Abstract
Background: Acetylcholinesterase (AChE) is an important neurotransmitter hydrolase in invertebrate and vertebrate nervous systems. The number of AChEs is various among invertebrate species, with different functions including the ‘classical’ role in terminating synaptic transmission and other ‘non-classical’ roles. Methods: Using rapid amplification of cDNA ends (RACE) technology, a new putative AChE-encoding gene was cloned from Pardosa pseudoannulata, an important predatory natural enemy. Sequence analysis and in vitro expression were employed to determine the structural features and biochemical properties of this putative AChE. Results: The cloned AChE contained the most conserved motifs of AChEs family and was clearly clustered with Arachnida AChEs. Determination of biochemical properties revealed that the recombinant enzyme had the obvious preference for the substrate ATC (acetylthiocholine iodide) versus BTC (butyrylthiocholine iodide). The AChE was highly sensitive to AChE-specific inhibitor BW284C51, but not butyrylcholinesterase-specific inhibitor tetraisopropyl pyrophosphoramide (ISO-OMPA). Based on these results, we concluded that a new AChE was identified from P. pseudoannulata and denoted as PpAChE5. Conclusion: Here we report the identification of a new AChE from P. pseudoannulata and increased the AChE number to five in this species. Although PpAChE5 had the biggest Vmax value among five identified AChEs, it showed relatively low affinity with ATC. Similar sensitivity to test insecticides indicated that this AChE might serve as the target for both organophosphorus and carbamate insecticides.
Highlights
As a dominating neurotransmitter hydrolase, acetylcholinesterases (AChEs) extensively exist in invertebrate and vertebrate nervous systems, and effectively hydrolyse acetylcholine to maintain the normal nerve conduction
In addition to four AChEs (PpAChE1-4) we identified from P. pseudoannulata, one new putative ace gene was found in P. pseudoannulata transcriptome and was confirmed by polymerase chain reaction
Phylogenetic tree of PpAChE5 with PpAChE1-4 and AChEs from other species was constructed, and it clearly showed that PpAChE5 has a relatively close evolutionary relationship with Arachnida
Summary
As a dominating neurotransmitter hydrolase, acetylcholinesterases (AChEs) extensively exist in invertebrate and vertebrate nervous systems, and effectively hydrolyse acetylcholine to maintain the normal nerve conduction. AChEs may have multiple non-cholinergic functions in organisms, and different AChEs in one species may have various physiological functions [9]. The generation of distinct multiple AChE isoforms may occur via ace gene duplications and alternative splicing, and different structural and functional. Acetylcholinesterase (AChE) is an important neurotransmitter hydrolase in invertebrate and vertebrate nervous systems. The number of AChEs is various among invertebrate species, with different functions including the ‘classical’ role in terminating synaptic transmission and other ‘non-classical’ roles. Methods: Using rapid amplification of cDNA ends (RACE) technology, a new putative AChE-encoding gene was cloned from Pardosa pseudoannulata, an important predatory natural enemy. The AChE was highly sensitive to AChE-specific inhibitor BW284C51, but not butyrylcholinesterase-specific inhibitor tetraisopropyl pyrophosphoramide (ISO-OMPA)
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