GDP-mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei

Helen Denton, Stewart Fyffe, Terry K Smith

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

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).

Original languageEnglish
Pages (from-to)603-614
Number of pages12
JournalBiochemical Journal
Volume425
Issue number3
DOIs
Publication statusPublished - 1 Feb 2010

Keywords

  • essentiality
  • guanidine diphosphomannose pyrophosphorylase
  • glycosylphosphatidylinositol
  • N-glycosylation
  • Trypanosoma brucei
  • variant surface glycoprotein
  • AFRICAN SLEEPING SICKNESS
  • DE-NOVO SYNTHESIS
  • LEISHMANIA-MEXICANA
  • SACCHAROMYCES-CEREVISIAE
  • BIOSYNTHETIC-PATHWAY
  • SALMONELLA-ENTERICA
  • ANTIGENIC VARIATION
  • N-GLYCOSYLATION
  • DRUG TARGET
  • VITAMIN-C

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