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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

Substrate specificity of TbUAP. Recombinant TbUAP-His6 was incubated with UTP and different sugar-1-phosphate substrates, as indicated, and the products were analyzed by HPLC. A sugar nucleotide product (UDP-GlcNAc) was observed using GlcNAc-1-P (A) but not without GlcNAc-1-P (B) or with GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate (C-E, respectively).
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fig1: Substrate specificity of TbUAP. Recombinant TbUAP-His6 was incubated with UTP and different sugar-1-phosphate substrates, as indicated, and the products were analyzed by HPLC. A sugar nucleotide product (UDP-GlcNAc) was observed using GlcNAc-1-P (A) but not without GlcNAc-1-P (B) or with GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate (C-E, respectively).

Mentions: Enzymatic Activity of TbUAP—The activity and substrate specificity of TbUAP was assessed by incubating recombinant TbUAP-His6 with UTP and GlcNAc-1-P, GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate and analyzing the products by HPLC. Using GlcNAc-1-P as the substrate, a single UV-absorbing peak that co-eluted with authentic UDP-GlcNAc was observed (Fig. 1A). No sugar nucleotide product was observed in the absence of TbUAP-His6 (Fig. 1B) or when GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate was used as a substrate (Fig. 1, C-E). These data show that GlcNAc-1-P is the preferred substrate of TbUAP under these conditions.


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)

Substrate specificity of TbUAP. Recombinant TbUAP-His6 was incubated with UTP and different sugar-1-phosphate substrates, as indicated, and the products were analyzed by HPLC. A sugar nucleotide product (UDP-GlcNAc) was observed using GlcNAc-1-P (A) but not without GlcNAc-1-P (B) or with GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate (C-E, respectively).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Substrate specificity of TbUAP. Recombinant TbUAP-His6 was incubated with UTP and different sugar-1-phosphate substrates, as indicated, and the products were analyzed by HPLC. A sugar nucleotide product (UDP-GlcNAc) was observed using GlcNAc-1-P (A) but not without GlcNAc-1-P (B) or with GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate (C-E, respectively).
Mentions: Enzymatic Activity of TbUAP—The activity and substrate specificity of TbUAP was assessed by incubating recombinant TbUAP-His6 with UTP and GlcNAc-1-P, GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate and analyzing the products by HPLC. Using GlcNAc-1-P as the substrate, a single UV-absorbing peak that co-eluted with authentic UDP-GlcNAc was observed (Fig. 1A). No sugar nucleotide product was observed in the absence of TbUAP-His6 (Fig. 1B) or when GalNAc-1-P, Glc 1-phosphate, or Gal 1-phosphate was used as a substrate (Fig. 1, C-E). These data show that GlcNAc-1-P is the preferred substrate of TbUAP under these conditions.

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