The role and characterization of phospholipase A(1) in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei

Gregory S. Richmond, Terry K Smith

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18 Citations (Scopus)

Abstract

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 A, (TbPLA(1)). In the present study, we show 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 approx. 3-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 that 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 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. TbPLA(1) 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 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.

Original languageEnglish
Pages (from-to)319-329
Number of pages11
JournalBiochemical Journal
Volume405
DOIs
Publication statusPublished - 15 Jul 2007

Keywords

  • interfacial activation
  • lipid metabolism
  • lysophosphatidylcholine
  • phospholipase
  • Trypanosoma brucei
  • BLOOD-STREAM FORMS
  • SACCHAROMYCES-CEREVISIAE
  • AFRICAN TRYPANOSOMES
  • KINETIC-ANALYSIS
  • MIXED MICELLES
  • BOVINE TESTIS
  • GENE
  • ACID
  • PURIFICATION
  • EXPRESSION

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