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.
Richmond
Gregory S
GS
Wellcome Trust Biocentre, Division of Biological Chemistry and Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Smith
Terry K
TK
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000096:biosynthesis*; D051379:Mice; D010261:Paraoxon / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054466:Phospholipases A1; D021241:Spectrometry, Mass, Electrospray Ionization; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000378:metabolism*; D014353:Trypanosomiasis, African / Q000097:blood
D002413:Cations, Divalent; D008244:Lysophosphatidylcholines; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D010741:Phospholipases A; D054466:Phospholipases A1; D010261:Paraoxon