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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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Electrospray mass spectrometry of sVSG221 from wild type and TbUAP conditional  mutant cells. Aliquots of sVSG221 from wild type cells (A) and TbUAP conditional  mutant cells grown in the presence of tetracycline (B) and the absence of tetracycline for 48 h (C) were analyzed by positive ion electrospray mass spectrometry, and the data were processed by Baysian protein reconstruction to produce mass graphs of isobaric glycoforms. Models of some of the principal glycoforms are indicated, and the compositions of the detected glycoforms are shown in Table 3.
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fig9: Electrospray mass spectrometry of sVSG221 from wild type and TbUAP conditional mutant cells. Aliquots of sVSG221 from wild type cells (A) and TbUAP conditional mutant cells grown in the presence of tetracycline (B) and the absence of tetracycline for 48 h (C) were analyzed by positive ion electrospray mass spectrometry, and the data were processed by Baysian protein reconstruction to produce mass graphs of isobaric glycoforms. Models of some of the principal glycoforms are indicated, and the compositions of the detected glycoforms are shown in Table 3.

Mentions: To analyze this further, the sVSG samples were analyzed by electrospray mass spectrometry (Fig. 9). Wild type and TbUAP conditional mutant cells grown under permissive conditions gave the expected glycoform mass ranges for sVSG221 (16, 44) (Table 3). However, sVSG from the TbUAP conditional mutant grown under nonpermissive conditions for 48 h displayed two discrete sets of sVSG glycoforms, corresponding to the two bands seen by SDS-PAGE. One set was similar to that of wild type sVSG, except that it lacks the higher molecular weight glycoforms that contain five GlcNAc residues (Table 3). The other set of glycoforms have masses consistent with the absence of oligomannose structures at the C-terminal (Asn-428) N-glycosylation site (consistent with the Endo H resistance of the lower VSG band on SDS-PAGE). Analysis of the glycopeptide fraction of a Pronase digest of the mutant VSG sample, prepared and analyzed by electrospray tandem mass spectrometry according to Ref. 54, revealed a range of Asn-263 and Asn-428 N-linked glycopeptide and Ser-433 GPI glycopeptide species4 similar to those of wild type VSG (supplemental Fig. 13). These results rule out the possibility that lower molecular weight VSG glycoforms arise from changes at the N-linked and GPI glycosylation sites that happen to be equivalent in mass to the loss of the oligomannose structures from Asn-428.


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)

Electrospray mass spectrometry of sVSG221 from wild type and TbUAP conditional  mutant cells. Aliquots of sVSG221 from wild type cells (A) and TbUAP conditional  mutant cells grown in the presence of tetracycline (B) and the absence of tetracycline for 48 h (C) were analyzed by positive ion electrospray mass spectrometry, and the data were processed by Baysian protein reconstruction to produce mass graphs of isobaric glycoforms. Models of some of the principal glycoforms are indicated, and the compositions of the detected glycoforms are shown in Table 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Electrospray mass spectrometry of sVSG221 from wild type and TbUAP conditional mutant cells. Aliquots of sVSG221 from wild type cells (A) and TbUAP conditional mutant cells grown in the presence of tetracycline (B) and the absence of tetracycline for 48 h (C) were analyzed by positive ion electrospray mass spectrometry, and the data were processed by Baysian protein reconstruction to produce mass graphs of isobaric glycoforms. Models of some of the principal glycoforms are indicated, and the compositions of the detected glycoforms are shown in Table 3.
Mentions: To analyze this further, the sVSG samples were analyzed by electrospray mass spectrometry (Fig. 9). Wild type and TbUAP conditional mutant cells grown under permissive conditions gave the expected glycoform mass ranges for sVSG221 (16, 44) (Table 3). However, sVSG from the TbUAP conditional mutant grown under nonpermissive conditions for 48 h displayed two discrete sets of sVSG glycoforms, corresponding to the two bands seen by SDS-PAGE. One set was similar to that of wild type sVSG, except that it lacks the higher molecular weight glycoforms that contain five GlcNAc residues (Table 3). The other set of glycoforms have masses consistent with the absence of oligomannose structures at the C-terminal (Asn-428) N-glycosylation site (consistent with the Endo H resistance of the lower VSG band on SDS-PAGE). Analysis of the glycopeptide fraction of a Pronase digest of the mutant VSG sample, prepared and analyzed by electrospray tandem mass spectrometry according to Ref. 54, revealed a range of Asn-263 and Asn-428 N-linked glycopeptide and Ser-433 GPI glycopeptide species4 similar to those of wild type VSG (supplemental Fig. 13). These results rule out the possibility that lower molecular weight VSG glycoforms arise from changes at the N-linked and GPI glycosylation sites that happen to be equivalent in mass to the loss of the oligomannose structures from Asn-428.

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