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The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation.

Stokes MJ, Güther ML, Turnock DC, Prescott AR, Martin KL, Alphey MS, Ferguson MA - J. Biol. Chem. (2008)

Bottom Line: Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite.The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed.The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, The Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

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Related in: MedlinePlus

Measurement of sugar nucleotides in the TbUAP conditional  mutant under permissive and nonpermissive conditions. Representative liquid chromatography-tandem mass spectrometry chromatograms of sugar nucleotides extracted from the TbUAP conditional  mutant before (A) and after (B) withdrawal of tetracycline for 48 h. The upper chromatogram in each panel shows the peaks corresponding to GDP-Man and the GDP-Glc internal standard, and the lower chromatogram shows the peak corresponding to UDP-GlcNAc.
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fig5: Measurement of sugar nucleotides in the TbUAP conditional mutant under permissive and nonpermissive conditions. Representative liquid chromatography-tandem mass spectrometry chromatograms of sugar nucleotides extracted from the TbUAP conditional mutant before (A) and after (B) withdrawal of tetracycline for 48 h. The upper chromatogram in each panel shows the peaks corresponding to GDP-Man and the GDP-Glc internal standard, and the lower chromatogram shows the peak corresponding to UDP-GlcNAc.

Mentions: Sugar Nucleotide Levels in the TbUAP Conditional Null Mutant—To analyze the effect of the selective removal of TbUAP gene expression on parasite UDP-GlcNAc levels, sugar nucleotides were extracted from the TbUAP conditional mutant under permissive and nonpermissive conditions, chromatographed as described in (35), and quantitated by the method described in Ref. 19. Briefly, sugar nucleotides were extracted from T. brucei, separated by reverse phase HPLC and quantitated by multiple reaction monitoring tandem mass spectrometry using an internal standard (GDP-glucose), a sugar nucleotide that is not found in trypanosomes. The multiple reaction monitoring approach exploits characteristic transitions between precursor and product ions to identify specific metabolites in complex mixtures. For example UDP-GlcNAc gives rise to an [M-H]- precursor ion at m/z 606 that fragments to produce a major product ion of [UDP-H2O]- at m/z 385. Thus, a chromatogram of the mass transition 606 → 385 is highly selective for UDP-GlcNAc. Similarly, GDP-Man and GDP-Glc can be monitored using the [GDP-Hex]- to [GDP-H2 O]- transition of m/z 604 → 424. Representative chromatograms (Fig. 5) illustrate the dramatic reduction in UDP-GlcNAc levels, relative to the GDP-Glc internal standard, in the TbUAP conditional mutant after 48 h in the absence of tetracycline. These data and the effects on other sugar nucleotides are summarized in (Table 2). The levels of UDP-Glc, UDP-Gal, and GDP-Man in the TbUAP conditional cell line under permissive conditions agree reasonably well with the wild type levels determined previously (19). However, even under permissive conditions, the level of UDP-GlcNAc is significantly lower in the conditional mutant (16 pmol/1 × 107 cells) than in the wild type (80 pmol/1 × 107 cells). The reduced level may be because TbUAP expression is no longer under the control of its endogenous promoter but rather under the control of the procyclin promoter in the pLew100-TbUAP ectopic copy. Thus, the lower growth rate of the mutant under permissive conditions may be a result of the reduced level of UDP-GlcNAc. Under nonpermissive conditions, the cells stopped dividing between 48 and 60 h and died, by cell lysis, by around 72 h. At 48 h, the level of UDP-GlcNAc was 2.9 pmol/1 × 107, <5% of wild type levels.


The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation.

Stokes MJ, Güther ML, Turnock DC, Prescott AR, Martin KL, Alphey MS, Ferguson MA - J. Biol. Chem. (2008)

Measurement of sugar nucleotides in the TbUAP conditional  mutant under permissive and nonpermissive conditions. Representative liquid chromatography-tandem mass spectrometry chromatograms of sugar nucleotides extracted from the TbUAP conditional  mutant before (A) and after (B) withdrawal of tetracycline for 48 h. The upper chromatogram in each panel shows the peaks corresponding to GDP-Man and the GDP-Glc internal standard, and the lower chromatogram shows the peak corresponding to UDP-GlcNAc.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2414269&req=5

fig5: Measurement of sugar nucleotides in the TbUAP conditional mutant under permissive and nonpermissive conditions. Representative liquid chromatography-tandem mass spectrometry chromatograms of sugar nucleotides extracted from the TbUAP conditional mutant before (A) and after (B) withdrawal of tetracycline for 48 h. The upper chromatogram in each panel shows the peaks corresponding to GDP-Man and the GDP-Glc internal standard, and the lower chromatogram shows the peak corresponding to UDP-GlcNAc.
Mentions: Sugar Nucleotide Levels in the TbUAP Conditional Null Mutant—To analyze the effect of the selective removal of TbUAP gene expression on parasite UDP-GlcNAc levels, sugar nucleotides were extracted from the TbUAP conditional mutant under permissive and nonpermissive conditions, chromatographed as described in (35), and quantitated by the method described in Ref. 19. Briefly, sugar nucleotides were extracted from T. brucei, separated by reverse phase HPLC and quantitated by multiple reaction monitoring tandem mass spectrometry using an internal standard (GDP-glucose), a sugar nucleotide that is not found in trypanosomes. The multiple reaction monitoring approach exploits characteristic transitions between precursor and product ions to identify specific metabolites in complex mixtures. For example UDP-GlcNAc gives rise to an [M-H]- precursor ion at m/z 606 that fragments to produce a major product ion of [UDP-H2O]- at m/z 385. Thus, a chromatogram of the mass transition 606 → 385 is highly selective for UDP-GlcNAc. Similarly, GDP-Man and GDP-Glc can be monitored using the [GDP-Hex]- to [GDP-H2 O]- transition of m/z 604 → 424. Representative chromatograms (Fig. 5) illustrate the dramatic reduction in UDP-GlcNAc levels, relative to the GDP-Glc internal standard, in the TbUAP conditional mutant after 48 h in the absence of tetracycline. These data and the effects on other sugar nucleotides are summarized in (Table 2). The levels of UDP-Glc, UDP-Gal, and GDP-Man in the TbUAP conditional cell line under permissive conditions agree reasonably well with the wild type levels determined previously (19). However, even under permissive conditions, the level of UDP-GlcNAc is significantly lower in the conditional mutant (16 pmol/1 × 107 cells) than in the wild type (80 pmol/1 × 107 cells). The reduced level may be because TbUAP expression is no longer under the control of its endogenous promoter but rather under the control of the procyclin promoter in the pLew100-TbUAP ectopic copy. Thus, the lower growth rate of the mutant under permissive conditions may be a result of the reduced level of UDP-GlcNAc. Under nonpermissive conditions, the cells stopped dividing between 48 and 60 h and died, by cell lysis, by around 72 h. At 48 h, the level of UDP-GlcNAc was 2.9 pmol/1 × 107, <5% of wild type levels.

Bottom Line: Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite.The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed.The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, The Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

Show MeSH
Related in: MedlinePlus