Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

Sujatha Manthri, M. Lucia S. Guther, Luis Izquierdo, Alvaro Acosta-Serrano, Michael A. J. Ferguson

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    Abstract

    We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man(9)GlcNAc(2)from Man(9)GlcNAc(2)-PP-Dol while others receive Man(5)GlcNAc(2) from Man(5)GlcNAc(2)-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the alpha-mannosyltransferase that converts Man(5)GlcNAc(2)-PP-Dol to Man(6)GlcNAc(2)-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man(9)GlcNAc(2) and processed no further than Man(5)GlcNAc(2) in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man(5)GlcNAc(2)-PP-Dol is the only route to complex N-glycans in T. brucei and that Man(9)GlcNAc(2)-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man(5)GlcNAc(2) or Man(9)GlcNAc(2) transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.

    Original languageEnglish
    Pages (from-to)367-383
    Number of pages17
    JournalGlycobiology
    Volume18
    Issue number5
    DOIs
    Publication statusPublished - May 2008

    Keywords

    • VARIANT SURFACE GLYCOPROTEIN
    • PHOSPHATIDYLINOSITOL MEMBRANE ANCHOR
    • ASPARAGINE-LINKED OLIGOSACCHARIDES
    • ACIDIC REPETITIVE PROTEIN
    • BLOOD-STREAM FORM
    • CONCANAVALIN-A
    • STRUCTURAL-CHARACTERIZATION
    • MUTANTS RESISTANT
    • PROCYCLIC FORM
    • BIOSYNTHESIS

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