Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

Fatma M. A. El-garj， Mustafa F.F. Wajidi， Silas W. Avicor

Molecular Entomology Research Group， School of Distance Education， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia



Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

Fatma M. A. El-garj， Mustafa F.F. Wajidi， Silas W. Avicor✉

Molecular Entomology Research Group， School of Distance Education， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia

ARTICLE INFO

Article history:

in revised form 10 June 2016

Accepted 15 July 2016

Available online 20 October 2016

Aedes aegypti

Clone

Cytochrome P450 pseudogene

CYP4H44P

Bioinformatics

Objective: To clone cytochrome P450 from Aedes aegypti （Ae. aegypti） and determine the characteristics using bioinformatics tools. Methods: Cytochrome P450 of Ae. aegypti was amplified using polymerase chain reaction， cloned and sequenced. Evolutionary relationship of the sequence was inferred and bioinformatics tools were used to predict subcellular localisation， signal peptide， transmembrane helix， phosphorylation， O-glycosylation，secondary and tertiary structures of the deduced protein. Results: Polymerase chain reaction rather amplified a cytochrome P450 pseudogene which was named CYP4H44P （GenBank accession number KF779932）. The pseudogene has 1537 nucleotides and an open reading frame of 335 amino acids containing cytochrome P450 motifs except the WxxxR motif. It is highly homologous to CYP4H28 and CYP4H28v2. Phylogenetic analysis and evolutionary divergence showed strong clustering with CYP4H28 alleles and least divergence from the alleles respectively. The deduced protein was predicted to be found in the cytoplasm and likely to be phosphorylated but devoid of signal peptide， transmembrane helix and O-glycosylated sites. The secondary and tertiary structures were also generated. Conclusions: A cytochrome P450 pseudogene， CYP4H44P was cloned from Ae. aegypti. The pseudogene is homologous with CYP4H28 alleles and seems to have recently diverged from this group. Isolating this pseudogene is an important step for evaluating its biological role in the mosquito and for the evolutionary analysis of Ae. aegypti CYPs.

Document heading doi: 10.1016/j.apjtm.2016.07.024

1. Introduction

Cytochrome P450 monooxygenases （CYPs） are an important enzymatic super group performing diverse functions in different organisms［1，2］. The group is made up of different clans which are subdivided into families［1］. CYPs perform a broad range of functions in insects， including insecticide resistance and activities related with insect physiology［1］. CYP genes and pseudogenes havebeen identified in insect genomes. Pseudogenes constitute about 3.1% of the CYPs in the genome of the yellow fever mosquito，Aedes aegypti （Ae. aegypti ）［3］. Although similar to functional genes， pseudogenes are regarded to have lost the ability to code for functional proteins［4，5］. They are grouped into three types， namely processed pseudogenes， duplicated pseudogenes and unitary pseudogenes［6，7］. Processed pseudogenes are formed as a result of retrotransposition of mRNA into the genome［6，7］. The duplicated pseudogenes are formed when functional genes duplicate， and one of the duplicates undergoes mutation and becomes non-functional while unitary pseudogenes arise when there is a disruptive mutation in the coding genes of functional proteins［4，6，7］. Despite being presumed as not coding for functional proteins， pseudogenes can produce transcriptional products and perform several roles in organisms［6-8］. During the evolutionary process， pseudogenes are presumed to be under less conservation constraints compared to functional genes and hence are useful in analysing the evolutionaryhistory of genomes and functional genes［6］. Interest in pseudogenes has grown and the possibility of artificially synthesising functional translational products from them has even been hypothesised［9］.

Ae. aegypti is a vector of arthropod borne viruses such as chikungunya， dengue， yellow fever and Zika. Routine control of the vector using insecticides has contributed to CYP-mediated insecticide resistance. Consequently， CYPs of this mosquito have been studied for their functional roles in insecticide resistance［3，10］. In the course of isolating CYP gene fragments from Ae. aegypti［11］，a processed CYP pseudogene was identified. This study describes isolation of the pseudogene and the predicted characteristics of its deduced protein. Identification of the CYP pseudogene will be useful in evolutionary analysis of functional CYPs of Ae. aegypti and in future studies to determine its biological role in the mosquito.

2. Material and methods

2.1. RNA extraction

Total RNA was extracted from fourth instar larvae （0.25 g） of a reference Ae. aegypti strain［12］ as described in［11］. The RNA was qualitatively and quantitatively analysed by electrophoresis in a 1% agarose-formaldehyde gel and using a Nanodrop spectrophotometer respectively.

2.2. cDNA synthesis and amplification reaction

Synthesis of cDNA from RNA （5 μg） was performed using RevertAid™ Premium First Strand cDNA Synthesis Kit（Fermentas®） as per the manufacturer's instructions. The cDNA was used in a polymerase chain reaction （PCR） with a pair of primers（Table 1） as previously described［11］ in a PTC-100™ Programmable Thermal Controller （MJ Research）.

