title: Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family.
abstract: A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood. We have previously shown for several peptide-preferring isoforms that the presence of a remote Thr-O-GalNAc, 6-17 residues from a Ser/Thr acceptor site, may enhance overall catalytic activity in an N- or C-terminal direction. This enhancement varies with isoform and is attributed to Thr-O-GalNAc interactions at the lectin domain. We now report on the glycopeptide substrate utilization of a series of glycopeptide (human-ppGalNAc-T4, T7, T10, T12 and fly PGANT7) and peptide-preferring transferases (T2, T3 and T5) by exploiting a series of random glycopeptide substrates designed to probe the functions of their catalytic and lectin domains. Glycosylation was observed at the -3, -1 and +1 residues relative to a neighboring Thr-O-GalNAc, depending on isoform, which we attribute to specific Thr-O-GalNAc binding at the catalytic domain. Additionally, these glycopeptide-preferring isoforms show remote lectin domain-assisted Thr-O-GalNAc enhancements that vary from modest to none. We conclude that the glycopeptide specificity of the glycopeptide-preferring isoforms predominantly resides in their catalytic domain but may be further modulated by remote lectin domain interactions. These studies further demonstrate that both domains of the ppGalNAc-Ts have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation.
authors.affiliation: Department of Chemistry.
authors.forename: Leslie
authors.initials: L
authors.lastname: Revoredo
authors.affiliation: Copenhagen Center for Glycomics (CCG), Departments of Cellular and Molecular Medicine and Dentistry, Faculty of Health Sciences, University of Copenhagen, Copenhagen N DK-2200, Denmark.
authors.forename: Shengjun
authors.initials: S
authors.lastname: Wang
authors.affiliation: Copenhagen Center for Glycomics (CCG), Departments of Cellular and Molecular Medicine and Dentistry, Faculty of Health Sciences, University of Copenhagen, Copenhagen N DK-2200, Denmark.
authors.forename: Eric Paul
authors.initials: EP
authors.lastname: Bennett
authors.affiliation: Copenhagen Center for Glycomics (CCG), Departments of Cellular and Molecular Medicine and Dentistry, Faculty of Health Sciences, University of Copenhagen, Copenhagen N DK-2200, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
authors.affiliation: Developmental Glycobiology Section.
authors.forename: Kelly G
authors.initials: KG
authors.lastname: Ten Hagen
authors.affiliation: Section on Biological Chemistry, Department of Health and Human Services, NIDCR, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Tabak
authors.affiliation: Department of Chemistry Department of Pediatrics and Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA txg2@cwru.edu.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Gerken
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain; D005643:Fucose / Q000031:analogs & derivatives / Q000737:chemistry; D006020:Glycopeptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000235:genetics; D009077:Mucins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D006020:Glycopeptides; D037102:Lectins; D009077:Mucins; D005643:Fucose; C059377:N-acetylfucosamine; D012799:Sialyltransferases
keywords: glycoprotein biosynthesis; glycosyltransferase; lectin; mucin