The Anopheles gambiae glutathione transferase supergene family: annotation, phylogeny and expression profiles
- Yunchuan Ding†1,
- Federica Ortelli†1, 2,
- Louise C Rossiter1, 3,
- Janet Hemingway1 and
- Hilary Ranson1Email author
© Ding et al; licensee BioMed Central Ltd. 2003
Received: 11 June 2003
Accepted: 13 August 2003
Published: 13 August 2003
Twenty-eight genes putatively encoding cytosolic glutathione transferases have been identified in the Anopheles gambiae genome. We manually annotated these genes and then confirmed the annotation by sequencing of A. gambiae cDNAs. Phylogenetic analysis with the 37 putative GST genes from Drosophila and representative GSTs from other taxa was undertaken to develop a nomenclature for insect GSTs. The epsilon class of insect GSTs has previously been implicated in conferring insecticide resistance in several insect species. We compared the expression level of all members of this GST class in two strains of A. gambiae to determine whether epsilon GST expression is correlated with insecticide resistance status.
Two A. gambiae GSTs are alternatively spliced resulting in a maximum number of 32 transcripts encoding cytosolic GSTs. We detected cDNAs for 31 of these in adult mosquitoes. There are at least six different classes of GSTs in insects but 20 of the A. gambiae GSTs belong to the two insect specific classes, delta and epsilon. Members of these two GST classes are clustered on chromosome arms 2L and 3R respectively. Two members of the GST supergene family are intronless. Amongst the remainder, there are 13 unique introns positions but within the epsilon and delta class, there is considerable conservation of intron positions. Five of the eight epsilon GSTs are overexpressed in a DDT resistant strain of A. gambiae.
The GST supergene family in A. gambiae is extensive and regulation of transcription of these genes is complex. Expression profiling of the epsilon class supports earlier predictions that this class is important in conferring insecticide resistance.
Glutathione transferases (GSTs) are a diverse family of dimeric proteins found in almost all living organisms. Originally studied for their role in detoxification of endogenous and xenobiotic compounds, they have since been found to have additional important roles as transport proteins and in protection against oxidative stress . Each GST subunit consists of two domains, each containing two binding sites, the G site and the H site. The highly conserved G site binds the tripeptide glutathione and is largely composed of amino acid residues found in the N-terminal domain. The H-site or substrate binding site is more variable in structure and is largely formed from residues at the C-terminal .
Purification of independent homogenous GST preparations with differing substrate specificities indicated the presence of multiple forms of GSTs . Subsequently, the availability of N-terminal sequence data led to the recognition of five classes of cytosolic GSTs in mammals, the alpha, mu, pi, theta and sigma classes [2, 4, 5] and an additional, structurally unrelated membrane bound microsomal class . Recently the advent of large scale EST and full genome sequencing projects has led to a marked increase in the number of GST classes recognized. Some of these, such as the omega and zeta classes are represented in a wide range of species [7, 8], whereas others, such as the mammalian kappa class , the insect epsilon class  and the plant tau and phi clases  have a more restricted distribution.
Most of these GST classes are encoded by multigene families. Alternative splicing [12, 13] and the formation of heterodimers , can add a further level of heterogeneity to this enzyme family. With this level of diversity, assigning physiological functions to individual GSTs is a complex task, but progress towards this goal can be greatly facilitated by the process of cataloguing the number of genes within the supergene family. Armed with this information, details of expression profiles, induction mechanisms, tissue distribution etc. can be accurately obtained enabling biologically important questions to be addressed. Automatic annotation algorithms applied to assembled eukaryotic genomes provide projections of the sizes of gene families within a species. Using these tools the numbers of GST genes is estimated at 10 in Saccharomyces cerevisiae, 57 in Caenorhabditis elegans, 43 in Drosophila melanogaster, 37 in Anopheles gambiae, 46 in Arabidopsis thaliana and 40 in Homo sapiens . Careful manual annotation is essential to confirm these predicted numbers. This process has led to revised sizes of the GST supergene family in A. thaliana and A. gambiae to 48 and 31 respectively [11, 16]. (This gene count in A. gambiae includes three genes encoding putative microsomal GSTs but these will not be discussed further in this report).
To facilitate the functional characterization of insect GSTs, we have studied the annotation of each member of this supergene family in the mosquito A. gambiae. RT-PCR experiments demonstrate that all but one of the predicted GST genes are actively transcribed in adult mosquitoes and that alternative splicing of two GST genes contributes additional diversity. We compare this GST supergene family with that of a second Diptera, D. melanogaster and identify classes of GSTs that are conserved between the species and other classes that have undergone independent radiation.
The majority of studies on insect GSTs have focused on their role in conferring insecticide resistance (e.g. [17–19]) and, more recently, in protecting against cellular damage by oxidative stress [20, 21]. Reactive oxygen species can be produced in response to infection by pathogens, and this phenomenon has been implicated in the defense mechanism of mosquitoes against malaria parasites . A. gambiae, is therefore an ideal species in which to study the role of GSTs in both of these biological processes. This species is the major vector of malaria in Africa and as such is responsible for over 1 million deaths each year . Efforts to control the disease by targeting the mosquito populations have relied on treatment of Anopheles breeding and resting sites with insecticides. The organochlorine DDT was the insecticide of choice for malaria control for much of the latter half of the 20th century and is still employed in public health campaigns today but, in many malarious regions, resistance has rendered DDT-based control programmes ineffectual. In A. gambiae, DDT resistance is associated with increased GST activity . Genetic mapping using microsatellite markers has located two loci associated with DDT resistance in A. gambiae . By aligning the cytogenetic position of these resistance loci with the in situ position of physically mapped GST genes we previously identified two candidate resistance-associated GSTs . Analysis of the draft genome sequence of A. gambiae identified a further six GST genes within this region of the genome . We now report that the expression of multiple members of this gene cluster is elevated in DDT resistant insects.
Results and Discussion
Classification of A. gambiae GSTs
Summary of the A. gambiae GST family. The length of the GSTd6 gene and putative translation are not known (N.K.).
No. of transcripts detected
Length of putative protein(s)
Length of gene (bp)
Genebank Accession number
219, 210, 217, 210
The two largest GST classes in A. gambiae are the insect specific delta and epsilon classes with 12 and 8 members respectively. Support for the monophyly of these two classes is low in the tree shown although, when the 218 residue alignment is used, the bootstrap values are more supportive (data not shown). Criteria for inclusion in a particular class is based primarily on amino acid sequence identity and phylogenetic relationship, but chromosomal location and immunological properties, where known, were taken into account. Thus GSTe8, although sharing less than 29% amino acid identity with other members of the epsilon class, is found immediately adjacent to the seven epsilon GSTs on chromosome 3R, is immunologically related  and, in the majority of the trees, formed a weakly supported monophyletic group with the seven bona fide epsilon GSTs. Thus this GST was classified as the eighth member of the epsilon class . Three GSTs, GSTu1, GSTu2 and GSTu3, are outliers from the major delta GST clade (Figure 2). The phylogenetic relationship of these GSTs to the remainder of the family was not consistent between the different trees. Furthermore, these GSTs share less than 37% amino acid identity with other members of the A. gambiae delta GST class (pairwise amino acid identities between the three tentative delta class GSTs and the remainder of the class range from 22.1% between GSTd4 and GSTu2 to 36.3% identity between GSTd11 and GSTu3) and are not physically clustered with the majority of the delta class GSTs on chromosome 2L. Thus it is possible that these GSTs may belong to an as yet unrecognised GST class (or classes) and in the absence of clarifying immunological or biochemical data these GSTs have been designated as unclassified (denoted by a 'u').
With the exception of the sigma class, all of the non-insect specific classes are expanded in D. melanogaster relative to A. gambiae. Neither species has any sequence related to the mitochondrial kappa class found in mammals . The endogenous function of these mammalian GST classes in insects has not been clearly resolved and therefore the significance of the difference in size of these classes in the two Diptera examined, is, at present, unknown.
Amplification of A. gambiae cDNAs
Transcripts from 27 of the 28 A. gambiae GST genes were detected in fourth instar larvae or one-day-old adults by RT-PCR. Expression of GSTd9 was not detected in any life stage and thus it is possible that this gene represents a silent pseudogene, as suggested earlier . A second putative pseudogene is GSTd6. Utilisation of the first inframe stop codon for this gene (as present in the genome sequence database) would generate a transcript with 885 bp of coding sequence (> 130 bp longer than any of the other cytosolic GST genes in this species) and the putative translation would encompass a string of 14 glutamine residues. We therefore sequenced the intergenic region between GSTd6 and the neighbouring gene, GSTd11 to detect any possible frame shifts or sequencing errors that may have masked the stop codon but none were detected. A GSTd6 transcript of 666 bp was amplified by RT-PCR but attempts at 3' RACE have so far failed to detect the 3' end of this transcript.
Intron positions and sizes
There are thirteen unique intron sites and 42 introns within the A. gambiae GST supergene family. The majority of the introns (28) are phase 0 introns, i.e. the intron does not interrupt a codon. There is a considerable conservation of intron positions within the different GST classes (Figure 2) and one intron is found in 17 GST genes spanning three different classes. Interestingly this conserved intron, found approximately 50 amino acid residues from the N-terminal, is also the splice site for the alternative transcripts of GSTs1 and GSTd1 (see below). It has been proposed, by proponents of the 'introns-early' hypothesis, that different exons correspond to different domains in a protein . The highly conserved GST intron however, splits the N-terminal domain (roughly residue 1–80). In addition, the phase of this intron is not conserved between the different classes, and thus the classification of this as an ancient intron is not well supported.
Clustering of GST genes in the genome
In both A. gambiae and D. melanogaster local duplications in the epsilon and delta GST families have led to independent expansions of these gene classes. Subsequent diversification of these enzymes has presumably facilitated the adaptation of the two Diptera to their different ecological niches.
The sigma class in D. melanogaster is also represented by a single gene, DmGSTs1. The Drosophila GST has an N-terminal extension of 46 amino acid residues relative to the Anopheles ortholog. This N-terminal extension is not essential for catalytic activity and may play a role in attaching the D. melanogaster protein to indirect flight muscles . The intron positions of DmGSTS1 and A gambiae GSTs1-1 are conserved indicating a common ancestor for these genes. Local duplication of the carboxyl exons of the A. gambiae GSTs1 gene presumably occurred subsequent to the speciation event separating the two Diptera.
Alternative splicing as a means of increasing the level of heterogeneity of GST subunits appears to be a rare phenomenon outside of the genus Anopheles, having, as far as we are aware, only been reported in a GST from the nematode Onchocerca volvulus . Two human cDNA clones encoding alternative transcripts of a mu class GST have been detected but both are incomplete and thus unlikely to encode functional GSTs .
Expression profiling of epsilon GSTs
Quantitative PCR results of A. gambiae epsilon GST genes. The transcript copy number or gene copy number was determined using cDNA or gDNA respectively. The copy numbers were normalised for variations in initial template concentration by dividing each sample by the copy number of the ribosomal protein gene, S7. The final column shows the ratio of the transcript or gene copy number between the resistant ZAN/U and insecticide susceptible Kisumu strain. Statistically significant differences (*p < 0.05, **p < 0.01) between ZAN/U and Kisumu are indicated.
Kisumu Strain Normalised cDNA copy number × 102(± S.D.)
ZAN/U Strain Normalised cDNA copy number × 102(± S.D.)
Ratio of Copy Number (ZAN/U:KISUMU)
1.50 ± 0.320
4.52 ± 1.054
1.66 ± 0.408
12.92 ± 4.125
0.15 ± 0.100
0.49 ± 0.133
1.13 ± 0.299
2.82 ± 0.795
0.08 ± 0.050
0.10 ± 0.065
0.58 ± 0.223
1.20 ± 0.551
0.03 ± 0.010
0.34 ± 0.152
0.48 ± 0.162
0.62 ± 0.541
Kisumu strain Normalised gDNA copy number. (± S.D.)
ZAN/U strain Normalised gDNA copy number. (± S.D.
Ration of Copy Number (ZAN/U:Kisumu)
0.29 ± 0.125
0.32 ± 0.092
0.32 ± 0.088
0.51 ± 0.144
0.22 ± 0.129
0.29 ± 0.073
0.28 ± 0.098
0.21 ± 0.081
There was a large variation in the number of transcripts detected for each of the individual GSTs within both the susceptible and resistant strains. For example, the normalised copy number of GSTe2 transcripts was over 26-fold greater than that of GSTe3 in the resistant strain (Table 2). This indicates that the basal expression of individual GSTs within a cluster is independently regulated. To confirm this result, we repeated the qPCR for four of the GSTs using gDNA as a template. If the differences seen with cDNA reflect genuine differences in GST transcript copy number as we hypothesised, then the genomic copy number for each of the four GSTs would be approximately the same. Figure 9 and Table 2 support our hypothesis. Furthermore, as the genomic qPCR was carried out on DNA extracted from both susceptible and resistant mosquitoes, the results shown in Figure 9B support our unpublished data from Southern blots demonstrating that the increases in GST transcript levels seen in the resistant strain is not due to gene amplification (F. Ortelli, unpublished data).