2.3. 3'- and 5'-rapid amplification of cDNA ends （RACE）

2.3.1. 3'-RACE

The primers 3R1 （0.5 μL of 10 μM） and Oligo（dT）25V （0.5 μL of 10 μM） were used in a PCR containing cDNA （2 μL）， OneTaq Hot Start 2X Master Mix with Standard Buffer （12.5 μL） and sterile distilled water （9.5 μL） at 94 ℃ for 5 min， 7 cycles of （94 ℃/30 s， 43 ℃/30 s and 72 ℃/1 min）， 27 cycles of （94.0 ℃/30 s， 51.5 ℃/30 s and 72.0℃/1 min）， and finally at 72 ℃/10 min. The product was used as template （2 μL） in a nested PCR using 3R1N and Oligo（dT）25V as primers with similar reaction volume and thermal condition as stated above.

2.3.2. 5'-RACE

The SMARTer™ RACE cDNA Amplification Kit （Clontech®）was used to obtain the 5'-RACE Ready cDNA according to the manufacturer's instructions. 5'-RACE PCR was performed using Advantage® GC 2 PCR Kit （Clontech®） according to the manufacturer's instructions at 5 cycles of 94 ℃/30 s and 72 ℃/3 min， followed by 5 cycles of （94 ℃/30 s， 68 ℃/30 s and 72 ℃/3 min） and then 30 cycles of （94 ℃/30 s， 66 ℃/30 s and 72 ℃/3 min）.

2.4. Electrophoresis and purification

The amplified product was electrophoresed in a 1% agarose gel and viewed under ultra-violet light. The product was purified using the Wizard® SV Gel and PCR Clean-Up System （Promega®） according to the manufacturer's instructions.

2.5. Cloning and plasmid extraction

The purified product was ligated into a pGEM®-T Easy Vector（Promega®） in an insert: vector ratio of 3:1. The ligation reaction consisting of 3 μL purified fragment， 1 μL of pGEM®-T Easy Vector， 5 μL of 2X rapid ligation buffer and 1 μL of T4 DNA Ligase（3 Weiss units/μL） was incubated overnight at 4 ℃. The ligated product was cloned into competent Escherichia coli JM 109 after heat shock［13］. The cells were spread on agar plates containing ampicillin（100 μg/mL）/X-gal （40 μg/mL）/IPTG （0.5 mM） and incubated overnight at 37 ℃ for blue/white colonies. Clones were screened by streaking them on fresh ampicillin/X-gal/IPTG agar plates and incubated overnight at 37 ℃. Plasmids were extracted from positive transformants using the Wizard® Plus SV Minipreps DNA purification System （Promega®） according to the manufacturer's instructions and restriction-digested with EcoRI to confirm that the insert DNA was present in the plasmid.

2.6. Sequencing

Sanger sequencing was performed by First Base Laboratories Sdn Bhd with the universal primers SP6 and T7 using the BigDye® Terminator v3.1 Cycle Sequencing Kit （Applied Biosystems®） and separating DNA fragments in an ABI PRISM 3730xl Genetic Analyzer（Applied Biosystems®）. Sequence similarity was performed in the National Centre for Biotechnology Information （NCBI） database using Basic Local Alignment Search Tool （BLAST）.

Table 1Primers for partial cDNA amplification， RACE PCR and isolation of full length CYP4H44P.

2.7. Verification of full length cDNA

To verify that the 3'- and 5'- RACE products were from the same cDNA， the full length cDNA was amplified using the Advantage® GC 2 PCR Kit （Clontech®）. The cycling condition was as follows；94 ℃/3 min， 30 cycles of 94 ℃/20 s， 55 ℃/20 s and 72 ℃/2 min and finally 72 ℃/10 min. Purification， cloning and sequencing of the product was as described in subsections 2.4 to 2.6.

2.8. Multiple sequence alignment and phylogenetic analysis

Sequence identity search was performed using BLAST. CYP sequences with homology to the query sequence were retrieved from the NCBI database and used for multiple sequence alignment with Clustal Omega［14］. The alignment file was used to compute pairwise evolutionary distances and construct a maximum-likelihood phylogenetic tree based on the Tamura-Nei model［15］ with 1000 bootstraps in MEGA6［16］.

2.9. Bioinformatics analysis

PSORT Ⅱ （http://psort.hgc.jp/form2.html） was used to predict subcellular localisation of the deduced protein. Detection of potential signal peptide and transmembrane helix were also predicted using PrediSi （http://www.predisi.de/） and TMHMM Server v. 2.0（http://www.cbs.dtu.dk/services/TMHMM/）. Phosphorylation and O-glycosylation sites were determined using NetPhos 2.0 Server（http://www.cbs.dtu.dk/services/NetPhos/） and DictyOGlyc 1.1（http://www.cbs.dtu.dk/services/DictyOGlyc/） respectively， while GOR IV （https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl？page=/ NPSA/npsa_gor4.html） was used to predict the secondary structure. Homology modelling was performed with the automated modelling mode in SWISS-MODEL［17］ to obtain the three dimensional （3D）model of the deduced protein.. The model was subjected to structure validation using PROCHECK［18］ and ProSA-web［19］.

3. Results

3.1. Sequence and homology of pseudogene

Amplification of the full length cDNA yielded a product of approximately 1.5kb （Figure 1A）. Sequencing of the product after cloning and plasmid extraction showed that it has 1 537 nucleotides （nt） with a deduced open reading frame of 1 008 nt， which translated into 335 amino acids （aa） （Figure 1B）. The sequence was classified as a pseudogene and assigned the name CYP4H44P by the P450 Nomenclature Committee. It has 449 and 80 nt in the 5'-and 3'-Untranslated Regions， a stop codon（TAA） starting at the 1 455th nt position， a polyadenylation signal（AATAAA） and a 26 nt long poly A tail. The translated sequence contains CYP motifs like ExxR （E202VLR205）， PERF （P256ERF259），FxxGxxxCxG （F275SVGARNCIG284） and the 13-residue sequence（E134VDTFMFEGHDTT146） of family 4 CYPs （Figure 2）.P

Figure 1. Gel electrophoresis of PCR products and sequence of CYP4H44P.（A） Amplified products after PCR to verify full length cDNA. Lane 1: 1 kb DNA ladder. Lane 2: CYP4H44P cDNA. Lane 3: CYP fragments. （B）CYP4H44P sequence consisting of 1 537 nucleotides coding for 335 amino acids. Motifs found in CYP and CYP family 4 genes have been underlined.

Table 2Estimated evolutionary divergence between CYP sequences.

Figure 2. Molecular phylogenetic analysis conducted in MEGA6［17］ using the Maximum Likelihood method based on the Tamura-Nei model［16］.

The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree（s） for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood （MCL） approach， and then selecting the topology with superior log likelihood value. The tree is drawn to scale， with branch lengths measured in the number of substitutions per site. The analysis involved 10 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 1 380 positions in the final dataset. Species name and GenBank accession number of the CYPs are as follows； CYP4H28 （Ae. aegypti，XM_001656716）， CYP4H28v2 （Ae. aegypti， KC481237）， CYP4H28v3 （Ae. aegypti， KF779931）， CYP4H30 （Ae. aegypti， XM_001656715）， CYP4H34（Cx. quinquefasciatus， JQ001927）， CYP4H42v1 （Ae. albopictus， KF029763），CYP4H43 （Ae. albopictus， KF029765）， CYP4D4v2 （Musca domestica，EF615001）， CYP9J26 （Ae. aegypti， XM_001649047）.

The nt sequence of CYP4H44P is 99% identical with CYP4H28［Query cover （QC）=93%］ and CYP4H28v2 （QC=99%）. The CYP4H44P aa sequence also has an identity of 99% with CYP4H28（QC=100%） and CYP4H28v2 （QC=100%）.

3.2. Evolutionary relationship

The phylogenetic tree （Figure 2） showed the relationship between the pseudogene and other family 4 CYP genes with CYP9J26 as outgroup. The closest evolutionary relationship of the pseudogene was with the CYP4H28 alleles， clustering with a bootstrapping support of 100%. CYP4H44P and the CYP4H28 group were closely related to CYP4H34 of Culex quinquefasciatus （Cx. quinquefasciatus）（99% bootstrap value） than the cluster of CYP4H42v1 and CYP4H43 of Aedes albopictus （Ae. albopictus） and CYP4H30 of Cx. quinquefasciatus （73% bootstrap value）. The computed estimated evolutionary divergence of CYP4H44P from the other sequences indicated that it was least diverged from CYP4H28 and CYP4H28v2（Table 2）.

3.3. Bioinformatics analysis

Figure 3. Predicted features in CYP4H44P-deduced protein.（A） Signal peptide； （B） Transmembrane helix； （C） Phosphorylated sites； （D） O-glycosylated sites.

