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

Lysophospholipids are ubiquitous intermediates in a variety of metabolic and signalling pathways in eukaryotic cells. We have reported recently that lysoglycerophosphatidylcholine (lyso-GPCho) synthesis in the insect form of the ancient eukaryote Trypanosoma brucei is mediated by a novel phospholipase A₁ (TbPLA₁). In the present study, we show that despite equal levels of TbPLA₁ gene expression in wild-type insect and bloodstream trypomastigotes, both TbPLA₁ enzyme levels and lysoGPCho metabolites are approx. 3-fold higher in the bloodstream form. Both of these parasite stages synthesize identical molecular species of lysoGPCho. TbPLA₁ null mutants in the bloodstream form of the parasite are viable, but are deficient in lysoGPCho synthesis, a defect that can be overcome by the expression of an ectopic copy of TbPLA₁. The biochemical attributes of TbPLA₁-mediated lysoGPCho synthesis were examined in vitro using recombinant TbPLA₁. Although TbPLA₁ possesses an active-site serine residue, it is insensitive to serine-modifying reagents, such as di-isopropyl fluorophosphate and PMSF, a characteristic shared by lipases that possess lid-sheltered catalytic triads. TbPLA₁ does not require 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 TbPLA₁ 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.