(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Metazoa: NE > Eumetazoa: NE > Bilateria: NE > Protostomia: NE > Ecdysozoa: NE > Panarthropoda: NE > Arthropoda: NE > Mandibulata: NE > Pancrustacea: NE > Hexapoda: NE > Insecta: NE > Dicondylia: NE > Pterygota: NE > Neoptera: NE > Holometabola: NE > Diptera: NE > Nematocera: NE > Psychodomorpha: NE > Psychodoidea: NE > Psychodidae: NE > Phlebotominae: NE > Phlebotomus [genus]: NE > Phlebotomus [subgenus]: NE > Phlebotomus papatasi: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MEIRGIVVTTMRPFTGIHSGVDQMFVICLLSLLGVMCQLAEGRHHDLSNT QSFKSGPKHIASVEAAAVSVLGESTLEVSSESDDTIFTPYLGHGDAVRVV DAELGTLEREGVSAGSDGTSQPRRRNISRRESNPDAEENDPLIVTTDKGK VRGVTLTSPTGKKVDAWMGIPYAQPPVGALRFRHPRPAERWSGILNATTP PNTCVQIVDTLFGDFPGATMWNPNTNLTEDCLYINVAVPHPRPKNSPVML WIFGGGFYSGTSTLDVYDHRTLVAEENIILVSMQYRVASLGFLYLGTPDA PGNAGLFDQHLALRWVRNNIHRFGGDPTRVTLFGESAGAVSVSMHLLSSL SHDLFQRAILQSGSPTAPWALITRDEAINRTLRLAEAVECPHNRDELSEV LECLRSRDAKQLVNNEWNNLGICEFPFVPVVDGSFLDESPQRAMATGRFK KTDILTGSNTEEGYYFIIYYLTELLRKEEGITVTREEFLKAVRELNPYVN GAVRQAIVFEYTDWTDPDNAHSNRDALDKMVGDYHFTCNVNEFAHRYAEE GNNVYMYLYTHRTKANPWPRWTGVMHGDEINYVFGEPLNPSLTYTDEEKE FSRRIMRYWVNFAKTGNPNPGFVSNLPDWPKHTAHGRQYMELGLNTTYLG RGPRLRQCAFWKKYLPQLMAATIENSSTKNCTNVGNQFVRNPNFSIPTTL LVILGILSVN
Background Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation.MethodsTargeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi.ResultsRecombinant PpAChE1 containing the G119 substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC).ConclusionsThe finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC inverted question mark AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.
Title: Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties Temeyer KB, Brake DK, Tuckow AP, Li AY, Perez de Leon AA Ref: Parasit Vectors, 6:31, 2013 : PubMed
ABSTRACT: BACKGROUND: Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. METHODS: A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. RESULTS: A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis.Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. CONCLUSIONS: Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.
