Genome Organization, Phylogenies, Expression Patterns, and Three-Dimensional Protein Models of Two Acetylcholinesterase Genes from the Red Flour Beetle

Abstract

Since the report of a paralogous acetylcholinesterase (AChE, EC3.1.1.7) gene in the greenbug (Schizaphis graminum) in 2002, two different AChE genes (Ace1 and Ace2) have been identified in each of at least 27 insect species. However, the gene models of Ace1 and Ace2, and their molecular properties have not yet been comprehensively analyzed in any insect species. In this study, we sequenced the full-length cDNAs, computationally predicted the corresponding three-dimensional protein models, and profiled developmental stage and tissue-specific expression patterns of two Ace genes from the red flour beetle (Tribolium castaneum; TcAce1 and TcAce2), a globally distributed major pest of stored grain products and an emerging model organism. TcAce1 and TcAce2 encode 648 and 604 amino acid residues, respectively, and have conserved motifs including a choline-binding site, a catalytic triad, and an acyl pocket. Phylogenetic analysis show that both TcAce genes are grouped into two insect Ace clusters and TcAce1 is completely diverged from TcAce2, suggesting that these two genes evolve from their corresponding Ace gene lineages in insect species. In addition, TcAce1 is located on chromosome 5, whereas TcAce2 is located on chromosome 2. Reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR analyses indicate that both genes are virtually transcribed in all the developmental stages and predominately expressed in the insect brain. Our computational analyses suggest that the TcAce1 protein is a robust acetylcholine (ACh) hydrolase and has susceptibility to sulfhydryl agents whereas the TcAce2 protein is not a catalytically efficient ACh hydrolase.

Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum

“…These results clearly indicate that AChE1 is essential for insect survival, presumably by regulating cholinergic neurotransmission, whereas AChE2 does not seem to play a major role in the neurotransmission in T. castaneum . The respective cholinergic and non-cholinergic functions of AChE1 and AChE2 are consistent with our large-scale protein simulation studies suggesting that T. castaneum AChE1 is a robust ACh hydrolase, whereas T. castaneum AChE2 is not a catalytically efficient ACh hydrolase, because the entrance of the active site of the AChE2 model refined by multiple molecular dynamics simulations appeared to be reversed relative to that of the corresponding AChE1 model and because ACh does not adopt the fully extended conformation and its carbonyl oxygen atom is not placed in the oxyanion hole in the AChE2 model15.…”

“…castaneum 15. Significant differences of biochemical properties between the two Ace products heterologously expressed in the baculovirous-infected cells have also been reported in the African malaria mosquito ( Anopheles gambiae )22, the wild silkworm ( Bombyx mandarina )23, and C. felis 21.…”

“…In view of the cholinergic and non-cholinergic functions of two AChEs in T. castaneum , we suggest that any insect-specific anticholinesterase insecticides designed for insect pest control should target the AChE encoded by Ace1 rather than Ace2 unless the insect species possessing only Ace2 . This notion is promoted by our findings that AChE1 in T. castaneum and many other insect species possesses a cysteine residue at the opening of the AChE active site but this cysteine residue is absent in fish and mammalian AChEs and insect AChE215273738. This insect AChE1-specific cysteine residue allows design of new chemicals that irreversibly inhibit AChE1 for insect pest control by conjugation of the chemicals to the cysteine residue27.…”

“…However, the T. castaneum AChE1 gene ( TcAce1 ) resides on chromosome 5, whereas the T. castaneum AChE2 gene ( TcAce2 ) is on chromosome 2. Our large-scale protein simulation studies suggest that T. castaneum AChE1 is a robust acetylcholine hydrolase, whereas AChE2 is not a catalytically efficient acetylcholine hydrolase15.…”

Expression of acetylcholinesterase 1 is associated with brood rearing status in the honey bee, Apis mellifera

“…From an evolutionary perspective, however, the physiological function of AmAChE1 does not seem to be limited to defense. In Tribolium castaneum , a minor catalytic enzyme (AChE2) has been revealed to play a non-cholinergic role in female reproduction, embryo development and offspring growth2122, suggesting that non-neuronal AChE is likely involved in various physiological functions.…”