Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei.
We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNA(2 from Man5GlcNAc2-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 Man5GlcNAc2-PP-Dol to Man6GlcNAc2-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 Man9GlcNAc2 and processed no further than Man5GlcNAc2 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 Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.
Manthri
Sujatha
S
The Division of Biological Chemistry and Drug Discovery, The Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Güther
M Lucia S
ML
Izquierdo
Luis
L
Acosta-Serrano
Alvaro
A
Ferguson
Michael A J
MA
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017353:Gene Deletion; D006031:Glycosylation; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry / Q000235:genetics
D009844:Oligosaccharides; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C058642:mannosyl(5)-N-acetyl(2)-glucose; D008364:Mannosyltransferases