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Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases.

Izquierdo L, Schulz BL, Rodrigues JA, Güther ML, Procter JB, Barton GJ, Aebi M, Ferguson MA - EMBO J. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, The College of Life Sciences, University of Dundee, Dundee, UK.

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 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.

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Animal infectivity studies with STT3ARNAi and STT3BRNAi cell lines. (A) T. brucei TbSTT3A,B,C+/−-STT3ARNAi cells or (B) TbSTT3A,B,C+/−-STT3BRNAi cells present in the blood of mice dosed with (white bar) and without doxycycline (grey bar) for 2 days before and throughout the 3 day infection. (C) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3ARNAi plus doxycycline (lanes 4–6) and TbSTT3A,B,C+/−-STT3ARNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody. (D) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3BRNAi plus doxycycline (lanes 4–6), and TbSTT3A,B,C+/−-STT3BRNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody.
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f7: Animal infectivity studies with STT3ARNAi and STT3BRNAi cell lines. (A) T. brucei TbSTT3A,B,C+/−-STT3ARNAi cells or (B) TbSTT3A,B,C+/−-STT3BRNAi cells present in the blood of mice dosed with (white bar) and without doxycycline (grey bar) for 2 days before and throughout the 3 day infection. (C) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3ARNAi plus doxycycline (lanes 4–6) and TbSTT3A,B,C+/−-STT3ARNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody. (D) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3BRNAi plus doxycycline (lanes 4–6), and TbSTT3A,B,C+/−-STT3BRNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody.

Mentions: We next investigated whether the TbSTT3A/TbSTT3B redundancy seen in culture was also evident in animal infections. Although both RNAi cell lines grew well in doxycycline-free mice, the infections in the presence of RNAi-inducing doxycycline were very low in both cases (Figure 7A and B). Western blot analysis of the VSG221 from the few surviving cells showed that they were expressing significant amounts of wild-type N-glycosylated VSG221 (Figure 7C and D). Most likely these cells proliferated because they had lost some of the effects of the doxycycline-induced TbSTT3 knockdown, a well-known phenomenon in T. brucei RNAi experiments. These data suggest that both mechanisms of protein N-glycosylation are independently required for parasite growth in mice.


Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases.

Izquierdo L, Schulz BL, Rodrigues JA, Güther ML, Procter JB, Barton GJ, Aebi M, Ferguson MA - EMBO J. (2009)

Animal infectivity studies with STT3ARNAi and STT3BRNAi cell lines. (A) T. brucei TbSTT3A,B,C+/−-STT3ARNAi cells or (B) TbSTT3A,B,C+/−-STT3BRNAi cells present in the blood of mice dosed with (white bar) and without doxycycline (grey bar) for 2 days before and throughout the 3 day infection. (C) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3ARNAi plus doxycycline (lanes 4–6) and TbSTT3A,B,C+/−-STT3ARNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody. (D) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3BRNAi plus doxycycline (lanes 4–6), and TbSTT3A,B,C+/−-STT3BRNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2722254&req=5

f7: Animal infectivity studies with STT3ARNAi and STT3BRNAi cell lines. (A) T. brucei TbSTT3A,B,C+/−-STT3ARNAi cells or (B) TbSTT3A,B,C+/−-STT3BRNAi cells present in the blood of mice dosed with (white bar) and without doxycycline (grey bar) for 2 days before and throughout the 3 day infection. (C) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3ARNAi plus doxycycline (lanes 4–6) and TbSTT3A,B,C+/−-STT3ARNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody. (D) Purified sVSG221 of wild-type cells grown in culture (lanes 1–3, as a control), and TbSTT3A,B,C+/−-STT3BRNAi plus doxycycline (lanes 4–6), and TbSTT3A,B,C+/−-STT3BRNAi minus doxycycline (lanes 7–9) cells isolated from mouse blood, digested or not, as indicated, with EndoH or PNGaseF were subjected SDS–PAGE and western blot with anti-VSG221 antibody.
Mentions: We next investigated whether the TbSTT3A/TbSTT3B redundancy seen in culture was also evident in animal infections. Although both RNAi cell lines grew well in doxycycline-free mice, the infections in the presence of RNAi-inducing doxycycline were very low in both cases (Figure 7A and B). Western blot analysis of the VSG221 from the few surviving cells showed that they were expressing significant amounts of wild-type N-glycosylated VSG221 (Figure 7C and D). Most likely these cells proliferated because they had lost some of the effects of the doxycycline-induced TbSTT3 knockdown, a well-known phenomenon in T. brucei RNAi experiments. These data suggest that both mechanisms of protein N-glycosylation are independently required for parasite growth in mice.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, The College of Life Sciences, University of Dundee, Dundee, UK.

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 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.

Show MeSH
Related in: MedlinePlus