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 gene, were obtained from fourth instar larvae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these three 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 shows that adGST1-2 versus agGST1-4, adGST1-3 versus agGST1-5 and adGST1-4 versus agGST1-3 have 85, 92 and 85% amino acid sequence identity, respectively, which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 ml of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit 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-2,4-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly 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 significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adGST1-4. 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 identical sequence in the N-terminal 45 amino acids, these GSTs show distinct substrate specificities, kinetic properties and inhibition properties modulated by the differences in the C-terminus.
Jirajaroenrat
K
K
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, 73170, Nakorn Pathom, Thailand.
Pongjaroenkit
S
S
Krittanai
C
C
Prapanthadara
L
L
Ketterman
A J
AJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D026023:Permethrin; D011722:Pyrethrins / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D011722:Pyrethrins; D011993:Recombinant Fusion Proteins; D026023:Permethrin; D005982:Glutathione Transferase