall A.R.
      • Hegele R.A.
      • Kastelein J.J.
      • Schadt E.E.
      • Rotter J.I.
      • Boerwinkle E.
      • Strachan D.P.
      • Mooser V.
      • Stefansson K.
      • Reilly M.P.
      • Samani N.J.
      • Schunkert H.
      • Cupples L.A.
      • Sandhu M.S.
      • Ridker P.M.
      • Rader D.J.
      • van Duijn C.M.
      • Peltonen L.
      • Abecasis G.R.
      • Boehnke M.
      • Kathiresan S.
      ). Obviously, there is a need for characterizing the peptide substrate specificity of each isoform to further elucidate their specific targets and mechanism of action. This information is critical for our understanding of the biological roles and significance of the ppGalNAc T family of transferases and mucin-type O-glycosylation in general.
      Our laboratory has recently reported the use of a series of oriented random peptide and glycopeptide substrate libraries for quantitatively determining the amino acid residue preferences of the catalytic domains of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 (
      • Perrine C.L.
      • Ganguli A.
      • Wu P.
      • Bertozzi C.R.
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      • Gerken T.A.
      ,
      • Gerken T.A.
      • Raman J.
      • Fritz T.A.
      • Jamison O.
      ,
      • Gerken T.A.
      • Ten Hagen K.G.
      • Jamison O.
      ). In this study, we extend our studies to three additional members of the family, ppGalNAc T3, T5, and T12, with potential roles in human disease, utilizing two previously reported random peptide substrates and an additional new substrate capable of obtaining preferences for neighboring nonglycosylated Ser residues (Table 1). With these substrates, unique substrate preference data for all amino acid residues except Thr, Trp, and Cys have now been obtained for the following six mammalian ppGalNAc Ts: T1, T2, T3, T5, T10, and T12. Our findings have revealed both common and unique features among the transferases characterized to date. The most striking was the very similar pattern of enhancements for those residues C-terminal to the site of glycosylation, particularly enhancements at +1 and +3 for Pro, found in all the ppGalNAc Ts characterized except the glycopeptide preferring ppGalNAc T10 (
      • Cheng L.
      • Tachibana K.
      • Zhang Y.
      • Guo J.
      • Kahori Tachibana K.
      • Kameyama A.
      • Wang H.
      • Hiruma T.
      • Iwasaki H.
      • Togayachi A.
      • Kudo T.
      • Narimatsu H.
      ,
      • Pratt M.R.
      • Hang H.C.
      • Ten Hagen K.G.
      • Rarick J.
      • Gerken T.A.
      • Tabak L.A.
      • Bertozzi C.R.
      ). A structural analysis suggests these enhancements arise from interactions with a common conserved Trp residue found in these transferases. In contrast, residues N-terminal to the site of glycosylation show a range of enhancements that are isoform-specific, with elevated preferences for Pro, Val, Ile, and Tyr being the most common. Further analysis revealed that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue preferences varied among transferases with ppGalNAc T1 and T2 preferring the most acidic substrates, with ppGalNAc T5 and T3 preferring the most basic. We also show that these observations are consistent with the homology modeled structures of the ppGalNAc Ts. Thus, the peptide sequence and overall charge serve to modulate the peptide substrate specificity of each ppGalNAc T. Coupled with the variable sensitivity of each isoform to prior substrate glycosylation (
      • Gerken T.A.
      • Gilmore M.
      • Zhang J.
      ,
      • Gerken T.A.
      • Tep C.
      • Rarick J.
      ,
      • Pratt M.R.
      • Hang H.C.
      • Ten Hagen K.G.
      • Rarick J.
      • Gerken T.A.
      • Tabak L.A.
      • Bertozzi C.R.
      ,
      • Hassan H.
      • Reis C.A.
      • Bennett E.P.
      • Mirgorodskaya E.
      • Roepstorff P.
      • Hollingsworth M.A.
      • Burchell J.
