cDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus

Abstract

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H-type catalytic subunits, which are inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl-binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl-binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS-7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G 2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine-glycan-phosphatidylinositol-anchored G 2 forms in vivo. ChE1 also possesses an ethanolamine-glycan-phosphatidylinositol-anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.

Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: the roles of two cysteines in the catalytic gorge of the enzyme

“…is the closest living invertebrate relative of the vertebrates (Satoh et al 2001). Previously, we found that amphioxus (Branchiostoma lanceolatum and Branchiostoma floridae) possesses two AChEs, which we call ChE1 and ChE2 to distinguish them from the AChE of the vertebrates (Sutherland et al 1997;McClellan et al 1998;Pezzementi et al 1998). Interestingly, ChE2 has two free cysteines in the catalytic gorge: one in the vicinity of the acyl pocket, Cys310, which is homologous to the free cysteine in aphids and mosquitoes; and another, Cys466, which is adjacent to the choline-binding site.…”

Molecular characterization of an acetylcholinesterase from the hemichordate Saccoglossus kowalevskii

“…predicted protein with 25% identity and 31% similarity to ColQ from T. marmorata (NCBI Reference Sequence: XP_798050.2; Tu et al, 2012). Likewise, there is also no evidence for the presence of asymmetric forms of ChEs in acraniate deuterostomes or in protostomes, with the possible exception of the cephalochordate B. floridae (McClellan et al, 1998). However, it has recently been found that soluble ChEs are evidently assembled by PRAD fragments derived from a variety of proline-rich intracellular proteins (Li et al, 2008;Biberoglu et al, 2012Biberoglu et al, , 2013, apparently via WAT-PRAD interactions (Larson et al, 2014).…”

“…In amphioxus (Branchiostoma floridae), a cephalochordate, six putative ChE genes are present (Johnson and Moore, 2012a). cDNAs for two of these ChEs have been characterized: ChE1 is an atypical ChE, and ChE2 resembles the AChE of protostomes (Sutherland et al, 1997;McClellan et al, 1998;Pezzementi et al, 2003), the other major lineage of bilaterian animals (Lowe, 2008). A number of the other putative amphioxus ChEs genes have sequence characteristics typical of craniate AChE.…”

Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: A reconsideration of the implications for insecticide design

“…has two AChEs, cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) (McClellan et al, 1998). ChE2 has two free cysteines in the catalytic gorge: one in the acyl pocket near the top of the catalytic gorge, Cys310, homologous to the free cysteine in aphids and mosquitoes; and another, Cys466, adjacent to the choline-binding site near the bottom of the gorge.…”