Publications

• Tetramerization of glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei.

  Armah D.A., Mensa-Wilmot K.

  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 n ... >> More

  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. << Less

  J Biol Chem 275:19334-19342(2000) [PubMed] [EuropePMC]

• A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein.

  Hereld D., Krakow J.L., Bangs J.D., Hart G.W., Englund P.T.

  The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (19 ... >> More

  The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol. << Less

  J Biol Chem 261:13813-13819(1986) [PubMed] [EuropePMC]

• Mutagenesis study of the glycosylphosphatidylinositol phospholipase C of Trypanosoma brucei.

  Carnall N., Webb H., Carrington M.

  The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cy ... >> More

  The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis. Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition. In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC. The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H. EMBO J 1995;14:3855-3863). The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue. << Less

  Mol Biochem Parasitol 90:423-432(1997) [PubMed] [EuropePMC]