A tetrameric acetylcholinesterase from the parasitic nematode Dictyocaulus viviparus associates with the vertebrate tail proteins PRiMA and ColQ

https://doi.org/10.1016/j.molbiopara.2011.10.005Get rights and content

Abstract

Dictyocaulus viviparus causes a serious lung disease of cattle. Similar to other parasitic nematodes, D. viviparus possesses several acetylcholinesterase (AChE) genes, one of which encodes a putative neuromuscular AChE, which contains a tryptophan (W) amphiphilic tetramerization (WAT) domain at its C-terminus. In the current study, we describe the biochemical characterization of a recombinant version of this WAT domain-containing AChE. To assess if the WAT domain is biologically functional, we investigated the association of the recombinant enzyme with the vertebrate tail proteins, proline-rich membrane anchor (PRiMA) and collagen Q (ColQ), as well as the synthetic polypeptide poly-l-proline. The results indicate that the recombinant enzyme hydrolyzes acetylthiocholine preferentially and exhibits inhibition by excess substrate, a characteristic of AChEs but not butyrylcholinesterases (BChEs). The enzyme is inhibited by the AChE inhibitor, BW284c51, but not by the BChE inhibitors, ethopropazine or iso-OMPA. The enzyme is able to assemble into monomeric (G1), dimeric (G2), and tetrameric (G4) globular forms and can also associate with PRiMA and ColQ, which contain proline-rich attachment domains (PRADs). This interaction is likely to be mediated via WAT-PRAD interactions, as the enzyme also assembles into tetramers with the synthetic polypeptide poly-l-proline. These interactions are typical of AChET subunits. This is the first demonstration of an AChET from a parasitic nematode that can assemble into heterologous forms with vertebrate proteins that anchor the enzyme in cholinergic synapses. We discuss the implications of our results for this particular host/parasite system and for the evolution of AChE.

Highlights

D. viviparus has an AChE with a C-terminus containing a WAT domain. ► The AChE is able to assemble into G1a, G2a, and G4na forms. ► The AChE is able to associate with the vertebrate tail proteins PRiMA and ColQ. ► The AChE is able to associate with the synthetic polypeptide poly-l-proline. ► These associations are mediated by WAT–PRAD interactions.

Introduction

Acetylcholine (ACh) is the major excitatory neurotransmitter controlling motor activities in nematodes [1], [2], and the enzyme which hydrolyzes and inactivates acetylcholine, acetylcholinesterase (AChE), is thus essential for regulation of cholinergic transmission. Nematodes are known to express a number of molecular forms of AChEs which, in contrast to vertebrate AChEs, are encoded by distinct genes. Of the nematodes, most detail is available on AChEs of the free-living worm, C. elegans, in which three genes are known to encode distinct enzymes, with a fourth gene thought to be non-functional [3], [4]. Of these enzymes, C. elegans ACE-1 is an amphiphilic tetramer which associates with a hydrophobic non-catalytic subunit, whereas C. elegans ACE-2 and ACE-3 are GPI-linked amphiphilic dimers. The genes that encode these proteins display distinct anatomical transcription patterns in neurons and musculature, suggestive of predominantly non-redundant functions [5], [6].

The bovine lungworm, Dictyocaulus viviparus, is a pathogenic parasitic nematode that inhabits the trachea and main stem bronchi of cattle and causes the disease parasitic bronchitis. Homologues of C. elegans ace-1 and ace-2 have been isolated from D. viviparus, and their predicted proteins contain C-terminal sequences similar to the C. elegans enzymes, suggesting that the parasite AChEs have comparable means of membrane anchorage. Thus, D. viviparus AChE-1 (Dv-ACE-1) is predicted to be a tetrameric form of AChE capable of associating with a non-catalytic structural subunit [7], whereas D. viviparus AChE-2 (Dv-ACE-2) is predicted to be a membrane-bound glycophosphatidylinositol-linked (GPI-linked) dimer [8]. In addition, D. viviparus expresses at least two other AChEs (Dv-sACE-1 and Dv-sACE-2) which are secreted and apparently monomeric [9].

The nature of the molecular forms produced by AChE, be they monomers, dimers, or tetramers, depends in large part on the nature of the carboxyl terminus of the enzyme [10]. Nematode secreted AChE monomers (AChES) have a truncated C-terminus, while GPI-linked AChE dimers (AChEH) possess a hydrophobic C-terminus, which is cleaved upon the addition of the GPI anchor. Tetrameric AChE molecules have a highly conserved tryptophan-containing amphiphilic C-terminus, which mediates tetramer formation and also association with non-catalytic structural proteins. In invertebrates, including nematodes, this non-catalytic protein and its function have not been identified [4], [11], [12]. In contrast, in vertebrates, tetrameric AChE (AChET) associates with collagen Q (ColQ), which stabilizes the enzyme and anchors it to the basal lamina, and with the proline-rich membrane anchor (PRiMA), which is required for the intracellular processing and anchoring of AChE in the plasma membrane of neurons [13], [14], [15], [16]. This association is mediated by an interaction between the tryptophan (W) amphiphilic tetramerization (WAT) domain of the C-terminus of tetrameric AChE with proline-rich attachment domains (PRADs) present in ColQ and PRiMA [10]. Previously, we cloned a cDNA (Dv-ace-1) for a putative AChET from D. viviparus [7]. Here, we report the kinetic and pharmacological characteristics of the Dv-ACE-1 AChE enzyme, its molecular forms, and its association with the vertebrate tail proteins ColQ and PRiMA, and the synthetic polypeptide poly-l-proline. We also discuss the implications of these interactions with regard to their role in the cholinergic nervous system of D. viviparus.

Section snippets

Sequence analysis

Sequences were aligned with Clustal X and phylogenetic analysis by the neighbor-joining method [17]. Amphipathic α-helical properties and helical wheels were determined by the method of Zidovetzki et al. [18].

Expression of cDNAs for AChE, ColQ, and PRiMA; incubations with poly-l-proline

The cDNA for the AChE, Dv-ACE-1 (GenBank Accession Number DQ375489), that was cloned previously as described in Matthews et al. [7], was sub-cloned into the expression vector, pcDNA3.1. COS-7 monkey cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle medium

Sequence analysis

We compared the sequence for Dv-ACE-1 to the sequences of the other AChEs from D. viviparus and the AChE from Torpedo californica. The sequences of Dv-ACE-1 and the other AChEs from D. viviparus contain the catalytic triad and disulfide bonds characteristic of all ChEs; however, Dv-ACE-1 differs from the other D. viviparus AChEs and from vertebrate AChEs, as represented by T. californica AChE, in a number of ways. Vertebrate AChEs have 14 aromatic amino acid residues lining the catalytic gorge

Discussion

From the current studies, it is clear that Dv-ACE-1 hydrolyzes ATCh preferentially and is sensitive to the AChE inhibitor, BW284c51, but not to BChE inhibitors. It was also observed that the Dv-ACE-1 produces G1a, G2a, and G4na molecular forms and that the tetramers can assemble into complexes with PRiMA, ColQ, and poly-l-proline. As prior sequence analysis indicated that the enzyme is an AChET possessing a WAT domain, association of this enzyme with poly-l-proline, PRiMA, and ColQ is most

Acknowledgement

This research was partially supported by an Academic Research Enhancement Award (1 R15 GM072510-01) from the National Institutes of Health to L.P.

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