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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*

  • 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; E-mail: .
    Affiliations
    From the Departments of Pediatrics (W. A. Bernbaum Center for Cystic Fibrosis Research),

    Biochemistry, and

    Chemistry, Case Western Reserve University, Cleveland, Ohio 44106,
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  • Leslie Revoredo
    Affiliations
    Chemistry, Case Western Reserve University, Cleveland, Ohio 44106,
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  • Joseph J.C. Thome
    Footnotes
    Affiliations
    From the Departments of Pediatrics (W. A. Bernbaum Center for Cystic Fibrosis Research),
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  • Lawrence A. Tabak
    Affiliations
    the Section on Biological Chemistry, NIDCR, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892,
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  • Malene Bech Vester-Christensen
    Affiliations
    the Copenhagen Center for Glycomics (CCG), Departments of Cellular and Molecular Medicine and Dentistry, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark,
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  • Henrik Clausen
    Affiliations
    the Copenhagen Center for Glycomics (CCG), Departments of Cellular and Molecular Medicine and Dentistry, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark,
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  • Gagandeep K. Gahlay
    Affiliations
    the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, and
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  • Donald L. Jarvis
    Affiliations
    the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, and
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  • Roy W. Johnson
    Affiliations
    the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
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  • Heather A. Moniz
    Affiliations
    the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
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  • Kelley Moremen
    Affiliations
    the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01-CA78834 (to T. A. G.) and P41-GM103390 and P41-RR005351 (to James Prestegard (Complex Carbohydrate Center, University of Georgia) and K. M. and D. L. J.) and a National Institutes of Health, NIDCR, intramural program grant (to L. A. T.). This work was also supported by Danish National Research Foundation Grant DNRF107 (to H. C.).
    2 Present address: Dept. of Microbiology and Immunology, Columbia University Graduate School of Arts and Sciences at the College of Physicians and Surgeons, Columbia University, New York, NY 10027.
Open AccessPublished:May 20, 2013DOI:https://doi.org/10.1074/jbc.M113.477877
      Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.
      Background: ppGalNAc transferases, which initiate O-glycosylation, possess a poorly understood lectin domain.
      Results: The lectin domain directs glycosylation in an N- or C- terminal direction in an isoform-specific manner.
      Conclusion: Unanticipated isoform-specific directionality was revealed for modification of glycopeptide substrates.
      Significance: A novel mechanism of controlling of mucin type O-glycosylation has been discovered based on tethered lectin domains specifying N- or C-terminal modification of glycopeptide substrates.

      Introduction

      Mucin type protein O-glycosylation, as defined by the α-GalNAc-O-Ser/Thr linkage, is one of the most common types of protein glycosylation found in higher organisms. This modification is initiated in the Golgi by a large family (∼20 members in mammals and about half the number in the fly and Caenorhabditis elegans (see Ref.
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      for a review) of polypeptide GalNAc transferases (ppGalNAc Ts)
      The abbreviations used are: ppGalNAc T, UDP-α-GalNAc:polypeptide N-α-acetylgalactosaminyltransferase; PTH, phenylthiohydantoin amino acid derivative of Edman sequencing; Ni-NTA, nickel-nitrilotriacetic acid; GPIV, GPIV-C, GPV, and GPV-C, random (glyco)peptides as defined in Table 1; HDPE, high density polyethylene.
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      Mucin type O-glycans have typically been associated with so-called mucin domains, where high numbers of Ser and Thr residues are glycosylated, producing a chemical- and protease-resistant extended peptide structural motif. Glycoproteins containing O-glycosylated mucin domains commonly function in the protection of the cell surface and the modulation of cell-cell interactions and hence play important roles, for example in inflammation, the immune response, metastasis, and tumorigenesis (
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      • Berois N.
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      • Barrios E.
      • Capandeguy L.
      • Douc-Rasy S.
      • Valteau-Couanet D.
      • Bénard J.
      • Osinaga E.
      GALNT9 gene expression is a prognostic marker in neuroblastoma patients.
      ). Except for the known role of ppGalNAc T3 in modulating the cleavage and inactivation of FGF23, the mechanisms for causing disease by the other transferase isoforms are still uncertain.
      Recent studies have further demonstrated the critical role of O-glycosylation in vertebrate and invertebrate development (
      • Xia L.
      • Ju T.
      • Westmuckett A.
      • An G.
      • Ivanciu L.
      • McDaniel J.M.
      • Lupu F.
      • Cummings R.D.
      • McEver R.P.
      Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans.
      ,
      • ten Hagen K.G.
      • Zhang L.
      • Tian E.
      • Zhang Y.
      Glycobiology on the fly. Developmental and mechanistic insights from Drosophila.
      ,
      • Tabak L.A.
      The role of mucin-type O-glycans in eukaryotic development.
      ,
      • Tran D.T.
      • Zhang L.
      • Zhang Y.
      • Tian E.
      • Earl L.A.
      • Ten Hagen K.G.
      Multiple members of the UDP-GalNAc. Polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila.
      ,
      • Tran D.T.
      • Ten Hagen K.G.
      Mucin-type O-glycosylation during development.
      ). In the fly, several ppGalNAc T isoforms are required to complete development (
      • Tran D.T.
      • Zhang L.
      • Zhang Y.
      • Tian E.
      • Earl L.A.
      • Ten Hagen K.G.
      Multiple members of the UDP-GalNAc. Polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila.
      ), whereas in the mouse, ppGalNAc T1 modulates salivary gland organogenesis (
      • Tian E.
      • Hoffman M.P.
      • Ten Hagen K.G.
      O-Glycosylation modulates integrin and FGF signalling by influencing the secretion of basement membrane components.
      ). Interestingly, the loss of the elongating transferase, T-synthase (which adds a β-Gal to the 3-position of the peptide GalNAc) leads to embryonic lethality in the mouse (
      • Xia L.
      • Ju T.
      • Westmuckett A.
      • An G.
      • Ivanciu L.
      • McDaniel J.M.
      • Lupu F.
      • Cummings R.D.
      • McEver R.P.
      Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans.
      ). Although a few target proteins have been implicated in the above studies, little is known about the actual site(s) of glycosylation and their specificity, and even less is known of the actual molecular mechanism(s) leading to their biological function.
      Structurally, members of the ppGalNAc T family (except h-ppGalNAc T20 (
      • Raman J.
      • Guan Y.
      • Perrine C.L.
      • Gerken T.A.
      • Tabak L.A.
      UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases. Completion of the family tree.
      )) possess a unique two-domain architecture consisting of a transmembrane tethered N-terminal catalytic domain linked via a short flexible segment to a C-terminal ricin-like lectin domain containing three potential carbohydrate binding sites (see Fig. 1 for the crystal structure of ppGalNAc T2 with bound EA2 peptide substrate (
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase-2.
      )). Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. Our recent studies utilizing short random (glyco)peptide substrates have shown that the ppGalNAc Ts possess specific binding preferences that vary among isoforms for peptide and even GalNAc-O-Ser/Thr-containing glycopeptide substrates (
      • Gerken 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 (ppGalNAc T) family of glycosyltransferases.
      ,
      • Gerken T.A.
      • Raman J.
      • Fritz T.A.
      • Jamison O.
      Identification of common and unique peptide substrate preferences for the UDP-GalNAc. Polypeptide α-N-acetylgalactosaminyltransferases T1 and T2 (ppGalNAc T1 and T2) derived from oriented random peptide substrates.
      ,
      • Perrine C.L.
      • Ganguli A.
      • Wu P.
      • Bertozzi C.R.
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      • Gerken T.A.
      The glycopeptide preferring polypeptide-GalNAc transferase-10 (ppGalNAc T10), involved in mucin type-O-glycosylation, has a unique GalNAc-O-Ser/Thr binding site in its catalytic domain not found in ppGalNAc T1 or T2.
      ). Interestingly, we and others have shown that the placement of a neighboring GalNAc-O-Ser/Thr residue near a glycosylation site produces a range of transferase-specific effects (i.e. a relative inhibition of glycosylation (i.e. ppGalNAc T1 and T2 (
      • Gerken T.A.
      • Gilmore M.
      • Zhang J.
      Determination of the site-specific oligosaccharide distribution of the O-glycans attached to the porcine submaxillary mucin tandem repeat. Further evidence for the modulation of O-glycan side chain structures by peptide sequence.
      ,
      • Gerken T.A.
      • Tep C.
      • Rarick J.
      Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5′-diphosphate-α-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferases T1 and T2. Kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats.
      )), an alteration or shift in glycosylation site (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.
      The lectin domain of UDP-N-acetyl-d-galactosamine. Polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities.
      ,
      • Hanisch F.G.
      • Reis C.A.
      • Clausen H.
      • Paulsen H.
      Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides.
      ,
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      )), or even a large apparent rate enhancement (i.e. requirement for glycosylated substrate) (ppGalNAc T7 and T10 (
      • Perrine C.L.
      • Ganguli A.
      • Wu P.
      • Bertozzi C.R.
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      • Gerken T.A.
      The glycopeptide preferring polypeptide-GalNAc transferase-10 (ppGalNAc T10), involved in mucin type-O-glycosylation, has a unique GalNAc-O-Ser/Thr binding site in its catalytic domain not found in ppGalNAc T1 or T2.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      ,
      • Bennett E.P.
      • Hassan H.
      • Hollingsworth M.A.
      • Clausen H.
      A novel human UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates.
      ,
      • 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.
      Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T10.
      )). Interestingly, the alteration in site preference observed for ppGalNAc T2 and T4 resides in their lectin domain (
      • Hassan 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.
      ,
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      ), whereas the nearly absolute glycopeptide requirement of ppGalNAc T10 resides completely in its catalytic domain (
      • Perrine C.L.
      • Ganguli A.
      • Wu P.
      • Bertozzi C.R.
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      • Gerken T.A.
      The glycopeptide preferring polypeptide-GalNAc transferase-10 (ppGalNAc T10), involved in mucin type-O-glycosylation, has a unique GalNAc-O-Ser/Thr binding site in its catalytic domain not found in ppGalNAc T1 or T2.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      ). Recent studies characterizing the properties of the lectin domains of ppGalNAc T2 and T3 further suggest that they may recognize glycopeptide sequence context (
      • Pedersen J.W.
      • Bennett E.P.
      • Schjoldager K.T.
      • Meldal M.
      • Holmér A.P.
      • Blixt O.
      • Cló E.
      • Levery S.B.
      • Clausen H.
      • Wandall H.H.
      Lectin domains of polypeptide GalNAc-transferases exhibit glycopeptide binding specificity.
      ) and only glycopeptides having O-linked α-GalNAc residues (
      • Yoshimura Y.
      • Nudelman A.S.
      • Levery S.B.
      • Wandall H.H.
      • Bennett E.P.
      • Hindsgaul O.
      • Clausen H.
      • Nishimura S.
      Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates.
      ), respectively. Because, to date, there have been no truly systematic studies of ppGalNAc T glycopeptide substrate utilization, we have now extended our studies to the function of the lectin domain, utilizing a series of random glycopeptides containing N- or C- terminally placed GalNAc-O-Thr residues. Our results show that the presence and the N- or C-terminal location of a GalNAc-O-Thr site in these glycopeptides are indeed important determinants of the overall catalytic activity and specificity of these enzymes, which can significantly differ between transferase isoforms. Because of the large differences in glycosylation rates observed for some isoforms between an N- or C-terminally placed GalNAc-O-Thr, we believe we have now uncovered another level of control of mucin type O-glycosylation that will further advance our understanding of the regulation of this important modification.
      Figure thumbnail gr1
      FIGURE 1Crystal structure of ppGalNAc T2 with bound peptide substrate EA2 (
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase-2.
      ) showing the tethered catalytic and lectin domains. Observed EA2 residues (5STTPAPTTK13) bound to the catalytic domain are space-filled (blue, N-terminal Ser; red, C-terminal Lys; purple, Pro; brown, Ser or Thr). The α-lectin subdomain Asp458 residue that is required for glycopeptide binding activity is space-filled in pink (
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Pedersen J.W.
      • Bennett E.P.
      • Schjoldager K.T.
      • Meldal M.
      • Holmér A.P.
      • Blixt O.
      • Cló E.
      • Levery S.B.
      • Clausen H.
      • Wandall H.H.
      Lectin domains of polypeptide GalNAc-transferases exhibit glycopeptide binding specificity.
      ) (see ).

      DISCUSSION

      Among eukaryote glycosyltransferases, the ppGalNAc T family is unique by possessing a separate carbohydrate binding lectin domain. Interestingly, such carbohydrate binding modules are common to bacterial glycoside hydrolases, serving to both increase local enzyme-substrate concentrations and to impart substrate specificity to nominally nonspecific catalytic domains by targeting the enzyme to specific substrates or substrate features (
      • Wakinaka T.
      • Kiyohara M.
      • Kurihara S.
      • Hirata A.
      • Chaiwangsri T.
      • Ohnuma T.
      • Fukamizo T.
      • Katayama T.
      • Ashida H.
      • Yamamoto K.
      Bifidobacterial α-galactosidase with unique carbohydrate-binding module specifically acts on blood group B antigen.
      ,
      • Cuskin F.
      • Flint J.E.
      • Gloster T.M.
      • Morland C.
      • Baslé A.
      • Henrissat B.
      • Coutinho P.M.
      • Strazzulli A.
      • Solovyova A.S.
      • Davies G.J.
      • Gilbert H.J.
      How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity.
      ,
      • Boraston A.B.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      Carbohydrate-binding modules. Fine-tuning polysaccharide recognition.
      ,
      • Hervé C.
      • Rogowski A.
      • Blake A.W.
      • Marcus S.E.
      • Gilbert H.J.
      • Knox J.P.
      Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects.
      ). Numerous studies on the ppGalNAc Ts have indeed demonstrated that their lectin domains serve similar functions to modulate glycopeptide substrate recognition and specificity (
      • Hassan 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.
      ,
      • Hanisch F.G.
      • Reis C.A.
      • Clausen H.
      • Paulsen H.
      Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides.
      ,
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      ,
      • Bennett E.P.
      • Hassan H.
      • Hollingsworth M.A.
      • Clausen H.
      A novel human UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates.
      ,
      • 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.
      Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T10.
      ), but to date there has been no systematic study of the family against a common set of (glyco)peptide substrates. In this work, we have characterized eight nonglycopeptide-requiring ppGalNAc T isoforms against the series of random (glyco)peptide substrates listed in Table 1. Our findings have unambiguously revealed that prior GalNAc-O-Thr(Ser) substrate glycosylation can be recognized by these transferases in a specific N- or C-terminal direction that varies with ppGalNAc T isoform (see FIGURE 3, FIGURE 4). Thus, for all eight transferases, at least one of the glycopeptide substrates, GPIV and/or GPV, shows significantly elevated activity over its nonglycosylated control. We attribute this N- or C-terminal specificity (i.e. elevated activity) to the binding of the glycosylated Thr residue of the substrate to the lectin domain in such a manner that the acceptor region of the substrate is oriented at the catalytic domain for optimal glycosylation. These findings strongly suggest that the tethered lectin domain on the ppGalNAc Ts may be mobile and that its location relative to the catalytic domain varies among isoforms. Such domain mobility is supported by the superimposition of the x-ray crystal structures of ppGalNAc T1, T2, and T10, as shown in Fig. 6B (
      • Fritz T.A.
      • Raman J.
      • Tabak L.A.
      Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase-2.
      ,
      • Fritz T.A.
      • Hurley J.H.
      • Trinh L.B.
      • Shiloach J.
      • Tabak L.A.
      The beginnings of mucin biosynthesis. The crystal structure of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase-T1.
      ,
      • Kubota 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 α-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).
      ). For those transferases with similar N- and C-terminal glycopeptide enhancements (Fig. 4), it is possible that the lectin domain may be sufficiently mobile that it can enhance glycosylation from both an N- and C-terminal direction. Alternatively, the lectin domain may serve to equally increase the N- and C-terminal glycopeptide substrate concentrations by a simple bind and release mechanism, as shown for the glycoside hydrolases (
      • Wakinaka T.
      • Kiyohara M.
      • Kurihara S.
      • Hirata A.
      • Chaiwangsri T.
      • Ohnuma T.
      • Fukamizo T.
      • Katayama T.
      • Ashida H.
      • Yamamoto K.
      Bifidobacterial α-galactosidase with unique carbohydrate-binding module specifically acts on blood group B antigen.
      ,
      • Cuskin F.
      • Flint J.E.
      • Gloster T.M.
      • Morland C.
      • Baslé A.
      • Henrissat B.
      • Coutinho P.M.
      • Strazzulli A.
      • Solovyova A.S.
      • Davies G.J.
      • Gilbert H.J.
      How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity.
      ,
      • Boraston A.B.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      Carbohydrate-binding modules. Fine-tuning polysaccharide recognition.
      ,
      • Hervé C.
      • Rogowski A.
      • Blake A.W.
      • Marcus S.E.
      • Gilbert H.J.
      • Knox J.P.
      Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects.
      ). Further evidence for a highly flexible lectin domain may be found in the broad distribution of glycosylation that is observed in the Xaa region of both the glycosylated and nonglycosylated substrates (see FIGURE 7, FIGURE 8). The only significant alteration in distribution is observed for ppGalNAc T3 glycosylating its nonpreferred glycopeptide substrate GPIV (Fig. 7). This suggests that the lectin domain of this transferase is involved to some extent in directing the glycosylation of this substrate. Obviously, further studies are necessary to fully understand the dynamics of the lectin domain of these transferases.
      TABLE 1ppGalNAc T lectin domain probing random (glyco)peptide substrates
      (Glyco)peptideSequence
      GPIVGAGAXXXXXXXXXXXXZZZZZT*ZZZZZAG
      GPIV-CGAGAXXXXXXXXXXXXZZZZZAZZZZZAG
      GPVGAGAZZZZZT*ZZZZZXXXXXXXXXXXXAG
      GPV-CGAGAZZZZZAZZZZZXXXXXXXXXXXXAG
      In an attempt to correlate the different N- and C-terminal glycopeptide specificities to transferase peptide sequence we have compared the sequences of their linker domain and selected lectin domain motifs, as shown in Table 2. From the alignment of the linker domains, no obvious differences in length or sequence can readily account for the different behavior observed within a class (i.e. ppGalNAc T2 and T14, which prefer glycopeptide GPIV, are only 1 residue shorter than T16, which shows preferences for both glycopeptide substrates, whereas the linker domains of ppGalNAc T1 and T13 are identical except for two conservative substitutions, although they show different glycopeptide specificities). By contrast, the linker domains of ppGalNAc T3 and T6 are only 50% identical but have the same elevated GPV preferences.
