title: Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant.
abstract: 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.
  
  
  METHODS
  Targeted 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.
  
  
  RESULTS
  Recombinant PpAChE1 containing the G119S 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).
  
  
  CONCLUSIONS
  The finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → 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.
authors.affiliation: Agricultural Research Service, U. S. Department of Agriculture, Knipling-Bushland U.S. Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX, 78028-9184, USA. kevin.temeyer@ars.usda.gov.
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Temeyer
authors.affiliation: Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, 2055 Mowry Road, PO Box 100009, Gainesville, FL, 32610-00009, USA. tongf@epi.ufl.edu.
authors.forename: Fan
authors.initials: F
authors.lastname: Tong
authors.affiliation: Molsoft LLC, 3366 North Torrey Pines Court, Suite 300, La Jolla, CA, 92037, USA. max@molsoft.com.
authors.forename: Maxim M
authors.initials: MM
authors.lastname: Totrov
authors.affiliation: Agricultural Research Service, U. S. Department of Agriculture, Knipling-Bushland U.S. Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX, 78028-9184, USA. alex.tuckow@ars.usda.gov.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Tuckow
authors.affiliation: Department of Chemistry, Virginia Tech, 900 West Campus Drive, 480 Davidson Hall, Blacksburg, VA, 24061-0001, USA. qchen@csufresno.edu.
authors.forename: Qiao-hong
authors.initials: QH
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Virginia Tech, 900 West Campus Drive, 480 Davidson Hall, Blacksburg, VA, 24061-0001, USA. pcarlier@vt.edu.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Carlier
authors.affiliation: Agricultural Research Service, U. S. Department of Agriculture, Knipling-Bushland U.S. Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX, 78028-9184, USA. Beto.perezdeleon@ars.usda.gov.
authors.forename: Adalberto A
authors.initials: AA
authors.lastname: Pérez de León
authors.affiliation: Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, 2055 Mowry Road, PO Box 100009, Gainesville, FL, 32610-00009, USA. jbquist@epi.ufl.edu.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Bloomquist
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002800:Cholinesterase Inhibitors / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010691:Phlebotomus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D002800:Cholinesterase Inhibitors; D019476:Insect Proteins; D000110:Acetylcholinesterase