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 secreted by the nematode parasite Nippostrongylus brasiliensis. The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic 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 active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, 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 invertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2. 5 mM and was highly sensitive to both active site and "peripheral" site inhibitors. Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection.
Hussein
A S
AS
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom.
Chacón
M R
MR
Smith
A M
AM
Tosado-Acevedo
R
R
Selkirk
M E
ME
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D007421:Intestine, Small / Q000469:parasitology; D008297:Male; D008969:Molecular Sequence Data; D009559:Nippostrongylus / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D017206:Strongylida Infections / Q000201:enzymology / Q000469:parasitology; D013379:Substrate Specificity; D014101:Torpedo
D002800:Cholinesterase Inhibitors; D018076:DNA, Complementary; D000110:Acetylcholinesterase