As a final test of the reliability of our qPCR results we calculated the expected copy number of a single copy gene in 10.4 ng of A. gambiae genomic DNA (the starting amount of gDNA template in the qPCR reactions with Kisumu DNA). The theoretical copy number (33,120) was within an order of magnitude of the range of values obtained (11,993–17,205).
The insect GST supergene family encodes a diverse set of proteins. The availability of the full genome sequence for two insect species has enabled the full extent of this protein family in insects to be realised. Multiple members of the epsilon class are upregulated in a DDT resistant strain of A. gambiae and it is proposed that this class plays a major role in the detoxification of xenobiotics. However, little is known about the endogenous substrates of insect GSTs. Functional genomics approaches will no doubt contribute to our understanding of the role of individual GSTs in insects and perhaps then the reason for the extensive diversity of this enzyme family will become clear.
The DDT resistant ZAN/U strain of Anopheles gambiae s.s. originated from a field population collected from Zanzibar, Tanzania, in 1982. Adults from this strain have been maintained under regular selection pressure by exposure to Whatmans no.1 filter papers impregnated with 4% DDT according to standard WHO methods . The Kisumu strain is susceptible to insecticides and originates from Kisumu in Western Kenya.
Annotation of A. gambiae GST genes
Members of the GST supergene family were identified in the A. gambiae genome by BLAST searches  using multiple representative sequences from each GST class as query sequences. The sequences retrieved from the genome were manually annotated to predict transcription initiation and termination sites and intron/exon boundaries using BlastX comparisons of putative amino acid translations . Primers pairs were designed to amplify the full length of the coding sequence of each gene from A. gambiae cDNA.
Total RNA was extracted from individual mosquitoes using the TRI reagent (SIGMA), according to the manufacturer's instructions. The RNA was treated with DNase to remove any contaminating genomic DNA and the mRNA was reverse transcribed into cDNA using superscript II (GIBCO BRL) and an oligo (dT) adapter primer (5'-GACTCGAGTCGACATCGA(dT)17-3'). The PCR conditions for amplifying GST cDNAs were determined empirically for each GST. Products of the expected size were subcloned into pGEM T-easy vectors (Promega) and used as templates for sequencing. At least three independent clones were sequenced for each GST. Sequencing reactions were performed using Beckman chemistry and the resultant products analysed on a Beckman CEQ800 capillary sequencer.
Primer sequences and PCR conditions for amplification of epsilon class GST genes by quantitative PCR.
Forward Primer (5' to 3')
Reverse Primer (5' to 3')
cDNA amplicon (bp)
gDNA amplicon (bp)
Annealing/Detection Temp (°C)
The experiment was repeated on genomic DNA samples extracted from approx 1 g of Kisumu or ZAN/U strains as described previously . For the GST genes for which the initial primers spanned introns, new plasmids were constructed containing the genomic fragment of the gene.
Putative amino acid sequences of the GSTs were aligned using ClustalW . The alignment was manually truncated as described in the Results and Discussion section. Evolutionary distances were calculated using the Jukes-Cantor algorithm  and phylogenetic trees were determined by the neighbor-joining method  with TREECON  or by parsimony methods using MEGA2 .
YD and LR carried out the expression analysis. FO participated in the sequencing and performed the majority of the data analysis. JH conceived the study and participated in its design. HR contributed to the sequencing and analysis of data, drafted the manuscript and coordinated the project. All authors read and approved the final manuscript.
1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, GST: glutathione transferase
Y. Ding was supported by a PhD fellowship award from the late Lord Leverhulme. F. Ortelli and L. Rossiter were supported by a Wellcome Trust project grant held by Ranson and Hemingway. H. Ranson is a Royal Society Dorothy Hodgkin Research Fellow. We are very grateful to A. Dana and F.H. Collins for sharing EST data from A. gambiae prior to publication.
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