Vontas and Janet Hemingway Liverpool School of Tropical Medicine, UK Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase AO was
Trang 1Molecular characterization of the amplified aldehyde oxidase
Michael Coleman, John G Vontas and Janet Hemingway
Liverpool School of Tropical Medicine, UK
Primary structural information including the complete
nucleotide sequence of the first insect aldehyde oxidase (AO)
was obtained from the common house mosquito Culex
quinquefasciatus(Say) through cloning and sequencing of
both genomic DNA and cDNA The deduced amino-acid
sequence encodes a 150-kDa protein of 1266 amino-acid
residues, which is consistent with the expected monomeric
subunit size of AO The Culex AO sequence contains a
molybdopterin cofactor binding domain and two
iron–sul-fur centres A comparison of the partial sequences of AO
from insecticide resistant and susceptible strains of C
quin-quefasciatusshows two distinct alleles of this enzyme, one of
which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae The elevated enzyme can be seen as a separate band
on polyacrylamide gel electrophoresis The role of AO in xenobiotic oxidation in mammals and the partial inhibition
of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance Keywords: aldehyde oxidase; mosquito; insecticide resis-tance
Culex mosquitoes are major vectors of filariasis and
Japanese encephalitis as well as a general biting nuisance
Amplification of nonspecific esterases accounts for > 90%
of known insecticide resistance in Culex populations In the
C pipienscomplex, distribution of amplified esterase alleles
is geographically restricted, except for esta21 and estb21,
which are coamplified on a single DNA amplicon and occur
world-wide The rapid spread of this amplicon through
Culex populations already containing alternative esterase
alleles on resistance-associated amplicons suggests that the
esta21/estb21amplicon has a very strong selective advantage
over other esterase-based resistance mechanisms [1] A
comparison of esterases from resistant C quinquefasciatus
strains with different amplified esterases suggests no
selec-tive advantage of esta21 and estb21 over other resistant
strains based on their enzyme activity alone [2] The selective
advantage observed for strains, such as PelRR, with this
amplicon, must therefore be due to some other factor
Recently, we have reported that the PelRR amplicon
contains a third complete gene, putatively aldehyde oxidase
(AO) [3] It is possible that either the AO or esterases on the
amplicon affect mosquito viability in the presence of filarial
parasites, as we have shown that parasite survival and
insecticide resistance status are highly negatively correlated
[4], which may contribute to the lack of correlation between
mosquito biting rates, prevalence of microfilaraemia and
disease which has been noted in several studies [5]
Alternatively, the AO may have a direct role in insecticide resistance
Cytochrome P450s have traditionally been thought of as the sole enzyme system associated with increased levels of insecticide oxidation However, in mammals oxidation of xenobiotics by the molybdopterin family of enzymes is now well documented [6,7] AO is a molybdenum-containing enzyme belonging to this family The tissue localization and physiological roles of this enzyme are still not fully understood The broad substrate specificity of AO makes
it a useful mammalian prodrug activator [8,9], and AO functions in herbivores to protect them against plant toxins [6] The amplification of AO in insecticide-resistant insects may therefore have a functional significance, which has been overlooked to date
There is no known sequence data for AO in insects, although the enzyme has a number of diverse physiological functions [10–12] and its distribution patterns in Drosophila [13,14] and Musca domestica have been studied histochem-ically [15] Here we report the first genomic, cDNA and deduced protein sequences of AO from the mosquito Culex quinquefasciatus, demonstrate that the allele present on the amplicon is expressed in insecticide-resistant insects and that
it interacts with insecticides
E X P E R I M E N T A L P R O C E D U R E S
Mosquito strains
C quinquefasciatuslarvae were collected from Peliyagoda, Sri Lanka in 1984 This population, which had been under fenthion selection pressure for several years, was selected in the laboratory to produce two strains: an insecticide susceptible strain, PelSS, with the nonamplified esterases esta3 and estb12, and an organophosphorus insecticide-resistant strain, PelRR, with the two coamplified esterases esta21/estb21[16,17] The C quinquefasciatus strain TemR
Correspondence to J Hemingway, Liverpool School of Tropical
Medicine, Pembroke Place, Liverpool L3 5QA, UK.
Fax: + 44 151 7088733, Tel.: + 44 151 7089393,
E-mail: hemingway@liverpool.ac.uk
Abbreviations: AO, aldehyde oxidase; ALDH, aldehyde
dehydrogen-ase; XDH, xanthine dehydrogenase.
(Received 18 July 2001, revised 14 November 2001, accepted 16
November 2001)
Trang 2was obtained from G Georghiou, University of
Califor-nia, Riverside, USA TemR is resistant to
organophos-phates due to the amplification of a single esterase gene,
estb11[18]
Genomic DNA sequence
A genomic library of PelRR fourth-instar larvae was
constructed in the kGEM-11 vector (Promega) and probed
with a partial esta21cDNA as previously described [19,20]
The sequence downstream of the esta21 gene from the
resultant positive bacteriophage clone (A2) suggested a third
ORF with high homology to the molybdenum containing
enzymes xanthine dehydrogenase (XDH) and AO [3]
Bacteriophage A2 was produced for analysis by inoculating
400 mL of Luria–Bertani broth [0.1% (w/v) bacto-tryptone,
0.05% (w/v) bacto yeast extract, 0.1% (w/v) NaCl] with
6 mL of Escherichia coli LE392 culture, grown overnight in
Luria–Bertani broth + 0.2% maltose and incubated for
20 min, 37°C, 225 r.p.m The culture was inoculated with
109 plaque forming units of bacteriophage A2 After
allowing the mixture to stand for 20 min at 37°C, the
culture was grown at 37°C, 225 r.p.m for 6 h Chloroform
(2 mL) was added to lyse any remaining cells Ten grams
NaCl, RNase 1 mgÆmL)1 and DNase 1 mgÆmL)1 were
added and the mixture incubated for 1 h at room
temperature The cell debris was removed by centrifugation
at 12 000 g, 4°C for 10 min 10% (w/v) of poly(ethylene
glycol) 6000 was gently dissolved into the supernatant, and
the mixture incubated at 4°C for 10 min and resuspended
in 5 mL SM [0.58% (w/v) NaCl, 0.2% (w/v) MgSO4, 5%
1MTris/HCl pH 7.5, 0.01% gelatin]
Chloroform extraction was carried out and the
superna-tant removed CsCl2(0.75 g per mL) was dissolved into the
supernatant and the mixture centrifuged at 100 000 g,
10°C for 24 h The DNA band was dialysed overnight
against 10 mM NaCl, 50 mM Tris/HCl pH 8.0, 10 mM
MgCl2 EDTA After dialysis, proteinase K (50 mgÆmL)1)
and SDS (0.5%) were added and the mixture incubated at
65°C for 1 h Phenol and chloroform extractions were
carried out before precipitating the DNA in ethanol and
resuspending in Tris/HCl pH 8.0
Restriction digests and subcloning of the A2 insert was
undertaken to analyse the AO sequence A2 was digested
with BamHI and SacI and run on a 1% agarose gel
(Bio-Rad) The three resultant bands were extracted from the gel
with the Wizard DNA Clean-up System (Promega) and
subcloned into pBluescript (Stratagene) The ligation
prod-ucts were used to transform E coli XL-1Blue (Stratagene)
and recombinant plasmids were isolated from amplified
bacterial colonies using a standard miniprep method
(Qiagen)
PCR was used to produce sequence spanning across the
three subcloned fragments Primer X6 (5¢-GGTGTACA
ACGTGCAGGA-3¢) and Y4 (5¢-GAGCGAGAACGAG
CCGGAAC-3¢) were used to PCR between plasmids
AO1 and AO2 Primer Y6 (5¢-GCCGAAATGTGATTAT
TTG-3¢) and A1 (5¢-TTAGCCCGAACCGCGGCC-3¢)
were used to PCR across plasmids AO2 and AO3 These
PCR products were ligated into pGEMT-easy (Promega)
and positive colonies were selected and prepared as above
A contig of the complete bacteriophage insert was made by
combining these sequence data
Synthesis of cDNA and sequencing of AO Total RNA was isolated from 1 g of fourth instar larval PelRR using TRI reagent (Sigma) according to the man-ufacturer’s instructions Reverse transcription of first strand cDNA from mRNA was accomplished with SuperScriptTM
RT (Gibco BRL) according to the manufacturers instruc-tions with an oligo(dT) adaptor primer [5¢-GACTCG AGTCGATCGA-(dT)17-3¢]
