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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 30, 2002 as doi:10.1096/fj.01-0683fje. |
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The School of Biology, University of Leeds, Leeds, West Yorkshire, UK, LS2 9JT
2Correspondence: The School of Biology, University of Leeds, Leeds, West Yorkshire, UK, LS2 9JT. E-mail: a.m.agnew{at}leeds.ac.uk
SPECIFIC AIMS
We previously described a novel cholinergic system that regulates glucose uptake through the plasma membrane that forms the outermost surface of the parasitic blood fluke Schistosoma. In this paper we address whether the primary structure and functional characteristics of the acetylcholinesterase (AChE) component of this system differs from other AChEs with more conventional functions and whether there are sufficient molecular distinctions between the AChEs of human host and parasite to exploit this molecule as a target of rationally designed drugs or vaccines.
PRINCIPAL FINDINGS
1. Determination of the primary structure and derived amino acid sequence of an AChE from Schistosoma hematobium (ShAChE)
S. hematobium cDNA was screened by PCR using degenerate primers derived from highly conserved sequences within AChEs. The ShAChE cDNA comprises a single open reading frame of 2067 bp, a 5' untranslated region of >84 bp, a 123 bp 3' untranslated region, and a poly(A) tail (GenBank accession number AF279462). The predicted open reading frame encodes a protein of 689 amino acids, including a putative signal peptide of 25 residues and 5 potential N-glycosylation sites. An alignment of amino acid sequences (Fig. 1
) shows that the ShAChE sequence possesses the key features common to AChEs (by convention, Torpedo AChE numbering is shown in parentheses throughout). Ten of the 14 functionally important aromatic residues of Torpedo AChE that line the catalytic gorge are conserved or conservatively substituted in ShAChE, including W132 (W84), which is essential for substrate binding. Residues F(288) and F(290), which are conserved in vertebrates, have been implicated in determining substrate specificity. All invertebrate AChEs characterized so far conserve or conservatively substitute F(290) whereas residues corresponding to F(288) are nonaromatic. ShAChE is the first AChE reported that replaces F(290) with the nonaromatic residue V345 and the first invertebrate AChE that conserves F(288). Overall, the ShAChE protein shares 
30% identity and 40% similarity with AChEs from diverse organisms.
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2. Immunolocalization studies confirmed that the identified AChE corresponds to that present on the parasite surface and in muscle
ShAChE was immunolocalized in both intact live worms and unfixed frozen sections using polyclonal mouse antibodies raised against a region unique to the parasite esterase. Immunofluorescence on worm sections was localized to the worm surface and the muscle tissues located immediately beneath. Confocal microscopy of intact live worms showed clear labeling of the worm surface alone (Fig. 2
).
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3. Despite its role in a novel mechanism, recombinant ShAChE has functional characteristics typical of a wide range of neuronal and non-neuronal AChEs
Expression of functionally active enzyme was achieved by intracytoplasmic injection of Xenopus oocytes with cRNA encoding the ShAChE open reading frame. ShAChE purified from the oocyte incubation medium appeared as a single band of 116 kDa in SDS-PAGE indicating significant post-translational modification from the 80 kDa primary product. This is consistent with the size of immunoprecipitated native ShAChE previously described as 110 and 76 kDa products.
Substrate preference was typical of other AChEs, showing a fivefold greater rate of hydrolysis of 1 mM acetylthiocholine (ATCh) over 1 mM butyrylthiocholine (BuTCh). Excess substrate inhibition was observed at ATCh concentrations >10 mM. KM values of 199 ± 9.8 µM with ATCh and 399 ± 2.5 µM with BuTCh (mean±SE, n=3) were obtained using substrate concentrations from 0.125 to 8 mM. ShAChE activity was highly sensitive to the classical AChE inhibitors BW284c51 and eserine as well as to dichlorvos, the active metabolite of metrifonate, an organophosphate and known schistosomicide. ShAChE showed no sensitivity to the butyrylcholinesterase inhibitor iso-OMPA up to a concentration of 0.1 mM; at 1 mM, <20% inhibition was observed. It was notably insensitive to the peripheral anionic site (PAS) inhibitor propidium iodide (KI=22±3.7 vs. 1.4 µM for recombinant human AChE), a property likely attributable to the absence of key residues in the region of the PAS.
4. Schistosome AChE is attached to membranes by a GPI anchor but primary sequence data show that the carboxyl terminus sequence is distinct from all known GPI-anchored molecules and may represent a novel GPI addition signal
AChE subunits can assemble into well-characterized forms depending on their carboxyl-terminal sequence, which determines oligomerization and anchoring. Sedimentation analysis suggests that only the GPI-anchored dimeric globular form of AChE exists in schistosomes. Consistent with this, only a single cDNA sequence was isolated despite extensive screening of multiple libraries. A surprising feature of the deduced sequence of the ShAChE carboxyl terminus was the absence of a conventional GPI addition signal: a hydrophilic region of 8–12 amino acids, followed by a hydrophobic stretch of 8–20 residues (Fig. 1)
. The ShAChE carboxyl terminus is truncated and hydrophilic, more like that of secreted subunits. However, all known secreted AChEs are monomeric and lack the carboxyl-terminal cysteine residue necessary for dimerization that is present in ShAChE. Being truncated and devoid of aromatic residues, the ShAChE carboxyl terminus does not conform to any other known form and thus may reflect a novel mode of post-translational signal for GPI anchoring.
5. The schistosome AChE possesses distinctions from human host AChE that can be exploited for the design of specific inhibitors/vaccines
Overall, the schistosome and human AChEs share 33% identity and 47% similarity. The active site is conserved, but other functionally significant regions of these molecules are highly divergent. These include several regions implicated in the PAS and the putative back door. The PAS is involved in excess substrate binding and interaction with certain inhibitors, and is thought to be the docking point for the substrate before it enters the catalytic gorge. Several peptide stretches in Torpedo AChE have been implicated in the PAS, most notably 251-NLNCNLNSDEELIH-264 and 270-KPQELIDVEWNVL-283. The equivalent sequences in human AChE differ from those in ShAChE by 86% and 100%, respectively, with the human enzyme containing a four amino acid insertion in the former peptide.
There is evidence that AChEs may have a back door that allows the exit of reaction products without interfering with passage of substrate through the catalytic gorge, thus explaining the capacity of this enzyme to hydrolyze substrate at a very high rate. In this proposed mechanism, the movement of residues W(84), V(129), and G(441) causes a channel to open, allowing the escape of reaction products. Studies of Electrophorus AChE have also implicated the peptide (441)-EKRLNYTLEEERLSR-(455) in regulating back door function. The residues W(84), V(129), and G(441) are conserved in human and schistosome AchEs, but the regions surrounding them differ considerably. For example, the equivalent of the above regulatory peptide (441)-(455) differs by 73% between human and schistosome AChEs, and the ShAChE peptide also contains a five amino acid insertion.
CONCLUSIONS
The best-known function for AChE is the regulation of access of acetylcholine (ACh) to acetylcholine receptors (AChRs) at cholinergic synapses, but the presence of AChE in bacteria, plants, and nonexcitable tissues of complex organisms suggests it has a wide range of alternative functions. One example is Schistosoma, which has a remarkable non-neuronal cholinergic system on the parasite surface that regulates glucose scavenging from the host bloodstream.
This system consists of nicotinic AChRs (nAChR) and AChE. In vitro exposure of the parasites to ACh alters their rate of glucose uptake, an effect that is concentration dependent and can be ablated by inhibition of either the surface AChE or nAChRs. Glucose is known to be transported by conventional GLUT 1-like transporters in this membrane, but exactly how the opening of nAChR affects the rate of glucose uptake is not yet known. The function for AChE in this system is assumed to be that of limiting the interaction of ACh with its receptor since inhibition of AChE results in an effect that mimics ligand excess. In vivo, the ACh is likely derived from the host bloodstream since the optimal effect varies between parasites of humans and cattle according to the respective host’s circulating ACh levels. Thus, this appears to be a conventional interaction between cholinergic molecules, but one that has a unique consequence.
This system and its component molecules are of interest for two principal reasons. 1) Obtaining the molecular structure and functional characteristics of ShAChE was a first requirement for understanding this cholinergic system. 2) Schistosoma is responsible for >200 million human infections worldwide. Currently, control of schistosomiasis is reliant on a single drug, praziquantel, but there is evidence of emerging drug resistance and a recognized need for novel treatments and prophylactics.
We earlier showed that AChE on the schistosome surface is the target of the organophosphate drug metrifonate. This drug is not widely used because it does not distinguish well between host and parasite AChE and thus is toxic. Since AChE is a known target of an effective drug and has an important function on the parasite surface, we consider that surface AChE is a priority target of rationally designed drugs and/or vaccines. Several functionally important regions of the PAS and back door of ShAChE are sufficiently different from human AChE to allow design of schistosome-specific inhibitors. We are assessing the ability of antibodies raised against the ShAChE PAS and back door to inhibit enzyme function and thus compromise parasite survival in vivo.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0683fje; to cite this article, use FASEB J. (January 30, 2002) 10.1096/fj.01-0683fje 
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cysteines forming three intrachain disulfide bonds; shaded, amino acids conserving or conservatively substituting the 14 aromatic residues lining the catalytic gorge of Torpedo AChE; # residues forming salt bridges. Overlined bridges: the sequence chosen for the generation of specific antibodies for immunolocalization studies. Carboxyl-terminal peptides are marked by an arrow and the GPI addition signal of Torpedo AChE is doubly underlined. The alignment was compiled using CLUSTALW.
