Characterization of a novel serotonin receptor from Caenorhabditis elegans: cloning and expression of two splice variants.

Serotonin [5-hydroxytryptamine (5-HT)] modulates feeding activity, egg-laying, and mating behavior in the free-living nematode, Caenorhabditis elegans. We have cloned a novel receptor cDNA from C. elegans (5-HT2Ce) that has high sequence homology with 5-HT2 receptors from other species. When transiently expressed in COS-7 cells, 5-HT2Ce exhibited 5-HT binding activity and activated Ca2+-mediated signaling in a manner analogous to other 5-HT2 receptors. However, 5-HT2Ce displayed unusual pharmacological properties, which resembled both 5-HT2 and 5-HT1-like receptors but did not correlate well with any of the known 5-HT2 subtypes. Two splice variants of 5-HT2Ce that differ by 48 N-terminal amino acids were identified. The two isoforms were found to have virtually identical binding and signaling properties but differed in their levels of mRNA expression, with the longer variant being four times more abundant than the shorter species in all developmental stages tested. Taken together, the results describe two variants of a novel C. elegans 5-HT receptor, which has some of the properties of the 5-HT2 family but whose pharmacological profile does not conform to any known class of receptor.

Cloning, sequencing, and developmental expression levels of a novel glutamate-gated chloride channel homologue in the parasitic nematode Haemonchus contortus.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics such as ivermectin (IVM) and the milbemycins such as moxidectin (MOX). The importance of this interaction between macrocyclic lactones and GluCls is strengthened by the recent genetic evidence that GluCls are involved in IVM and MOX resistance in the parasitic nematode Haemonchus contortus (1). We have cloned two full length GluCl putative alpha-subunit cDNAs from H. contortus (HcGluCla and b) that exhibit different sized ligand binding domains. Phylogenetic analysis of the conserved regions of the amino acid sequence of HcGluCla suggests that it is a member of the GluCl family but forms a distinct subbranch within this family. The expression level of HcGluCla was examined in different developmental stages of H. contortus (eggs, L3, and adults) and found to be significantly downregulated in eggs compared to adults.

Cloning and characterization of a novel form of tyrosine hydroxylase from the human parasite, Schistosoma mansoni.

Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni. We have cloned a novel S. mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Two subsets of SmTH transcripts were identified, one of which carries the S. mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S. mansoni SL. The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa. Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme. Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme. This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation. This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.