Abstract
We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC50 values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense.
Highlights
Acetylcholinesterase (AChE, EC 3.1.1.7) is a critical enzyme in the cholinergic synapses and neuromuscular junctions of both vertebrates and invertebrates that regulates the level of the neurotransmitter acetylcholine and terminates nerve impulses [1]
Recent studies have shown that two different ace loci have been cloned from various insect species, such as the cotton aphid Aphis gossypii [10], the greenbug Schizaphis graminum [11], the diamondback moth Plutella xylostella [12] and the German cockroach B. germanica [13], while only one type of AChE has been discovered in cyclorrhaphan flies [14], including Drosophila melanogaster [15] and Musca domestica [16]
Expression Patterns of AmAChEs in Various Tissues To determine the tissue-specific expression profiles of two AmAChEs, native-polyacrylamide gel electrophoresis (PAGE) was performed on proteins extracted from six tissues of forager bees, and their AChE activities were visualized by activity staining (Fig. 1B)
Summary
Acetylcholinesterase (AChE, EC 3.1.1.7) is a critical enzyme in the cholinergic synapses and neuromuscular junctions of both vertebrates and invertebrates that regulates the level of the neurotransmitter acetylcholine and terminates nerve impulses [1]. Studies on the evolution of ChE, including AChE and BChE, suggest that true ChEs, with highly selective substrate specificity, appear in the early bilaterians [9] Genes for both AChE and BChE are usually present in most lineages of vertebrates, whereas duplications of the ace gene encoding AChE are observed in a few lineages such as nematodes, arachnids and insects. Insects that are resistant to OP and CB insecticides possess point mutations in the ace gene that are responsible for target site insensitivity [20,21]. Based on these findings, it was proposed that AChE1 is likely the major AChE involved in synaptic transmission in insects possessing both AChE1 and AChE2 [19,22,23,24]
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