GDP-mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei.
A putative GDP-Man PP (guanidine diphosphomannose pyrophosphorylase) gene from Trypanosoma brucei (TbGDP-Man PP) was identified in the genome and subsequently cloned, sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 mumol/min per mg of protein and Km values of 67 muM and 12 muM for GTP and mannose 1-phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi (RNA interference) of the cytosolic TbGDP-Man PP showed that mRNA levels were reduced to ~20% of wild-type levels, causing the cells to die after 3-4 days, demonstrating that TbGDP-Man PP is essential in the bloodstream form of T. brucei and thus a potential drug target. The RNAi-induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential GPI (glycosylphosphatidylinositol) anchors. The RNAi-induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high-mannose Man9GlcNAc2 N-glycosylation at Asn428 and formation of complex N-glycans at Asn263.
Denton
Helen
H
Biomolecular Sciences Research Complex, The North Haugh, The University, St Andrews, Fife KY16 9ST, Scotland, UK.
Fyffe
Stewart
S
Smith
Terry K
TK
D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010641:Phenotype; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014353:Trypanosomiasis, African / Q000097:blood*
D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase