A tryptophan amphiphilic tetramerization domain-containing acetylcholinesterase from the bovine lungworm, Dictyocaulus viviparus.

Acetylcholine (ACh) is one of an array of neurotransmitters used by invertebrates and, analogous to vertebrate nervous systems, acetylcholinesterase (AChE) regulates synaptic levels of this transmitter. Similar to other invertebrates, nematodes possess several AChE genes. This is in contrast to vertebrates, which have a single AChE gene, transcripts of which are alternatively spliced to produce different types of the enzyme which vary at their C-termini. Parasitic nematodes have a repertoire of AChE genes which include those encoding neuromuscular AChEs and those genes which code for secreted AChEs. The latter proteins exist as soluble monomers released by the parasite during infection and these AChE are distinct from those enzymes which the nematodes use for synaptic transmission in their neuromuscular system. Thus far, Dictyocaulus viviparus is the only animal-parasitic nematode for which distinct genes that encode both neuromuscular and secreted AChEs have been defined. Here, we describe the isolation and characterization of a cDNA encoding a putative neuromuscular AChE from D. viviparus which contains a tryptophan amphiphilic tetramerization (WAT) domain at its C-terminus analogous to the common 'tailed' AChE form found in the neuromuscular systems of vertebrates and in the ACE-1 AChE from Caenorhabditis elegans. This enzyme differs from the previously isolated, D. viviparus neuromuscular AChE (Dv-ACE-2), which is a glycosylphosphatidylinositol-anchored variant analogous to vertebrate 'hydrophobic' AChE.

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.

Heterologous expression and enzymatic properties of a selenium-independent glutathione peroxidase from the parasitic nematode Brugia pahangi.

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).

Extracellular and cytoplasmic CuZn superoxide dismutases from Brugia lymphatic filarial nematode parasites.

We have isolated full-length cDNAs encoding two distinct types of CuZn superoxide dismutases (SODs) from the filarial nematode parasite Brugia pahangi. The derived amino acid sequences suggested that one class of cDNAs represented a cytoplasmic form of SOD and the second class represented an extracellular (EC) variant. The predicted proteins were highly homologous to each other, but the sequence of the latter contained an additional 43 residues at the N terminus, the first 16 of which were markedly hydrophobic, and four potential sites for N-linked glycosylation. Western blotting (immunoblotting) with an antiserum to a partial SOD expressed in Escherichia coli revealed two proteins with estimated molecular masses of 19 and 29 kDa. Digestion with N-glycanase indicated that the latter protein corresponded to the EC form, as it possessed N-linked oligosaccharide chains at three sites, leaving a peptide backbone with an estimated molecular mass of 22 kDa, which was consistent with the additional 27 amino acids predicted from the cDNA sequence. Gel filtration indicated that both enzymes were dimeric in their native forms, in contrast to the human EC-SOD, which is tetrameric. Comparison of the primary structure of the parasite EC-SOD with that of the human EC enzyme revealed two major differences: the N-terminal extension of the parasite enzyme was shorter by 25 residues, and it also lacked the C-terminal charged extension which mediates binding to cell surface sulfated proteoglycans. Lavage of Mongolian jirds infected intraperitoneally with Brugia malayi resulted in the recovery of filarial CuZn SODs, principally the EC form, indicating that this form of SOD is secreted in vivo. This EC enzyme may contribute to parasite persistence by neutralizing superoxide generated by activated leukocytes, thus acting as both an antioxidant and an anti-inflammatory factor.