Primers were designed to the putative 5¢, F1 (5¢-ATG GAAGTCATATTTACGAT-3¢) and 3¢, F2 (5¢-TTG TAGTTTAAACTGTTC-3¢) ends of AO based on the genomic sequence The 50-lL PCR reaction contained
20 ng of first strand cDNA, 150 ng of each primer, 0.5 mM
dNTPs, 2.5 mM MgCl2, 1.25 U Pfu DNA polymerase (Stratagene) 5 U of Amplitaq Gold DNA polymerase (PerkinElmer) and Taq DNA polymerase buffer After one cycle of 95°C for 10 min to activate the Amplitaq, 35 cycles
of amplification were carried out as follows: 95°C, 45 s;
50°C, 45 s; 72 °C, 7.5 min
3¢ RACE RACE was used to obtain the 3¢ UTR of AO Primer A20 (5¢-CCGAGAACTTGATCTACAG-3¢) designed to the 3¢ end of the cDNA was used in conjunction with an adaptor primer (5¢-GACTCGAAGTCGACATCGA-3¢) in a PCR reaction The PCR reaction was carried out as above, without Pfu DNA polymerase and with an extension time
of 2 min
5¢ PCR The partial 5¢ UTR was obtained using a primer designed from the genomic DNA 102 base pairs upstream of the transcription start codon, UTR5 (5¢-GCACTGTTTAACT CAGTTCG-3¢) and a primer (X7) designed to the 5¢ end of the cDNA (5¢-TCCTGCACGTTGTACACC-3¢) The PCR reaction was carried out as for the 3¢ RACE
PCR products were isolated using a Wizard DNA
Clean-up System (Promega) and subcloned into pGEMT-Easy (Promega) for sequencing
DNA sequencing Initially the inserts were sequenced using universal M13 forward and reverse primers complimentary to the plas-mids Internal primers were synthesized based on this initial sequence data Sufficient primers were synthesized to allow sequencing of both strands of the AO gene at least twice Sequencing was carried out with an ABI Automatic Sequencer (PerkinElmer) Sequence data were analysed with theDNASTAR(Lasergene) program
Genomic Southern blots Genomic DNA was extracted by the method of Vaughan
et al.[21] Ten milligrams of PelRR, PelSS and TemR DNA was digested with EcoRV restriction enzyme and the products separated by electrophoresis on a 0.8% agarose gel The DNA was denatured, neutralized [22] and trans-ferred to a nylon membrane (Amersham) using the Hybaid Vacu-Aid The DNA was fixed to the membrane with UV
Ó FEBS 2002 AO from C quinquefasciatus (Eur J Biochem 269) 769
Trang 3light Membranes were prehybridized at 65°C for 1 h in
hybridization buffer (6· NaCl/Cit, 0.1% (w/v) SDS, 0.1%
(w/v) sodium pyrophosphate, 5% poly(ethylene glycol),
5· Denhardt’s solution) The genomic clone AO1 was
digested with HincII and PstI Products were separated by
electrophoresis and the 0.65-kb band with high homology to
AO was extracted from the agarose using a WizardTMkit
(Promega) It was labelled with 32P (specific activities >
2· 106c.p.m.Æmg)1) by random priming with a Pharmacia
oligonucleotide labeling kit and used as a probe The probe
was hybridized to the phage DNA overnight at 60°C in
hybridization buffer Final washes were in 0.1· NaCl/Cit,
0.1% (w/v) SDS at 60°C for 45 min
Aldehyde oxidase assay
Individual mosquitoes were assayed for AO activity by a
method adapted from Moura & Barata [23] and Mira et al
[24] Briefly, individual larvae were homogenized in 40 lL
potassium phosphate buffer pH 7.8 with 1 mM EDTA
Two replicates of 10 lL were transferred to a microplate
and 200 lL of reaction mixture containing 0.1 mgÆmL)1
phenazine methosulfate, 0.1 mgÆmL)1
2,6-dichloroindophe-nol, 50 lMallopurinol, and 0.1 mMof a ‘neat’ mixture of
aldehyde substrates (1 : 1, v/v, acetaldehyde/benzaldehyde)
was added AO activity was determined by measuring the
rate of 2,6-dichloroindophenol reduction at 600 nm
(e ¼ 21 mM )1Æcm)1) as aldehyde is enzymatically
oxi-dized Kinetics were read immediately, by following the
decrease in absorbance at 650 nm for 5 min Specific
activities are given in UÆmg)1protein where a unit
corre-sponds to 1 lmol of 2,6-dichloroindophenol reduced per
min, under the assay conditions used All assays were
compared to controls of identical composition lacking
substrate (aldehydes) or homogenate
Measurement of aldehyde dehydrogenase (ALDH)
activity
The ALDH assay was performed by the method of Tasayco
& Prestwich [25], under anaerobic conditions A 1-mL
aliquot of potassium phosphate buffer pH 7.8 saturated
with nitrogen, containing 1 mM NAD+ and 10–15 mg
protein of pooled crude homogenate was added to a 2-mL
cuvette Four milliliters of 10 mMaldehyde in ethanol was
added anaerobically and the appearance of NADH
(e ¼ 6.22 mM )1Æcm)1) was recorded continuously for
10 min at 340 nM Specific activities are given in UÆmg
protein)1 A unit corresponds to the production of 1 nmol
NADH in 1 min
Protein assay
Protein content was determined by the method of Bradford
[26] Protein values in mgÆmL)1 were calculated from a
standard curve of absorbance of known concentrations of
bovine serum albumin
Inhibition of AO activity by pesticides and inhibitors
Crude homogenates for the pesticide and effector
experi-ments were prepared in ice-cold 0.1M phosphate buffer
(pH 7.8) with 5% (v/v) glycerol Stopped time inhibition
assays were performed Solutions of AO inhibitors and various pesticides were prepared in either phosphate buffer
or acetonitrile depending on their solubility (acetonitrile concentration of the medium never exceeded 1%, v/v) Each effector was preincubated with the crude homogenate for
15 min at 20°C AO or ALDH residual activity were then measured as described above in the presence of each effector, except that phenazine methosulfate was omitted from the AO activity reaction mixture
Electrophoresis Electrophoresis of native protein samples was performed in
a Phastsystem (Pharmacia) Crude homogenates were prepared as described above Two microliters of each sample ( 5 lg protein) was applied to a 8–25% gradient Phastgel (Pharmacia) with standard molecular mass mark-ers (Sigma) and subjected to native PAGE Phastsystem (400 V, 10 mA, 2.5 w, 10°C, 390 Vh)
Gels were divided to visualize standard proteins by Comassie Blue R250 staining and AO activity bands using a formazan staining solution, prepared according to Tasayco
& Prestwich [25] Briefly, 20 mg Nitro Blue tetrazolium, 0.8 mg phenazine methosulfate, 50 mg allopurinol (to inhibit xanthine oxidase and XDH) and 1 mL of aldehyde substrates (acetaldehyde, benzaldehyde, Dimethyl-amino-benzaldehyde or heptaldehyde), were added in 50 mL 0.1M
Tris/borate buffer, pH 8.0 with 1 mM EDTA All native gels were compared to controls of identical composition with aldehyde omitted
R E S U L T S
A complete ORF (ORF) coding for a putative AO enzyme was obtained from genomic DNA of the C quinquefascia-tusmosquito strain PelRR (Fig 1) The computer-based analysis of the 5¢ flanking region of the AO gene identified several potential transcription factor-binding sites Amongst them were three Barbie boxes located at)1137 to )1122, ) 666 to )652 and )539 to )525 to this ORF These sequences have been previously found in the promoter regions of most GSTs with the core sequence of AAAG common in all of them [27,28] This element might be responsible for the induction of the GST genes by the drug phenobarbital and may play a role in drug resistance during cancer treatment The common arthropod initiator sequence, TCAGT, occurs at both positions 13–18 (AI1) and 25–29 (AI2), with a possible TATA box located at)12
to)3, relative to the +1 of the 5¢ UTR, suggesting that this ORF codes for a functional gene
Primers designed to the 5¢ and 3¢ ends of the AO genomic DNA sequence were used to obtain a full-length cDNA of
