Characterization of Novel Biogenic Amine Receptors In

Characterization of novel biogenic amine receptors in

the human bloodfluke Schistosoma mansoni

Fouad Sufian El-Shehabi

Doctorate of Philosophy

Institute of Parasitology

McGill University

Montreal, Quebec, Canada

A thesis submitted to McGill University in partial fulfilment of the

requirements of the degree of Doctorate of Philosophy

August 2009

 Fouad El-Shehabi 2009

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ABSTRACT

The genome of the human bloodfluke Schistosoma mansoni encodes 18 putative biogenic amine-like G-protein-coupled receptors (GPCRs). These receptors are potential targets for the development of antischistosomal drugs. One of these sequences, SmGPR-1 (formerly SmGPCR), was previously cloned and was identified as a histamine receptor. In this study, we expanded the functional analysis of SmGPR-1 by studying its expression and tissue distribution both at the RNA and protein levels in different developmental stages of the parasite. In the second part of the study, we cloned and characterized two structurally related receptors, named SmGPR-2 and SmGPR-3. Bioinformatics analyses showed that the three receptors are members of a new clade of biogenic amine GPCRs and are characterized in part by the absence of a highly conserved aspartate (Asp3.32) of the third transmembrane domain. Like SmGPR-1, our first cloned receptor, SmGPR-2, was activated by histamine and its developmental expression at the mRNA level was similar to that of SmGPR-1, both receptors being upregulated in young schistosomula. However, their tissue localization was different. SmGPR-1 was enriched in the tegument, subtegumental musculature and the suckers, whereas SmGPR-2 was associated with neurons of the subtegumental plexuses. The distribution of these receptors correlated with that of histaminergic neurons, which were also detected in the subtegumental neuronal plexuses, the innervation of the suckers, elements of the central nervous system and transverse commissures. These studies suggest that histamine is an important neurotransmitter system in schistosomes. The third receptor investigated in this study, SmGPR-3, was not responsive to histamine but rather was found to have broad specificity for catecholamines, particularly dopamine and related metabolites. In vitro assays of cultured schistosomula revealed that many of the ligands that interact with SmGPR-3 also have strong effects on larval motility, including dopamine, which caused significant inhibition of movement. The results

suggest that SmGPR-3 is probably one of several catecholamine receptors controlling motor function in these parasites. The pharmacological analysis of SmGPR-2 and SmGPR-3 showed they are atypical histaminergic and dopaminergic receptors, respectively.

ABRÉGÉ

Au génome de Schistosoma mansoni, un parasite sanguin de l‟homme, on retrouve 18 récepteurs putatifs à amine biogène couplés aux protéines G (RCPG). Ces récepteurs ont un potentiel thérapeutique contre les infections aux schistosomes. La séquence SmGPR-1 (anciennement SmGPCR) a déjà été clonée et identifiée comme un récepteur à l‟histamine. Une analyse fonctionnelle plus poussée de SmGPR-1 est l‟objet de cette thèse. L‟analyse de taux d‟ARNm et de protéines à différents stades de développement du parasite a servi à l‟étude de l‟expression et la répartition tissulaire de SmGPR-1. Deux récepteurs similaires, de par leur structure, le SmGPR-2 et le SmGPR-3 ont été identifiés, clonés et caractérisés lors de cette étude. Suite à des analyses bioinformatiques, ces trois récepteurs ont révélé leur appartenance à une nouvelle variante de récepteurs à amine biogène couplés aux protéines G caractérisés par l‟absence d‟aspartate conservé (Asp3.32) dans le troisième domaine transmembranaire. Tout comme SmGPR-1, le récepteur SmGPR-2 est activé par l‟histamine, et l‟expression de l‟ARNm est similaire à celle de SmGPR-1, les deux récepteurs étant régulés à la hausse chez les jeunes schistosomes. Toutefois, ils sont localisés à différents endroits, SmGPR-1 se retrouve dans le tégument, la musculature subtégumentaire et les ventouses, tandis que SmGPR-2 est associé aux plexus nerveux subtégumentaires. La localisation de ces récepteurs est similaire à celle des neurones histaminergiques que l‟on retrouve dans les plexus nerveux subtégumentaires, l‟innervation des ventouses, dans certains éléments du système nerveux central et les commissures transversales. Il semblerait que l‟histamine soit un important système neurotransmetteur du schistosome. Le troisième récepteur identifié, SmGPR-3, n‟est pas activé par l‟histamine, mais semble démontrer une spécificité étendue aux catécholamines et tout particulièrement à la dopamine et ses métabolites. Des essais in vitro sur des cultures de jeunes schistosomes ont révélé que plusieurs des ligands qui interagissent avec SmGPR- 3 perturbent considérablement la mobilité des larves, incluant la dopamine qui

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provoque une inhibition motrice importante. Ces résultats suggèrent que SmGPR- 3 est probablement un des récepteurs à la catécholamine régulant la fonction motrice de ces parasites. L‟analyse pharmacologique de SmGPR-2 et SmGPR-3 démontre qu‟ils sont respectivement des récepteurs histaminergiques et dopaminergiques atypiques.

ACKNOWLEDGEMENTS

Since I came to this great country as an immigrant, I was searching for universities that contain departments/institutes of Parasitology. I was lucky to get the opportunity to work my research at the Institute of Parasitology of McGill and under the supervision of Professor Paula Ribeiro. Her valuable contributions, suggestions and continuous encouragement, helped me a lot to improve my technical and writing skills. Dr. Ribeiro is a great supervisor with a superb attitude toward her research team. She always listens to her students for any technical problem, giving a superior advice and providing an excellent guidance to resolve it. I would like to say: Thank you Paula very much for your support, excellent recommendations and critiques, especially during the preparation of manuscripts and this dissertation. This thesis would not have been possible without Paula‟s encouragement and support. I really enjoyed my work of Schistosoma mansoni GPCRs project with a knowledgeable scientist like Professor Ribeiro. I would not stop saying thank you Paula for these wonderful years I spent in your lab. I would like to extend my appreciation to all present and past lab-colleagues. Aisha Mousa, Dr. Joseph Nabhan, Serge Dernovici, Nicholas Patocka, Dr. Amira Taman, Laura Sayegh, Dr. Christelle Bouchard, Kristi Bangs, Kevin Macdonald and Dr. Fadi Hamdan. A special thank to Christiane Trudeau, for her excellent assistance in providing French translating of the thesis‟s abstract. During exciting years of this study, I made a friendship with many wonderful people at the Institute of Parasitology. Special thanks to my good friends Dr.Omar Alqawi, Dr. Aws Abdul-Wahid and Ms. Kathy Keller. I also would like to thank Mr. Gorden Bingham, Mrs. Shirley Mongeau, Elizabeth Ruizlancheros, Dr. Manami Nishi, Dr. Cristina Gheorghiu, Yovany Moreno, Alireza Shaneh, Houtan Moshiri, Dr. Mike Osei-Atweneboana, Dr. Jeff Eng, Smriti Kala, Hamed Shaterinajafabadi, Vijayaraghava Rao, Maurice Odiere, Catherine Bourguinat, Sonia Edaye and Dr. Hiren Banerjee.

I also want to show deep appreciation and respect to all the faculty staff at the fascinating institute in the past and current, especially Dr. Timothy Geary, Dr. Roger Prichard, Dr. James Smith, Dr. Marilyn Scott, Dr. Gaetan Faubert, Dr. Reza Salavati, Dr. Robin Beech, Dr. Terry Spithill, Dr. Elias Georges and Dr. Florence Dzierszinski. Finally, I am truly grateful to my dad Sufian and my sisters Basma, Eman and Fida for their unlimited love and encouragement. This thesis is dedicated to the souls of my mother and my grandmother (May ALLAH SWT Bless all of them, Ameen).

THESIS OFFICE STATEMENT

Candidates have the option, subject to the approval of their Department, of including, as part of their thesis, copies of the text of a paper(s) submitted for publication, or the clearly-duplicated text of a published paper(s) provided that these copies are bound as an integral part of the thesis.

If this option is chosen, connecting texts, providing logical bridges between the different papers are mandatory.

The thesis must still conform to all other requirements of the “Guidelines Concerning Thesis Preparation” and should be in a literary form that is more than a mere collection of manuscripts published or to be published. The thesis must include, as separate chapters or sections: (1) a Table of Contents, (2) a general abstract in English and French, (3) an introduction which clearly states the rationale and objectives of the study, (4) a comprehensive general review of the background literature to the subject of the thesis, when this review is appropriate, and (5) a final overall conclusion and/or summary.

Additional material (procedural and design data, as well as descriptions of the equipment used) must be provided where appropriate and in sufficient detail (e.g. in appendices) to allow a clear and precise judgement to be made of the importance and originality of the research reported in the thesis.

In the case of manuscripts co-authored by the candidate and others, the candidate is required to make an explicit statement in the thesis as to who contributed to such work and to what extent; supervisors must attest to the accuracy of such claims at the Ph.D. Oral defence. Since the task of the examiners is made more difficult in these cases, it is in the candidate‟s interest to make perfectly clear the responsibilities of all the authors of the co-authored papers.

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STATEMENT OF CONTRIBUTIONS

The experimental work presented in this thesis was designed and performed by the author, under the supervision of Dr. Paula Ribeiro. In manuscript I, the RT- PCR analysis of SmGPCR mRNA expression in miracidium and sporocyst developmental stages of Schistosoma mansoni were conducted in the laboratory of Dr. Timothy Yoshino (Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA) by Dr. Jon Vermeire.

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STATEMENT OF ORIGINALITY

The following aspects described in this thesis are considered contributions of original knowledge:

Manuscript I

In this study, I conducted the first investigation of the subunit organization of SmGPCR (SmGPR-1), tissue distribution and expression levels in different developmental stages of Schistosoma mansoni. SmGPR-1 is a previously described histamine receptor of S. mansoni. The study shows that SmGPR-1 forms a stable but non-covalent dimer, which may be physiologically relevant. SmGPR-1 mRNA expression occurs in all developmental stages of the parasite but it is upregulated in the parasitic forms, mainly in schistosomula. Immunolocalization studies in adult parasites and larvae suggest potential roles for SmGPR-1 in sensory structures of the tegument, the body wall musculature and the innervation of the suckers.

Manuscript II

In this study, I used bioinformatics tools to search the S. mansoni genome database for SmGPR1-like receptors. We identified seven receptors, one of which was cloned (SmGPR-2) and was characterized as a second histamine receptor in this parasite. We also report that SmGPR-2 has an atypical agonist and antagonist profile, which is markedly different from that of mammaian receptors. Expression of SmGPR-2 mRNA is very similar to that SmGPR-1 in that they are both upregulated in young schistosomula. At the protein level, SmGPR-2 is localized in the neuronal plexuses of the subtegumental region in close proximity to histaminergic neurons. This report provides the first evidence for an abundance of histaminergic neurons in the nervous system of S. mansoni, including elements of the central nervous system, the innervation of the suckers and the subtegumental

neuronal plexuses. The results show that histamine is an important neurotransmitter in schistosomes.

Manuscript III

In the last part of the study, we cloned a third SmGPR-1 related receptor, SmGPR-3, and characterized it in vitro. Despite the structural similarity to the HA receptors of S. mansoni, SmGPR-3 was not activated by HA. Functional expression studies in yeast identified a broad specificity for catecholamine ligands. The agonist/antagonist assays show that SmGPR-3 is an atypical dopamine receptor that can also recognize catecholamine metabolites, such as epinine and metanephrine, while many common dopaminergic ligands have no effect. This is the first example of a cloned receptor being activated by these catechol derivatives. The study also shows that DA, epinine, metanephrine and other ligands of SmGPR-3 have strong effects on the motility of S. mansoni schistosomula in culture. The results suggest that SmGPR-3 may play an important role in the control of motor function in these parasites.

Appendix

In addition to the work presented in the body of the thesis, I developed and tested a new method of RNA interference (RNAi) in cultured S. mansoni schistosomula. The method is based on the use of short interfering RNAs (siRNAs) and a liposome-based reagent for transfection of the larvae. The technique was used in an unrelated study to knockdown a schistosome proteasome subunit (Nabhan, J.F., El-Shehabi, F., Patocka, N., Ribeiro, P., 2007. The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies. Exp Parasitol 117, 337-347). The method is also being used in RNAi studies of SmGPR receptors.

TABLE OF CONTENTS

THESIS OFFICE STATEMENT ...... VII STATEMENT OF CONTRIBUTIONS ...... VIII STATEMENT OF ORIGINALITY ...... IX TABLE OF CONTENTS...... XI LIST OF TABLES ...... XVI LIST OF FIGURES ...... XVII LIST OF ABBREVIATIONS ...... XIX INTRODUCTION ...... 1 CHAPTER I (Literature Review) ...... 3 1 The parasite Schistosoma mansoni ...... 4 1.1 biology and life cycle ...... 4 1.2 Pathology and immune evasion mechanisms ...... 6 1.3 The nervous system of S. mansoni ...... 8 1.4 Neurotransmitters in S. mansoni and other flatworms ...... 9 2 Histamine and its receptors ...... 12 2.1 HA biosynthesis and its roles in vertebrates ...... 12 2.2 Histamine GPCR receptors in vertebrates ...... 13 2.3 Histamine in non-helminthic invertebrates ...... 15 2.4 Histamine in platyhelminths: presence and biological roles ...... 16 2.5 Inactivation of HA signaling ...... 19 3 Catecholamines and their receptors ...... 21 3.1 Catecholamine biosynthesis and degradation ...... 21 3.2 DA receptors in vertebrates ...... 22 3.3 Catecholamines in the invertebrates ...... 27 4 G protein-coupled receptors (GPCRs) ...... 32 4.1 The structure ...... 32 4.2 Numbering of residues in GPCRs ...... 34 4.3 Ligand binding in GPCRs ...... 35 XI

4.4 Switching “on” GPCR-mediated signals ...... 36 4.5 The heterotrimeric G-proteins ...... 37 4.6 Receptor desensitization ...... 38 4.7 GPCR dimerization: Is it an artifact or biologically relevant? ...... 39 4.8 GPCR interacting proteins (GIPs) ...... 41 4.9 GPCRs of schistosomes and other platyhelminths ...... 41 References ...... 43 CHAPTER II (manuscript I)...... 66 Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni ...... 66 ABSTRACT ...... 67 1. Introduction ...... 68 2. Materials and methods ...... 70 2.1. Parasites ...... 70 2.2. Production of a polyclonal anti-SmGPCR antibody ...... 72 2.3. IFA-confocal microscopy of SmGPCR-transfected cells ...... 73 2.4. IFA-confocal microscopy of larval and adult stages of S. mansoni ...... 73 2.5. SDS–PAGE and Western blots ...... 74 2.6. Immunoprecipitation of SmGPCR ...... 75 2.7. Quantitative PCR analyses ...... 76 2.8. Other methods ...... 78 3. Results ...... 78 3.1. Production of the anti-SmGPCR polyclonal antibody ...... 78 3.2. SmGPCR forms multiple species in transfected HEK293 cells and schistosomes ...... 79 3.3. Confocal immunofluorescence analysis of SmGPCR in S. mansoni ...... 80 3.4. Quantitative RT-PCR analysis of SmGPCR mRNA expression . 81

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4. Discussion ...... 82 Acknowledgments ...... 89 References ...... 90 Figures ...... 97 CONNECTING STATEMENT 1 ...... 104 CHAPTER III (manuscript II) ...... 105 Histamine signalling in Schistosoma mansoni: Immunolocalization and characterization of a new histamine receptor (SmGPR-2) ...... 105 Abstract ...... 106 1. Introduction ...... 107 2. Materials and methods ...... 109 2.1. The parasite ...... 109 2.2. Cloning of S. mansoni SmGPR-2 ...... 109 2.3. Yeast functional expression assays ...... 110 2.4. Quantitative PCR analyses ...... 111 2.5. Immunolocalization studies ...... 112 2.6. Other methods ...... 114 3. Results ...... 114 3.1. SmGPR-2 belongs to a cluster of novel amine-like receptors .. 114 3.2. Functional assays: SmGPR-2 is a second histamine receptor of S. mansoni ...... 116 3.3. SmGPR-2 expression is upregulated in schistosomula ...... 117 3.4. In situ localization of histamine (HA) in S. mansoni ...... 118 3.5. Confocal immunofluorescence analysis of SmGPR-2 in S. mansoni ...... 119 4. Discussion ...... 119 Acknowledgements ...... 126 References ...... 127 Figures ...... 132

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CONNECTING STATEMENT 2 ...... 141 CHAPTER IV (manuscript III) ...... 142 Characterization of a novel catecholamine receptor (SmGPR-3) in the bloodfluke Schistosoma mansoni ...... 142 Abstract ...... 143 1. Introduction ...... 144 2. Materials and methods ...... 147 2.1 The parasite ...... 147 2.2 Cloning of S. mansoni SmGPR-3 ...... 147 2.3 Yeast functional expression assays ...... 148 2.4 Measurements of motor activity ...... 149 3. Results ...... 150 3.1 SmGPR-3 belongs to a new clade of BA receptors ...... 150 3.2 Functional assays: SmGPR-3 is a catecholamine receptor of S. mansoni ...... 151 3.3 Antagonist assays: SmGPR-3 has a novel pharmacological profile ...... 152 3.4 In vitro motility assays ...... 153 4. Discussion ...... 155 Acknowledgements ...... 160 References ...... 160 Tables ...... 167 Figures ...... 168 CHAPTER V (Discussion and Conclusions) ...... 177 Reference ...... 186 APPENDIX ...... 188 RNA interference (RNAi) studies of SmGPR receptors in cultured Schistosoma mansoni schistosomula ...... 188 1. Introduction ...... 189 2. Materials and Methods ...... 190 XIV

2.1. dsRNA synthesis and production of siRNA...... 190 2.2 Labelling of siRNA ...... 191 2.3. Transfection with siRNA ...... 191 2.4. Quantitative PCR analyses ...... 192 3. Results ...... 193 4. Discussion ...... 195 References ...... 196 Figures ...... 200

LIST OF TABLES

CHAPTER I (Literature Review) Table 1: Effect of neurotransmitters on the flatworms ...... 11 Table 2: The four mammalian HA receptor (H1-H4) classes,...... 14

CHAPTER IV (manuscript III) Characterization of a novel catecholamine receptor (SmGPR-3) in the bloodfluke Schistosoma mansoni Table 1: Pairwise alignments of SmGPR receptors...... 167

Appendix RNA interference (RNAi) studies of SmGPR receptors in cultured Schistosoma mansoni schistosomula Table 1: Primers used in dsRNA/siRNA production and real-time quantative PCR (qPCR) analysis...... 199

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LIST OF FIGURES

CHAPTER I (Literature Review) Figure 1: Life cycle of the bloodfluke Schistosoma ...... 5 Figure 2: The biosynthesis of histamine...... 13 Figure 3: HA role in photoreception in the arthropod eye...... 16 Figure 4: Evolutionary tree of histidine decarboxylase (HDC) in both vertebrate and invertebrates...... 19 Figure 5: The three histamine inactivation strategies...... 20 Figure 6: The biosynthesis of three naturally occurring catecholamines from tyrosine.. .. 23 Figure 7: Major degradation pathways of catecholamines...... 26 Figure 8: The heptahelical structure of three major families of GPCRs (A-C)...... 33

CHAPTER II (manuscript I) Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni Figure 1: Production of an anti-SmGPCR polyclonal antibody ...... 97 Figure 2: Western blot and immunoprecipitation analyses of SmGPCR...... 98 Figure 3: Localization of SmGPCR in larval stages of S. mansoni...... 99 Figure 4: Localization of SmGPCR in adult worms...... 101 Figure 5: Developmental expression of SmGPCR in S. mansoni...... 103

CHAPTER III (manuscript II) Histamine signalling in Schistosoma mansoni: Immunolocalization and characterization of a new histamine receptor (SmGPR-2) Figure 1: Dendogram analysis of biogenic amine GPCRs...... 132 Figure 2: SmGPR-1-like receptors lack the conserved aspartate (D 3.32) of transmembrane domain 3...... 133 Figure 3: Functional expression studies of SmGPR-2 (Smp_043340) in yeast...... 135 Figure 4: Pharmacological studies of SmGPR-2...... 136 Figure 5: SmGPR-2 has an atypical drug profile...... 137 Figure 6: Developmental expression of SmGPR-2 in S. mansoni...... 138 Figure 7: Localization of histamine (HA) in adult S. mansoni...... 139 Figure 8: Localization of SmGPR-2 in S. mansoni...... 140

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CHAPTER IV (manuscript III) Characterization of a novel catecholamine receptor (SmGPR-3) in the bloodfluke Schistosoma mansoni Figure 1: Phylogenetic tree of biogenic amine GPCRs...... 168 Figure 2: SmGPR-3 is a class A GPCR...... 170 Figure 3: Functional expression studies of SmGPR-3 in yeast...... 172 Figure 4: Antagonist effects on SmGPR-3 activity...... 173 Figure 5: Effects of SmGPR-3 agonists on intact schistosomula...... 175 Figure 6: In vitro drug effects on the motor activity of S. mansoni schistosomula: ...... 176

Appendix RNA interference (RNAi) studies of SmGPR receptors in cultured Schistosoma mansoni schistosomula Figure 1: Two strategies for PCR mediated in vitro transcription of dsRNA ...... 200 Figure 2: Gel analysis of dsRNA and ssRNA...... 201 Figure 3: Intake of siRNA by cultured schistosomula...... 202 Figure 4: SmGPCR silencing in the cultured schistosomula...... 203

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LIST OF ABBREVIATIONS

1-metHA: 1-methylhistamine 3-AT: 3-Amino-1, 2, 4-Triazole 5-HT: 5-hydroxytryptamine (serotonin) 7-TMs: seven transmembrane domains A: adrenaline (epinephrine) BA: biogenic amine cDNA: complementary deoxyribonucleic acid CNS: Central nervous system COMT: catechol-O-methyl transferase DA: dopamine DTT: Dithiothreitol el: extracellular loop (GPCR structure) ELISA: enzyme linked immunesorbent assay EPN: Epinine, (deoxyepinephrine) ex / em: excitation and emission wavelengths FITC: Fluorescein isothiocyanate GAPDH: glyceraldehyde 3-phosphate dehydrogenase GPCR: G protein-coupled receptor HA: histamine HRP: horseradish peroxidase IFA: Immunofluorescence assay IgG: immunoglobulin G il: intracellular loop (in GPCR structure) IP: Immunoprecipitation L-DOPA: L-3,4-dihydroxyphenylalanine mRNA: messenger ribonucleic acid MTN: Metanephrine, (3-O-methyl-epinephrine) NA: noradrenaline (norepinephrine)

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NT: neurotransmitter one-way ANOVA: one-way analysis of variance PBS: phosphate buffer solution PBST: phosphate buffer solution + TritonX-100 PCR: polymerase chain reaction PFA: paraformaldehyde PNMT: phenylethanolamine N-methyltransferase PNS: Peripheral nervous system qPCR: quantitative PCR RNAi: RNA-interference rRNA: Ribosomal RNA RT: reverse transcription S. mansoni: Schistosoma mansoni SC medium: synthetic complete medium SDS–PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis ssDNA: single stranded deoxyribonucleic acid TH: tyrosine hydroxylase TRITC: tetramethylrhodamine B isothiocyanate YPD medium: Yeast extract-peptone-dextrose medium

INTRODUCTION

Parasitic flatworms of the genus Schistosoma are important human parasites and the causative agents of schistosomiasis, a debilitating disease that afflicts 210 million people worldwide. Among five human species, S. mansoni is responsible for > 90% of all infections. Praziquantel is the drug of choice but the recent emergence of resistant strains threatens the effectiveness of the drug. It is important to learn more about the basic biology of these organisms and to identify new targets for antischistosomal drugs. Like all other platyhelminths, schistosomes do not have a body cavity (i.e. they are acoelomate), circulating body fluid or classical endocrine system. Instead, they depend on the nervous system for cellular communication, coordination of movement, metabolism, reproduction and other functions that are essential for parasite survival. Thus, the schistosome nervous system is ideally suited for drug targeting. Many neurotransmitters (NTs) have been identified in schistosomes, both classical (small) transmitters and neuropeptides. The most common of the small transmitters are biogenic amines (BAs), which are derived from aromatic amino acids (tryptophan, tyrosine) or histidine and share a protonated amino group. Although they act as NTs, BAs can serve as hormones and neuromodulators as well. Serotonin (5-hydroxytryptamine, 5-HT) is the most ubiquitous BA in flatworms, including schistosomes, and is present throughout the central and peripheral nervous systems, suckers, body wall musculature, and reproductive structures. 5-HT has many important functions in flatworms, particularly in the regulation of carbohydrate metabolism and motor activity. Aside from 5HT, flatworms have histamine and catecholamines (dopamine and noradrenaline) within their nervous system. These BAs have also been implicated in the control of movement but less is known about their mode of action. With few exceptions, most BAs exert their effects by binding to cell surface G-protein coupled receptors (GPCRs). Although there is good pharmacological evidence for

the existence of BA receptors in flatworms, very few of these GPCRs have been cloned or characterized at the molecular level. The genome of S. mansoni encodes 18 putative biogenic amine GPCRs. Among these sequences are seven amine-like receptors that have no vertebrate or invertebrate orthologues but are also present in other Schistosoma species (e.g. S. japonicum). These seven receptors are closely related and seem to have evolved from a common ancestor, forming a new BA receptor clade. One of these sequences, SmGPCR, was cloned previously from S. mansoni and shown to be activated by histamine. SmGPCR (also called SmGPR-1 in this thesis) was the first histamine GPCR ever cloned in invertebrates and became the prototype for this new clade. The goal of this study is to investigate structural, functional and pharmacological properties of these novel receptors. The specific objectives are: First, to continue the characterization of SmGPCR/SmGPR-1 by studying its subunit organization, RNA expression pattern in different life cycle stages and tissue localization in the intact parasite (chapter II). Our goal is to learn more about the function of this histamine receptor by examining when it is expressed during the course of development and where it is located in the organism. Second, to clone and characterize two other members of this new clade, namely SmGPR-2 and SmGPR-3 (chapters III and IV). The main goal of these studies is to identify the natural ligand for each receptor and to investigate their pharmacological profiles, using a variety of receptor agonists and antagonists. As discussed later, the results suggest these are atypical aminergic receptors that can bind not only common BAs but their metabolites as well. Third, we examine the tissue distribution of histaminergic neurons in S. mansoni and compare it to that of histamine receptors, using specific antibodies. (Chapter III). Finally, we test the effects of various BA agonists and antagonists on parasite motility in vitro (Chapter IV). Together, these studies provide important new information on the mechanism of BA signalling in S. mansoni.

CHAPTER I

Literature Review

1 The parasite Schistosoma mansoni

1.1 biology and life cycle

Schistosoma species are blood-dwelling dioecious parasites that possess 7 pairs of autosomal chromosomes and 1 pair of sex chromosomes where the female schistosome is heterogametic, bearing ZW chromosomes while the male is homogametic or ZZ (Short, 1983). The S. mansoni nuclear genome has >360 megabases (MB) and consists of 11,809 putative genes, encoding for 13,197 transcripts in 72 families (Berriman et al., 2009). Male schistosomes develop normally with no morphological difference, whether they are isolated from a single-sex or bisexual infection. In contrast, female schistosomes require growth signals from their male partners to develop properly. Females show underdeveloped immature reproductive systems and are stunted when they are collected from a single-sex infection (Kunz, 2001; LoVerde et al., 2004). Schistosomes exhibit an unusual double (heptalaminate) membrane in some of its developmental stages namely, schistosomula and the adult worm (McLaren and Hockley, 1977) or the common trilaminate membrane with glycocalyx in the other stages.

The major five species of the genus Schistosoma that infect human are: S. mansoni, S. haematobium, S. japonicum, S. intercalatum and S. mekongi. The life cycle patterns of these five species are similar (Figure 1) except for the genus of the snail intermediate host, which varies among the species, the final location within the definitive host and the spine morphology of the egg. The latter is considered to be a diagnostic tool to identify the schistosoma species. The life cycle of S. mansoni has been described in detail elsewhere (Gryseels et al., 2006).The female adult Schistosoma (Fig 1 A) produces hundreds to thousands of eggs per day. These eggs (Fig 1 B) contain embryos which secrete proteolytic enzymes that assist the migration of the eggs from the lumen of the bladder (S. haematobium) and to be excreted in the urine or they migrate from the intestine

(other spp. including S. mansoni) and are excreted in the feces (Gryseels et al., 2006). When the eggs reach the freshwater and in the presence of light, they hatch and release the short-lived, free swimming ciliated larvae, named miracidia (Fig 1 C). With the aid of light and chemical stimuli, the miracidia search for the specific intermediate snail host (Biomphalaria glabrata in the case of S. mansoni) and

Figure 1: Life cycle of the bloodfluke Schistosoma (modified from Gryseels et al., 2006).

penetrates through the snail‟s foot epithelium (Olds and Dasarathy, 2001). Inside the snail (Fig 1 D), the miracidium sheds its ciliated glycocalyx, develops to the mother sporocyst and reproduces asexually to one or more generations of daughter sporocysts, which mature in the snail‟s liver (Olds and Dasarathy, 2001). These daughter sporocysts finally develop into cercariae with characteristic bifurcated tails. The latter start emerging from the snail‟s mantle 30-45 days post 5

infection (Fig 1 E). These short-lived, free-swimming tailed larvae are the infective stage of man and are equipped with glands near the acetabulum that secrete mucus-like substances and proteolytic enzymes to facilitate attachment and penetration of the skin. During skin penetration, the tail is lost in the dermis and within 3-4 hours the cercarial body is fully transformed into schistosomula. The preacetabular and post-acetabular gland contents are fully discharged and the original trilaminate glycocalyx is changed into a heptalaminate double lipid bilayer as an adaptation to the new serum and salt-rich host environment (Stirewalt, 1974; Wiest et al., 1988). To prevent host immune attack, the parasitic schistosomulae take host components and attach them on their surface and gain access to the host‟s veins within the first two days. They circulate in the blood to the heart and then the lungs in the 5-7days post infection (Miller and Wilson, 1980). In intestinal schistosomes (all human schistosome spp. except S. haematobium), schistosomulae reach the heart from the lungs via pulmonary capillaries and then to liver where they mature within 4-6 weeks in the portal veins and eventually they migrate to the mesenteric veins and mate (Olds and Dasarathy, 2001).The average life span of the adult worms is 3-5 years but they can live longer (over 30 years) in the definitive host (Gryseels et al., 2006).

1.2 Pathology and immune evasion mechanisms

The bloodfluke Schistosoma infects over 200 million people in tropical and subtropical zones and causes schistosomiasis. Although the disease does not manifest complications in the majority of infected people, approximately 10% of schistosome-infected individuals develop serious disease (Kheir et al., 1999; Forrester and Pearce, 2006). It was estimated that the number of deaths directly attributable to schistosomiasis in sub-Saharan Africa is at 280,000 per annum (van der Werf et al., 2003), making it second only to malaria among parasitic diseases as a cause of human suffering. Schistosome eggs are the primary source of morbidity and schistosomiasis is caused mainly by the immune response to tissue- trapped eggs. Two problems are associated with schistosome eggs: First, the high 6

fecundity of the adult female worms results in production of as many as 300 eggs/day in S. mansoni and up to 3000 eggs/day in S. japonicum (Moore and Sandground, 1956). Second, while many eggs are excreted out the body, about half of them become trapped in vital tissues, particularly the liver, resulting in portal hypertension, periportal fibrosis and the serious clinical manifestations of intestinal schistosomiasis, such as hepatosplenomegaly (LoVerde et al., 2004).

To stop the disease successfully, the human immune system should respond to the vulnerable pre-adult stages (Wilson, 1987) since there is no evidence of immune clearance after the development of the adult parasitic stage (Agnew et al., 1993). The parasite uses host immune components for its own benefit and development since both parasite fecundity and egg excretion are reduced in severe combined immunodeficiency (SCID) mice that lack functional B- and T-cells (Ozaki et al., 1997). CD4+ T-cells influence S. mansoni development by secreting interleukin-7 (Wolowczuk et al., 1999; Davies et al., 2001; Saule et al., 2005; Blank et al., 2006). The tegument of the human parasite stages expresses several receptors that bind to the host‟s components. For example, the tegumental expression of ser/thr kinases SmRK1 and SmRK2, which bind host-derived, transforming growth factor β, (TGFβ) (Davies et al., 1998; Forrester et al., 2004). The different stages of schistosomes have developed various mechanisms of evasion, which enable them to survive and overcome the definitive host immunity. For instance, cercariae secrete the acetabular gland proteases including elastase for quick skin penetration and they undergo a complete transformation into schistosomula within 3-4 hours by shedding the glycocalyx, evacuation of the acetabular glands and formation of the heptalaminated tegument. Schistosomula have the ability to produce several prostaglandins (PG) including PGD2 and PGE2, which interfere with the migration of dendritic cells (Angeli et al., 2001). Moreover, the skin stage schistosomula secrete a 23 kDa protein that induce apoptosis of skin T lymphocytes and was termed S. mansoni-derived apoptosis- inducing factor (Chen et al., 2002). On the other hand, the lung stage

schistosomula produce PGE2 which stimulates IL-6; the latter facilitates parasite migration within the lung tissue by diminishing the inflammation in that organ (reviewed by Forrester and Pearce, 2006). By the time they reach the lung stage, the growing schistosomula are resilient to the host‟s antibody and complement- mediated cellular cytotoxicity that can kill the newly transformed schistosomula.

The adult stage is more vulnerable to the host‟s immune system but they evade attack by masking their immunogenic surface proteins with host‟s antigens (Forrester and Pearce, 2006). Another way of immune evasion is by shedding parasite surface antigens (Pearce et al., 1986). Although both Th1 and Th2 are subsets of the CD4+ T-lymphocytes, Th1 produces interferon γ (INF-γ) and usually is stimulated by intracellular pathogens, whereas Th2 is activated by helminths and synthesizes IL-4, IL-5, IL-10 and IL-13 (reviewed by Forrester and Pearce, 2006). In schistosomiasis, both Th1/Th2 are working but there is a preferential activation of Th1 in the pro-inflammatory stages and it shifts into Th2 as the parasite starts to produce eggs (Grzych et al., 1991; Pearce et al., 1991). IL-4 and IL-10 are important for the Th1 to Th2 shift and when IL-4 is absent, as in IL-4 deficient mice, Th1 will continue even after egg production. Unless they die and their antigens are exposed to the host immune system, the intact adult bloodflukes usually cause very little damage to their host. As already noted, it is the eggs that cause most of the pathology including liver cirrhosis, fibrosis and granuloma formation (Andrade and Cheever, 1995; Wilson et al., 2007). The eggs are resistant to the host‟s proteases, thanks to glycine rich protein of the eggshell, which is cross linked by tyrosine residues (Cordingley, 1987). Schistosomes can contribute to cancer, especially in S. haematobium infection (Christie et al., 1986; Botelho et al., 2009).

1.3 The nervous system of S. mansoni

Flatworms do not possess endocrine or circulatory systems and since they are acelomates (i.e. lack a body cavity), they rely on their nervous system for

coordination of all major activities such as feeding, reproduction, movement and development (Halton and Maule, 2004; Ribeiro et al., 2005). The basic plan of the nervous system in S. mansoni is similar to other flatworms, which is composed of both a central nervous system (CNS) and a peripheral nervous system (PNS). The former consists of anterior bilobed cerebral ganglia connected by ring-like commissures and three pairs of longitudinal nerve cords (LNC). The latter are connected by multiple transverse commissures, which gives the CNS of flatworms the appearance of a ladder or orthogonal shape (Halton and Maule, 2004; Maule et al., 2006). The PNS consists of nerve plexuses that are present in the subtegumental region and are associated with the digestive and the reproductive systems (Ribeiro et al., 2005; Maule et al., 2006). The PNS supplies the body wall muscles and provides neuron input to sensory and attachment organs (Halton and Maule, 2004). The association between nerves and muscle in these worms is extremely important and the somatic muscle consists of several layers, including an outer circular muscle layer, inner longitudinal muscle fibers and fewer diagonal muscles that run beneath the longitudinal muscles (Mair et al., 2000; Mair et al., 2003; Halton and Maule, 2004b). Circular and longitudinal muscle fibers are responsible for the elongation and shortening of the animal, respectively, while the diagonal muscles are required for bending (Halton and Maule, 2004b). The body wall musculature is innervated by neuronal plexuses that are enriched in the subtegumental region.

1.4 Neurotransmitters in S. mansoni and other flatworms

The definition of a neurotransmitter (NT) is a chemical that is synthesized in the presynaptic neuron, stored in synaptic vesicles and is released into the synaptic cleft upon the arrival of the action potential. In the synapse, it binds and activates a specific receptor that is expressed in the postsynaptic neuron to conduct the nerve impulse. NTs include neuropeptides, amino acids, biogenic amines and other chemicals like acetylcholine and diffusible gases such as nitric oxide NO and carbon monoxide CO. The different NTs detected in flatworms are 9

summarized in Table 1. The biogenic amines (BAs) constitute a class of NTs that are derived from aromatic amino acids or histidine. They include 5- hydroxyl tryptamine (5-HT), also called serotonin, which is derived from tryptophan, histamine (HA) derived from histidine, and the catecholamines, dopamine (DA), noradrenaline (NA), which are tyrosine derivatives. Since they bind and activate several receptors present in the flatworms, NTs can induce different physiological effects, many of which have been reported (Halton and Maule, 2004; Ribeiro et al., 2005; Maule et al., 2006). Table 1 summarizes the effects of NTs in platyhelminths, including schistosomes. In general, serotonin acts as a stimulator, increasing motility, muscle contraction, tissue regeneration and energy production. In contrast, acetylcholine is myoinhibitory in the majority of flatworms, except for the free-living turbellarian Bdelloura candida, where it is myoexcitatory (see the review of Halton and Maule, 2004). Moreover, ACh enhances glucose transport via nicotinic-like acetylcholine receptors (nAChR) that are present in the tegument of some schistosome species (Camacho and Agnew, 1995). Glycine (Gly) and γ-Amino butyric acid (GABA) are inhibitory neurotransmitters while glutamate induces myoexcitation (Table 1). Neuropeptides can modulate neuronal activity, enhance muscle contraction and affect egg development (Maule et al., 1989; Marks et al., 1997). The diffusible gas nitric oxide NO acts as a NT in flatworms and modulates feeding behavior in planarians (Eriksson, 1996), reproduction and development in H. diminuta and S. mansoni (Gustafsson et al., 1996; Kohn et al., 2001) and also has a potential myoinhibitory role in F. hepatica (Gustafsson et al., 2001). Additional detail on the functional roles of these transmitters can be found in several recent reviews (Halton and Maule, 2004; Ribeiro et al, 2005; Maule et al., 2006). Here we will focus our attention on those NTs that are relevant to the thesis, in particular catecholamine and histamine in S. mansoni.

Table 1: Effect of neurotransmitters on the flatworms (modified from Ribeiro et al, 2005) Neurotransmitter Species Activity

Fh2,23, Sm5,8, 25, Hd6,24, Hc22 ↓ contraction (flaccid paralysis)

Acetylcholine Bc26, Pl11 ↑ contraction (ACh) Dg27 Hypokinesia

Sm & Sh28, 29 ↑ glucose transport

Sm 1,3,7, 8, 10,13, Fh12,13, Hd6,, Pl11, Dm, ↑ contraction and motility Hc22 Serotonin (5-HT) Dm15,16 , Pt37 ↑ tissue regeneration16, ↓ RNA synthesis37

Sm14, Fh12,13, Hd47 ↑ metabolism (glycogenolysis), worm migrational response

Histamine (HA) Sm20, 21, Hd6,39, Hc45 , Dd46 ↑ motility, regulation of reproduction45, modulates the excretory duct permeability46, worm (distal) migrational response39.

Dm4, Fh2 ↑ contraction, ↑ motility (DA)

Catecholamines Sm 3,5,7, 8,17 , Fh2 ↑worm length, ↓ contraction (DA, NA and A) motility (NA & A), ineffective3

Dg18,19,27 Hyperkinesia

Pt37, 38 ↑ regeneration and ↑ RNA synthesis

Glycine Na31 ↓ electrical evoked activity (spike)

GABA Na31 ↓ electrical evoked activity (spike)

Glutamate Sm40, Hd9, ↑ contraction

Gf 30 ↑ spike

Neuropeptides 32 Fh43,44, Sm33,44, PI11, Bc42, Dm4 ↑ contraction, myoexcitatory43, neuronal functioning and egg development

Nitric Oxide (NO) Dt34, Hd35, Sm36, Fh41 Feeding role34, neuronal signalling, reproduction and development35-36 , myoinhibitory role41

Turbellarians: Pl, Procerodes littoralis; Na, Notoplana acticola; Dg, Dugesia gonocephala; Bc, Bdelloura candida; Dt, Dugesia tigrina; Pt, Polycelis tenuis

Monogeneans: Dm, Diclidophora merlangi

Trematodes: Fh, Fasciola hepatica; Sm, Schistosoma mansoni; Sh, Schistosoma haematobium, Dm, Diclidophora merlangi; Hc, Haplometra cylindracea

Cestodes: Hd, Hymenolepis diminuta; Gf, Gyrocotyle fimbriata; Dd, Diphyllobothrium dendriticum.

1. Boyle et al. 2000 17. Tomosky et al. 1974 33. Day et al. 1994b

2. Holmes &Fairweather, 1984 18. Venturini et al. 1989 34. Eriksson 1996

3. Hillman & Senft, 1973 19. Palladini et al. 1996 35. Gustafsson et al, 1996

4. Maule et al. 1989 20. Ercoli et al. 1985 36. Kohn et al, 2001

5. Mellin et al. 1983 21. Mousa, 2002 37. Franquinet & Martelly 1981

6. Sukhdeo et al. 1984 22. McKay et al. 1989 38. Franquinet, 1979

7. Pax et al. 1981 23. Sukhdeo et al. 1986 39. Mettrick and Podesta 1982

8. Pax et al. 1984 24. Thompson et al. 1986 40. Miller et al. 1996

9. Thompson & Mettrick, 1989 25. Day et al. 1996 41. Gustafsson et al, 2001

10. Day et al. 1994a 26. Blair & Anderson, 1993 42. Johnston et al, 1996

11. Moneypenny et al. 2001 27. Buttarelli et al. 2000 43. Marks et al, 1997.

12. Mansour, 1979 28. Camacho & Agnew, 1995 44. Marks et al, 1995

13. Mansour, 1984 29. Camacho et al. 1995 45 Eriksson et al 1996

14. Rahman & Mettrick, 1985 30. Keenan & Koopowitz, 1982 46.Wikgren et al, 1990

15. Martelly& Franquinet, 1984 31. Keenan et al. 1979 47. Cho and Mettrick, 1982

16. Saitoh et al. 1996 32. see Halton & Maule, 2004

2 Histamine and its receptors

2.1 HA biosynthesis and its roles in vertebrates

Histamine (HA, also 2,4-Imidazolyl ethylamine) is synthesized from histidine via a single decarboxylation reaction catalyzed by the enzyme histidine decarboxylase (HDC, EC 4.1.1.22) (Fig. 2). Histamine and its precursor amino acid histidine exist in two isomers, based on the location of the double bond that 12

shifts between the two nitrogens of the imidazole ring. The closer nitrogen atom to the side chain is named pros π and the second is called tele  (Black and Ganellin, 1974). In humans, HA is present within the nervous system and cells of the immune system, particularly mast cells and basophils. HA regulates various body functions, including digestive, immune, circulatory and respiratory. It acts as an inflammatory mediator in allergic reactions, including asthma. It lowers blood pressure due to its vasodilation properties and controls the heart rate as well. In the nervous system, HA functions as a neurotransmitter, both in the CNS and periphery. Histaminergic neurons control release of gastric acid in the stomach and also stimulate the smooth muscles of the bronchi and the intestinal tract to contract.

Figure 2: The biosynthesis of histamine. Histamine is synthesized from histidine via a decarboxylation reaction catalyzed by histidine decarboxylase (HDC). The two π and  tautomers of histamine are shown.

2.2 Histamine GPCR receptors in vertebrates

In mammals including humans, the diverse effects of HA are due to the presence of four main classes of receptors, H1-H4, which are expressed in different tissues (Hill et al., 1997; O'Reilly et al., 2002). Although they all belong to the superfamily of GPCRs (see the GPCR, section 4), the histaminergic receptors H1

and H2 are structurally unrelated with ≤ 30% sequence homology. H1 receptors have a long third intracellular loop while H2 receptors have shorter il3 and relatively longer C-terminal tails (Rangachari, 1998). In contrast, H3 and H4 receptors are related (O'Reilly et al., 2002) with at least twenty different H3 spliced forms (Coge et al., 2001; Strakhova et al., 2008; van Rijn et al., 2008).

Table 2: The four mammalian HA receptor (H1-H4) classes, modified from (Hill et al., 1997; O'Reilly et al., 2002).

HA affinity for G-protein tissue expression Action class HA (Kd)

2+ Heart, CNS, most smooth ↑ [Ca ], ↑IP3, ↑NO muscles (s. m.), brochoconstriction, HHigh Gα q/11 endothelium, airway s. m., motion sickness, allergic 1 (nM range) lymphocytes, GI, Adr. symptoms, endothelial medulla, placenta. cell contraction

Hlow Stomach parietal cells, ↑HCl secretion, ↑cAMP, 2 CNS, heart, neutrophils, s. m. relaxation, inhibits (µM range) Gα s uterus, suppressor T cells. lymphocyte function, ↓ firing rate.

HHigh CNS and to a lesser extent ↓ cAMP, decreases NT 3 PNS (to lung, heart, GI). release (HA, NA, 5HT (nM range) Gα i/o and ACh)

HHigh Mast cells, basophils, ↓ cAMP, chemotaxis (in 4 eosinophils, thymus, eosinophils) (nM range) Gα i/o spleen, B. marrow and small intestine

Recently, the first alternatively spliced variants of human H4 receptor were cloned and were found to play a dominant negative role when they heterodimerize with the human H4 receptor (van Rijn et al., 2008). The four mammalian HA receptors possess different binding affinities, different signaling pathways and tissue localization as illustrated in Table 2. 14

2.3 Histamine in non-helminthic invertebrates

The role of HA in invertebrates is still poorly understood. The best evidence of histaminergic activity comes from studies of arthropods. HA serves as a photoreceptor neurotransmitter in insects (Hardie, 1987; Nassel et al., 1988) as both the compound eyes and the ocelli “simple eyes” of insects are enriched in histamine. HA was shown to play a NT role in all three major groups of arthropods: insects, such as cockroaches, locusts, crickets, honey bee, blowflies, and Drosophila (Nassel et al., 1988; Monastirioti, 1999; Nassel, 1999), crustaceans (Callaway and Stuart, 1999; Stuart, 1999) and chelicerata such as the horseshoe crab, Limulus (Battelle et al., 1991) and spiders (Schmid and Becherer, 1999). Light depolarizes the arthropod photoreceptors, allowing them to release HA (Stuart et al., 2007). As a photoreceptor transmitter, histamine acts on ligand- gated chloride channels (Nassel, 1999). Once it is released from the presynaptic photoreceptor in the presence of light, HA acts as an inhibitory transmitter on postsynaptic chloride channels (Figure 3) and causes hyperpolarization of the postsynaptic interneuron (Stuart, 1999; Stuart et al., 2007). The genes encoding for HA-gated Cl- channels were cloned from the fruit fly (Gengs et al., 2002; Gisselmann et al., 2002; Witte et al., 2002; Zheng et al., 2002). The pharmacology of the HA-gated Cl- channel in arthropods is related to the mammalian H2 receptor but the proteins are structurally unrelated (Hardie, 1987; Stuart, 1999).

The arthropod ionotropic receptor is a member of the superfamily of Cys- loop ligand-gated channels and shares homology with GABAA and glycine-gated chloride channels (Hardie, 1989; Hatton and Yang, 2001; Schnizler et al., 2005). HA-gated channels have not been identified in mammals and may be invertebrate- specific. In addition to photoreception, HA inhibits NT release from the presynaptic olfactory lobe in the spiny lobster (Wachowiak et al., 2002) and serves as a central transmitter in the cerebral ganglia of the marine gastropod Aplysia californica (Weinreich et al., 1975; Prell and Green, 1986), fresh water Lymnaea stagnalis (Turner et al., 1980; Hegedus et al., 2004) and the terrestrial 15

mollusc Helix pomatia (Hegedus et al., 2004). The receptors involved in these effects have not been identified yet.

Figure 3: HA role in photoreception in the arthropod eye. When light depolarizes the photoreceptor, it induces the release of HA, which acts as an inhibitory NT in the postsynaptic interneurons. The effect of HA is mediated by ligand-gated Cl-channels and causes hyperpolarization of the postsynaptic membrane (Stuart 1999).

2.4 Histamine in platyhelminths: presence and biological roles

The first report of HA and HDC in flatworm extracts was published over forty five years ago (Mettrick and Telford, 1963). HA was found in significant amounts in crude extracts of several parasitic worms, including the trematodes Mesocoelium monodi and Fasciola hepaticaand the cestode Oochoristica ameivae (Mettrick and Telford, 1963). The level of HA in M. monodi was nearly 200 times greater than its toad host (Bufo marinus) and the parasite was found to have high levels of the HA biosynthetic enzyme, HDC, suggesting the source of HA was the parasite itself (Mettrick and Telford, 1963). On the other hand, as a part of the same study, the researchers detected small amounts of HA present in F. hepatica and Oochoristica ameivae, which were probably derived from the host (Mettrick and Telford, 1963). In a separate study, the amphibian trematode, Haplometra

cylindracea was reported to have an exceptionally high content of endogenous histamine and the amine was confirmed by HPLC, HDC assay and immunolocalization (Eriksson et al., 1996). The latter method demonstrated widespread distribution of histaminergic neurons in the nervous system of the fluke, both in the CNS and peripheral elements. HA immunoreactivity was found throughout the cerebral ganglia, the longitudinal nerve cords, the innervation of the suckers, the musculature of the pharynx, subtegumental neuroplexuses and the reproductive system. According to Eriksson et al. (1996), the tissue level of HA in Haplometra cylindracea is 800 pmol/mg, nearly 50% higher than that of the related amphibian fluke, M. monodi (520 pmol/mg), both of which are among the highest HA levels ever reported in the animal kingdom. It was suggested that HA could play important roles in sensory and motor neurons of H. cylindracea, as well as in the regulation of reproduction of the parasite (Eriksson et al., 1996). In the plerocercoid larvae of the fish cestode Diphyllobothrium dendriticum, HA was immunolocalized in the ganglia in the scolex and in the nerve cords and in the cells associated with the excretory system (Wikgren et al., 1990). The authors of this study speculated that HA could influence the permeability of the excretory ducts by acting on the smooth muscle lining of the duct wall (Wikgren et al., 1990). Since cestodes lack a digestive system, their excretory system may play an important role in food absorption and distribution (Lindroos and Gardberg, 1982). Thus HA can modulate the physiology in the excretory system in cestodes. HA was also identified in the excretory system-associated nerves of the free living turbellarian Microstomum lineare and in the NS of another turbellarian, Polycelis nigra (Wikgren et al., 1990). HA modulated the peristaltic-like motility of the strobila of the rat cestode Hymenolepis diminuta in a dose-dependent manner (Sukhdeo et al., 1984). At a low concentration of 10-9 M, HA significantly enhanced movement of the posterior strobila while it had myoinhibitory effect at higher concentrations of 10-6 M and 10-3 M (Sukhdeo et al., 1984). Moreover, when HA (40 mg / animal) was administered orally to H. diminuta-infected rats, it caused redistribution of the tapeworms, which were seen to migrate to the distal 17

portion of the small intestine (Mettrick and Podesta, 1982). Like the trematode F. hepatica and the cestode Oochoristica ameivae (Mettrick and Telford, 1963), histamine is derived from the host in H. diminuta, taken up by diffusion rather than by a carrier mediated mechanism and is metabolized by an endogenous diamine oxidase to imidazole acetic acid (Yonge and Webb, 1992).

In S. mansoni, HA increased motor activity in cercariae in a dose-dependent manner, while antihistaminic H1 drugs caused paralysis to both the larvae and the adult worms (Ercoli et al., 1985; Mousa, 2002; Ribeiro et al., 2005). HA at high concentration can reverse the paralysis effect of the drugs (Ercoli et al., 1985; Mousa, 2002). In 2002, a first HA- responsive GPCR was cloned from S. mansoni and named SmGPCR (Hamdan et al., 2002). A distinctive feature of SmGPCR (also named SmGPR-1 in this thesis is that it lacks an important functional residue, an aspartate, located in the TM3 region (D3.32), which it highly conserved in all BA receptors of vertebrate and invertebrate origin. This aspartate is one of the principal ligand binding sites and its side-chain carboxylic group forms an ionic interaction with the protonated amino moiety of the various amine ligands. In SmGPCR, D3.32 is replaced with asparagine and a reverse mutation N3.32D did not change receptor activity, suggesting that position 3.32 is not directly involved in ligand binding (Hamdan et al., 2002). An orthologue of SmGPCR was identified in S. japonicum with accession # AAX28307 and was found to bear N3.32 as well. Recently, after the S. mansoni genome database was completed, a total of 6-7 genes encoding putative biogenic amine GPCRs were shown to be structurally related to SmGPCR and were described as SmGPR1-like receptors. The majority of these receptors carry the same N3.32 substitution (see chapters 3 and 4 for more detail) and are believed to constitute a new clade of BA receptors. As discussed later, one of the major objectives of this thesis is to determine if any of these SmGPCR-related receptors is similarly activated by HA.

Despite the evidence in support of a histaminergic signaling system, HA has not yet been detected in the nervous system of schistosomes and very little is

known about HA biosynthesis or inactivation in this parasite. Histaminase (diamine oxidase) was reported in cercariae and adults of S. mansoni (Schwabe and Kilejian A., 1968). In addition, the S. mansoni genome contains a putative

Figure 4: Evolutionary tree of histidine decarboxylase (HDC) in both vertebrate and invertebrates. The putative HDC of S. mansoni is marked by an arrow. The protein alignment sequence and tree construction were analyzed using CLC sequence viewer (6.0.2) with the UPGMA algorithm and bootstrap analysis of 1000 replicates. histidine decarboxylase HDC encoding gene (Smp_135230, accession #CAZ30088) that shows significant homology with aromatic amino acid decarboxylases and HDCs from other species (Fig. 4). Cloning and studying the biochemistry of schistosomal HDC will shed light on whether the parasite can adequately synthesize its endogenous HA or whether it must obtain the amine from the host.

2.5 Inactivation of HA signaling

HA signaling can be terminated through desensitization and internalization of HA receptors, by transporters or enzymatically (Beaven, 1982; Rangachari, 1998). Two major enzymatic pathways (Figure 5) are involved (1) diamine oxidase catalyzes the conversion of HA to imidazole acetaldehyde and

subsequently to imidazole acetic acid and (2) histamine methyl-transferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine (SAM) to HA, producing the main HA metabolite, N-tele- methylhistamine (N-

Figure 5: The three histamine inactivation strategies modified from (Rangachari, 1998). When HA is terminated enzymatically, three enzymes (shaded) in two pathways can convert HA into various metabolites.

methylHA). The latter serves as a substrate for a second enzyme N-methylHA oxidase to produce an acetic acid derivative (Fig. 5). In some invertebrates, however, glutamyl-histamine was found to be the major product of histamine metabolism in Aplysia (Stein and Weinreich, 1983) while it is converted into imidazole acetic acid in the cestode H. diminuta (Yonge and Webb, 1992). Desensitization (i.e. attenuation of the response in the presence of the ligand) occurs when ser/thr kinases phosphorylate residues in the cytoplasmic side of HA 20

receptors. This causes the phosphorylated receptor to be internalized and eventually degraded, such that the number of receptors available to bind HA is diminished (for more explanation, see GPCR section). Histamine can also be inactivated by specific Na+-dependent HA-transporters, which remove the amine from extracelular spaces to terminate the signaling (Corbel and Dy, 1994; Corbel et al., 1997).

3 Catecholamines and their receptors

Having reviewed the properties of histamine, I will now discuss the other BA system of interest to this thesis, the catecholamines. This next section will review mechanisms of catecholamine biosynthesis and degradation as well as receptor- mediated roles in vertebrates and invertebrates, with particular emphasis on flatworms.

3.1 Catecholamine biosynthesis and degradation

Catecholamines are derived from the aromatic amino acid tyrosine (Fig. 6). The first and rate-limiting reaction in catecholamine biosynthesis is mediated by tyrosine hydroxylase (TH, EC 1.14.16.2), which produces 3, 4-dihydroxy phenylalanine (DOPA). The subsequent decarboxylation of DOPA, catalyzed by an aromatic amino acid decarboxylase (AADC, EC 4.1.1.28) synthesizes the first catecholamine in the pathway, dopamine (DA). DA, in turn, is converted to noradrenaline (NA) by the activity of dopamine- β -hydroxylase (DBH) and NA is further metabolized by phenylethanolamine-N-methyltransferase (PNMT) to produce adrenaline (A) in the presence of the electron donor ascorbic acid (Levine et al., 1985). An alternative, though minor biosynthetic route converts DA to deoxyepinephrine, also known as epinine (EPN), which is then hydroxylated to adrenaline (Laduron et al., 1974). Once released from the cell, catecholamines are rapidly inactivated by carrier-mediated reuptake followed by enzymatic degradation. In the case of DA, the amine is degraded through the sequential activities of monoamine oxidase (MAO) and catechol-O-methyl-transferase 21

(COMT) to produce either 3,4,-dihydroxyphenylacetic acid (DOPAC) or 3- methyxytyramine (3-MT), depending on the pathway, and homovanillic acid (HVA) (Fig. 7). NA and A are converted to normethanephrine (NMT) and metanephrine (MTN), respectively, through the activity of COMT or are oxidatively deaminated by MAO to produce dihydroxymandelic acid. (Fig. 7 i and ii). The DA synthesis pathway is widely conserved in both invertebrates and vertebrates (Kumer and Vrana, 1996).

3.2 DA receptors in vertebrates

3.2.1 DA receptor classes, structure and expression

The effects of DA in vertebrates are mediated by multiple metabotropic membrane receptors, all of which are members of the GPCR superfamily. The importance of studying DA receptors is due to their association with many human pathologies such as Parkinson‟s disease and schizophrenia, cardiovascular diseases, hypertension and drug addiction (Seeman and Van Tol, 1994; Sokoloff et al., 1995). In vertebrates, DA receptors belong to two classes (D1-like and D2- like) based on structural homology, pharmacology, ligand binding affinity and their modulation of adenylyl cyclase (AC) activity and cAMP accumulation. Despite their common specificity for DA, these two classes of receptors are structurally distinct; they share about the same level of sequence homology as they do with other biogenic amine receptors (Callier et al., 2003). D1 activates AC and increases production of cAMP whereas D2 inactivates AC and causes a decrease in cAMP concentration (Callier et al., 2003). In mammals, the D1 class is further subdivided into two receptor subtypes (D1/D1A and D5/D1B), which share

80% identity in their TMs and additional subtypes (D1C and D1D) are present in non-mammalian vertebrates (Dearry et al., 1990; Monsma et al., 1990; Sunahara et al., 1991; Tiberi et al., 1991; Sugamori et al., 1994; Demchyshyn et al., 1995; Lamers et al., 1996; Le Crom et al., 2004).

Dopamine D2 receptors include three subtypes D2, D3 and D4 that share between 53-75% homology within the TM regions (Bunzow et al., 1988; Sokoloff et al., 1990; Van Tol et al., 1991 and reviewed by Missale et al., 1998). In both classes, D1 and D2 are the most abundant subtypes (Missale et al., 1998; Callier et

Figure 6: The biosynthesis of three naturally occurring catecholamines from tyrosine.