PSORT Ⅱ software predicted that the CYP4H44P-deduced protein had the following probabilities of localisation； 60.9% （cytoplasmic），17.4% （nuclear）， 13.0% （mitochondrial）， 4.3% （vacuolar） and 4.3%（vesicles of secretory system）. Signal peptide， transmembrane helix and O-glycosylated sites were not predicted in the deduced protein （Figure 3）. However， sixteen （16） phosphorylated sites were predicted in the deduced protein （Figure 3）. The predicted secondary structure was made up of alpha helices （35.52%）， randomcoils （47.46%） and extended strands （17.01%）. The 3D structure was modelled using CYP3A4 （PDB ID: 4D6Z） as template. This template was a human CYP and had 34.74% sequence identity with CYP4H44P. Ramachandran plot analysis of the model showed that 85.2， 12.0， 2.1 and 0.7% of the residues were in the most favoured，additional allowed， generously allowed and disallowed regions respectively （Figure 4）. Validation of the model using the ProSA z-score indicated a model of good quality with a value of -5.81（Figure 4）.

4. Discussion

This study isolated a processed CYP pseudogene， CYP4H44P， from Ae. aegypti larvae. The CYP4H44P sequence has been deposited at the GenBank database with the accession number KF779932. The translated sequence has several CYP motifs including the signature haem-binding motif FxxGxxxCxG （F275SVGARNCIG284）［1，20］ but lacks the WxxxR motif due to its truncated N-terminal region. The WxxxR motif is presumed to interact with the propionate of haem to form a charge pair［1］. However， translation of the 5'-Untranslated Region shows that nt sequences from the 315-329th position code for this motif but this is upstream of the start codon （A450TG452）in this sequence. CYP4H44P is highly identical to CYP4H28 and CYP4H28v2 but has a shorter aa sequence in the coding region than the other functional products due to its truncated 5' region. Truncation and structural loss in CYP4H44P might have led to its classification as a pseudogene.

Phylogenetic and evolutionary divergence analyses indicated that CYP4H44P is closely related to the CYP4H28 group. Genes such as CYP4H28 and CYP4H28v2 are induced by xenobiotics［10，21］ so the close evolutionary relationship between these and CYP4H44P suggests that CYP4H44P could have possessed a similar trait if it was a functional gene. The high sequence identity and evolutionary relatedness of CYP4H44P to the functional CYP4H28 alleles imply that divergence of the pseudogene is likely to be recent［22］. Five CYP pseudogenes have been identified in the Ae. aegypti genome but none belongs to the family 4 CYP group［3］. To the authors' best knowledge， this is the first family 4 CYP pseudogene from Ae. aegypti. The family 4 CYP is a functionally diverse group with a broad range of functions［1］ and although CYP4H44P may presumptively be enzymatically non-functional， it will be interesting to determine its biological significance since biological functions have been reported for some pseudogenes［7］.

The predicted localisation of the pseudogene-deduced protein to the cytoplasm is similar to the predicted cytosolic localisation of pseudogene-deduced proteins in Shidhi et al.［9］. The protein was also predicted to have several phosphorylated sites. Phosphorylation of functional CYPs regulates protein activity［23］； hence，phosphorylation may help to modulate activity of the pseudogenededuced protein if it was synthesised. The fold of the 3D model is akin to the conserved fold of CYPs［20］. The model has 0.7% of its residues in the disallowed regions， which is comparable to predicted CYP models of Tribolium castaneum， which had between 1.1% and 2.2% of residues in the disallowed regions［24］. Quality assessment using validation tools indicated that the model was of good quality and compares well with predicted models of proteins deduced from pseudogenes［9］.With the predicted possibility of synthesising functional proteins from pseudogenes［9］， the predicted characterisation of the pseudogene in this study offers useful insight in this regard.

Figure 4. 3D model and validation results of CYP4H44P-deduced protein.（A） 3D model with labelled secondary structures； （B） Ramachandran plot analysis； （C） ProSA z-score.

In conclusion， a processed CYP pseudogene （CYP4H44P） was isolated from Ae. aegypti. The pseudogene is evolutionary related with CYP4H28 alleles and appears to have recently diverged fromthis group. Bioinformatics tools were used to characterise the deduced protein and the predicted 3D model indicates that it has the conformational fold of CYPs. Since pseudogenes may not be functionally defunct as previously thought， identification of this pseudogene provides a platform for investigating its functional role in the mosquito.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

This work was funded by the Malaysian Ministry of Education（ERGS 203/PPJAUH/6730097） and Universiti Sains Malaysia （RU 1001/PPJAUH/815095）.

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13 May 2016

Fatma M. A. El-garj， Molecular Entomology Research Group， School of Distance Education， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia. Tel: +60174074032

E-mail: fatmagorj@yahoo.com

Molecular Entomology Research Group， School of Distance Education， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia.

Tel: +60142401941

E-mail: wintuma@live.com； swavicor@usm.my

Foundation project: This work was funded by the Malaysian Ministry of Education（ERGS 203/PJJAUH/6730097） and Universiti Sains Malaysia （RU grant 1001/ PJJAUH/815095）.