      Numerous studies of the ricin lectin α-, β-, and γ-subdomain repeats across multiple organisms have revealed several binding motifs, including the CLD and QXW sequences (
      • Fujimoto Z.
      • Kuno A.
      • Kaneko S.
      • Yoshida S.
      • Kobayashi H.
      • Kusakabe I.
      • Mizuno H.
      Crystal structure of Streptomyces olivaceoviridis E-86 β-xylanase containing xylan-binding domain.
      ,
      • Imberty A.
      • Piller V.
      • Piller F.
      • Breton C.
      Fold recognition and molecular modeling of a lectin-like domain in UDP-GalNac:polypeptide N-acetylgalactosaminyltransferases.
      ,
      • Hazes B.
      The (QxW)3 domain. A flexible lectin scaffold.
      ,
      • Maveyraud L.
      • Niwa H.
      • Guillet V.
      • Svergun D.I.
      • Konarev P.V.
      • Palmer R.A.
      • Peumans W.J.
      • Rougé P.
      • Van Damme E.J.
      • Reynolds C.D.
      • Mourey L.
      Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.
      ). Mutagenesis studies of the ppGalNAc T lectin domains have shown that the Asp residue of the α-subdomain CLD motif is typically required for lectin binding activities (i.e. ppGalNAc T1-D444A (
      • Pedersen J.W.
      • Bennett E.P.
      • Schjoldager K.T.
      • Meldal M.
      • Holmér A.P.
      • Blixt O.
      • Cló E.
      • Levery S.B.
      • Clausen H.
      • Wandall H.H.
      Lectin domains of polypeptide GalNAc-transferases exhibit glycopeptide binding specificity.
      ,
      • Raju T.S.
      • Davidson E.A.
      Role of sialic acid on the viscosity of canine tracheal mucin glycoprotein.
      ,
      • Tenno M.
      • Saeki A.
      • Kézdy F.J.
      • Elhammer A.P.
      • Kurosaka A.
      The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites.
      ), ppGalNAc T2-D458H (
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Pedersen J.W.
      • Bennett E.P.
      • Schjoldager K.T.
      • Meldal M.
      • Holmér A.P.
      • Blixt O.
      • Cló E.
      • Levery S.B.
      • Clausen H.
      • Wandall H.H.
      Lectin domains of polypeptide GalNAc-transferases exhibit glycopeptide binding specificity.
      ), and ppGalNAc T3-D519H (
      • Pedersen J.W.
      • Bennett E.P.
      • Schjoldager K.T.
      • Meldal M.
      • Holmér A.P.
      • Blixt O.
      • Cló E.
      • Levery S.B.
      • Clausen H.
      • Wandall H.H.
      Lectin domains of polypeptide GalNAc-transferases exhibit glycopeptide binding specificity.
      ,
      • Yoshimura Y.
      • Nudelman A.S.
      • Levery S.B.
      • Wandall H.H.
      • Bennett E.P.
      • Hindsgaul O.
      • Clausen H.
      • Nishimura S.
      Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates.
      ) are lectin-inactivating mutants). A similar mutation in the ppGalNAc T1 β-ricin subdomain (D484A) only modestly decreases lectin-modulated activity (
      • Tenno M.
      • Saeki A.
      • Kézdy F.J.
      • Elhammer A.P.
      • Kurosaka A.
      The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites.
      ,
      • Tenno M.
      • Kézdy F.J.
      • Elhammer A.P.
      • Kurosaka A.
      Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1.
      ), whereas mutation of the γ-ricin subdomains of ppGalNAc T1 and T2 (D525A and D541A, respectively) shows no effects on their lectin domain activities (
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Tenno M.
      • Saeki A.
      • Kézdy F.J.
      • Elhammer A.P.
      • Kurosaka A.
      The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites.
      ,
      • Tenno M.
      • Kézdy F.J.
      • Elhammer A.P.
      • Kurosaka A.
      Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1.
      ). Mutation of the α-subdomain of ppGalNAc T4 (D459H) also eliminates its lectin activity (
      • Hassan 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.
      ). As shown in Table 2, the β-subdomains of ppGalNAc T2, T14, T16, T3, T6, and T5 and the γ-subdomains of T3 and T6 lack this Asp. Several transferases possess Glu instead of Asp (i.e. ppGalNAc T14 and T16 in the α-subdomain and T16 and T5 in the γ-domain); whether these domains can bind GalNAc is presently unknown. The β-subdomains of ppGalNAc T6 and T5 lack the QXW motif having EEW and LKW and would be expected to be inactive. Interestingly, the co-crystal structure of ppGalNAc T10 shows GalNAc-O-Ser bound to its β-subdomain (having canonical motifs CFD and QLW) (
      • Kubota 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 α-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).
