mutation in 336 found in sitav-ACHE1 and 131 in pluxy-ACHE1, likely to be important. The four mutations are present in OP-resistant strain Tuxpan Tx11(R). Other mutations E55G, E60K, P78T, T219A, E238G, A260T, N333S, A349V, T362A, K363R, M426V, T437A, Q488R, I493T, R549H, E552Q, W571F, T576A, N583D where found in strains Deutch5(S), SR4(R,) SR11(R)or Tx11(R) but were present either in both resistant and sensitive strains or unrelated to paraoxon insensitivity
Kinetic parameters
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References:
Title: Baculoviral Expression of Presumptive OP-Resistance Mutations in BmAChE1 of Rhipicephalus (Boophilus) microplus (Ixodida: Ixodidae) and Biochemical Resistance to OP Inhibition Temeyer KB, Schlechte KG, McDonough WP Ref: Journal of Medical Entomology, 56:1318, 2019 : PubMed
The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), transmits bovine babesiosis and anaplasmosis, and is endemic to Mexico, Latin and South America. Rhipicephalus (B.) microplus infestations within the United States are a continuing threat to U.S. cattle producers. An importation barrier between Texas and Mexico keeps the ticks from re-entering the United States. All cattle imported into the United States are dipped in an organophosphate (OP) acaricide and hand inspected for presence of ticks. Tick resistance has developed to most available acaricides, including coumaphos, the OP used in the cattle dip vats. OP-resistance can result from one or more mutations in the gene encoding the enzyme, acetylcholinesterase (AChE), resulting in production of an altered AChE resistant to OP inhibition. Previous research reported a large number of BmAChE1 mutations associated with OP resistance. We report baculovirus expression of recombinant tick BmAChE1 (rBmAChE) enzymes containing a single resistance-associated mutation, to assess their contribution to OP inhibition resistance. Surprisingly, of the naturally occurring BmAChE1 resistance-associated mutations, only D188G resulted in markedly reduced sensitivity to OP-inhibition suggesting that OP-insensitivity in BmAChE1 may result from the D188G mutation, or may possibly result from multiple mutations, each contributing a small decrease in OP sensitivity. Furthermore, an OP-insensitivity mutation (G119S) found in mosquitoes was expressed in rBmAChE1, resulting in 500-2000-fold decreased sensitivity to OP inhibition. Recombinant BmAChE1 with the G119S mutation demonstrated the lack of any structural prohibition to broad and high-level OP-insensitivity, suggesting potential increases in tick OP-resistance that would threaten the U.S. importation barrier to ticks.
The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 muM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control.
Title: Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus Temeyer KB, Pruett JH, Olafson PU Ref: Vet Parasitol, 172:114, 2010 : PubMed
Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3, were previously identified as presumptively encoding acetylcholinesterases (AChEs), but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus. Biochemical characterization of the recombinant proteins supports classification of rBmAChE1, rBmAChE2, and rBmAChE3 as AChEs (E.C.3.1.1.7), as evidenced by (i) substrate preference for acetylthiocholine, (ii) inhibition by eserine, BW284c51, and the organophosphates (OPs) malaoxon and paraoxon, (iii) insensitivity to iso-OMPA, and (iv) rapid hydrolysis of acetyl-beta-methyl-thiocholine. Unlike reports for insect AChEs, we did not observe substrate inhibition of activity at acetylthiocholine concentrations as high as 40 mM, however, product inhibition was apparent at 10-100 microM choline in agreement with properties reported for the catalytic domain of Anopheles gambiae acetylcholinesterase-1. Substrate affinity and V(max) values were highest for rBmAChE1 proteins, and one rBmAChE1 enzyme (Tx11, derived from the OP-resistant strain Tuxpan), was insensitive to paraoxon and exhibited a greatly reduced V(max) near that of rBmAChE2. To date, recombinant BmAChE1 and BmAChE3 enzymes with reduced sensitivity to OP-inhibition have been cloned and expressed from OP-resistant strains. The presence of at least three genes expressing AChEs in R. (B.) microplus, at least two of which contain mutations expressed as OP-insensitive enzymes, strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character.
