Elsevier

Methods in Enzymology

Volume 401, 2005, Pages 226-241
Methods in Enzymology

Mosquito Glutathione Transferases

https://doi.org/10.1016/S0076-6879(05)01014-1Get rights and content

Abstract

The glutathione transferases (glutathione S‐transferases, GSTs) are a diverse family of enzymes involved in a wide range of biological processes, many of which involve the conjugation of the tripeptide glutathione to an electrophilic substrate. Relatively little is known about the endogenous substrates of mosquito GSTs, and most studies have focused on their role in insecticide metabolism, because elevated levels of GST activity have been associated with resistance to all the major classes of insecticides. In addition, there is growing interest in the role of this enzyme family in maintaining the redox status of the mosquito cell, particularly in relation to vectorial capacity.

Most GSTs are cytosolic dimeric proteins, although a smaller class of microsomal GSTs exists in insects, mammals, and plants. Each GST subunit has a G site that binds glutathione and a substrate‐binding site or H site. There are more than 30 GST genes in mosquitoes. Additional diversity is contributed by alternative splicing to produce GSTs with differing substrate specificities. In this review, we first discuss the diversity of insect GST enzymes and their mode of action before focusing on the various functions that have been attributed to specific mosquito GSTs.

Introduction

The glutathione S‐transferases (GSTs) are important components of the detoxification pathway in almost all organisms. They act as phase II detoxifying enzymes, conjugating glutathione to products of metabolism or xenobiotics, thereby increasing their solubility and aiding their excretion from the cell. The diverse ranges of substrates recognized by GSTs is partly due to the broad substrate specificities of some individual enzymes but is largely attributable to the extensive nature of this enzyme family in most eukaryotic organisms. The expansion in the number of GST subunits identified has been matched by a parallel expansion in the functions attributed to this enzyme family. GSTs are now recognized as playing important roles in protecting cells from the harmful effects of oxidative stress, cell signaling pathways, intracellular transport, and several biosynthetic pathways.

The GSTs from the major African malaria vector, Anopheles gambiae have been the most extensively studied of all the mosquito GST families. The complete draft genome sequence for this species was determined in 2002 (Holt et al., 2002), enabling the full extent of the mosquito GST gene family to be recognized for the first time (Ranson et al., 2002). At the time of writing, genome sequencing of the dengue vector, Aedes aegypti, is in progress and that for the lymphatic filariasis vector, Culex quinquefasciatus, is expected to follow shortly. Several GST genes have already been identified in the unassembled sequence data from Ae. aegypti, some of which have been further characterized (Lumjuan et al., 2005). Initial observations on the similarities and differences between the GST supergene families in Anopheles and Aedes are discussed in the following. To date, little is known about the GSTs of Cx quinquefasciatus, but the ability of this species to survive in more highly polluted water than other mosquitoes may lead us to anticipate greater GST diversity.

Section snippets

Nomenclature

GSTs were first identified in rat livers in 1961 (Booth 1961, Combes 1961). It was quickly realized that multiple enzymes were present in rats and in other mammals, and different studies categorized GSTs according to their order of elution from affinity columns, substrate specificity, or immunological cross‐reactivity. Eventually, in 1992, a unifying system of nomenclature was applied to the mammalian GSTs that classified each subunit to a different class, designated by a Greek letter,

Classification of Mosquito GST Genes

At least six classes of soluble cytosolic insect GSTs have been identified in insects plus a single membrane‐bound microsomal class (Ranson et al., 2002). Criteria for inclusion in a particular class are primarily based on amino acid sequence identity and phylogenetic relationship, but chromosomal location and immunological properties, when known, are also taken into account. Thus, GSTE8 was assigned to the Epsilon class, despite sharing less than 28% amino acid identity with other members of

Epsilon Class GSTs

The Epsilon class is also a large, insect‐specific class. Eight members have been identified in A. gambiae and to date, three have been identified in the EST sequence database of Ae. aegypti. Most of the Epsilon GSTs characterized have low levels of activity with the model substrate, 1 chloro 2,4, dinitrobenzene (CDNB), which is usually used to detect GST activity, and many are not retained by glutathione‐based affinity matrices. Both of these factors may explain the relatively recent discovery

