Baculovirus Expression, Biochemical Characterization and Organophosphate Sensitivity of RBmAChE1, RBmAChE2, and RBmAChE3 of Rhipicephalus (Boophilus) Microplus

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.