Glutathione S-transferase in cestodes

Abstract

Glutathione S-transferases (GSTs) are multifunctional proteins widely distributed as isomeric forms in both the plant and animal kingdom [ 11. They can function as enzymes, using the glutathione thiolate anion as co-substrate to interact with a multitude of electrophilic second substrates; a number of GST isoenzymes may also be intracellular binding proteins for hydrophobic ligands [2,3]. A major advance in the understanding of GST biology was the division of mammalian cytosolic isoenzymes into three species-independent families, Alpha, Mu and Pi, using differences in biochemical and physical properties 141. GSTs arc associated with Phase I1 detoxification of exogenously derived (xenobiotics) and endogenously derived toxic compounds including secondary products of lipid peroxidation [ 11. A number of GST isoenzymes may have more specific functions with physiological substrates, including a role in leukotriene synthesis 151 and as intracellular transport proteins for haem 161. The GSTs have been implicated in the development of drug resistance in mammalian tumours [7], insects 181, fungi 191 and plants [ 101. This report summarizes our investigations into the possible roles of GSTs in cestodes. The majority of the GST activity was detected in the cytosolic fraction of the cestode cell. The cytosolic GST activity in Motiieziu expunsu and Schistocephulus solidus (plerocercoid) was purified by glutathione-affinity chromatography 11 I I and apparent isoenzymes were resolved by chromatofocusing. The cytosolic GST activity in Hymenolepsis diminutu did not bind to the glutathione-affinity column until a high molecular mass factor was removed from the sample preparation by hydroxyapatite chromatography or by chromatofocusing. Four apparent near neutral/acidic GST isoenzymes were resolved from the three cestodes by chromatofocusing and purity was confirmed by SDS/polyacrylamide-gel electrophoresis and analytical isoelectric focusing. The major GSTs of M . expansa and S. solidus (plerocercoid) cytosol showed a relationship to the Mu group of mammalian GSTs in terms of substrate specificity and inhibitor sensitivity, including sensitivity to the Mu inhibitor markers Cibacron Blue and triphenyltin chloride and significant catalytic activity with a Mu marker substrate trans-4phenyl-3-buten-2-one. The N-terminal of the major M . expansa and S . solidus (plerocercoid) GST subunits had sequence homology with Mu group enzymcs. The major GST of H . dimitiirru cytosol showed no clear biochemical relationship to any one mammalian GST family. The standard conjugation assay for the major GST in the three cestodes was inhibited by a number of commercially available anthelmintics, including members of the substituted phenyl and benzimidaiole group o f compounds with I,,, (50% inhibition of standard y) values in the micromolar range. Binding of the anthel ics to the major GST in M . expunsa was confirmed by competitive fluorescence spectroscopy [ 121. There was n o evidence, using a thiol utilization method [ 131, that the major GST of M. expurisu could conjugate the anthelmintics with glutathione. Free radicals that arise from host immuno-effector cells may have a role in the immune rejection of a number of parasites 141. Lipid peroxidation can result from free-radicalinitiated chain reactions [ 151 and mammalian GSTs can conjugate 4-hydroxyalkenals 161, established secondary products of lipid peroxidation [ 171. The major GSTs of the cestodes M. expansa, S. solidus (plerocercoid) and H. diminutu conjugated members of the alk-2-enal and alka2,4-dienal reactive carbonyl series [ 181, also known secondary products of lipid peroxidation [ 171. The cestode GSTs may help provide a ‘final line of defence’ against host immune and/or chemotherapeutic attack by conjugating toxic secondary products of lipid peroxidation or by passively detoxifying anthelmintics. Selective inhibition of the cestode enzymes could tip the molecular balance in favour of the host. A potential chemotherapeutic platform may exist as cestodc GSTs do not show a strong biochemical relationship to the Pi family GSTs, which, at least in humans, are the most abundant GST type in extrahepatic tissues [ 191. The mechanisms for the intracellular regulation of cestode and other helminth GSTs is currently under investigation.