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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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Mass spectrometric analyses of intact mature sVSG221 glycoforms before and after selective knockdown of TbSTT3A and TbSTT3B expression. Samples of sVSG221 from (A) wild type cells, (B) TbSTT3A,B,C+/−, (C) TbSTT3A,B,C+/−-STT3ARNAi minus Tet, (D) TbSTT3A,B,C+/−-STT3ARNAi plus Tet, (E) TbSTT3A,B,C+/−-STT3BRNAi minus Tet and (F) TbSTT3A,B,C+/−-STT3BRNAi plus Tet were analysed by ES-MS. The spectra show the masses of the various glycoforms of the intact mature sVSG221 molecules. The inset cartoons represent our interpretation of those glycoform masses in terms of the ranges of N-glycans present at each of the two N-glycosylation sites. These assignments combine additional data from the ES-MS and ES-MS/MS analyses of Pronase glycopeptides from the same sVSG221 preparations (Supplementary Figure S3; Table SIV). In the inset cartoons, endo-H-resistant N-glycans are in white and endo-H-sensitive N-glycans are in black. Circles and squares (filled and open) represent mannose and N-acetylglucosmaine residues, respectively, and grey circles represent galactose residues.
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f2: Mass spectrometric analyses of intact mature sVSG221 glycoforms before and after selective knockdown of TbSTT3A and TbSTT3B expression. Samples of sVSG221 from (A) wild type cells, (B) TbSTT3A,B,C+/−, (C) TbSTT3A,B,C+/−-STT3ARNAi minus Tet, (D) TbSTT3A,B,C+/−-STT3ARNAi plus Tet, (E) TbSTT3A,B,C+/−-STT3BRNAi minus Tet and (F) TbSTT3A,B,C+/−-STT3BRNAi plus Tet were analysed by ES-MS. The spectra show the masses of the various glycoforms of the intact mature sVSG221 molecules. The inset cartoons represent our interpretation of those glycoform masses in terms of the ranges of N-glycans present at each of the two N-glycosylation sites. These assignments combine additional data from the ES-MS and ES-MS/MS analyses of Pronase glycopeptides from the same sVSG221 preparations (Supplementary Figure S3; Table SIV). In the inset cartoons, endo-H-resistant N-glycans are in white and endo-H-sensitive N-glycans are in black. Circles and squares (filled and open) represent mannose and N-acetylglucosmaine residues, respectively, and grey circles represent galactose residues.

Mentions: We studied the roles of the TbSTT3 genes in trypanosomes using inducible RNA interference (RNAi) and glycoprotein structure analysis. Our principal reporter molecule is the VSG coat glycoprotein of the parasite that, in the variant 221 clone used in our studies, contains two N-glycosylation sites (see Figure 2A, inset): one at Asn263 occupied by small, biantennary, endoglycosidase-H (EndoH)-resistant paucimannose and complex structures, which originate from the transfer of Man5GlcNAc2 from the Man5GlcNAc2-PP-Dol precursor, and one at Asn428 occupied by conventional EndoH-sensitive triantennary oligomannose structures, which originate from the transfer of Man9GlcNAc2 from Man9GlcNAc2-PP-Dol (Jones et al, 2005; Manthri et al, 2008). Thus, analysis of VSG221 N-glycosylation allows us to simultaneously assess the effects of genetic or chemical perturbations on both mechanisms of protein N-glycosylation in this organism (Jones et al, 2005; Urbaniak et al, 2006; Manthri et al, 2008; Stokes et al 2008). Cell-surface VSG can be purified in a soluble form (sVSG221) after osmotic shock, a process that releases VSG from the parasite surface by the action of endogenous GPI-specific phospholipase C that cleaves the dimyristoylglycerol lipid component of the VSG GPI anchor (Cross, 1975, 1984; Cardoso de Almeida and Turner, 1983; Ferguson et al, 1985). This cell surface-derived sVSG221 is amenable to glycoform analysis as an intact glycoprotein by electrospray–mass spectrometry (ES-MS) and as Pronase glycopeptides by ES-MS and ES-MS/MS (Jones et al, 2005; Urbaniak et al, 2006; Manthri et al, 2008; Stokes et al 2008). In addition, the N-glycosylation status of both cell surface-derived sVSG221 and of newly synthesised (presumably ER-associated) VSG221, which is not released by osmotic shock (Ferguson et al 1986), can be assessed by EndoH and PNGaseF digestion and analysis by SDS–PAGE and Coomassie blue staining or anti-VSG221 western blot, respectively.