      ). The ricin subdomain motifs of ppGalNAc T5 would be expected to have the weakest lectin binding activities because all three subdomains lack the critical Asp residue, although, in its γ-subdomain, the Asp is replaced by a Glu. Nevertheless, because ppGalNAc T5 displays clear glycopeptide specificities, one or more of its lectin subdomains (perhaps its γ-subdomain) must possess significant binding activity to provide glycopeptide enhancements. Generally, glycopeptide binding to of any of the ppGalNAc Ts lectin domains has been difficult to directly detect, further suggesting its weak nature (
      • 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.
      The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc. Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
      ,
      • Pedersen J.W.
      • Bennett E.P.
      • Schjoldager K.T.
      • Meldal M.
      • Holmér A.P.
      • Blixt O.
      • Cló E.
      • Levery S.B.
      • Clausen H.
      • Wandall H.H.
      Lectin domains of polypeptide GalNAc-transferases exhibit glycopeptide binding specificity.
      ,
      • Yoshimura Y.
      • Nudelman A.S.
      • Levery S.B.
      • Wandall H.H.
      • Bennett E.P.
      • Hindsgaul O.
      • Clausen H.
      • Nishimura S.
      Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates.
      ). As with the analysis of the linker domains, we conclude that we cannot discern any obvious correlation between the likely activities of specific lectin subdomain motif and a given transferase's observed glycopeptide specificity.
      From the above discussion of the lectin domain motifs listed in Table 2, it is likely, for all but ppGalNAc T5, that glycopeptide binding would occur predominantly at the α-subdomain. As shown in the superimposed crystal structures of the ppGalNAc Ts in Fig. 6B, the critical Asp residue of the CLD motif in the α-subdomain (highlighted in the structures) is found in a wide range of positions relative to the catalytic domain. One could easily assume that the conformational flexibility of the lectin domain relative to the catalytic domain in solution would be greater than that observed in Fig. 6B. Thus, both the structural and experimental results are consistent with a highly mobile lectin domain, whose dynamics and relative position with respect to the catalytic domain varies widely with transferase isoform. Whether additional domain-domain interactions may modulate the relative positions of the lectin domain to the catalytic domain is presently unknown, but it seems highly plausible that such interactions could assist in the positioning of the lectin domain. Future studies will address these questions.

      Prior Evidence for the Lectin Domain Targeting N- or C-terminal Glycopeptide Glycosylation

      The glycosylation of the Muc 7 peptide (PTPSATT7PAPPSS13S14APPET19T20AAK) by the subclass Ia transferases ppGalNAc T1 and T13 has been examined by Zhang et al. (
      • Zhang Y.
      • Iwasaki H.
      • Wang H.
      • Kudo T.
      • Kalka T.B.
      • Hennet T.
      • Kubota T.
      • Cheng L.
      • Inaba N.
      • Gotoh M.
      • Togayachi A.
      • Guo J.
      • Hisatomi H.
      • Nakajima K.
      • Nishihara S.
      • Nakamura M.
      • Marth J.D.
      • Narimatsu H.
      Cloning and characterization of a new human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes Tn antigen.
      ). Interestingly, they observed that although Thr7 was initially glycosylated by both transferases, only ppGalNAc T13 was capable of further glycosylating the boldface C-terminal Ser and Thr residues of the peptide. These findings are entirely consistent with the transferase glycopeptide preferences summarized in Fig. 4, where ppGalNAc T1 prefers to glycosylate sites N-terminal of a prior site of glycosylation, whereas ppGalNAc T13 is capable of glycosylating sites both N- and C-terminal of a prior site of glycosylation. Raman et al. (
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      ) have extensively characterized the glycosylation of a series of MUC5AC glycopeptides by ppGalNAc T2 and its catalytic domain. Based on comparative binding studies, it was concluded that ppGalNAc T2 bound glycopeptide when the acceptor site was 10 residues N-terminal of an existing site of GalNAc glycosylation but not a glycopeptide with the reverse orientation and that this was due to the presence of the lectin domain, an observation consistent with our studies of ppGalNAc T2 (see Fig. 4).
      Note that Raman et al. (
      • Raman J.
      • Fritz T.A.
      • Gerken T.A.
      • Jamison O.
      • Live D.
      • Liu M.
      • Tabak L.A.
      The catalytic and lectin domains of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.
      ) have also proposed in their kinetic analysis that the ppGalNAc T2 lectin domain may also direct glycosylation to sites C-terminal of a GalNAc-O-Thr residue by assisting in product release rather than assisting in substrate binding.
      Likewise, studies on the glycan binding requirements of the lectin domain of ppGalNAc T3 suggest that this transferase utilizes its lectin domain to glycosylate residues C-terminal of the site of prior glycosylation (
      • Yoshimura Y.
      • Nudelman A.S.
      • Levery S.B.
      • Wandall H.H.
      • Bennett E.P.
      • Hindsgaul O.
      • Clausen H.
      • Nishimura S.
      Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates.
      ), again in keeping with our findings for ppGalNAc T3. ppGalNAc T3 was previously shown to be the only isoform capable of glycosylating the proprotein processing region of FGF23 (IHFNT171PIPR RHT178R↓SAEDD) (
      • Kato K.
      • Jeanneau C.
      • Tarp M.A.
      • Benet-Pagès A.
      • Lorenz-Depiereux B.
      • Bennett E.P.
      • Mandel U.
      • Strom T.M.
      • Clausen H.
      Polypeptide GaINAc-transferase T3 and familial tumoral calcinosis. Secretion of FGF23 requires O-glycosylation.
      ,
      • Ichikawa S.
      • Sorenson A.H.
      • Austin A.M.
      • Mackenzie D.S.
      • Fritz T.A.
      • Moh A.
      • Hui S.L.
      • Econs M.J.
      Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.
      ,
      • Garringer H.J.
      • Fisher C.
      • Larsson T.E.
      • Davis S.I.
      • Koller D.L.
      • Cullen M.J.
      • Draman M.S.
      • Conlon N.
      • Jain A.
      • Fedarko N.S.
      • Dasgupta B.
      • White K.E.
      The role of mutant UDP-N-acetyl-α-d-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 in regulating serum intact fibroblast growth factor 23 and matrix extracellular phosphoglycoprotein in heritable tumoral calcinosis.
      ), and in unpublished work,
      K. Kato, C. Jeanneau, I. Dar, E. P. Bennett, K. T. Schjoldager, A. Benet-Pagès, T. M. Strom, and H. Clausen, unpublished results.
      Kato et al. have found that ppGalNAc T3 glycosylates the Thr178 site in a lectin-dependent manner by first glycosylating Thr171 in a lectin-independent manner, both in in vitro enzyme assays and ex vivo in CHO cells stably transfected with ppGalNAc T3 variants. The Thr171 site is also glycosylated by several other ppGalNAc-T isoforms (
      • Kato K.
      • Jeanneau C.
      • Tarp M.A.
      • Benet-Pagès A.
      • Lorenz-Depiereux B.
      • Bennett E.P.
      • Mandel U.
      • Strom T.M.
      • Clausen H.
      Polypeptide GaINAc-transferase T3 and familial tumoral calcinosis. Secretion of FGF23 requires O-glycosylation.
      ). Our previous work (
      • Gerken 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 (ppGalNAc T) family of glycosyltransferases.
      ) utilizing transferase specific preferences, obtained from random peptide studies, also suggests that Thr171 would be a moderate to good substrate for ppGalNAc T1, T2, and T3, whereas Thr178 would not be a substrate for ppGalNAc T1 or T2 and would be only a modest substrate for ppGalNAc T3 (see Table 2 of Ref
      • Gerken 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 (ppGalNAc T) family of glycosyltransferases.
      ). This suggests that the prior glycosylation of Thr171 may serve to target ppGalNAc T3 glycosylation of Thr178, 7 residues C-terminal of Thr171. The glycosylation distribution plots for ppGalNAc T3 for glycopeptide GPV (Figs. 7C and 8D) are entirely consistent with this, showing a peak of glycosylation 8–9 residues C-terminal of the original site of glycosylation. By contrast, the prior glycosylation of Thr171 would fail to enhance the glycosylation of Thr178 by ppGalNAc T1 and T2 because these transferases possess the reverse glycopeptide specificities. Thus, glycosylation of Thr171 enhances the glycosylation of Thr178 by ppGalNAc T3 while effectively reducing the activities of those transferases with the reverse glycopeptide preferences. Thus, prior glycosylation of Thr171 serves as a targeting switch or enhancer for the glycosylation of Thr178, whose glycosylation is required to inhibit the proprotein convertase cleavage of FGF23 in vivo and in vitro (
      • Schjoldager K.T.
      • Vester-Christensen M.B.
      • Goth C.K.
      • Petersen T.N.
      • Brunak S.
      • Bennett E.P.
      • Levery S.B.
      • Clausen H.
      A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing.
      ,
      • Kato K.
      • Jeanneau C.
      • Tarp M.A.
      • Benet-Pagès A.
      • Lorenz-Depiereux B.
      • Bennett E.P.
      • Mandel U.
      • Strom T.M.
      • Clausen H.
      Polypeptide GaINAc-transferase T3 and familial tumoral calcinosis. Secretion of FGF23 requires O-glycosylation.