      • Taylor-Papadimitriou J.
      • Clausen H.
      ,
      • Hanisch F.G.
      • Reis C.A.
      • Clausen H.
      • Paulsen H.
      ,
      • Wandall H.H.
      • Irazoqui F.
      • Tarp M.A.
      • Bennett E.P.
      • Mandel U.
      • Takeuchi H.
      • Kato K.
      • Irimura T.
      • Suryanarayanan G.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      ), a wide range of unique and specific substrate preferences is achieved across the ppGalNAc T family of transferases. We further demonstrate that for several of the transferases, these preferences can be used to predict isoform-specific glycosylation patterns consistent with previously reported experimental data.
      TABLE 1ppGalNAc transferase random substrates utilized in this work
      PeptideSequenceNo. of unique sequences
      GAGAXXXXXTXXXXXAGAGK
      P-VIX = Gly, Ala, Pro, Val, Leu, Tyr, Glu, Gln, Arg, HisRef
      • Gerken T.A.
      • Ten Hagen K.G.
      • Jamison O.
      10.0 × 109
      P-VIIX = Gly, Ala, Pro, Ile, Met, Phe, Asp, Asn, Arg, LysRef
      • Gerken T.A.
      • Ten Hagen K.G.
      • Jamison O.
      10.0 × 109
      P-VIIIX = Gly, Ala, Pro, Val, Tyr, Glu, Asn, Ser, Arg, Lys10.0 × 109

      DISCUSSION

      In this study, we have extended our earlier studies of ppGalNAc T1, T2, and T10 to three additional transferases, ppGalNAc T3, T5, and T12, which have been variously implicated in human disease (
      • Bennett E.P.
      • Chen Y.W.
      • Schwientek T.
      • Mandel U.
      • Schjoldager K.B.
      • Cohen S.M.
      • Clausen H.
      ,
      • Simmons A.D.
      • Musy M.M.
      • Lopes C.S.
      • Hwang L.Y.
      • Yang Y.P.
      • Lovett M.
      ,
      • Wood L.D.
      • Parsons D.W.
      • Jones S.
      • Lin J.
      • Sjöblom T.
      • Leary R.J.
      • Shen D.
      • Boca S.M.
      • Barber T.
      • Ptak J.
      • Silliman N.
      • Szabo S.
      • Dezso Z.
      • Ustyanksky V.
      • Nikolskaya T.
      • Nikolsky Y.
      • Karchin R.
      • Wilson P.A.
      • Kaminker J.S.
      • Zhang Z.
      • Croshaw R.
      • Willis J.
      • Dawson D.
      • Shipitsin M.
      • Willson J.K.
      • Sukumar S.
      • Polyak K.
      • Park B.H.
      • Pethiyagoda C.L.
      • Pant P.V.
      • Ballinger D.G.
      • Sparks A.B.
      • Hartigan J.
      • Smith D.R.
      • Suh E.
      • Papadopoulos N.
      • Buckhaults P.
      • Markowitz S.D.
      • Parmigiani G.
      • Kinzler K.W.
      • Velculescu V.E.
      • Vogelstein B.
      ,
      • Guda K.
      • Moinova H.
      • He J.
      • Jamison O.
      • Ravi L.
      • Natale L.
      • Lutterbaugh J.
      • Lawrence E.
      • Lewis S.
      • Willson J.K.
      • Lowe J.B.
      • Wiesner G.L.
      • Parmigiani G.
      • Barnholtz-Sloan J.
      • Dawson D.W.
      • Velculescu V.E.
      • Kinzler K.W.
      • Papadopoulos N.
      • Vogelstein B.
      • Willis J.
      • Gerken T.A.
      • Markowitz S.D.
      ,
      • Guo J.M.
      • Chen H.L.
      • Wang G.M.
      • Zhang Y.K.
      • Narimatsu H.
      ). In addition, we have introduced a unique random peptide substrate, not previously described, capable of providing preferences for free Ser residues flanking the site of glycosylation for all six transferases (Fig. 1). Thus, by utilizing the oriented random peptide substrates given in Table 1 (P-VI, P-VII, and P-VIII), we have systematically characterized the peptide substrate specificities of 6 of the 20 ppGalNAc Ts for all nonglycosylated amino acid residues except Cys, Trp, and Thr (FIGURE 2, FIGURE 3). Remarkably, we observe that the C-terminal preferences for the majority of the isoforms are very similar, i.e. having a nearly identical TP(G/A)P-like motif. We concluded that this motif is principally governed by the presence of conserved Trp and Phe residues in the peptide-binding site of the catalytic domain (Trp-282, Phe-280, and Phe-361 in ppGalNAc T2) (supplemental Fig. S1) and that for ppGalNAc T10, which lacks these residues, this preference is lost. On this basis, we suggest that the remaining relatively uncharacterized transferases containing the analogous residues would likely possess similar C-terminal preferences, although those transferases, i.e. ppGalNAc T7, T8, T9, T18, and T20 and WBSCR17 (GALNTL3), lacking these specific residues would not. Indeed, similar to ppGalNAc T10, ppGalNAc T7, and T20 are glycopeptide-preferring transferases that have significant activity only against GalNAc-containing glycopeptide substrates (
      • Bennett E.P.
      • Hassan H.
      • Hollingsworth M.A.
      • Clausen H.
      ,
      • Peng C.
      • Togayachi A.
      • Kwon Y.D.
      • Xie C.
      • Wu G.
      • Zou X.
      • Sato T.
      • Ito H.
      • Tachibana K.
      • Kubota T.
      • Noce T.
      • Narimatsu H.
      • Zhang Y.
      ). The (S/T)PXP motif is furthermore observed in the data base analysis of O-glycosylation sites (
      • Elhammer A.P.
      • Poorman R.A.
      • Brown E.
      • Maggiora L.L.
      • Hoogerheide J.G.
      • Kézdy F.J.
      ,
      • Gupta R.
      • Birch H.
      • Rapacki K.
      • Brunak S.
      • Hansen J.E.
      ,
      • Chen Y.Z.
      • Tang Y.R.
      • Sheng Z.Y.
      • Zhang Z.
      )6 further confirming the commonality of the C-terminal preferences of the majority of the ppGalNAc T isoforms.
      As shown in FIGURE 2, FIGURE 3, transferase specificity against nonglycosylated substrates principally arises from differential recognition at the N-terminal positions of the site of glycosylation with the −1 position showing the most selectivity. Thus, ppGalNAc T2 is distinguished by its elevated −1 Pro enhancement and ppGalNAc T3 and T1 by their Val preferences, although ppGalNAc T12 is distinguished by the presence of Tyr enhancements from the −1 to −3 sites. In addition, we observed that specificity is further modulated by the overall charge of the peptide substrate; ppGalNAc T3 and T5 prefer more basic substrates and ppGalNAc T1 and T2 more acidic substrates. As shown in Fig. 4, these trends were roughly correlated to the isoelectric point and surface charge of the catalytic domain of each transferase.
      We have further demonstrated that the preferences can be used to roughly predict rates and sites of glycosylation for a number of common substrates for ppGalNAc T1, T2, and T3 (Fig. 5). More importantly, the preferences predict the isoform-specific glycosylation patterns of the FGF23 peptide and of a HSV-1 gC1 peptide (Table 2). We have further demonstrated the possibility that the ppGalNAc T1 and T-synthase preferences together can predict the site-specific glycosylation of a critical Thr residue in mouse and human PSGL1 (Fig. 6). Although the initial published work on ppGalNAc T5 and T12 do not fully agree with our enhancement value predictions (supplemental Tables S3 and S4), we have shown that for ppGalNAc T5 the enhancements indeed approximate the experimental glycosylation patterns (Fig. 7). We suggest that many of the discrepancies, particularly with the short peptide substrates, arise from end effects,10 the presence of multiple glycosylation sites, and potential conformational effects. It should be further noted that by the nature of the random peptide approach it is possible that highly cooperative specific sequences with strong preferences could go undetected. It is anticipated that these, if present, will be revealed in future studies. Nevertheless, the general success of the transferase-specific random peptide preference values to roughly predict sites of glycosylation will significantly advance our ability to understand site-specific mucin-type O-glycosylation. This is likely to lead to our ability to identify and/or confirm isoform-specific glycosylation sites.
