Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Luis Izquierdo, Benjamin L. Schulz, Joao A. Rodrigues, Maria Lucia S. Guther, James B. Procter, Geoffrey J. Barton, Markus Aebi (Lead / Corresponding author), Michael A. J. Ferguson (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    82 Citations (Scopus)

    Abstract

    Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Delta stt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man(5)GlcNAc(2) to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis. The EMBO Journal (2009) 28, 2650-2661. doi: 10.1038/emboj.2009.203; Published online 23 July 2009 Subject Categories: microbiology & pathogens; proteins

    Original languageEnglish
    Pages (from-to)2650-2661
    Number of pages12
    JournalEMBO Journal
    Volume28
    Issue number17
    DOIs
    Publication statusPublished - 2 Sep 2009

    Keywords

    • glycosylation
    • oligosaccharyltransferase
    • STT3
    • Trypanosoma
    • VARIANT SURFACE GLYCOPROTEIN
    • N-GLYCOSYLATION
    • LEISHMANIA-MAJOR
    • AFRICAN TRYPANOSOMES
    • REPETITIVE PROTEIN
    • MEMBRANE ANCHOR
    • LINKED GLYCANS
    • IN-VIVO
    • GENE
    • STT3

    Cite this

    @article{910950f58a9d4bd3866c79b5335d00a6,
    title = "Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases",
    abstract = "Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Delta stt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man(5)GlcNAc(2) to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis. The EMBO Journal (2009) 28, 2650-2661. doi: 10.1038/emboj.2009.203; Published online 23 July 2009 Subject Categories: microbiology & pathogens; proteins",
    keywords = "glycosylation, oligosaccharyltransferase, STT3, Trypanosoma, VARIANT SURFACE GLYCOPROTEIN, N-GLYCOSYLATION, LEISHMANIA-MAJOR, AFRICAN TRYPANOSOMES, REPETITIVE PROTEIN, MEMBRANE ANCHOR, LINKED GLYCANS, IN-VIVO, GENE, STT3",
    author = "Luis Izquierdo and Schulz, {Benjamin L.} and Rodrigues, {Joao A.} and Guther, {Maria Lucia S.} and Procter, {James B.} and Barton, {Geoffrey J.} and Markus Aebi and Ferguson, {Michael A. J.}",
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    language = "English",
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    pages = "2650--2661",
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    Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases. / Izquierdo, Luis; Schulz, Benjamin L.; Rodrigues, Joao A.; Guther, Maria Lucia S.; Procter, James B.; Barton, Geoffrey J.; Aebi, Markus (Lead / Corresponding author); Ferguson, Michael A. J. (Lead / Corresponding author).

    In: EMBO Journal, Vol. 28, No. 17, 02.09.2009, p. 2650-2661.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

    AU - Izquierdo, Luis

    AU - Schulz, Benjamin L.

    AU - Rodrigues, Joao A.

    AU - Guther, Maria Lucia S.

    AU - Procter, James B.

    AU - Barton, Geoffrey J.

    AU - Aebi, Markus

    AU - Ferguson, Michael A. J.

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    AB - Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Delta stt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man(5)GlcNAc(2) to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis. The EMBO Journal (2009) 28, 2650-2661. doi: 10.1038/emboj.2009.203; Published online 23 July 2009 Subject Categories: microbiology & pathogens; proteins

    KW - glycosylation

    KW - oligosaccharyltransferase

    KW - STT3

    KW - Trypanosoma

    KW - VARIANT SURFACE GLYCOPROTEIN

    KW - N-GLYCOSYLATION

    KW - LEISHMANIA-MAJOR

    KW - AFRICAN TRYPANOSOMES

    KW - REPETITIVE PROTEIN

    KW - MEMBRANE ANCHOR

    KW - LINKED GLYCANS

    KW - IN-VIVO

    KW - GENE

    KW - STT3

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