Histamine signalling in Schistosoma mansoni: Immunolocalisation and characterisation of a new histamine-responsive receptor (SmGPR-2)

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Abstract

In parasitic platyhelminthes, including Schistosoma mansoni, biogenic amines play several important roles in the control of motility, metabolism and reproduction. A bioinformatics analysis of the S. mansoni genome identified approximately 16 full-length G protein-coupled receptors (GPCRs) that share significant homology with aminergic receptors from other species. Six of these sequences are structurally related to SmGPR-1 (formerly SmGPCR), a previously described histamine receptor of S. mansoni, and constitute a new clade of amine-like GPCRs. Here we report the cloning of a second member of this clade, named SmGPR-2. The full-length receptor cDNA was expressed in Saccharomyces cerevisiae and shown to be activated by histamine and 1-methylhistamine, whereas other common biogenic amines had no significant effect. Antagonist assays showed that SmGPR-2 was inhibited by classical biogenic amine antagonists but the pharmacological profile was unlike those of known mammalian histamine receptors. Confocal immunolocalisation studies revealed that SmGPR-2 was expressed in the nervous system and was particularly enriched in the subtegumental neuronal plexus of adult S. mansoni and larvae. The ligand, histamine, was found to be widely distributed, mainly in the peripheral nervous system including the subtegumental plexus where the receptor is also expressed. Finally, SmGPR-2 was shown to be developmentally regulated at the RNA level. Quantitative PCR studies showed it was up-regulated in the parasitic stages compared with cercaria and expressed at the highest level in young schistosomula. The widespread distribution of histamine and the presence of at least two receptors in S. mansoni suggest that this transmitter is an important neuroactive substance in schistosomes.

Introduction

Schistosoma mansoni (Platyhelminthes, Trematoda) is a major cause of human schistosomiasis, a disease that afflicts over 200 million people worldwide. S. mansoni exists where its intermediate host, the freshwater snail Biomphalaria glabrata, is available, notably in Africa, the Middle East, South America and the Caribbean. Praziquantel is the drug of choice for treatment of schistosomiasis but drug-resistant strains have emerged and thus alternative chemotherapeutic agents should be designed and tested (Fallon and Doenhoff, 1994, Ismail et al., 1994, William et al., 2001). Many pharmaceutical drugs exert their effects by interacting with G protein-coupled receptors (GPCRs) (Wise et al., 2002, Eglen, 2005), in particular Family A (Rhodopsin-like) GPCRs, which include the vast majority of small transmitter and hormone receptors. While a few GPCRs have been cloned from schistosomes (Hoffmann et al., 2001, Hamdan et al., 2002, Pearson et al., 2007, Taman and Ribeiro, 2009), there are many more predicted sequences in the S. mansoni gene database that have yet to be characterised (Berriman et al., 2009). These GPCRs are potentially good targets for new anti-schistosomal drugs, especially if their pharmacological profiles prove to be parasite-specific.

Biogenic amines (BAs) are derivatives of amino acids (tryptophan, tyrosine or histidine) and act as neurotransmitters, hormones and modulators. They include such ubiquitous substances as serotonin (5-hydroxytryptamine, 5HT), catecholamines (dopamine and noradrenaline) and histamine (HA). In platyhelminthes, BAs play many vital roles in metabolism, the control of motility and therefore survival within the host (Ribeiro et al., 2005, Maule et al., 2006, Ribeiro and Geary, 2010). The most widespread and best studied BA is 5HT. Serotonergic neurons are distributed abundantly in the CNS and peripheral nervous system (PNS) of every flatworm tested to date, including S. mansoni. Moreover, 5HT is strongly myoexcitatory (Day et al., 1994, Pax et al., 1996, Walker et al., 1996, Ribeiro et al., 2005, Maule et al., 2006) and there is evidence both for endogenous biosynthesis (Hamdan and Ribeiro, 1999) and carrier-mediated transport (Boyle and Yoshino, 2005, Patocka and Ribeiro, 2007). By comparison, little is known about other BAs, particularly HA. HA is variably distributed among parasitic flatworms. Some species are capable of endogenous HA biosynthesis and have very high tissue levels of the amine (Mettrick and Telford, 1963, Eriksson et al., 1996), whereas in other parasites HA is present at low levels and may be entirely of host origin (Yonge and Webb, 1992). The biological role of HA in flatworms is unclear but it probably affects the musculature and the outcome is concentration-dependent. It was reported that HA significantly modulates movement in the posterior region of the strobila in Hymenolepis diminuta (Sukhdeo et al., 1984) and stimulates motility in S. mansoni (Ercoli et al., 1985). HA-containing neurons innervate the somatic musculature and the suckers in some species (Wikgren et al., 1990, Eriksson et al., 1996), which further supports a role in the control of muscle function and movement. The distribution of HA neurons in S. mansoni has not been investigated.

Previously, a GPCR from S. mansoni, named SmGPR-1 (formerly SmGPCR), was cloned in our laboratory and was shown to be selectively activated by HA (Hamdan et al., 2002). Further analysis of this receptor revealed that it was expressed in the tegument and musculature of larval and adult parasites (El-Shehabi et al., 2009). Following completion of the S. mansoni genome project, we detected several new sequences that are structurally related to SmGPR-1. Bioinformatics analyses suggest these sequences have evolved from a common ancestor and constitute a new structural type of BA receptor. Given their novelty, we have adopted the system of classification used for human orphan GPCRs and designated these sequences as S. mansoni GPR receptors (SmGPR). In the present study, we report the cloning, functional analysis and immunolocalisation of a new member of this clade, named SmGPR-2. The results indicate that SmGPR-2 is a second histaminergic receptor of S. mansoni and is expressed in close proximity to HA-containing neurons in the subtegumental neuronal plexus. We further demonstrate that histaminergic neurons are abundantly distributed in schistosomes, suggesting that HA is an important neuroactive system in this parasite.

Section snippets

The parasite

B. glabrata snails infected with a Puerto Rican strain of S. mansoni were kindly provided by Dr. Fred Lewis, Biomedical Research Institute, Rockville, MD, USA. S. mansoni cercaria were collected 35–45 days p.i. (Lewis et al., 1986, Lewis et al., 2001) and were mechanically transformed to produce schistosomula (Basch, 1981) as described by El-Shehabi et al. (2009). In vitro transformed schistosomula were cultured at 37 °C and 5% CO2 in OPTI-MEM I medium (Invitrogen) supplemented with 10% FBS,

SmGPR-2 belongs to a cluster of novel amine-like receptors

A bioinformatics search of the S. mansoni GeneDB identified a sequence that was closely related to SmGPR-1 (SmGPCR; Accession #AF031196; Smp_043260), a previously described HA receptor of S. mansoni (Hamdan et al., 2002). The new predicted receptor cDNA was cloned from adult S. mansoni by RT-PCR, verified by DNA sequencing, submitted to the GenBank (Accession #GQ397114; Smp_043340) and was designated SmGPR-2. BLAST analyses of the general protein database at NCBI confirmed the identity of this

Discussion

Previously, our laboratory described the first HA-responsive receptor of S. mansoni, named SmGPR-1 (SmGPCR; Hamdan et al., 2002). In the present study, we report the cloning and expression of a structurally related receptor, which we have named SmGPR-2. The bioinformatics analysis identified two S. japonicum sequences and a total of six orphan receptors in the genome of S. mansoni that share high homology with SmGPR-2. These sequences do not align within the known clades of the biogenic amine

Acknowledgements

The authors would like to thank Dr. J. Broach (Princeton University, NJ, USA), who kindly provided us with the yeast expression strains. We also thank Dr. Fred Lewis (Biomedical Research Institute, Rockville, MD, USA), who supplied the infected snails. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to P.R.

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