Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis

We have isolated a full-length cDNA encoding an acetylcholinesterase secret ed by the nematode parasite Nippostrongylus brasiliensis. The predicted pro tein is truncated in comparison with acetylcholinesterases from other organ isms such that the carboxyl terminus aligns closely to the end of the catal ytic domain of the vertebrate enzymes. The residues in the catalytic triad are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic residues which line the ac tive site gorge in the Torpedo enzyme are substituted by nonaromatic residu es, corresponding to Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met), High level expression was obtained via secretion from Pichia pastoris. The purified enzyme behaved as a monomeric hydrophilic species. Although of inv ertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and sho wed minimal activity against butyrylthiocholine, It displayed excess substr ate inhibition with acetylthiocholine at concentrations over 2.5 mM and was highly sensitive to both active site and "peripheral" site inhibitors, Nor thern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection.