We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasiteNippostrongylus 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.  Acetylcholine (ACh)1 is the major excitatory neurotransmitter which regulates motor functions in the nematode Caenorhabditis elegans (1), and cholinergic motor neurons have been identified in both free-living (1, 2) and parasitic (3, 4) nematode species. In contrast to vertebrates (5) and insects (6), C. elegans possesses multiple genes which encode acetylcholinesterases (AChEs). Genetic studies originally defined three separate genes, ace-1, ace-2, andace-3 (7-9) which encoded different catalytic forms of AChE (10), although recent data resulting from cDNA cloning indicate that C. elegans possesses at least four separate genes for AChE (11).  In addition to possessing cholinergic motor neurons, many parasitic nematodes secrete AChE into the culture medium when maintained in vitro (12-14). A nonneuronal origin for these enzymes has been confirmed by enzyme- and immunohistochemistry, which demonstrated that they are synthesized by and secreted from specialized amphidial and secretory glands (13, 15-17). This remarkable phenomenon is exhibited predominantly by nematodes which inhabit the gastrointestinal tract of their vertebrate hosts and has provoked much discussion on the putative physiological role of these enzymes (18, 19).  Analysis of the AChE secreted by the human hookworm Necator americanus indicated that the enzyme existed as a nonamphiphilic dimer (G2 na form) (20), whereas both G1 na and G2 na forms were identified in secreted products of Trichostrongylus colubriformis, a nematode parasite of sheep (21). We are utilizingNippostrongylus brasiliensis, the adult stages of which colonize the jejunum of rats, as a model system in which to study AChE secretion and have recently purified and characterized three variants of secreted AChE (22). These variants (designated A, B, and C) are all G1 na forms, with apparent masses of 74, 69, and 71 kDa, respectively, when resolved by SDS-polyacrylamide gel electrophoresis. Substrate and inhibitor specificities defined them as true AChEs rather than pseudocholinesterases. Although they show broadly similar enzymatic properties, they can be distinguished by subtle differences in molecular mass, inhibitor profiles, and excess substrate inhibition and display distinct and characteristic mobilities on nondenaturating PAGE (22).  These forms are differentially expressed by N. brasiliensisduring residence in the small intestine of the rat. Thus, parasites which have entered the duodenum from the stomach by three days postinfection exclusively secrete isoform A. Following moulting to the adult stage and migration to a more distal position in the jejunum however, a switch to expression of forms B and C occurs, with form B progressively becoming the most abundant secreted enzyme (22-24). In this report, we have derived amino-terminal amino acid sequence of form B and utilized this to isolate a full-length cDNA clone. High level expression of a recombinant form of the enzyme was obtained via secretion from the methylotrophic yeast Pichia pastoris. The unusual primary structure of the enzyme is discussed in relation to the enzymatic properties of the recombinant enzyme and to the structure and properties of AChEs from other organisms.

العنوان

The Journal of Biological Chemistry, 274, 9312-9319, 1999 Apr 2

الناشر

--

بلد الناشر

فلسطين

نوع المنشور

Both (Printed and Online)

المجلد

--

السنة

1999

الصفحات

--