두 곤충 아세틸콜린에스터레이즈의 진화적 기원과 양상 및 구조적 보전과 분화 분석

Abstract

Acetylcholinesterase (AChE) plays a pivotal role in the synaptic transmission in the cholinergic nervous system of most animals, including insects. Insects possess two distinct acetylcholinesterases (AChE1 vs. AChE2), which are encoded by two paralogous loci originated from the duplication that occurred long before the radiation of insects. In this study, phylogenetic analysis and structural modeling were performed to understand when the ace duplication occurred and what structural features have been associated with the differentiation of two AChEs during evolution. The phylogenetic analysis was conducted for the AChE-like genes from all known lower animals with their genome sequenced together with all known arthropod ace1 and ace2 orthologs, including those from a number of insects that were newly cloned in this study. The result suggested that the last common ancestor of ace1 and ace2 shared its origin with those of platyhelminthes, which further implies that the lineage of arthropod ace1 and ace2 has undergone a divergent evolution along with those of platyhelminthes. In addition, it appears that the ace duplication event resulting in the split of the ace1 and ace2 clades occurred after the divergence of Ecdysozoa and Lophotrochozoa from their Protostomian common ancestor but before the split of Ecdysozoa into their descendents. The ace1 lineage showed a significantly lower evolutionary rate (d, or distance) and higher purifying selection pressure (dN/dS) compared to ace2 lineage, suggesting that the ace1 lineage has maintained relatively more essential functions following duplication. Given that the putative functional transition of ace in some Hymenopteran could not be explained by such difference in evolutionary rate, this event appears to have occurred in relatively recent time by only a few numbers of mutations resulting in dramatic alteration of AChE function. The amino acid sequence comparison between AChE1 and AChE2 from a wide variety of insect taxa revealed a high degree of sequence conservancy in the functionally crucial domains, suggesting that presence of strong purifying selection pressure over these essential residues. Interestingly, the EF-hand motif was mostly found in the AChE1 lineage but not in AChE2. In contrast, LRE-motif was partially conserved in the AChE2 lineage but not in AChE1. In addition, the AChE2-specific insertion domain appeared to have been introduced relatively more recently, perhaps during the radiation of insects, after the duplication. The comparison of five essential domains [i.e., the catalytic anionic site (CAS), peripheral anionic site (PAS), acyl binding pocket (ABP), oxyanion hole and catalytic triad] in the active-site gorge showed unique differences in amino acid residues of the PAS (Asp72 vs. Tyr72, Tyr121 vs. Met121

amino acid numbering of Torpedo californica AChE, hereafter) and the ABP (Cys288 vs. Leu288) between AChE1 and AChE2. Three-dimensional modeling of active-site gorge from insect AChEs with a particular focus on the PAS revealed that a subtle but consistent structural alteration in the active-site gorge topology was caused by the PAS amino acid substitution, likely resulting in a remarkable functional differentiation between two AChEs. Although ace1 appears to have evolved at significantly slower rates to retain its essential function, it is likely that a few specific amino acid substitutions in ace1 causing a dramatic reduction of enzyme activity may have occurred locally in more recent time and resulted in the functional transition from AChE1 to AChE2 as observed in some Hymenopteran insects.