Heterologous expression and characterization of alternatively spliced glutathione S-transferases from a single Anopheles gene

Three cDNA sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 ge ne, were obtained from fourth instar lan;ae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these thre e cDNAs share > 67% identity and the translated amino acid sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes s hows that adGST1-2 versus agGST1-4. adGST1-3 versus agGST1-5 and adGST1-4 v ersus agCST1-3 have 85, 92 and 85% amino acid sequence identity, respective ly. which confirms that orthologous isoenzymes occur across anopheline spec ies. These three proteins were expressed at high levels, approximately 15-2 0 mg from 200 mi of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The sub unit sizes of adGST1-2. adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,3-dinitrobenze ne (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedl y low activity with ethacrynic acid and p-nitrophenethyl bromide, adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was signif icantly different, being uncompetitive for adGST1-2, noncompetitive for adG ST1-3 and competitive for adCST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing id entical sequence in the N-terminal 35 amino acids, these GSTs show distinct substrate specificities-kinetic properties and inhibition properties modul ated by the differences in the C-terminus. (C) 2001 Elsevier Science Ltd. A ll rights reserved.