3798 nucleotides from fourth instar Culex larvae This cDNA sequence was completely homologous to the predicted exon regions of the genomic DNA sequence and the structure is superimposed on the genomic sequence in Fig 1 A presumptive polyadenylation site, AATAA, is located at 5884–5889 (7540 on genomic sequence) The cDNA ORF predicts a protein of 1266 amino-acid residues and an molecular mass of 150 kDa This is consistent with the expected monomeric subunit size for AO 3¢ RACE was used to obtain a 3¢ UTR of 67 nucleotides that includes the presumptive polyadenylation site A 102-bp 5¢ UTR was
Trang 4Ó FEBS 2002 AO from C quinquefasciatus (Eur J Biochem 269) 771
Trang 6obtained by PCR using primers designed to the genomic
DNA sequence
The cDNA encodes a protein with two predicted iron–
sulfur [2Fe)2S] centres between amino-acid residues 33–62
and 138–164 There is a highly conserved molybdopterin
cofactor binding site between residues 706–752 (Fig 2), and
a region with high homology to the NADH binding site of
various Drosophila enzymes [29] is located at 343–515 This
region of the PelRR AO sequence has little homology with
the related molybdopterin enzyme XDH and there is no
putative NAD+binding domain in the mosquito sequence,
as would be expected if this ORF coded for XDH The
PelRR AO has a high degree of homology to XDH from
Drosophila melanogaster(51%), Bombyx mori (51%) and
D subobscura(50%) which is expected, as AO and XDH
are closely related enzymes Assigning this gene as an AO is
supported by the three conserved active site centers and the
high homology to bovine AO (51%), human AO (51%) and
ArabidopsisAO (50%) (Fig 2)
A Southern blot of genomic DNA from various
mosquito strains (Fig 3), shows that this AO is amplified
in PelRR, in contrast to PelSS and the insecticide resistant
strain TemR, which has an amplicon containing estb11 The
complete AO ORF in PelRR is located on the same
insecticide resistance-associated amplicon as the esterase
genes esta21/estb21 The coding regions of the genomic AO
and esta21DNA sequences overlap at their extreme 3¢ ends
in the PelRR amplicon There are five introns within the
ORF All are small introns of < 200 bp with the exception
of intron 3 which is 1700 bp in length (Fig 1) A partial
sequence of an AO from PelSS DNA is conserved at the
3¢ end of the gene (329/330 nucleotides) and has lower
conservation in the mid region (263/276 nucleotides) when
PelRR and PelSS sequences are compared, suggesting that
the two strains carry different alleles of this gene The 3¢ end
of the gene contains the conserved active site centers hence
the lack of variability in this region is expected
On native PAGE gels it is difficult to distinguish between ALDH and AO [30] Allopurinol was used to inhibit xanthine oxidase and XDH bands which will stain with AO substrates Figure 4 indicates that the upper band is xanthine oxidase and the two lower bands in PelRR are either AO or ALDH Three bands occurred in PelRR adults
in contrast to two in PelSS when equal amounts of protein were loaded (Fig 5) Size estimation of the novel AO band was obtained under native conditions using its relative mobility and the linear regression equation for the molec-ular mass markers The apparent molecmolec-ular mass of the amplified AO was estimated in its native form as approx-imately 302 kDa, which is in line with the predicted molecular mass from the translated amino-acid sequence for a homodimeric AO, as recorded in other insects and mammals The novel AO band in PelRR is stage specific (Fig 6), with highest activity during the larval stages, peaking in the third and fourth instars and decreasing to low levels in the pupal and adult stages