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)

Mass spectrometric analyses of intact mature sVSG221 glycoforms before and after selective knockdown of TbSTT3A and TbSTT3B expression. Samples of sVSG221 from (A) wild type cells, (B) TbSTT3A,B,C+/−, (C) TbSTT3A,B,C+/−-STT3ARNAi minus Tet, (D) TbSTT3A,B,C+/−-STT3ARNAi plus Tet, (E) TbSTT3A,B,C+/−-STT3BRNAi minus Tet and (F) TbSTT3A,B,C+/−-STT3BRNAi plus Tet were analysed by ES-MS. The spectra show the masses of the various glycoforms of the intact mature sVSG221 molecules. The inset cartoons represent our interpretation of those glycoform masses in terms of the ranges of N-glycans present at each of the two N-glycosylation sites. These assignments combine additional data from the ES-MS and ES-MS/MS analyses of Pronase glycopeptides from the same sVSG221 preparations (Supplementary Figure S3; Table SIV). In the inset cartoons, endo-H-resistant N-glycans are in white and endo-H-sensitive N-glycans are in black. Circles and squares (filled and open) represent mannose and N-acetylglucosmaine residues, respectively, and grey circles represent galactose residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Mass spectrometric analyses of intact mature sVSG221 glycoforms before and after selective knockdown of TbSTT3A and TbSTT3B expression. Samples of sVSG221 from (A) wild type cells, (B) TbSTT3A,B,C+/−, (C) TbSTT3A,B,C+/−-STT3ARNAi minus Tet, (D) TbSTT3A,B,C+/−-STT3ARNAi plus Tet, (E) TbSTT3A,B,C+/−-STT3BRNAi minus Tet and (F) TbSTT3A,B,C+/−-STT3BRNAi plus Tet were analysed by ES-MS. The spectra show the masses of the various glycoforms of the intact mature sVSG221 molecules. The inset cartoons represent our interpretation of those glycoform masses in terms of the ranges of N-glycans present at each of the two N-glycosylation sites. These assignments combine additional data from the ES-MS and ES-MS/MS analyses of Pronase glycopeptides from the same sVSG221 preparations (Supplementary Figure S3; Table SIV). In the inset cartoons, endo-H-resistant N-glycans are in white and endo-H-sensitive N-glycans are in black. Circles and squares (filled and open) represent mannose and N-acetylglucosmaine residues, respectively, and grey circles represent galactose residues.
Mentions: We studied the roles of the TbSTT3 genes in trypanosomes using inducible RNA interference (RNAi) and glycoprotein structure analysis. Our principal reporter molecule is the VSG coat glycoprotein of the parasite that, in the variant 221 clone used in our studies, contains two N-glycosylation sites (see Figure 2A, inset): one at Asn263 occupied by small, biantennary, endoglycosidase-H (EndoH)-resistant paucimannose and complex structures, which originate from the transfer of Man5GlcNAc2 from the Man5GlcNAc2-PP-Dol precursor, and one at Asn428 occupied by conventional EndoH-sensitive triantennary oligomannose structures, which originate from the transfer of Man9GlcNAc2 from Man9GlcNAc2-PP-Dol (Jones et al, 2005; Manthri et al, 2008). Thus, analysis of VSG221 N-glycosylation allows us to simultaneously assess the effects of genetic or chemical perturbations on both mechanisms of protein N-glycosylation in this organism (Jones et al, 2005; Urbaniak et al, 2006; Manthri et al, 2008; Stokes et al 2008). Cell-surface VSG can be purified in a soluble form (sVSG221) after osmotic shock, a process that releases VSG from the parasite surface by the action of endogenous GPI-specific phospholipase C that cleaves the dimyristoylglycerol lipid component of the VSG GPI anchor (Cross, 1975, 1984; Cardoso de Almeida and Turner, 1983; Ferguson et al, 1985). This cell surface-derived sVSG221 is amenable to glycoform analysis as an intact glycoprotein by electrospray–mass spectrometry (ES-MS) and as Pronase glycopeptides by ES-MS and ES-MS/MS (Jones et al, 2005; Urbaniak et al, 2006; Manthri et al, 2008; Stokes et al 2008). In addition, the N-glycosylation status of both cell surface-derived sVSG221 and of newly synthesised (presumably ER-associated) VSG221, which is not released by osmotic shock (Ferguson et al 1986), can be assessed by EndoH and PNGaseF digestion and analysis by SDS–PAGE and Coomassie blue staining or anti-VSG221 western blot, respectively.

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