      TABLE 2Random peptide enhancement value products predict experimental transferase-specific sites of glycosylation and confirm the role of charged residues
      Figure thumbnail fx2
      Our work further demonstrates the uniqueness of the different ppGalNAc T isoforms. Although several isoforms appear to have similar enhancement value patterns (FIGURE 2, FIGURE 3), we find that when the enhancement products are obtained, clear differences between isoforms emerge. An example of this is shown for the Thr residues of the mucin domain of the mouse and human PSGL1 plotted in supplemental Fig. S3. It is clear from the plots that the patterns for the different isoforms can vary significantly from site to site. Of further interest, we find regions of very high and very low probabilities for all of the transferases examined thus far, suggesting that some regions of the peptide core may be designed to be more heavily O-glycosylated than others.
      Our understanding of the biological significance of ppGalNAc T substrate preferences remains incomplete. However, several human diseases and conditions are now known to be the result of aberrant O-glycosylation, and several are related to specific ppGalNAc T mutations (reviewed in Tabak (
      • Tabak L.A.
      )).7 With the explosion of data from genome-wide association studies, additional ppGalNAc T associations and biological processes will be uncovered (e.g. GALNT2 (ppGalNAc T2) is associated with the level of blood lipids (
      • Willer C.J.
      • Sanna S.
      • Jackson A.U.
      • Scuteri A.
      • Bonnycastle L.L.
      • Clarke R.
      • Heath S.C.
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      • Najjar S.S.
      • Stringham H.M.
      • Strait J.
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      • Davey-Smith G.
      • Shuldiner A.R.
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      • Cao A.
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      • Lakatta E.
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      • Boehnke M.
      • Schlessinger D.
      • Mohlke K.L.
      • Abecasis G.R.
      ,
      • Kathiresan S.
      • Melander O.
      • Guiducci C.
      • Surti A.
      • Burtt N.P.
      • Rieder M.J.
      • Cooper G.M.
      • Roos C.
      • Voight B.F.
      • Havulinna A.S.
      • Wahlstrand B.
      • Hedner T.
      • Corella D.
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      • Hedblad B.
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      ,
      • Teslovich T.M.
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      • Fouchier S.W.
      • Isaacs A.
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      • Bis J.C.
      • Aulchenko Y.S.
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      • Chambers J.
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      • Melander O.
      • Johnson T.
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      • Jin Go M.
      • Jin Kim Y.
      • Lee J.Y.
      • Park T.
      • Kim K.
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      • Twee-Hee Ong R.
      • Croteau-Chonka D.C.
      • Lange L.A.
      • Smith J.D.
      • Song K.
      • Hua Zhao J.
      • Yuan X.
      • Luan J.
      • Lamina C.
      • Ziegler A.
      • Zhang W.
      • Zee R.Y.
      • Wright A.F.
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      • Willemsen G.
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      • Waeber G.
      • Vollenweider P.
      • Voight B.F.
      • Vitart V.
      • Uitterlinden A.G.
      • Uda M.
      • Tuomilehto J.
      • Thompson J.R.
      • Tanaka T.
      • Surakka I.
      • Stringham H.M.
      • Spector T.D.
      • Soranzo N.
      • Smit J.H.
      • Sinisalo J.
      • Silander K.
      • Sijbrands E.J.
      • Scuteri A.
      • Scott J.
