Determinants of substrate specificity of a second non‐neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis

Abstract

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met).A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/ W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the`peripheral site' inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.

Parasitic nematodes—From genomes to control

“…Mining of this information can reveal candidate drug targets with characterized pathways that differ from the host having particular interest. Certain nematode protein sequences can be modeled, based on knowledge of structure and function from homologous proteins, to predict drug susceptibility or resistance (Geary et al, 1998;Hussein et al, 2000;Perbandt et al, 2005) or suggest routes to the design of protein inhibitors or activators (Ring et al, 1993). Nematodespecific proteins lacking known functions represent attractive avenues for research, although the utility of the proteins as targets for control requires determination.…”

High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification

“…in biosensor applications, a purified enzyme is highly advantageous. Up to now, usually two different methods are used for the purification of AChEs: affinity chromatography Heim et al, 1998;Sussmann et al, 1988;Villatte et al, 1998Villatte et al, , 2000 based on the fusion of a His 6 -Tag to the enzyme Heim et al, 1998;Hussein et al, 1999Hussein et al, , 2000 or the chemical modification of the support with reversible AChE inhibitors (Massoulie and Bon, 1976;Ralston et al, 1983) or ion exchange chromatography Heim et al, 1998).…”

“…AChE is counted among the fastest enzymes known up to now (Nair et al, 1994). The parasitic nematode Nippostrongylus brasiliensis, which colonizes the rat intestine, secretes three monomeric, hydrophilic AChEs encoded by separate genes (Hussein et al, 2000). Among them, the 69 kDa NbAChE B variant was used in this study.…”

Design of acetylcholinesterases for biosensor applications

“…Two approaches are usually applied for the purification of the enzymes: ion exchange chromatography (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998) and affinity chromatography (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998;Villatte et al, 1998;Villatte et al, 2000) based on the chemical modification of the support with reversible AChE inhibitors (Massoulie and Bon, 1976;Ralston et al, 1983) or on the addition of a His-tag to the enzyme (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998;Hussein et al, 1999;Hussein et al, 2000).…”