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 genonne and subsequently cloned. sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a V-max of 0.34 mu mol/min per mg of protein and K-m values of 67 mu M and 12 mu M 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 similar 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 Man(9)GlcNAc(2) N-glycosylation at Asn(428) and formation of complex N-glycans at Asn(263).