      ,
      • Garringer H.J.
      • Fisher C.
      • Larsson T.E.
      • Davis S.I.
      • Koller D.L.
      • Cullen M.J.
      • Draman M.S.
      • Conlon N.
      • Jain A.
      • Fedarko N.S.
      • Dasgupta B.
      • White K.E.
      The role of mutant UDP-N-acetyl-α-d-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 in regulating serum intact fibroblast growth factor 23 and matrix extracellular phosphoglycoprotein in heritable tumoral calcinosis.
      ).
      The control of site-specific mucin type O-glycosylation and the need for such a large family of initiating ppGalNAc Ts is not well understood.
      ppGalNAc T1 and T2 are ubiquitously expressed in nearly all mammalian tissues and cell lines, whereas the remaining ppGalNAc T isoforms studied in this work are selectively expressed (see Refs.
      • Bennett 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.
      ,
      • Steentoft C.
      • Vakhrushev S.Y.
      • Joshi H.J.
      • Kong Y.
      • Vester-Christensen M.B.
      • Schjoldager K.T.B.
      • Lavrsen K.
      • Dabelsteen S.
      • Pedersen N.B.
      • Marcos-Silva L.
      • Gupta R.
      • Paul Bennett E.
      • Mandel U.
      • Brunak S.
      • Wandall H.H.
      • Levery S.B.
      • Clausen H.
      Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.
      , and
      • Young Jr., W.W.
      • Holcomb D.R.
      • Ten Hagen K.G.
      • Tabak L.A.
      Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isoforms in murine tissues determined by real-time PCR. A new view of a large family.
      ). For example, ppGalNAc T3 is the dominant transferase expressed in the testis and is also highly expressed in the kidney (
      • Kato K.
      • Jeanneau C.
      • Tarp M.A.
      • Benet-Pagès A.
      • Lorenz-Depiereux B.
      • Bennett E.P.
      • Mandel U.
      • Strom T.M.
      • Clausen H.
      Polypeptide GaINAc-transferase T3 and familial tumoral calcinosis. Secretion of FGF23 requires O-glycosylation.
      ,
      • Young Jr., W.W.
      • Holcomb D.R.
      • Ten Hagen K.G.
      • Tabak L.A.
      Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isoforms in murine tissues determined by real-time PCR. A new view of a large family.
      ,
      • Bennett 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- α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy.
      ), and ppGalNAc T16 is more highly expressed than ppGalNAc T1 in the heart and brain (
      • Raman J.
      • Guan Y.
      • Perrine C.L.
      • Gerken T.A.
      • Tabak L.A.
      UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases. Completion of the family tree.
      ), whereas ppGalNAc T13 is specifically expressed in neurons (
      • Zhang Y.
      • Iwasaki H.
      • Wang H.
      • Kudo T.
      • Kalka T.B.
      • Hennet T.
      • Kubota T.
      • Cheng L.
      • Inaba N.
      • Gotoh M.
      • Togayachi A.
      • Guo J.
      • Hisatomi H.
      • Nakajima K.
      • Nishihara S.
      • Nakamura M.
      • Marth J.D.
      • Narimatsu H.
      Cloning and characterization of a new human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes Tn antigen.
      ). How the multiple transferases work in concert in glycosylating their target proteins is largely unknown, as is their regulation at both the protein and transcriptional levels, although, judging from observations in our laboratory and other laboratories, large differences in protein stabilities exist.
      In this work, we have identified a previously unappreciated level of control, whereby prior O-glycosylation is used to target and enhance the glycosylation of specific N- or C-terminal sites in an isoform-specific manner. Our studies and those of others strongly suggest that this N- or C- terminal selectivity is due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is highly mobile and isoform-dependent. This glycopeptide selectivity can provide an additional level of control or fidelity for the glycosylation of biologically significant sites and suggests that O-glycosylation in some instances may be exquisitely controlled. Furthermore, our observations that homologous ppGalNAc T isoforms within a given subfamily (that presumably have similar peptide substrate specificities; see Ref.
      • Gerken T.A.
      • Ten Hagen K.G.
      • Jamison O.
      Conservation of peptide acceptor preferences between Drosophila and mammalian polypeptide-GalNAc transferase orthologue pairs.
      ) may possess different N- or C-terminal glycopeptide preferences may help explain the large number of ppGalNAc T family members thereby maintaining peptide specificity while altering glycopeptide specificity. These studies clearly demonstrate that the biological control mucin type O-glycosylation is highly complex and that further structural, biochemical, and biological studies are necessary to fully understand this important modification.

      Acknowledgments

      We thank Oliver Jamison for initial assistance with designing the lectin peptide substrates and Dr. Vivian Yee for assistance with the molecular modeling.

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