title: 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.
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 null 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 null 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.
authors.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.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Stokes
authors.forename: M Lucia S
authors.initials: ML
authors.lastname: Güther
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Turnock
authors.forename: Alan R
authors.initials: AR
authors.lastname: Prescott
authors.forename: Kirstee L
authors.initials: KL
authors.lastname: Martin
authors.forename: Magnus S
authors.initials: MS
authors.lastname: Alphey
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D006031:Glycosylation; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D037121:Plant Lectins / Q000737:chemistry; D046188:Protein Modification, Translational / Q000502:physiology*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D037121:Plant Lectins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C034981:tomato lectin; D014537:Uridine Diphosphate N-Acetylglucosamine; C026146:N-acetylglucosamine-1-phosphate; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D000117:Acetylglucosamine