The specific AO activity of PelRR larval crude homo-genate using this assay was approximately fourfold higher than that in PelSS crude homogenates (Fig 7) However, this fourfold elevation in the aldehyde oxidizing activity can not be readily compared to the degree of the AO genomic amplification (which is estimated as being much higher), as a many fold AO upregulation may only increase the overall activity a few fold Adult PelRR and PelSS had similar levels of AO activity (data not shown), due to the lower expression of the amplified AO in this life stage, in comparison with other aldehyde oxidizing enzymes (such as ALDH)
The identification of the novel PelRR enzyme as an AO was confirmed by measuring AO substrate oxidation anaerobically These conditions suppress AO activity and any remaining enzymatic activity is due to ALDH PelRR and PelSS had similar ALDH specific activities (Table 1) Any differences between the activity of the two strains with
Fig 1 Nucleotide sequence of C quinquefasciatus PelRR AO genomic sequence, deduced after complete sequencing of both DNA strands (Accession
no AF 202953) A PelRR AO cDNA was also sequenced in both directions and exactly matched the underlined exon sequences of the genomic DNA The potential arthropod initiator sequences and poly A site are indicated as AI1, AI2 and PA, respectively Some of the primers used in PCR have been indicated The 5¢ UTR and 3¢ UTR sequences are also shown.
Ó FEBS 2002 AO from C quinquefasciatus (Eur J Biochem 269) 773
Trang 8AO substrates is therefore due to differences in AO activity The novel PelRR AO had a high affinity for the substrate heptaldehyde, which is a good AO, but poor ALDH substrate, supporting our earlier suggestion from anaerobic assays that this novel band is not an ALDH Both bands were prominent with acetaldehyde
Because the true kinetic parameters (Vmax or Km) and inhibition constants are not readily obtainable for a mixed
Fig 3 Southern blot of genomic DNA from the PelRR, PelSS and
TemR strains of Culex quinquefasciatus digested with EcoRV
demon-strating genomic amplification of AO in resistant PelRR, but not in
resistant TemR.
Fig 4 Specific staining and inhibition of the xanthine oxidase (XO)
stained band Equal crude homogenate protein samples from C
quin-quefasciatus PelRR adults were loaded to each well of a gradient
PhastGel (8–25%) and subjected to native PAGE Phastsystem The
formazan staining solution was prepared as described in materials and
methods, with acetaldehyde/benzaldehyde substrates and the xanthine
oxidase inhibitor allopurinol (lane 1) and the specific xanthine oxidase
substrate hypoxanthine (lane 2) The positions of the molecular mass
markers are indicated on the left.
Fig 5 The amplified AO stained band in the insecticide resistant
C quinquefasciatus PelRR strain (A) Native PAGE of the insecticide susceptible PelSS (lane 1) and the insecticide resistant PelRR (lane 2) Culex quinquefasciatus adults Equal amounts of crude homogenate of pooled samples were loaded to each well of a gradient PhastGel (8– 25%) and a neat mixture of aldehyde substrates [1 : 1 (v/v) acetalde-hyde/benzaldehyde] was used as substrate (B) The molecular mass was estimated by bilogarithmic plotting of molecular masses of the stan-dards against T%, which was the total polyacrylamide concentration reached by each protein after electrophoresis The standard markers were as follows: (a) urease (hexamer, M r 545 000); (b) urease (trimer,
M r 272 000); (c) albumin bovine serum (dimer, M r 132 000); (d) albumin bovine serum (monomer, M r 66 000); (e) albumin chicken egg (M r 45 000).
Fig 2 Comparison of the putative AO from PelRR with the amino-acid
sequences of bovine, human and maize AO with XDH from Drosophila
melanogaster Common residues between sequences are boxed.
Ó FEBS 2002 AO from C quinquefasciatus (Eur J Biochem 269) 775
Trang 9enzyme system a simple inhibition study with various AO inhibitors and pesticides was performed Table 2 shows the percentage inhibition for each chemical tested on the standard AO activity assay Methadone, a potent inhibitor
of rat AO, was the most potent inhibitor of PelRR AO activity All four pesticides at concentrations of 0.05– 0.1 mMproduced partial inhibition of AO activity, as did two commonly used herbicides at concentrations of 1 mM Only the triazine herbicides had an inhibitory effect on anaerobically measured ALDH activity (data not shown), suggesting that inhibition is due to interaction with the AO enzyme
D I S C U S S I O N
Cytochrome P450s have traditionally been considered as the only enzymes to oxidize insecticides Other oxidizing enzymes, such as AO and XDH have only recently been recognized as important in the oxidation of many drugs and xenobiotics [8] AO is capable of utilizing a wide range of substrates such as, N-heterocyclics, aldehydes (which includes a number of drugs), azo dyes and N-oxides Hepatic AO in humans mediates the oxidation of a large number of such compounds [31] Bovine AO is expressed at high levels in the liver and lungs and is implicated in the detoxification of environmental pollutants [32] The pres-ence of an amplified AO on the insecticide resistance-associated amplicon of C quinquefasciatus opens up the possibility that this enzyme, may play a role in insecticide
Table 2 Influence of pesticides and inhibitors on the AO activity of pooled C quinquefasciatus PelRR larvae AO activity was measured with a reaction mixture containing 2,6,dichloroindophenol and 1 : 1 acetaldehyde/benzaldehyde (0.1 m M ) The data are means ± SD of three separate experiments each of which was performed in duplicate.
Pesticides
Inhibitor concentration (m M )
Remaining activity (%) Herbicides
Insecticides
AO inhibitors
Table 1 ALDH specific activities in insecticide resistant PelRR and insecticide susceptible PelSS C quinquefasciatus larvae Assays were performed anaerobically and activities were estimated for pooled
C quinquefasciatus PelRR and PelSS fourth instar larvae.
Strain
ALDH activity (nmolÆmin)1Æmg protein)1)
Fig 6 AO enzymatic activity in different life stages of insecticide
resistant C quinquefasciatus PelRR Upper panel: AO specific activity
was measured in pooled crude homogenates of isogenic lines Activity
means were determined for each of three independent isogenic samples
at each time point Results are means ± SD Lower panel: native AO
stained bands from equal loading of crude homogenate proteins of
different life stages of an isogenic C quinquefasciatus PelRR line.
Fig 7 Ranges of AO activity from individual PelRR and PelSS larvae.
Ranges of AO specific activity from individual C quinquefasciatus
PelRR and PelSS fourth instar larvae, respectively (n is the number of
individuals tested).
Trang 10resistance This may account for the selective advantage of
insects carrying the esta21/estb21amplicon over those with
other esterase containing amplicons
An AO gene has been cloned from the
resistance-associated amplicon of the PelRR strain of C
quinquefas-ciatus It contains the three conserved active site centres
expected of an AO enzyme This is the first reported AO
sequence from any insect, hence absolute identification of
the enzyme through DNA sequence homology alone is
difficult [33] However, the Culex enzyme clearly differs
from XDH, a similar molybdenum containing enzyme in
this family, as it lacks the NAD+domain which is essential
for XDH activity The predicted amino-acid sequence of the
CulexORF encodes for two [2Fe)2S] centres, an NADH
binding site and a molybdenum binding domain, which is
consistent with the primary structure of AO from a range of
species There are several complete AO sequences on the
database; within these there is a high degree of homology
between human and bovine AO sequence, but little
homology between these and Arabidopsis AO [9,32] The
Culexsequence has similar levels of homology with all three
AOs
The lack of structural conservation between AO of
different species is suggested by Southern blots where
bovine AO cDNA probes did not cross hybridize with
Drosophila or toad DNA, but did cross hybridize with
lizard, chicken, mouse, rat and human DNA AO may be
less conserved than XDH, as a Southern blot using bovine
XDH cross-hybridized to DNA from all the organisms
above [9]
Identification of this enzyme as an AO is further
supported by the gene structure Three introns are
con-served in all 31 insect XDHs recorded to date and a fourth is
conserved in all but one insect species There are three
narrowly distributed novel introns, one in the medfly and
two in the Willistroni group of Drosophila, one of which is
shared by a second Drosophila group [35] All of these
introns, along with numerous others, are found in the
genomic DNA encoding mammalian XDHs and AOs Of
the five introns in the Culex AO genomic DNA, introns
1 and 5 occur in all known XDH and AO sequences Introns
3 and 4 occur in all mammalian AO and XDH sequences
but in no insect XDH sequence, and intron 2 is novel to this
CulexAO sequence (Fig 8)
The amplified AO occurs on the common esta21/estb21
amplicon, but does not occur on either of the two estb1
amplicons in the TemR or COL Culex strains [3] We have
previously shown a ladder of truncated AO bands in the
COL strain [3], and the current study shows TemR has no
amplified AO sequence, further confirming the differences
in the amplicons of these two strains despite both having an
amplified estb1[18,36]
To influence fitness of mosquitoes carrying the amplicon,
and play a role in insecticide detoxification, the amplified AO
needs to be expressed Multiple allelic variants of the esterase
genes occur in Culex, making it easy to identify transcripts
from the esterases on the insecticide resistance-associated
amplicons PCR analysis was undertaken to see whether a
similar level of allelic variation occurred in the AO locus
Comparison of the AO from resistant PelRR and susceptible
PelSS strains of Culex show that there is allelic variation at
this locus The amplified AO diverges significantly from its
nonamplified counterparts at its 5¢ end, although all allelic
variants are highly conserved at their 3¢ ends in line with other known AO sequences [32,34] The variability between the nonamplified and amplified alleles of AO from PelSS and PelRR, coupled with the complete homology between the PelRR genomic exons and the cDNA sequence from PelRR, suggests that the AO cDNA cloned from PelRR was transcribed from the amplicon and not from an un-amplified
AO elsewhere in the genome The strength of PCR product
in PelRR also suggests that the rates of AO expression are higher in this strain than in PelSS
The normal physiological role of AO in mosquitoes is, as yet, unknown, hence it is difficult to predict what effect if any the over-expression of this enzyme would have on the fitness of the mosquito carrying the AO containing ampli-con Histochemical studies on the patterns of AO content in imaginal wing discs in Minute mutants of hybrids of
D melanogaster [33] and hybrids of M domestica [15] suggest that it plays a role in larval development In male moths AO assists in the catabolism of pheromones for location of female moths [10,12] In some female moths their response to aldehydes in plant material is mediated by AO [12]
To further characterize the effects of the amplified AO,
we analysed AO activity in resistant PelRR and susceptible PelSS mosquitoes The active site of AO includes an extended lipophilic active site [37], which can accommodate the diphenyl ring systems of methadone and SKF-525A SKF-525A (Profidane) and its related analogues and methadone are potent inhibitors of rat liver cytsolic AO [38] Methadone was a potent inhibitor of mosquito AO activity, whilst SKF-525A was a poorer inhibitor
The absence of clear trends in structural requirements for substrates of AO, has been attributed to the flexibility of its substrate binding sites and its multiple productive orienta-tion [39] This makes AO an effective detoxifying enzyme for a broad range of substrates in higher vertebrates The broad substrate specificity of AO, including the oxidative or reductive metabolism of a wide variety of nitrogen or sulfur containing heterocyclic xenobiotics has been well docu-mented [6,7] This suggests that insecticides and their metabolites may make good substrates for AO AO from higher vertebrates is involved in the oxidative metabolism of neurotoxins [40], substituted quinazolines and pthalazines [41], chinhona antimalarials [42], purines and their ana-logues [39], quinoloinium cations and quinines [43,44],
Fig 8 Schematic diagram of the intron positions of PelRR AOs com-pared to AOs and XDHs from a range of species Introns at positions A and G are common to PelRR AO and insect XDHs Intron* is novel to the Culex AO, while the four remaining introns are at positions in common with mammalian AO and XDH.
Ó FEBS 2002 AO from C quinquefasciatus (Eur J Biochem 269) 777