      • Schlessinger D.
      • Sanna S.
      • Salomaa V.
      • Saharinen J.
      • Sabatti C.
      • Ruokonen A.
      • Rudan I.
      • Rose L.M.
      • Roberts R.
      • Rieder M.
      • Psaty B.M.
      • Pramstaller P.P.
      • Pichler I.
      • Perola M.
      • Penninx B.W.
      • Pedersen N.L.
      • Pattaro C.
      • Parker A.N.
      • Pare G.
      • Oostra B.A.
      • O'Donnell C.J.
      • Nieminen M.S.
      • Nickerson D.A.
      • Montgomery G.W.
      • Meitinger T.
      • McPherson R.
      • McCarthy M.I.
      • McArdle W.
      • Masson D.
      • Martin N.G.
      • Marroni F.
      • Mangino M.
      • Magnusson P.K.
      • Lucas G.
      • Luben R.
      • Loos R.J.
      • Lokki M.L.
      • Lettre G.
      • Langenberg C.
      • Launer L.J.
      • Lakatta E.G.
      • Laaksonen R.
      • Kyvik K.O.
      • Kronenberg F.
      • König I.R.
      • Khaw K.T.
      • Kaprio J.
      • Kaplan L.M.
      • Johansson A.
      • Jarvelin M.R.
      • Janssens A.C.
      • Ingelsson E.
      • Igl W.
      • Kees Hovingh G.
      • Hottenga J.J.
      • Hofman A.
      • Hicks A.A.
      • Hengstenberg C.
      • Heid I.M.
      • Hayward C.
      • Havulinna A.S.
      • Hastie N.D.
      • Harris T.B.
      • Haritunians T.
      • Hall A.S.
      • Gyllensten U.
      • Guiducci C.
      • Groop L.C.
      • Gonzalez E.
      • Gieger C.
      • Freimer N.B.
      • Ferrucci L.
      • Erdmann J.
      • Elliott P.
      • Ejebe K.G.
      • Döring A.
      • Dominiczak A.F.
      • Demissie S.
      • Deloukas P.
      • de Geus E.J.
      • de Faire U.
      • Crawford G.
      • Collins F.S.
      • Chen Y.D.
      • Caulfield M.J.
      • Campbell H.
      • Burtt N.P.
      • Bonnycastle L.L.
      • Boomsma D.I.
      • Boekholdt S.M.
      • Bergman R.N.
      • Barroso I.
      • Bandinelli S.
      • Ballantyne C.M.
      • Assimes T.L.
      • Quertermous T.
      • Altshuler D.
      • Seielstad M.
      • Wong T.Y.
      • Tai E.S.
      • Feranil A.B.
      • Kuzawa C.W.
      • Adair L.S.
      • Taylor Jr., H.A.
      • Borecki I.B.
      • Gabriel S.B.
      • Wilson J.G.
      • Holm H.
      • Thorsteinsdottir U.
      • Gudnason V.
      • Krauss R.M.
      • Mohlke K.L.
      • Ordovas J.M.
      • Munroe P.B.
      • Kooner J.S.
      • Tall A.R.
      • Hegele R.A.
      • Kastelein J.J.
      • Schadt E.E.
      • Rotter J.I.
      • Boerwinkle E.
      • Strachan D.P.
      • Mooser V.
      • Stefansson K.
      • Reilly M.P.
      • Samani N.J.
      • Schunkert H.
      • Cupples L.A.
      • Sandhu M.S.
      • Ridker P.M.
      • Rader D.J.
      • van Duijn C.M.
      • Peltonen L.
      • Abecasis G.R.
      • Boehnke M.
      • Kathiresan S.
      )). The ability to predict sites of O-glycosylation in a more precise manner will no doubt speed analysis of associations and help with the functional evaluation.

      Acknowledgments

      We acknowledge the helpful assistance of Joseph Thome, Jayalakshmi Raman, and Hazuki Miwa.

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