Identification of a developmentally and hormonally regulated Delta-Class glutathione S-transferase in rice moth Corcyra cephalonica ☆ Damodar Gullipalli a , Abul Arif a,b , Polamarasetty Aparoy a , Gavin J. Svenson c , Michael F. Whiting c , Pallu Reddanna a , Aparna Dutta-Gupta a, ⁎ a Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India b Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio, USA c Department of Integrative Biology, Brigham Young University, Provo, Utah, USA abstract article info Article history: Received 19 October 2009 Received in revised form 29 January 2010 Accepted 30 January 2010 Available online 4 February 2010 Keywords: Glutathione S-transferases Insect Juvenile hormone Post-embryonic development Glutathione S-transferases (GSTs) are a large family of multifunctional enzymes, known for their role in cellular detoxification. Here we report a cytosolic GST with optimal activity at alkaline pH (8.3) from the visceral fat body of late-last instar (LLI) larvae of a lepidopteran insect rice moth Corcyra cephalonica. All previously known GSTs are active between pH 6.0 to 6.5. Purification and characterization revealed the Corcyra cephalonica GST (CcGST) as a 23-kDa protein. HPLC and 2D analysis showed a single isoform of the protein in the LLI visceral fat body. Degenerate primer based method identified a 701-nucleotide cDNA and the longest open reading frame contained 216 amino acids. Multiple sequence and structural alignment showed close similarity with delta-class GSTs. CcGST is present mainly in the fat body with highest activity at the late-last instar larval stage. Juvenile hormone (JH) negatively inhibits the CcGST activity both ex vivo and in vivo. We speculate that high expression and activity of CcGST in the fat body of the late-last instar larvae, when endogenous JH titer is low may have role in the insect post-embryonic development unrelated to their previously known function. © 2010 Elsevier Inc. All rights reserved. 1. Introduction GSTs (EC 2.5.1.18) in insects are a ubiquitously present superfamily of enzymes that play key roles in detoxification of xenobiotics (including insecticides) and protection of cells against damage due to oxidative stress (Enayati et al., 2005; Giovanini et al., 2006; Li et al., 2008; Salinas and Wong, 1999; Udomsinprasert et al., 2005). They catalyze the conjugation of electrophilic compounds with thiol groups making the xenobiotics more water-soluble (Habig et al., 1974). GSTs also catalyze reactions in metabolic pathways not associated with detoxification (Sheehan et al., 2001). Most GSTs are cytosolic enzymes, present in both homo and heterodimeric forms with subunit masses of 23- to 28-kDa. Each subunit contains a specific glutathione (GSH)- binding site (G-site) in the highly conserved N-terminal region next to a non-specific electrophilic ligand-binding site (H-site). Phylogenetic analysis revealed the presence of at least six distinct classes of cytosolic GSTs in insects along with several unclassified genes (Enayati et al., 2005). GSTs are classified into two major subfamilies characterized by Ser (delta and theta classes) or Tyr (alpha, mu, pi and sigma classes), which plays an important role in lowering the pKa of the thiol group of bound GSH from 9.0, to approximately 6.0 to enhance the rate of nucleophilic attack of GSH towards electrophilic substrates (Caccuri et al., 1999; Wongsantichon and Ketterman, 2006). The majority of insect GSTs belongs to the delta and epsilon classes and lack alpha, mu and pi classes. In spite of low sequence homology among GST classes they have fairly similar tertiary structures, topography of active site and G-sites, and are induced after insecticide application for detoxification (Dirr et al., 1994; Enayati et al., 2005). Recent elucidation of the structure of an epsilon class GST from Anopheles gambaie provided critical explanation to high DDT-detoxification activity (Wang et al., 2008). The fat body of insects is the main metabolic centre, and performs a large number of complex cellular functions (Keeley, 1985). It is involved in detoxification/degradation of xenobiotics and protection from oxidative stress (Motoyama and Dauterman, 1980; Clark et al., 1984; Sawicki et al., 2003). Due to the role of GSTs in insecticide detoxification and other detoxifications such as O-dealkylation or O-dearylation of organophosphorous insecticides (Hayes and Pulford, 1995; Hayes et al., 1998), there is a significant increase in the number of well characterized insect GSTs. However, little is known about the role of GSTs of stored- grain pests, which are exposed to a variety of chemical formulations. As these pests live successfully under high stress conditions, characteriza- tion of these enzymes is critical to ascertain the basis for stress management. Studies on these groups of GSTs will yield important Comparative Biochemistry and Physiology, Part B 156 (2010) 33–39 Abbreviations: GST, glutathione S-transferase; VFB, visceral fat body; HGLFB, hind-gut associated lobular fat body. ☆ GenBank submission. The CcGST nucleotide sequences discussed in the paper have been submitted to the GenBank with accession number DQ234273. ⁎ Corresponding author. Tel.: +91 40 23010 052; fax: +91 40 23010 120. E-mail address: apdgsl@uohyd.ernet.in (A. Dutta-Gupta). 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.01.016 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb