The role and characterization of phospholipase A1 in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei

Gregory S Richmond (1) , Terry K Smith

1 Biological Chemistry and Molecular Microbiology

Lysophospholipids are ubiquitous intermediates in a variety of metabolic and signalling pathways in eukaryotic cells. We have recently reported that lysophosphatidylcholine (lysoGPCho) synthesis in the insect form of the ancient eukaryote Trypanosoma brucei is mediated by a novel phospholipase A 1} (TbPLA 1}). In this report it is shown that despite equal levels of TbPLA 1} gene expression in wild type insect and bloodstream trypomastigotes, both TbPLA 1} enzyme levels and lysoGPCho metabolites are approximately three fold higher in the bloodstream form. Both of these parasite stages synthesize identical molecular species of lysoGPCho. TbPLA 1} null mutants in the bloodstream form of the parasite are viable but are deficient in lysoGPCho synthesis, a defect which can be overcome by the expression of an ectopic copy of TbPLA 1}. The biochemical attributes of TbPLA 1}-mediated lysoGPCho synthesis were examined in vitro using recombinant TbPLA 1}. Although TbPLA 1} possesses a serine active site residue, it is insensitive to serine-modifying reagents such as DFP and PMSF, a characteristic shared by lipases that possess lid-sheltered catalytic triads. TbPLA 1} needs no metal co-factors for activity but it does require interfacial activation prior to catalysis. Results from size exclusion chromatography and binding kinetics analysis revealed that TbPLA 1} activation by Triton X-100/GPCho mixed micelle surfaces was not specific and did not require the pre-formation of a specific enzyme-substrate complex to achieve surface binding.

Mots clés: Life Sciences

Gregory S Richmond, Terry K Smith. The role and characterization of phospholipase A1 in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei. Biochemical Journal, 2007, 405 (2), pp.319-329. ⟨10.1042/BJ20070193⟩. ⟨hal-00478753⟩