Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases.
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 Deltastt3 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.
Izquierdo
Luis
L
Division of Biological Chemistry and Drug Discovery, The College of Life Sciences, University of Dundee, Dundee, UK.
Schulz
Benjamin L
BL
Rodrigues
João A
JA
Güther
Maria Lucia S
ML
Procter
James B
JB
Barton
Geoffrey J
GJ
Aebi
Markus
M
Ferguson
Michael A J
MA
D000818:Animals; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D006023:Glycoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins