Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei

Manuela Damerow, Joao A. Rodrigues, Di Wu, M. Lucia S. Güther, Angela Mehlert, Michael A. J. Ferguson (Lead / Corresponding author)

    Research output: Contribution to journalArticle

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    Abstract

    Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known ß1-4-galactosyltransferase or ß1-2- or ß1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian ß1-3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:a3-D-mannoside ß1-2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical "pseudohybrid" glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the ß3-glycosyltransferase family to catalyze ß1-2 linkages.

    Original languageEnglish
    Pages (from-to)9328-9339
    Number of pages12
    JournalJournal of Biological Chemistry
    Volume289
    Issue number13
    DOIs
    Publication statusPublished - 28 Mar 2014

    Fingerprint

    Trypanosoma brucei brucei
    Polysaccharides
    Glycosyltransferases
    Uridine Diphosphate
    Genes
    Parasites
    Uridine Diphosphate Sugars
    Mannosides
    Galactosyltransferases
    Sequence Alignment
    Biosynthesis
    Golgi Apparatus
    Rodentia
    Assays
    alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
    Genome
    Substrates
    Enzymes
    Growth
    Proteins

    Keywords

    • Blood
    • Cell Line
    • Glycosylation
    • Golgi Apparatus
    • Humans
    • Mutation
    • N-Acetylglucosaminyltransferases
    • Phenotype
    • Polysaccharides
    • Protein Transport
    • Substrate Specificity
    • Trypanosoma brucei brucei
    • Uridine Diphosphate

    Cite this

    @article{70f196a2514c4271b159c7ebce379330,
    title = "Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei",
    abstract = "Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known {\ss}1-4-galactosyltransferase or {\ss}1-2- or {\ss}1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian {\ss}1-3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:a3-D-mannoside {\ss}1-2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical {"}pseudohybrid{"} glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the {\ss}3-glycosyltransferase family to catalyze {\ss}1-2 linkages.",
    keywords = "Blood, Cell Line, Glycosylation, Golgi Apparatus, Humans, Mutation, N-Acetylglucosaminyltransferases, Phenotype, Polysaccharides, Protein Transport, Substrate Specificity, Trypanosoma brucei brucei, Uridine Diphosphate",
    author = "Manuela Damerow and Rodrigues, {Joao A.} and Di Wu and G{\"u}ther, {M. Lucia S.} and Angela Mehlert and Ferguson, {Michael A. J.}",
    year = "2014",
    month = "3",
    day = "28",
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    language = "English",
    volume = "289",
    pages = "9328--9339",
    journal = "Journal of Biological Chemistry",
    issn = "0021-9258",
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    Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei. / Damerow, Manuela; Rodrigues, Joao A.; Wu, Di; Güther, M. Lucia S.; Mehlert, Angela; Ferguson, Michael A. J. (Lead / Corresponding author).

    In: Journal of Biological Chemistry, Vol. 289, No. 13, 28.03.2014, p. 9328-9339.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei

    AU - Damerow, Manuela

    AU - Rodrigues, Joao A.

    AU - Wu, Di

    AU - Güther, M. Lucia S.

    AU - Mehlert, Angela

    AU - Ferguson, Michael A. J.

    PY - 2014/3/28

    Y1 - 2014/3/28

    N2 - Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known ß1-4-galactosyltransferase or ß1-2- or ß1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian ß1-3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:a3-D-mannoside ß1-2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical "pseudohybrid" glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the ß3-glycosyltransferase family to catalyze ß1-2 linkages.

    AB - Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known ß1-4-galactosyltransferase or ß1-2- or ß1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian ß1-3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:a3-D-mannoside ß1-2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical "pseudohybrid" glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the ß3-glycosyltransferase family to catalyze ß1-2 linkages.

    KW - Blood

    KW - Cell Line

    KW - Glycosylation

    KW - Golgi Apparatus

    KW - Humans

    KW - Mutation

    KW - N-Acetylglucosaminyltransferases

    KW - Phenotype

    KW - Polysaccharides

    KW - Protein Transport

    KW - Substrate Specificity

    KW - Trypanosoma brucei brucei

    KW - Uridine Diphosphate

    U2 - 10.1074/jbc.M114.555029

    DO - 10.1074/jbc.M114.555029

    M3 - Article

    C2 - 24550396

    VL - 289

    SP - 9328

    EP - 9339

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 13

    ER -