Abstract
Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is an integral membrane protein in the protozoan parasite Trypanosoma brucei. Enzyme activity appears to be suppressed in T. brucei, although the polypeptide is readily detectable. The basis for the apparent quiescence of GPI-PLC is not known. Protein oligomerization was investigated as a possible mechanism for post-translational regulation of GPI-PLC activity. An equilibrium between monomers, dimers, and tetramers of purified GPI-PLC was detected by molecular sieving and shown to be perturbed with specific detergents. Homotetramers dominated in Nonidet P-40, and dimers and monomers of GPI-PLC were the major species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3. 6-20-fold more active than the monomeric enzyme. Tetramer existence was confirmed by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species in T. brucei. Assembly of tetrameric GPI-PLC occurred when parasites were subjected to conditions known to activate the enzyme. In Leishmania where heterologous expression of GPI-PLC causes a GPI deficiency, the enzyme existed as a tetramer. Hence, oligomerization of GPI-PLC is associated with high enzyme activity both in vivo and in vitro.
Highlights
The physiological function of the enzyme remains unclear (4, 5)
variant surface glycoprotein (VSG) is not released actively by Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). This situation is in sharp contrast to the phenotype of Leishmania major or Trypanosoma cruzi that have been stably transfected with a cDNA for the T. brucei GPI-PLC (14, 15)
Treatment with 1% SDS caused GPI-PLC to run off the gel, most likely due to denaturation and net negative charge introduced by the detergent
Summary
Monomorphic T. brucei strain 427 bloodstream form was used in this work. Parasites were grown in rodents and harvested by chromatography on DE52 (20). Recombinant GPI-PLC—Purified GPI-PLC (620 ng) in 5 l of 75 mM Tris-HCl, pH 9.3, containing 1% Nonidet P-40 (6), was added to 95 l of the indicated running buffer (see figure legends) and incubated at 27 °C for 5 min before loading onto the specified column. 2.5 l of 1 M Tris-HCl, pH 7.5, was introduced, and after 5 min incubation on ice, 10 l of 5ϫ SDS-PAGE sample buffer added. TbGPI-PLC—[35S]Methionine-labeled GPI-PLC, from intact cells or lysates, was immunoadsorbed to protein A-Sepharose-mc2A6-6 beads (6) and eluted by heating at 90 °C for [2,3] min in 25 l of 2.5ϫ SDSPAGE sample buffer. A 30-l aliquot of each supernatant was subjected to SDS-PAGE (12% minigel) and processed for phosphorimaging or fluorographic detection with BioMaxTM MR film (Eastman Kodak Co.)
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