The Proteome of Insect Glutathione S-Transferases: its Response to Toxic Challenge

Abstract

Insects possess many different GST isoenzymes and a complete genome database available for Drosophila melanogaster has assisted with the identification of the isoenzymes. The model organism has a predicted 39 GST genes - the actual number of GSTs detected is much lower. By using a two stage purification strategy, a combination of Sephadex G-25 and different affinity matrices, this study had successfully isolated several GSTs. By using GSH-agarose (C3) GSTD1, GSTD3 and GSTS1 were purified. An extra isoform, namely CG16936 protein product was isolated with GSH-agarose (C12) and S-hexyl-GSH-agarose. All of the mentioned GST isoforms could be isolated with BSP/GSH-agarose together with additional GSTs of the Epsilon class, which were GSTE3, GSTE6, GSTE7 and GSTE9. Therefore, this matrix appeared to be very useful, as it was able to isolate the greatest number of expressed GST compared to that of the GSH-agarose affinity matrices. The matrix did however, have a tendency to bind non-GST proteins. By using a combination of GSH-agarose (C3) and QAE Sephadex A-25 or MonoP, GSTS1 could be homogenously isolated from the D-class GSTs. This has made it possible to show the occurrence of a 'ladder' of spots, identified as GSTS1, which at this point are assumed to be degradation products of the parent GSTS1. The study showed that GSTS1 is active towards lipid peroxidation products, confirming reports of its role in defense against oxidative stress. The kinetic analysis has indicated that GSTS1 acts by a rapid-equilibrium Bi-Bi mechanism. GSTD1 was the major GST expressed in fruitflies. It resolved into 5 spots with estimated pI values of 5.75, 6.02, 6.42, and 7.0 on the 2-D gel. A single pair mating experiment indicated that the generation of the isoforms was not related to the Presence of alleles. It could have been caused by artifactual modification. In parallel to isolation and identification of the expressed GSTs in the adult fruitfly, it was also of interest to ascertain which of the isoforms were responsive towards developmental, oxidative, sedative and insecticidal stress. Developmental studies showed that GSTD1, GSTD3, GSTS1and GSTE3 were all expressed in all developmental stages. The CG16936 and GSTE7 were only expressed during adult and pupal stages. GSTE6 and GSTE9 were only observed in adult flies. The present study also used several chemicals to induce changes in the Drosophila GSTs. The relative % volume of individual GST spots in a sample and the protein amount were compared between control and chemical-treated sample. Several GST isoforms were upregulated in response to oxidative stress. The relative % volume of GSTD1 (2.9-fold, p<0.05), GSTD3 (1.2-fold, p<0.05), GSTE6 (1.6-fold, p<0.05), and GSTE7 (2.5-fold, p<0.05), were significantly increased in Paraquat treated flies. The protein amount of GSTD1 (3.2-fold, p<0.05), GSTD3 (1.4-fold, p<0.05), GSTE6 (1.8-fold, p<0.05), and GSTE7 (2.8-fold, p<0.05) were also shown to be induced significantly. A significant decrease in % volume of GSTE9 was also reported (0.5 X fold, p<0.05). In contrast, when protein amount was compared there was no significant change was reported. No significant changes in expression were observed with GSTS1, CG16936 and GSTE3. Phenobarbital (PhB, 10 mM) resulted in change in the relative % volume of GSTD1 (1.4- fold, p<0.05), GSTD3 (1.5-fold, P<0.05), GSTE3 (1.5-fold, p<0.05), GSTE6 (2.0-fold, p<0.05) and GSTE7 (1.3-fold, p<0.05) significantly. The GSTD1 (2.0-fold, p<0.05), GSTD3 (1.5-fold, P<0.05), GSTE3 (2.1-fold, p<0.05), GSTE6 (2.8-fold, p<0.05) and GSTE7 (1.8-fold, p<0.05) proteins were significantly induced. The study also showed that the relative % volume of GSTS1 was down-regulated 0.7-fold (p<0.05). In contrast, the protein amount of GSTS1 in the control and PhB-treated sample showed no significant change in expression. Interestingly, the sedative agent had contributed to the expression of GSTD2. Under the acute treatment of methyl parathion (MP, lµM), it was shown that % volume of GSTD1, GSTE6 and GSTE7 were significantly up-regulated at 1.2-fold (p<0.05), 2.0-fold( p <0"0.5) and l.7-fold (p <0'05) change, respectively. The corresponding significant increase in protein amount for GSTD1 (1.3-fold, p<0.05), GSTE6 (2.2-fold, p<0.05) and GSTE7 (1.9-fold, p<0.05) were also reported. The quantitative analysis did not indicate change in protein expression in other GSTs. The isolation of glutathione S-transferases from adult Mussa domestica was also performed. The 2-D gel of the GSH-agarose (C3)-isolated sample resolved into six isoforms of same molecular weight (~24 kDa) and all were identified as GST class I (GSTT1) with predicted M1 and p1 values of 23.8 and 7.7, respectively. GSTT1 was shown to be an ortholog of the GSTD1. The study also identified Sigma Class GSTS, GST6A and GST6B. The developmental stage studies showed that the GSTT1, GST6A, GST6B, and GSTS were expressed in all stages.