Structure of Cytosolic GSTs

Cytosolic GSTs are dimeric proteins. Each subunit is composed of between 200 and 250 amino acid residues with typical molecular masses ranging from 20–28 kDa. Each GST subunit adopts a canonical GST fold of seven or eight alpha helices and four beta sheets to produce two distinct domains, the N‐ and C‐terminal domains. Five insect GSTs have been crystallized, including three from mosquitoes (Chen et al., 2003; Oakley et al., 2001). The structure of a Delta class GST, bound to the inhibitor

Microsomal GSTs

The microsomal GST class is evolutionarily and structurally distinct from the cytosolic class but catalyzes a similar range of reactions. Each subunit is composed of approximately 150 amino acids that assemble into four transmembrane alpha helices (Schmidt‐Krey et al., 2000). These form a trimeric protein with a molecular mass of approximately 50 KDa and a single substrate‐binding site. Three members of this class have been identified in A. gambiae.

Functions of Mosquito GSTs

The reaction most commonly associated with the GST enzyme family is glutathione conjugation. This is a key stage in the conversion of lipophilic compounds to water‐soluble metabolites that are more readily exported from the cell. Although it is well established that GSTs are involved in many other reactions besides conjugation, in reality, little is known about the endogenous substrates of mosquito GSTs. They presumably play a role in the metabolism of dietary components, such as plant

GSTs and Insecticide Resistance

GSTs can catalyze the detoxification of several major classes of insecticide. For example, they catalyze the conjugation of glutathione to organophosphate insecticides such as tetrachlorvinphos and parathion, resulting in their O‐dealkylation or O‐dearylation, and they catalyze the dehydrochlorination of the organochlorine insecticide, DDT (Clark and Shamaan, 1984). GSTs with peroxidase activity may confer protection against the secondary effects of insecticide exposure, such as oxidative

Regulation of Mosquito GST Expression

Many GSTs are differentially regulated in response to various inducers or environmental signals, and some are expressed in a tissue‐ or developmental‐specific manner. In A. gambiae, transcripts for all but one of the GST supergene family were detectable in 1‐day‐old adult mosquitoes by RT‐PCR (Ding et al., 2003), but preliminary data suggest that expression of some of these genes is restricted to certain tissues.

Some of the GST genes are tightly clustered within the mosquito genome. For

Summary

In summary, mosquito GSTs form a complex, multi‐class, family of enzymes that fulfil a raft of important housekeeping and protective roles within the insect. Their role in insecticide resistance is currently best understood, but our knowledge of the structure and function of this important enzyme class is expanding rapidly driven in part by recent advances in mosquito genome sequencing.

References (34)

  • BoardP.G. et al.

    Zeta, a novel class of glutathione transferases in a range of species from plants to humans

    Biochem. J.

    (1997)
  • BoothJ. et al.

    An enzyme from rat liver catalyzing conjugation with glutathione

    Biochem. J.

    (1961)
  • ChelvanayagamG. et al.

    Fly fishing for GSTs: A unified nomenclature for mammalian and insect glutathione transferases

    Chem. Bio. Interact.

    (2001)
  • ChenL. et al.

    Structure of an insect Delta‐class glutathione S‐transferase from a DDT‐resistant strain of the malaria vector Anopheles gambiae

    Acta Cryst.

    (2003)
  • CombesB. et al.

    A liver enzyme that conjugates sulfobromophthalein sodium with glutathione

    J. Clin. Invest.

    (1961)
  • DingY. et al.

    The Anopheles gambiae glutathione transferase family: Annotation, phylogeny and gene expression profiles

    BMC Genomics

    (2003)
  • DingY. et al.

    Characterisation of the promoters of Epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress

    Biochem J.

    (2005)
  • Cited by (167)

    View all citing articles on Scopus
    View full text