Molecular and Kinetic Properties of Two Acetylcholinesterases from the Western Honey Bee, Apis mellifera

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.

RNA interference: Applications and advances in insect toxicology and insect pest management

“…These findings strongly support that AChE1 plays a major role in cholinergic functions, and is the major target of anticholinesterase insecticides. However, more recent studies revealed that some insects express AChE2 as the major catalytic enzyme, rather than AChE1 [28,37,38]. Although the ability of AChE2 to function as a major catalytic enzyme does not necessarily indicate either its major function in cholinergic neurotransmission or as a major target for OP and CB insecticides, it would be interesting to investigate if AChE2 is a major enzyme responsible for cholinergic neurotransmission and serves as a major target for these insecticides by RNAi.…”

Validating the importance of two acetylcholinesterases in insecticide sensitivities by RNAi in Pardosa pseudoannulata, an important predatory enemy against several insect pests

“…However, ace2 showed relative more high expression than ace1 in some insects, e.g. Bombyx mori [4] and was confirmed as the major catalytic enzyme in synaptic transmission in Apis mellifera [5]. In our previous studies, two ace genes (Ppace1 and Ppace2) were cloned from the pond wolf spider Pardosa pseudoannulata, an important predatory enemy against several insect pests in most parts of Asia and showed different sensitivities to organophosphate and carbamate insecticides, when compared to some insect pests, such as the rice pest Nilaparvata lugens [6].…”

The Activity of Cholinesterases in Diapausing and Flying Red Mason Bees <I>Osmia bicornis</I> (Megachilidae)

“…Some authors have ruled out the presence of BChE in insects (see KIM et al 2012), whereas others measured its activity (DE VILLAR et al 1980;KULIEVA et al 1995). Recent research has validated the hypothesis proposed by GNAGEY et al (1987) who argued that insects possess AChE with substrate specificity that occupies the mid range between vertebrate AChE and BChE.…”

“…The above could be attributed to the fact that AChEs in insects are coded by two genes, ace 1 and ace 2, whose products, enzymes AChE1 and AChE2, have different functions (LI & HaN 2002;KIM & LEE 2013;XIAO et al 2014). KIM et al (2012) demonstrated that AChE2 is the key neurotransmission enzyme in honey bees. AChE1 probably has nonneuronal functions and protects bees against the harmful effects of xenobiotics, therefore, it plays a similar role to vertebrate BChE.…”

“…In many insects, AChE1 is characterized by much higher expression and activity in CNS, and is considered the major cholinesterase responsible for neurotransmission; AChE2 has lower activity and is less sensitive to xenobiotics (LI & HAN 2002;KIM & LEE 2013;XIAO et al 2014). A reverse role for both enzymes was observed in Apis mellifera by KIM et al (2012).…”