Tetrahydrobiopterin (BH4) is the cofactor required for activity of tyrosine hydroxylase in the rate limiting step of catecholamine biosynthesis. Another cofactor ascorbic acid is the elector donor for β-hydroxylation of DA into NA but it is not essential for the conversion of EPN to adrenaline (see Fig. 7 for more details). The coenzyme S-adenosyl methionine (SAM) donates a -CH3 group in the synthesis of adrenaline.

al., 2003). The receptors of the two classes are different in their protein structure and topology. For example receptors of the D1 class have a short third intracellular loop (il3) and very long C-terminal tail, while D2 class receptors are characterized by a very long il3 and a short cytoplasmic C-terminus (Chapter 4, Fig). Another difference is the G proteins that interact with these receptors. D1 receptors mainly interact with Gs and Golf class of Gα proteins while D2 GPCRs bind to Gi/Go proteins. Since D1 encoding genes are intronless, they can be isolated easily from genomic DNA. In contrast, it is more difficult to clone D2 genes from genomic DNA due to the presence of three introns (D4), five introns

(D3) and six introns in D2 receptors (reviewed by Missale et al., 1998). The D2 receptor has two isoforms (the longer D2L and the shorter D2S), which result from the alternative splicing in the long il3 region (Dal Toso et al., 1989; Monsma et al., 1989; Merchant et al., 1993; Xu et al., 2002; Jomphe et al., 2006). Moreover, the two isoforms of D2 inhibit AC and have similar pharmacology but D2S displays a higher affinity (i.e. stronger AC inhibition) than D2L when expressed in cultured cells (Dal Toso et al., 1989; Montmayeur and Borrelli, 1991). In contrast, the alternative splicing of the D3 results in a nonfunctional receptor (Giros et al., 1991; Fishburn et al., 1993).

In agnathans (jawless vertebrates), a D1 like sequence was identified but it was unrelated to any of the D1 receptors in other vertebrates. Three different D1 receptors (D1A - D1C) were identified in cartilaginous fish and amphibians (Sugamori et al., 1994). In teleost (bony) fish, an extra D1 receptor sequence was cloned (Cardinaud et al., 1997). Among the four receptors in D1 class, D1A is the most conserved and probably it is the most functionally important, while other receptors have been lost during evolution. For example D1D is absent in bony fish and amphibians (Callier et al., 2003). Due to the availability of specific antibodies, immunolocalization studies indicated that the different receptor subtypes exhibit discrete expression patterns within the nervous system. Among

D2-like receptors, D4 is less abundant than D2 but they do overlap in some regions

Figure 7: Major degradation pathways of catecholamines. (i). Methylation of dopamine (DA) by PNMT produces epinine (EPN); 3-O-methylation of noradrenaline (NA) and adrenaline (A) by Mg2+-dependent COMT produces normetanephrine (NMT) and metanephrine (MTN), respectively. (ii). Oxidative deamination of catecholamines by monoamine oxidase (MAO), followed by 3-O-methylation by COMT. SAM coenzyme is the donor of -CH3 group in the synthesis of all the five catecholamine metabolites EPN, MTN, NMT, HVA and VMA. The major DA metabolite is homovanillic acid (HVA).

of the brain, whereas D3 expression does not share the location of D2 (Khan et al.,

1998; Callier et al., 2003). Although they do not overlap, both D1 and D2 are expressed in the post-synaptic neurons and at the axon terminal of presynaptic neurons of the mammalian brain (Levey et al., 1993; Smiley et al., 1994).

3.2.2 Vertebrate DA receptor signalling and catecholamine binding domain

As noted earlier, D1-like (D1 and D5) receptors couple to Gαs/Gαolf, stimulate AC and cause accumulation of cAMP in the tissues that express them. The resulting activation of cAMP-dependent PKA causes phosphorylation of many cellular proteins and ultimately leads to a physiological response. However, some reports indicated the ability of D1-like receptors to couple with Gαi or Gαq, suggesting that alternative signaling pathways are possible (Kimura et al., 1995;

Wang et al., 1995). In addition, D1 can couple to Gαo and D5 to Gαz both of which are able to modulate ion (Ca2+, Na+ and K+) channels (Sidhu and Niznik, 2000; Perez et al., 2006). The two subtypes of D1 differ in several aspects. For instance,

D5 exhibits a higher affinity to DA and other agonists and a lower affinity to inverse agonists than D1. These changes are due to the differences in the cytoplasmic tail that regulates DA binding affinity and in receptor desensitization (Sugamori et al., 1998; Jackson et al., 2000).

Similar to the D1 class, D2-like receptors can modulate ion channels

(reviewed by Missale et al., 1998 and Callier et al., 2003). For example D2 receptors inhibit voltage-gated Ca2+ and activates K+ channels, which leads to hyperpolarization (Lledo et al., 1992; Greif et al., 1995) and D3 and D4 can inhibit 26

2+ Ca channels as well (Seabrook et al., 1994). D2 stimulates the release of arachidonic acid (AA) mediated by activation of phospholipase A2 and Protein Kinase C (Kanterman et al., 1991; Di Marzo et al., 1993; Bhattacharjee et al.,

2005). Similar to D2, D4 enhances AA release while D3 does not (McAllister et al., 1993; Chio et al., 1994; MacKenzie et al., 1994). In contrast, the activated D1 expressed in CHO cells did not stimulate AA release when it was expressed alone but when it was co-expressed with D2, it showed a synergetic effect on AA release greater than D2 alone (Piomelli et al., 1991). Site-directed mutagenesis and protein modeling indicated that catecholamine ligands bind within the transmembrane (TM) domains of their receptors, particularly TMIII, V and VI. The principal binding residues include Asp3.32 of TMIII (Strader et al., 1988), two serines of TMV (Wang et al., 1991; Cox et al., 1992; Mansour et al., 1992; Tomic et al., 1993) and a conserved phenylalanine present in TMVI. For more detail about GPCR structure, see section 4 in this review.

3.3 Catecholamines in the invertebrates

3.3. 1 Roles of catecholamines in the invertebrates

DA is a major neuroactive substance among invertebrates. It is present in every phylum tested to date and its range of activities is very broad. In the lobster, Homarus gammarus, DA was identified in the nervous system and was found to modulate the activity of swimming motor neurons (Barthe et al., 1989; Cournil et al., 1994). DA also has an excitatory effect on the cardiac ganglion of several crustaceans and it stimulates the ventilation motor neurons in the shore crab (Rajashekhar and Wilkens, 1992). Both DA and proctolin play a role in courtship behaviour of blue crabs (Wood et al., 1995; Wood and Derby, 1996) and DA increases Na+/K+-ATPase activity and influx of Na+ in the green crab Carcinus maenas, which subsequently increases the concentration of cAMP in the animal gills (Sommer and Mantel, 1991). It was shown that DA suppresses the immunity of several crustaceans like the giant river prawn Macrobrachium rosenbergii and

increases its susceptibility to bacterial infection (Li et al., 2005). In addition, DA was implicated in stress-induced immunosuppression in tiger shrimp (Chang et al., 2007) and served as an immunomodulator in Litopenaeus vannamei shrimp (Cheng et al., 2005). In other studies, both DA and NA induced hyperglycaemic responses in the crayfish Procambarus clarkii and in the giant river shrimp M. rosenbergii (Zou et al., 2003; Hsieh et al., 2006). Moreover, DA modulated Ca2+ channels in pyloric neurons of lobster stomatogastric ganglion (Johnson et al., 2003) and significantly reduced conductance of ionotropic glutamate receptors (IGluRs) of the postsynaptic stomatogastric ganglion in spiny lobsters (Cleland and Selverston, 1997). Aside from crustaceans, catecholamine signalling has been described in insects, nematodes and, as described below, flatworms. In insects, DA was identified in both neuronal and non-neuronal tissues of Drosophila melanogaster (fruit fly) to stimulate grooming, locomotion, sleeping/arousal regulation as well as to modulate the neuromuscular and motor neuron activities (Pendleton et al., 1996; Yellman et al., 1997; Cooper and Neckameyer, 1999; Kume et al., 2005; Pendleton et al., 2005). DA is one of the transmitters involved in the escape behavior of cockroaches (Goldstein and Camhi, 1991) and catecholamines other than DA could be involved in Drosophila development since DBH inhibitors significantly inhibited fly maturation (Pendleton et al., 1996). In C. elegans, dopamine is made in a few neurons located in the head region of the hermaphrodite and in the tail of the male (Sulston et al., 1975; Chase and Koelle, 2007). When applied exogenously, DA inhibits locomotion and egg laying in the nematode while D2 receptor antagonists activate egg laying and defecation (Weinshenker et al., 1995). Furthermore, dopamine mediates food- induced slowing in C. elegans, a locomotory response that enables the animal to slow down when it comes in contact with food so as to eat more efficiently (Sawin et al., 2000).

3.3. 2 DA receptors in non-flatworm invertebrates

Three DA receptors were cloned from the honey bee Apis mellifera, AmDOP1 (D1 type) is involved in signal processing of visual and olfactory information, AmDOP2 (D2 type) participates in bee maturation and AmDOP3 (D2-like) whose function is unknown (Blenau et al., 1998; Humphries et al.,

2003; Beggs et al., 2005). Recently, D1α and D1β receptors were cloned from the spiny lobster and both were found to mediate accumulation of cAMP (Clark and Baro, 2006). Both D1-like and D2-like receptors were cloned from Drosophila melanogaster. The first receptor was similar to the mammalian D1/D5 receptors, both structurally (70% homology) and pharmacologically, as it was activated by classical DA agonist SKF 38393 and was inhibited by antagonists such as butaclamol and flupenthixol (Gotzes et al., 1994; Sugamori et al., 1995). Two additional D1-like receptors were cloned from D. melanogaster, one of which (DAMB) was found to be distributed in the mushroom bodies and spatially co- expressed with AC (Feng et al., 1996; Han et al., 1996). In addition, a D2- like receptor (DD2R) was identified in D. melanogaster. DD2R is alternatively spliced and signals through a PTx-sensitive Gi/o-mediated pathway, consistent with the signaling mechanism of mammalian D2 receptors (Hearn et al., 2002). DA receptors have also been well characterized in C. elegans. The first DA receptor CeDOP1 was cloned in 2002 and showed similarities to human D1, D5, Drosophila DmDOP1 and honey bee AmDOP1(Suo et al., 2002) while a second receptor CeDOP2 that belonged to D2-like class was cloned and characterized in the next year (Suo et al., 2003). CeDOP2 undergoes alternative splicing in the third intracellular loop to give CeDOP2S and CeDOP2L isoforms, which were expressed in several neurons of the male tail (Suo et al., 2003). In 2005, another two DA receptors DOP-3 (D2-like) and DOP-4 (D1-like) were cloned from C. elegans. The former had a non-functional truncated spliced form, named DOP- 3nf, which lacked the TM6-TM7 and C-terminus and could not inhibit forskolin- stimulated cAMP formation (Sugiura et al., 2005). Functional studies revealed

that DOP-1 and DOP3 are expressed in motorneurons and act antagonistically to control worm motility (Chase et al., 2004). Recently, a subunit of dopamine-gated chloride channel (HcGGR3) was cloned and characterized from the ruminant gastrointestinal parasitic nematode Haemonchus contortus (Rao et al., 2009) and had a robust response to the biogenic amine DA when it was expressed in Xenopus laevis oocytes. This is the only known example of an ionotropic (non- GPCR) DA receptor and may be invertebrate-specific.

3.3. 3 Catecholamines in flatworms

The presence of catecholamines in flatworm tissues has been demonstrated by immunoassays using catecholamine-specific antibodies and a variety of biochemical assays (Ribeiro and Webb, 1983; Orido, 1989; Gustafsson and Eriksson, 1991; Eriksson et al., 1993). DA and NA were identified in multiple species of planarians (Joffe and Kotikova, 1991), in cestodes such as Diphyllobothrium dendriticum and H. diminuta (Ribeiro and Webb, 1983; Gustafsson and Eriksson, 1991; Eriksson et al., 1993) and in trematodes like S. mansoni, S. japonicum, F. hepatica (Gianutsos and Bennett, 1977; Orido, 1989). Some parasites can synthesize their own catecholamines. For example, the rate- limiting TH encoding gene was cloned from S. mansoni (Hamdan and Ribeiro, 1998) and the rat tapeworm H. diminuta can convert exogenously supplied tyrosine to DA (Ribeiro and Webb, 1983). Recently, TH and AADC were cloned from the free-living Dugesia japonica and were shown to co-localize in DA neurons (Nishimura et al., 2007). Functional studies have shown that catecholamines have neuromuscular effects in flatworms but the nature of the response varies depending on the species (reviewed by Maule et al., 2006). DA and NA induced muscle contraction when added to Diclidophora merlangi (Maule et al., 1989a) whereas in F. hepatica, DA was excitatory and NA inhibited motility (Holmes and Fairweather, 1984). In the case of S. mansoni, application of exogenous DA and NA increased the length of the body, (Mellin et al., 1983) and decreased the muscle tone of both circular and longitudinal muscle fibers, (Pax et 30

al., 1984). More recently, a DA receptor was cloned from S. mansoni (SmD2) and was localized in the subtegumental musculature (both cicular and longidutinal muscles) but not the CNS of the worm. This suggests that DA exerts at least some of its neuromuscular effects by acting directly on the musculature or associated innervation, rather than centrally located neurons. (Taman and Ribeiro, 2009). SmD2 resembles D2-like receptors from other species but signals through AC activation and elevates intracellular cAMP with no detectable change in Ca2+ levels, when expressed in HEK293 cells (Taman and Ribeiro, 2009). Since the somatic muscles of flatworms are of a smooth type (Halton and Maule, 2004a) which are usually inhibited by cAMP-mediated phosphorylation, the increase in cAMP may explain why DA causes muscle relaxation in schistosomes. More DA receptors are expected to be identified in S. mansoni, now that the genome sequence is completed. There are several putative BA receptors in the S. mansoni database that share some homology with DA receptors from other species and could prove to be dopaminergic if they are cloned and characterized.

Free-living planarians are the best examples of a dopaminergic system in flatworms. DA has profound locomotory effects in the free-living planarians (reviewed by Ribeiro et al., 2005; Nishimura et al., 2007) Different responses have been observed depending on which DA receptor is activated. Activation of D1-like receptors produces screw-like (SL) hyperkinesias while D2-like receptors cause a C-like (CL) posture (Venturini et al., 1989; Passarelli et al., 1999; Raffa et al., 2001). The classification of these receptors is based on pharmacological profiles, which closely resemble those of the mammalian D1- and D2-like prototypes (Venturini et al., 1989; Raffa et al., 2000; Raffa et al., 2001; Nishimura et al., 2007). Direct evidence of DA involvement in these behaviours came from recent RNAi studies targeting the biosynthetic enzyme, TH. (Nishimura et al., 2007). The control planarians, where TH was expressed at normal levels, showed normal hyperkinesias, whereas the RNAi treated animals lacked the response, indicating that endogenous DA signaling was required for these behaviours

(Nishimura et al., 2007). Aside from movement, DA and probably NA contribute to the control of tissue regeneration in the Planarian Polycelis tenvis (Franquinet, 1979) and in D. japonica (Nishimura et al., 2007).

4 G protein-coupled receptors (GPCRs)

4.1 The structure

GPCRs constitute the largest superfamily of transmembrane cell-surface receptors. Approximately 5% of the Caenorhabditis elegans genome encodes GPCRs. In humans, there are 720-800 GPCR genes, which is roughly equivalent to 1% - 2% of our genome (Krauss G., 2003; Wise et al., 2004; Bond and Ijzerman, 2006; Jacoby et al., 2006). They are so named because of their ability to couple to and to regulate the activity of heterotrimeric G-proteins (Gether, 2000). These receptors are activated by a diverse group of extracellular ligands like biogenic amines (serotonin, histamine, dopamine, epinephrine and others), peptides, glycoproteins, lipid derivatives, hormones, nucleotides, ions and even photons. On the intracellular side, GPCRs interact with GPCR kinase (GRK) and several GPCR interacting proteins (GIP) that control G protein coupling (Bockaert et al., 2004a; Tilakaratne and Sexton, 2005). A common structural feature among GPCRs is the existence of seven transmembrane (7TM) helices, which are connected by alternating extracellular loops and intracellular loops with the amino terminus located in the extracellular side, while the carboxy terminus is intracellular (Figure 8). When seen from the extracellular side, the 7TM helices are arranged sequentially in a counterclockwise fashion (Unger and Schertler, 1995; Unger et al., 1997). The loops and the termini are most divergent among GPCRs whereas the greatest degree of conservation is found within the TM regions (Krauss G., 2003). GPCRs are broadly classified according to three main families A, B and C (Gether, 2000). The classification is based principally on the size of the extracellular N-terminus and the presence of key functional residues.

Figure 8: The heptahelical structure of three major families of GPCRs (A-C). All GPCRs share the same topology of seven transmembrane (7-TM) domains that are connected by three extracellular loops el1-el3) and three intracellular loops (il1-il3), the N-terminus is located in the extracellular side while the C-terminus is cytoplasmic. The receptors also carry a conserved intramolecular disulfide bond between a pair of cysteines located in el1 and el2 (black residues). The receptors within each family are further characterized by the presence of specific signature motifs (e.g. DRY and NPxxY motifs in family A receptors) as well as the presence of highly conserved residues, especially in the TM domains (open circles). The size of N-terminal region is moderate in family A, longer with many disuldide linkages in family B and is extremely elongated in family C, where it forms a venus flytrap structure. The il3 is very long in most family A and B receptors but comparatively shorter in family C. The intracellular C-terminus is of variable length and is particularly long in family C receptors. This cartoon is adapted from Krauss G., 2003. G Protein-Coupled Signal Transmission Pathways (Chapter 5). In: Krauss G. (Ed) Biochemistry of Signal Transduction and Regulation.3rd Edition, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. pp-179-230).

Family A includes rhodopsin and approximately 90% of all known GPCRs. Family A (or rhodopsin-like) receptors are characterized in part by the presence of signature motifs near the cytoplasmic end of TM3 (E/DRY) and TM7 (NPxxY). These motifs are absent in receptors of other families, whereas the conserved prolines of family B GPCRs are different from those of family A receptors (Gether, 2000).

The three GPCR families share two disulfide forming cysteines in EL1 and EL2 (Fig.8). The resulting disulfide linkage helps to stabilize the conformation of the extracellular domain of the receptors and is important for activity (Gether, 2000; Krauss G., 2003). GPCRs possess at least one N-glycosylation site (Asn-X- Ser/Thr) at the N-terminal end. Glycosylation is the most common posttranslational modification of GPCRs and it contributes to trafficking of receptors to the plasma membrane, though the effect differs among GPCRs (Dong et al., 2007). For example, N-linked glycosylation is absolutely essential for proper targeting of the follicle stimulating hormone receptor (FSHR) to the cell surface and it also facilitates β2-AR export but has no effect on H2 histamine and M2-muscarinic receptors (Rands et al., 1990; van Koppen and Nathanson, 1990; Davis et al., 1995; Fukushima et al., 1995). Another posttranslational modification of note is the palmitoylation of Cys residues localized at the C-terminus. Palmitoylation occurs in some GPCRs of family A (Fig. 8) to anchor the C- terminus onto the membrane, causing the formation of an additional loop. Much of the information available on GPCR structure has come from a few crystal structures that were recently made available. Crystal images were obtained for the family A prototype, rhodopsin, first in low resolution ( 5-9°A) (Unger et al., 1997; Davies et al., 1996; Krebs et al., 1998) and later at higher resolution of 2.8 °A (Palczewski et al., 2000; Murakami et al., 2007). X-ray crystal images of the first ligand activated GPCR were obtained recently for human beta 2 adrenergic receptor with very high resolution of 3.5°A (Rasmussen et al., 2007) and 2.4°A (Cherezov et al., 2007).

4.2 Numbering of residues in GPCRs

To facilitate comparisons among GPCRs, researchers have adopted a numbering scheme based on the position of invariant residues within the TM regions (Ballesteros and Weinstein, 1995; Schwartz et al., 1995; Baldwin et al., 1997). The system, described by Ballesteros and Weinstein assigns the number 50 to the most conserved residue in each of the seven TM domains and the 34

designation is preceded by the TM number. Thus, for example, N1.50 describes the most conserved residue ( an asparagine) of TM1. For family A receptors, the seven reference residues are N (1.50), D (2.50), R (3.50), W (4.50), P (5.50), P (6.50) and P (7.50). All other residues within that TM region are numbered accordingly, either below or above 50, depending on their position relative to the reference. This numerical system is used throughout this thesis to describe the positions of key residues in S. mansoni receptors.

4.3 Ligand binding in GPCRs

The new crystallographic data, combined with site directed mutagenesis, biochemical and pharmacological studies are beginning to unveil the location of the ligand binding site in GPCRs. This location varies depending on the GPCR and the type of ligand. Class A GPCRs that bind small ligands (e.g. biogenic amines) have a binding crevice buried within transmembrane helices TM 3, 5, 6 and 7. In the case of biogenic amine receptors, the principal binding site is an aspartate (Asp3.32) located near the extracellular end of TM3 (see residue numbering system above). A salt bridge formed between the side-chain carboxylate of Asp3.32 and the protonated amino moiety of the biogenic amine ligand contributes the most important binding interaction (Strader et al., 1991; Befort et al., 1996; Kristiansen et al., 2000; Floresca and Schetz, 2004). With the exception of a few schistosome receptors described in this thesis, Asp3.32 is conserved in all biogenic amine receptors discovered to date and its contribution to binding has been demonstrated in histamine receptors (Gantz et al., 1992), serotonin receptors (Ho et al., 1992; Wang et al., 1993), dopamine receptors (Mansour et al., 1992) and acetylcholine (muscarinic) receptors (Spalding et al., 1994). Besides that salt bridge, there are hydrogen bond interactions between the –OH moieties of the ligand and serine residues (S5.43 and S5.46) present in TM5 or N6.55 of TM6 (Strader et al., 1989; Wieland et al., 1996). Additional interactions with aromatic residues of TM6 help to anchor the ring structure of the biogenic amine. Although most of the small agonists bind deeply in the binding crevice of 35

the TM domains, structurally unrelated antagonists may bind to a separate site that is closer to the surface in the ELs, as in the case of phentolamine, the α1B- antagonist (Zhao et al., 1996). Larger peptide ligands are thought to bind to both the extracellular loops and regions of the TM domains, though the precise location of the binding site is unknown. Ligands of family C (glutamate, GABA, Ca2+) bind to specific pockets located in the large extracellular N-terminal domain (Takahashi et al., 1993; Tones et al., 1995).

4.4 Switching “on” GPCR-mediated signals

Early GPCR studies based on alanine substitution mutagenesis identified several intramolecular interactions that serve to constrain the receptor in its inactive form in the absence of agonist (Kjelsberg et al., 1992). When an agonist binds to the receptor, these intramolecular interactions are disturbed, leading to a conformational change and receptor activation. Some of these intramolecular interactions are conserved among GPCRs and involve residues mainly of the TM regions. Of particular importance among family A receptors is a network of ionic interactions that are centered around the conserved arginine (Arg3.50) of the E/DRY motif. The arginine “cage” model suggests that Arg3.50 interacts with the adjacent aspartate (D3.49) at the end of TM3 and a neighbouring glutamate of TM6. These interactions stabilize the inactive conformation by holding TM3 and TM6 close together, which hinders interaction with the G protein (Ballesteros et al., 1998; Visiers et al., 2002; Fan et al., 2005). When the agonist binds to its site, the Arg3.50 “cage” is broken and the helices move further apart, enabling the receptor to become activated. Other residues of TM3, TM6 and TM7 play critical roles in the transition to the active state in several receptors including rhodopsin and the β2-adrenergic receptor (Elling et al., 1999; Gether, 2000; Ringkananont et al., 2006; Bhattacharya et al., 2008). Upon ligand activation, the conformational change of GPCR allows the intracellular loops, il2 and il3, as well as the proximal end of the C-terminus domain to interact with the Gα subunit of the heterotrimeric G-protein (Kobilka, 1992; Savarese and Fraser, 1992; Wess, 1997, 1998). 36

Some GPCRs can become spontaneously activated in the absence of agonist. These receptors have intrinsic constitutive activity that enables them to become active and to interact with downstream G-proteins. Constitutive activity is due to mutations or post-transcriptional events that disrupt the normal constraints on GPCR activation (Wade et al., 2001; Ladds et al., 2005). Some constitutively active mutants (CAMs) have been linked to genetic diseases, such as hypocalcaemia and hypercalciuria, which are caused by a Ca2+ sensing receptor CAM, and thyroid adenomas caused by a TSH receptor CAM (Parnot et al.,

2002). The B1B2X2B3 motif (where B is a basic residue and X is any amino acid) located in the junction between il3 and TM6 plays a role in stabilizing the receptor‟s inactive conformation. Mutations within this motif have been shown to increase constitutive activity of several GPCRs (Huang et al., 2001; Fan et al., 2005). It should be noted that most constitutively active receptors can still respond to agonists. Though their basal activity is high, the receptors can be further activated by their ligands, and thus exhibit both agonist-dependent and independent activities. This point is relevant to later discussions of schistosome receptors (see manuscript II).

4.5 The heterotrimeric G-proteins

With few exceptions, most GPCR signaling is mediated by heterotrimeric G-proteins. They belong to the GTPase superfamily and are capable of binding and hydrolyzing GTP due to endogenous GTPase activity. G-proteins serve as a “switch” in signal transduction because they can exchange GDP for GTP to turn signaling“ON” and hydrolyze GTP to turn it “OFF”. The binding of trimeric G proteins to an activated receptor triggers the GDP/GTP exchange and subsequent dissociation of GTP-bound Gα from the remaining Gβγ subunits. Both Gα and the βγ dimer can modulate the activity of various effectors (enzymes and ion channels). When the intrinsic GTPase activity of Gα subunit hydrolyzes the bound GTP to GDP, this allows the Gα subunit to reassemble with the dimer again, thus returning the G-protein to its heterotrimeric inactive form. 37

The heterotrimeric G-protein consists of three different subunits: α-subunit (39-46 KDa), which possesses the GDP/GTP binding domain and the intrinsic GTPase activity; β-subunit (36 KDa) and γ subunit (8 KDa). The three subunits show great structural diversity. There are 20 genes encoding different α-subunits, 5 genes for β-subunits and 12 for γ-subunits. This diversity of structures increases the complexity of GPCR signaling because it allows for many different combinations of G protein subunits. Not all possible combinations exist, however. Of the 1000 potential heterotrimers, only a small fraction has been detected in cells and many of these are tissue-specific (Krauss G., 2003). Although the βγ dimer modulates several effector proteins, the Gα subunit is responsible for most activities of the G protein. Gα subunits can be classified into four main classes based on their amino acid sequences: Gαs, Gαi, Gαq and Gα12 (Downes and Gautam, 1999).

4.6 Receptor desensitization

Under the conditions of long lasting stimulation (i.e. the presence of the agonist for prolonged time), the signal is either relayed in a weakened form or is completely blocked and no longer passed into the cell interior. The failure to respond is due to receptor desensitization and internalization, which occur when two different classes of protein kinases phosphorylate specific residues on the cytoplasmic side of the receptor. The first class includes Ser/Thr protein kinases, such as cAMP-dependent protein kinase (PKA) and PKC, both of which catalyze the phosphorylation of Ser/Thr residues of the cytoplasmic domain of the receptor. The second class includes G protein-coupled receptor protein kinases (GPK), which phosphorylate the agonist-bound receptor and allow the binding of intracellular β-arrestins. GPKs can phosphorylate the receptor only after it has been activated and the G protein is dissociated. The resulting Gβγ dimer binds to GPK and recruits the kinase to the membrane where it phosphorylates the receptor. GPK-dependent phosphorylation of GPCRs causes the formation of high-affinity sites for arrestin binding. Subsequently, the receptor is translocated 38

to the cell interior and β-arrestin serves as an adaptor for GPCR internalization via endocytosis. β-arrestin also binds to clathrin via clathrin-coated vesicles. Thus, the receptor is internalized in the membrane-associated form, dephosphorylated, and then transported back to the cell membrane via recycling endosomes or is targeted to the lysosome for destruction. These steps weaken signal transmission during conditions of long-lasting agonist stimulation by reducing the number of receptor sites available on the surface (Krauss G., 2003). In some GPCRs, the binding of the phosphorylated receptor to β-arrestin stimulates a MAPK cascade, where the arrestins function as scaffolding proteins. The protein kinases of the MAPK pathway relay the signal to the nucleus, eventually activating transcriptional factors. In this last example, arrestin has a dual role to allow cross-talking between the different signaling systems; it acts as a terminator of GPCR signaling and as an activator of MAPK signaling.

4.7 GPCR dimerization: Is it an artifact or biologically relevant?

Although it was thought for a long time that GPCRs were monomeric, there is increasing evidence that many receptors form functional dimers in vivo. Biophysical methods based on bioluminescence and fluorescence resonance energy transfer (BRET and FRET) have confirmed the existence of dimers and even larger oligomeric complexes in living cells (Jones et al., 1998; White et al., 1998; Angers et al., 2002). Some GPCRs form homodimers as in the case of dopamine D1 and D3 receptors (Ng et al., 1996; Nimchinsky et al., 1997; George et al., 1998), β2-adrenergic receptor (Hebert et al., 1996), δ-opioid receptor (Cvejic and Devi, 1997), Ca2+ sensing receptor and mGlu5 receptors (Romano et al., 1996; Bai et al., 1998). The dimerization mechanism varies depending on the receptor. For example, dimerization of the δ-opioid receptor occurs through the carboxy terminus since the deletion of a few amino acids in that region was sufficient to eliminate dimerization (Cvejic and Devi, 1997). In contrast, homodimerization of the β2-adrenergic and D2 receptors requires interactions between the TM regions, especially TM6, as determined by peptide competition 39

studies (Hebert et al., 1996; Ng et al., 1996). In the case of class C GPCRs, for example Ca2+ sensing and mGlu5 receptors, dimers are formed by intermolecular disulfide bonds present in the large N-terminal domain (Romano et al., 1996; Bai et al., 1998). Heterodimerization is less common but can also occur between closely related GPCRs, between unrelated GPCRs or even between a GPCR and a different protein (Tilakaratne and Sexton, 2005). The best example of a functional

GPCR heterodimer is the GABAB receptor. It consists of two related GABAB1 and

GABAB2 GPCRs, in which one of the receptors (GABAB1) possesses the ligand binding site and the other (GABAB2) couples to the G protein. GABAB1 has an endoplasmic reticulum retention signal that is masked when the dimer is formed. Thus only the dimer is able to reach the plasma membrane successfully and become activated (Bockaert et al., 2004a; Bockaert et al., 2004b; Dong et al., 2007; Duthey et al., 2002; Galvez et al., 2000; Galvez et al., 2001; Pagano et al., 2001). Another well established example of a GPCR heterodimer is the opioid receptor, which is formed by association of two δ-opioid and κ-opioid GPCRs. In this case, the individual monomers are fully functional but they can also heterodimerize to produce a new receptor that has distinct binding and functional properties, thus increasing signaling diversity (Jordan and Devi, 1999). More recent evidence suggests that some GPCRs may heterodimerize with non GPCR proteins to modify receptor activity in some defined way. This has been demonstrated for a calcitonin-like receptor (CLR) which can heterodimerize with different types of receptor activity modifying proteins (RAMPs). The interaction with RAMPs influences the folding, trafficking and importantly the pharmacology of some GPCRs of families B and C (McLatchie et al., 1998; Hay et al., 2006; Parameswaran and Spielman, 2006). In the case of the calcitonin receptor, that interaction is used to change ligand binding specificity; CRLC functions as a calcitonin receptor when bound to RAMP1, whereas the association with RAMP2, causes it to display of the properties of an adrenomedullin receptor (McLatchie et al., 1998; Mallee et al., 2002). In the same context, different tastes can be perceived from different association of taste receptors (T1R1, T1R2 and 40

T1R3). The sweet taste is the output of T1R2-T1R3 heterodimer or T1R3 homodimer whereas the umami taste results from the combination of T1R1-T1R3 (Li et al., 2002; Nelson et al., 2002; Zhao et al., 2003). Thus, GPCR dimerization increases receptor complexity to diversify ligand binding properties, to facilitate folding and trafficking during maturation.

4.8 GPCR interacting proteins (GIPs)

As new information emerges, it is becoming apparent that GPCRs can interact with a wide range of proteins besides the G-protein effector. These may be soluble or membrane-associated and are collectively described as GIPs (GPCR interacting proteins). The functions of GIPs include targeting of the receptors to specific subcellular compartments and control GPCR trafficking to and from the plasma membrane. GIPs also help GPCRs assemble into large functional complexes, called „receptosomes‟, and fine-tune their signaling properties (Bockaert et al., 2004b). One example of a GIP are the aforementioned β- arrestins, which bind to phosphorylated receptors and are involved in GPCR desensitization and internalization (Pfister et al., 1985). Other GIPs work in ER quality control as chaperones that interact with GPCRs (Kleizen and Braakman, 2004; Williams, 2006; Anukanth and Khorana, 1994; Siffroi-Fernandez et al., 2002; Mizrachi and Segaloff, 2004; Dong et al., 2007). The RAMP1- RAMP3 proteins described above are examples of GIPs that bind directly to GPCRs and modulate receptor activity (Dong et al., 2007).

4.9 GPCRs of schistosomes and other platyhelminths

To date, only a few platyhelminth GPCRs have been successfully cloned and characterized. Two serotonergic 5-HT1A (5HTLpla4 and 5HTLpla1) receptors were cloned from planarians (Creti et al., 1992; Saitoh et al., 1997), and a FMRFamide-like neuropeptide receptor was recently identified in the planarian, Girardia tigrina (Omar et al., 2007). Searching the literature for reports of GPCRs in Schistosoma parasites revealed only four articles to date, all of which were 41

derived from S. mansoni. These include studies of SmRHO, the light reactive rhodopsin receptor (Hoffmann et al., 2001), SmGPCR, histamine-responsive receptor (Hamdan et al., 2002), an orphan tegumental receptor Sm-7TM of unknown function (Pearson et al., 2007) and, more recently, the first demonstration of a dopamine receptor in schistosomes (Taman and Ribeiro, 2009).There is also evidence of a possible somatostatin-like receptor in S. mansoni, (SSTR-like GPCR) but the cDNA has yet to be cloned (Chatterjee et al., 2007). Despite the paucity of cloned sequences, a close examination of the S. mansoni genome identified as many as 92 potential GPCRs of which the vast majority (n= 82) are structurally related to rhodopsin-like Family A (Berriman et al, 2009). Among these putative GPCRs are 15-18 amine-like receptors, 24 peptide receptors and numerous orphan GPCRs. Based on a bioinformatics analysis of biogenic amine receptors, flatworm GPCRs resemble other invertebrate and vertebrate GPCR sequences, particularly in the TM domains, which share approximately 40%-55% identity. A computer-generated model of SmGPCR identified a typical GPCR topology with all the major characteristics described above. Family A signature motifs such as the E/DRY motif of TM3 and NPxxY in TM7, are present in the schistosome receptor, as are many of the residues involved in ligand binding and conformational activation (Ribeiro et al., 2005). Nevertheless, despite an overall conserved topology, many schistosome GPCRs have no mammalian or invertebrate orthologues and are considered to be schistosome-specific. By virtue of their unique structures, these receptors have potential for drug discovery against schistosomiasis. Three of these novel receptors will be described in this thesis.

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CHAPTER II (manuscript I)

Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni

Fouad El-Shehabia, Jon J. Vermeireb, Timothy P. Yoshinob, Paula Ribeiroa

a Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9

b Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Dr., Madison, WI 53706, USA

Experimental Parasitology 122 (2009) 17–27

ABSTRACT

A Schistosoma mansoni G-protein coupled receptor (SmGPCR) was previously cloned and shown to be activated by the biogenic amine, histamine. Here we report a first investigation of the receptor‟s subunit organization, tissue distribution and expression levels in different stages of the parasite. A polyclonal antibody was produced in rabbits against the recombinant third intracellular loop (il3) of SmGPCR. Western blot studies of the native receptor and recombinant protein expressed in HEK293 cells showed that SmGPCR exists both as a monomer (65 kDa) and an apparent dimer of ~130 kDa. These species were verified by immunoprecipitation of SmGPCR from S. mansoni extracts, using antibody that was covalently attached to agarose beads. Further investigation determined that the SmGPCR dimer was resistant to treatment with various detergents, 4 M urea and 0.1 M DTT but could be made to dissociate at acidic pH, suggesting the dimer is non-covalent in nature. Confocal immunofluorescence studies revealed significant SmGPCR immunoreactivity in sporocysts, schistosomula and adult worms but not miracidia. SmGPCR was found to be most widely expressed in the schistosomula, particularly the tegument, the subtegumental musculature and the acetabulum. In the adult stage we detected SmGPCR immunofluorescence mainly in the tubercles of male worms and, to a lesser extent, the body wall musculature. Localization in sporocysts was mainly confined to the tegument and cells within parenchymal matrices. A real-time quantitative reverse-transcription PCR analysis revealed that SmGPCR is upregulated at the mRNA level in the parasitic stages compared to the free-living miracidium and cercariae, and it is particularly elevated during early sporocyst and schistosomula development. The results identify SmGPCR as an important parasite receptor with potential functions in muscle and the tegument of S. mansoni.

Keywords: Schistosoma mansoni, Histamine, G-protein coupled receptor (GPCR), Neurotransmitter, Platyhelminth, Biogenic amines, Real-time qPCR, Confocal microscopy 67

1. Introduction

Biogenic amines are small signaling molecules, which are derived from the metabolism of amino acids and include such ubiquitous substances as serotonin (5-hydroxytryptamine: 5HT), catecholamines, phenolamines and histamine (HA). HA is an important neuroactive substance of both vertebrates and invertebrates. In mammals, it functions as a neurotransmitter of the central nervous system (CNS) (Panula et al., 1991; Airaksinen et al.,1991) and also has numerous effects outside the CNS, notably as a regulator of gastric acid secretion, a vasodilator and a mediator of immunity (Marone et al., 2001, 2003). Among invertebrates, HA has been implicated as a neurotransmitter or neuromodulator in the insect eye (Hardie, 1987; Callaway et al., 1989; Nassel et al., 1988) and the somatogastric ganglia of crustaceans (Claiborne and Selverston, 1984). These effects are mediated by cell surface HA receptors, the majority of which are members of the heptahelical G- protein coupled receptor (GPCR) superfamily and are structurally related to rhodopsin. Four different types of histaminergic GPCRs have been identified in mammals (H1–H4), one of which (H3) is alternatively spliced to produce additional variants. These receptors differ in their affinities for HA, signaling mechanisms, tissue distribution and physiological roles (Leurs et al., 1995; Lovenberg et al., 2000; Tardivel-Lacombe et al., 2000; Liu et al., 2000, 2001). Invertebrates have at least one type of HA-activated GPCR that shows modest homology with the mammalian H1 prototype (Hamdan et al., 2002a). In addition, there is increasing evidence that some of the effects of HA in invertebrates are mediated by ionotropic receptors. Studies of arthropods have identified an unusual HA-gated chloride channel of the Cys-loop superfamily that appears to be invertebrate-specific (Hong et al., 2006).

Parasitic flatworms employ a wide range of biogenic amines in their nervous system. Being acoelomates, platyhelminths lack a conventional endocrine system and rely instead on neuronal signaling to coordinate their activities. Much of this signaling is mediated by biogenic amines. The best characterized of these 68

substances, 5HT, has been shown to stimulate muscle contraction and to promote both glycogenolysis and glucose utilization, the overall effect being an increase in motor activity (Boyle et al., 2000; Ribeiro et al., 2005; Boyle and Yoshino, 2005). By comparison, very little is known about the role of HA in flatworms. What information there is available suggests that HA is localized within neuronal structures (Mettrick and Telford, 1963; Wikgren et al., 1990; Eriksson et al., 1996), consistent with a neuroactive role, and is synthesized endogenously (Eriksson et al., 1996), or taken up from the host via tegumental transport (Yonge and Webb, 1992). HA levels vary substantially among parasitic flatworms. Some species, including the bloodfluke Schistosoma mansoni, have low tissue levels of HA, whereas other parasites, notably the amphibian trematode, Haplometra cylindracea store HA at very high concentrations (Mettrick and Telford, 1963; Schwabe and Kilejian, 1968; Ercoli et al., 1985; Eriksson et al., 1996). The reason for this variation is not clear. A BLAST analysis of the S. mansoni genome database identified a potential orthologue of human histidine decarboxylase, the enzyme that synthesizes HA. This suggests that schistosomes have the ability to synthesize HA endogenously, just as they synthesize other biogenic amines (Hamdan and Ribeiro, 1998, 1999). It is possible the rate of synthesis is low or that HA is rapidly degraded once it is released. Schistosoma mansoni was reported to possess histaminase (diamine oxidase) activity, an enzyme that breaks down HA (Schwabe and Kilejian, 1968). A high rate of HA catabolism could explain why the basal level of this amine is so low in these animals. Though the function remains unclear, there is evidence to suggest that HA is an important modulator of neuromuscular function and movement among flatworms. Studies of H. cylindracea have shown that HA-containing neurons innervate all major bodies of muscle, including the subtegumental (body wall) musculature, ventral sucker and the muscle layers surrounding the alimentary and reproductive tracts (Eriksson et al., 1996). Other studies have shown that exogenously applied HA modulates the frequency of body wall contractions and influences motor activity both in cestodes and trematodes (Sukhdeo et al., 1984; Ercoli et al., 1985). Schistosoma 69

mansoni treated with anti-histaminic drugs, such as promethazine, are rapidly paralyzed and the paralysis is reversed by addition of HA (Ercoli et al., 1985), suggesting the amine has a positive effect on motility in this parasite. It is unclear at present whether HA exerts its effects by interacting with receptors located on the musculature or through some other mechanism that indirectly controls motility. The paralysis produced by the anti-histaminic drugs highlights the importance of HA receptors in these parasites, both with respect to motor control and as potential drug targets.

We have previously cloned a novel S. mansoni receptor (SmGPCR) that is specifically activated by HA when expressed heterologously in mammalian cells (Hamdan et al., 2002a, b). SmGPCR shares about the same level of sequence homology with all different types of biogenic amine GPCRs, including the histaminergic H1 type, but has no identifiable mammalian or invertebrate orthologues. This is consistent with the notion that schistosome neuroreceptors are structurally divergent and raises the interesting possibility that SmGPCR may be unique to these parasites. Here we describe a first investigation into the potential function of this receptor in S. mansoni. We have examined the pattern of developmental expression and tissue distribution both at the RNA and protein levels, using real-time qPCR, immunofluorescence and confocal microscopy. The results point to SmGPCR as an important schistosome receptor, which is upregulated in the parasitic stages and appears to be enriched in the tegument and neuromuscular structures.

2. Materials and methods

2.1. Parasites

All parasite stages used in this study were derived from a Puerto Rican (NMRI) strain of S. mansoni. Infected Biomphalaria glabrata snails were obtained from the Biomedical Research Institute, Rockville, Maryland, USA (Lewis et al., 1986) and were induced to shed cercariae approximately 45 days post-infection by 70

exposure to continuous light for 30 min at room temperature. For cercarial transformation and culturing of schistosomula we modified the original Basch protocol (Basch, 1981) as follows: Cercariae were collected by cooling at 4 C for 3 h and then vortexed at maximal speed for 2 min to detach the tails (Ramalho- Pinto et al., 1974; Gold and Flescher, 2000). The latter were subsequently removed by adding 70% Percoll (Sigma, Oakville, Ontario, Canada) prepared in minimal essential medium MEM (Gibco, Invitrogen, Canada) followed by centrifugation at 1700 rpm for 10 min at 5 C. The supernatant containing the tails was discarded and the remaining cercarial bodies were washed twice by repeated cycles of centrifugation (1200 rpm/10 min) and resuspension in MEM supplemented with 1 mg/ml streptomycin (Sigma) and 1000 U/ml penicillin (Sigma) and 0.25 µg/ml Fungizone (Invitrogen, Canada). The bodies were then gradually transferred to RPMI 1640 growth medium (Gibco) through three constitutive washes in a solution containing the same antibiotics as above and a mixture of MEM + RPMI 1640 at increasing ratios of 3:1, 1:1 and 1:3 v/v. Finally, the cercarial bodies were washed once in RPMI 1640 supplemented with antibiotics and transferred into RPMI 1640 supplemented with 10% heat- inactivated Fetal Bovine Serum FBS (Gibco), 100 U/ ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml Fungizone. The transformed schistosomula were cultured in 24-well plates in a humidified incubator set at 5% CO2/37 C and supplemented with fresh media every 3–5 days. Animals could be maintained under these conditions for up to 6 weeks with no apparent loss of viability. To obtain adult worms, 28-day old CD1 female mice were infected with freshly shed cercariae (~150 cercariae/mouse) by active penetration through the skin. Approximately 7–8 weeks postinfection the mice were sacrificed and the adult worms were collected by perfusion of the livers and mesenteric veins (Smithers and Terry, 1965; Carneiro and Lopes, 1986). Miracidia were hatched from eggs collected from the mouse livers approximately 7-week post-infection and transformed into mother sporocysts according to previously described methods (Yoshino and Laursen, 1995). Sporocysts were maintained at 26 C for 4- or 20- 71

days in complete SM culture medium (Ivanchenko et al., 1999) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 50 µg/ml streptomycin.

2.2. Production of a polyclonal anti-SmGPCR antibody

A fraction of the third intracellular loop (il3) of SmGPCR (Accession # AF031196, pos. 1330–1701) was amplified by PCR using primers (sense 5‟- CCGAATTCATGCCCGAACCAACAGA-3‟ and antisense 5‟- TAGCGGCCGCTGCAGTTTTTTGTTC-3‟) designed to incorporate EcoRI and NotI sites (underlined) at the 5‟ and 3‟ ends, respectively. The PCR product was cloned between the EcoRI and NotI sites of prokaryotic expression vector pET30a (Novagen, EMD Biosciences, San Diego, CA, USA) and expressed in Escherichia coli as an N-terminal 6X Histidine-tagged protein. The resulting SmGPCR-il3 recombinant protein was purified by metal chelation chromatography under denaturing conditions, using a commercial His.Bind kit (Novagen, EMD Biosciences, USA). The purification was verified by SDS– PAGE electrophoresis and Western blot analysis targeting the hexa-histidine tag, according to standard protocols. To produce the antibody we injected two female adult albino rabbits subcutaneously with 1 ml (2 mg) of the purified His-tagged SmGPCR-il3 mixed in 1:1 ratio with Freund‟s complete adjuvant (Sigma). Two boosters were done at 2-week intervals with 0.5 ml (1 mg) of the purified recombinant protein and 0.5 ml of incomplete Freund‟s adjuvant. The animals were sacrificed 2 weeks after the third injection and the serum was isolated. Pre- immune serum was obtained from each rabbit prior to injection of antigen. The antiserum was first evaluated by ELISA against the purified SmGPCR-il3 antigen and the titer was determined to be 1:50,000. The IgG fraction was subsequently purified by protein A sepharose affinity chromatography (Sigma, Canada), dialyzed against PBS, pH 7.4, and the protein concentration was adjusted to 5 mg/ml.

2.3. IFA-confocal microscopy of SmGPCR-transfected cells

The anti-SmGPCR-il3 antibody was initially tested in HEK293 (EBNA1) cells that were stably transfected with a codon-optimized SmGPCR cDNA (Hamdan et al., 2002b) tagged to a green fluorescence protein (EYFP) at the C-terminal end (kindly provided by Dr. F. Hamdan, University of Montreal). Cells stably expressing FLAG-SmGPCR-EYFP or untransfected HEK293E cells (0.5 x 106/ well) were seeded in six well plates and cultured in DMEM containing 5% heat- inactivated FBS, 20 mM HEPES, 10 µg/ml Zeocin and 0.25 µg/ml puromycin (Invitrogen, Burlington, Ontario, Canada). For immunofluorescence, the cells were fixed in cold (-20 C) methanol for 5 min, washed in PBS and blocked in 5% goat serum and 0.5% Triton X-100 in PBS for 1 h at room temperature. Anti- SmGPCR was prepared in the same blocking solution (1:100 dilution) and added for an additional hour at room temperature. This was followed by three washes in PBS and then 1 h incubation with rhodamine-labeled anti-rabbit IgG secondary antibody (1:300 dilution in blocking buffer). Cells were counterstained with 40,6- diamidino-2-phenylindole DAPI (Sigma, at 1:1000), mounted onto slides and examined using a BIO-RAD RADIANCE 2100 confocal laser scanning microscope equipped with Nikon E800 fluorescence microscope for confocal image acquisition and the LASERSHARP 2000 analyzing software package.

2.4. IFA-confocal microscopy of larval and adult stages of S. mansoni

Larval stages (miracidia, sporocysts and schistosomula) and in vivo-derived adult worms were washed twice in PBS, pH 7.4 and fixed in freshly prepared 4% paraformaldehyde (PFA; Sigma, Canada) in PBS at 4 C for 4 h, using end-over- end rotation. The fixative was changed twice after 1 and 3 h of incubation. Following fixation, the worms were washed twice in PBS and treated with 0.1 M glycine for 5–10 min to reduce autofluorescence. Alternatively, a cold acetone fixative was used (Thors and Linder, 2003). The samples were subsequently incubated for 24 h at 4 C in the same blocking permeabilizing solution described

above and treated with anti-SmGPCR IgG (1:100 in blocking solution) for 3–4 days at 4 C with gentle rotation, as described previously (Mair et al., 2000, 2003). Animals were washed three times in PBS and incubated in fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG (Chemicon, Temecula, CA) (1:300 in blocking solution) for 3 days at 4 C. When phalloidin (0.2 mg/ml) was used as a counter stain, 400 ng of tetramethylrhodamine B isothiocyanate (TRITC)-labeled phalloidin (Sigma, USA) was added 24 h after addition of the secondary antibody and the incubation was continued for two additional days at 4 C. After washing, the samples were mounted with anti-quench mounting medium (Sigma) and examined by confocal microscopy as described above. The following controls were routinely used in these studies: (1) omission of primary antibody, (2) replacement of primary antibody with pre-immune serum, (3) replacement of primary antibody with an irrelevant IgG and (4) pre-adsorption with an excess of the purified SmGPCR-il3 antigen. The protocol for pre-adsorption was as described previously (Rosin et al., 1998; Coling and Kachar, 2001).

2.5. SDS–PAGE and Western blots

Analysis of recombinant SmGPCR were done in HEK293 (EBNA1) cells transiently transfected with an N-tagged FLAG.SmGPCR expression pCEP4 plasmid (Hamdan et al., 2002a). Cells (106/100 mm dish) were transiently transfected with 3 µg of pCEP4.- FLAG.SmGPCR or empty pCEP4 plasmid (control), using the transfection agent FuGene 6 (Roche, Canada), according to the manufacturer‟s recommendations. For membrane protein extraction, we followed the protocol of Uberti et al. (2005) with the following modifications: HEK cells were homogenized in PBS containing a protease inhibitor cocktail (1:100 dilution, Sigma) and the homogenate was spun at 30,000g for 20 min at 4 C to isolate a crude membrane pellet. The pellet was solubilized in lysis buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 100 mM NH4SO4, 10% v/v glycerol and protease inhibitor cocktail) containing 2% CHAPS for 2 h at room temperature with gentle end-over-end rotation and then centrifuged at 16,000g for 10 min at 4 74

C to remove insoluble material. Aliquots of the resulting supernatant (2.5–3 µg protein) were subsequently prepared in SDS–PAGE sample buffer containing 100 mM DTT and incubated at 37 C for 20 min prior to loading onto a 4–12% Tris– Glycine precast gel (Invitrogen) for SDS–PAGE. Western blotting was done according to standard protocols, using either polyclonal rabbit anti-SmGPCR-IL3 (1:5000 dilution) or a monoclonal mouse anti-FLAG M2 antibody (1:5000 dilution) (Sigma, USA), followed by the appropriate horseradish peroxidase (HRP)- labeled secondary antibody (1:20,000 dilution). For analyses of schistosomal extracts, we homogenized 30–40 adult S. mansoni worms (mixed males and females) in the same 2% CHAPS buffer for 2 min on ice, using a handheld homogenizer. The resulting homogenates were similarly incubated for 2 h at room temperature with end-over-end rotation and centrifuged to remove insoluble material. Aliquots of the supernatant (5–6 µg protein) were subjected to SDS–PAGE and then Western blotted, as described above, using rabbit anti- SmGPCR (1:2500 dilution) and HRP-labeled goat anti-rabbit IgG as the secondary (1:20,000).

2.6. Immunoprecipitation of SmGPCR

Immunoprecipitations (IP) were performed with the Seize Primary Immunoprecipitation kit (Pierce, USA), according to the specifications of the manufacturer. The IP affinity column was prepared first by coupling approximately 400 µg of purified SmGPCR IgG to AminoLink Plus gel in the presence of sodium cyanoborohydride, followed by extensive washing, as described in the kit protocol. For the IP, a crude homogenate of adult S. mansoni was prepared as described above in the same homogenization buffer except that CHAPS was replaced with 1% Triton X-100. Aliquots of the crude extract were diluted with the „„Bind” buffer supplied with the kit (1:4 v/v), mixed with the IgG-linked gel and incubated overnight at 4 C with gentle rotation. After incubation, the gel was washed three to five times with a buffer containing 150 mM NaCl and 0.5% Triton X-100 and the bound proteins were eluted under 75

acidic conditions (pH 2.8). Unless otherwise specified, aliquots of the IP eluate were immediately neutralized to ~ pH 7.4 by addition of 1 M Tris–HCl, pH 9.5 (final concentration 50 mM) and then prepared in standard Laemmli SDS–PAGE sample buffer (pH 6.8) supplemented with 100 mM DTT (Laemmli, 1970). The gel and Western blot analyses of IP eluates were performed as described above for the crude schistosomal extracts. For experiments testing the effects of pH, aliquots of the IP eluate were adjusted to the desired final pH by addition of the appropriate buffer prior to SDS–PAGE. To test the effects of urea (4 M) and Triton X-100 (1%), the test substances were added to aliquots of the neutralized (pH 7.4) IP eluate at the indicated final concentrations. The samples were incubated for 15 min at room temperature after which they were prepared in reducing sample buffer and subjected to gel analysis and immunoblotting as above.

2.7. Quantitative PCR analyses

Total RNA was purified from S. mansoni cercariae, schistosomulae and adult worms, using RNeasy micro or mini kits (Qiagen, Mississauga, Ontario, Canada). The RNA was quantitated with a Nanodrop ND1000 spectrophotometer (Wilmington, USA) and equal amounts of RNA from the various developmental stages were used for reverse-transcription (RT). The RT was performed according to standard protocols in a 20 µl reaction volume containing purified total RNA (130–180 ng), 200U M-MLV reverse transcriptase (Invitrogen), 40U RNaseOUT ribonuclease inhibitor (Invitrogen), 0.5 µM oligo (dT)12–18 (or a gene-specific primer targeting SmGPCR positions 1282–1306: 5‟- GAGATGTCAAAGAAAATTCTCTATC-3‟), 0.5 mM dNTPs and 10 mM DTT in 1X first strand buffer (Invitrogen). The real-time qPCR was carried out with the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) in a final volume of 25 µl containing 2 µl of cDNA and 0.2 lM of each primer. The primers for qPCR were designed so as to amplify approximately 200 bp of either SmGPCR (Accession # AF031196) or S. mansoni GAPDH (Accession # 76

M92359), which was used as a housekeeping gene for data normalization. The SmGPCR primer pair was: 5‟-CATATTAAAGCGACACGTAAGC-3‟ (sense) and 5‟-TTGTGGTTGAGTAAACAACTCG-3‟ (antisense) and the GAPDH primers were: 5‟-GTTGATCTGACATGTAGGTTAG-3‟ (sense) and 5‟- ACTAATTTCACGAAGTTGT TG-3‟ (antisense). Standards consisting of various concentrations of plasmid pCIneo-SmGPCR were included in each experiment for subsequent quantitation of PCR data. The reactions were performed in a Rotor-Gene RG3000 instrument (Corbbett Research, Australia) and the cycling conditions were as follows: 50 C/2 min, 95 C/2 min followed by 45 cycles of 94 C/15 s; 53 C/30 s; 72 C/30 s. For comparison of steady-state SmGPCR transcript levels between miracidia and 4-day/20-day in vitro cultured sporocysts a similar procedural approach as that described above was used with the following modifications: After cDNA synthesis, real-time qPCR amplifications were performed in 96-well formatted optical tube strips and caps using a GeneAmp5700 qPCR apparatus (Applied Biosystems, Foster City, CA). SYBR Green reaction mixtures were identical to those described previously (Boyle et al., 2003), with cycle conditions consisting of: 95 C for 15 min, followed by 40 cycles of 15 s at 95 C and 60 s at 60 C. At the termination of all qPCR reactions, the generation of specific PCR products was confirmed by melting point dissociation curve analyses and DNA sequencing. Expression levels were determined according to the standard curve method (Bustin, 2000) and were normalized to the housekeeping genes GAPDH for cercarial, schistosomula and adult stages, and 18S rRNA for miracidia and sporocysts. The normalized data were then calculated as the fold-change in expression relative to the cercarial or miracidium stage, which was used as an arbitrary term of reference. To verify the quantification and for statistical analysis, the data were re-calculated by the comparative DDCT method (Livak and Schmittgen, 2001; Cikos et al., 2007) with identical results. For graphic purposes, all qPCR data are represented as fold- difference relative to transcript levels found in the miracidial or cercarial stage.

2.8. Other methods

Protein content was measured with a Lowry assay, using a commercial kit (BioRad). Indirect ELISA was performed in 96-well plates coated with purified recombinant SmGPCR il3 protein (0.25–25 µg/ml) and incubated with a serial dilution (1:250,000–1:50) of rabbit anti-SmGPCR antiserum or pre-immune serum, followed by incubation with a HRP-labeled secondary antibody (goat anti- rabbit IgG, 1:2000), according to standard protocols. Statistical comparisons of qPCR data were done with the Student t-test or a one-way ANOVA, followed by a Tukey pairwise comparison. A P 6 0.05 was considered statistically significant.

3. Results

3.1. Production of the anti-SmGPCR polyclonal antibody

A polyclonal antibody against SmGPCR was produced by immunizing rabbits with a purified fragment corresponding to the receptor‟s third intracellular loop (SmGPCR-il3, Fig. 1A). This region was selected because it is the most divergent among GPCRs and it has been used successfully to generate antibodies against other biogenic amine receptors (Levey et al., 1991; Rosin et al., 1993, 1998; Schiaffino et al., 1999; Zhou et al., 1999). A BLAST analysis of the SmGPCRil3 fragment against the S. mansoni genome database found no significant homology with other schistosome proteins, including other SmGPCR- like receptors. The antibody was first tested against the purified E. coli-expressed SmGPCR-il3 fragment by ELISA (not shown) and Western blot analysis (Fig. 1B). The Western blot identified a single band of the correct size, whereas no response could be seen with pre-immune serum or the pre-adsorbed antibody control, showing the interaction was specific. To test if the antibody was suitable for immunofluorescence analyses, and to verify its specificity, we performed a confocal analysis of a stably transfected cell line that expresses SmGPCR fused to a green fluorescent tag (EYFP) at the C-terminal end. The results (Fig. 1A and C) show that cells expressing SmGPCR-EYFP (green fluorescence signal) were also 78

labeled by the anti- SmGPCR antibody (red fluorescence), as shown by co- localization of the two signals (yellow fluorescence overlay). No immunolabeling was detected either in untransfected cells (data not shown) or transfected cells treated with antibody pre-adsorbed with purified SmGPCR-il3 (Fig. 1C).

3.2. SmGPCR forms multiple species in transfected HEK293 cells and schistosomes In subsequent studies we performed Western blots of the full length SmGPCR expressed in HEK293 cells (Fig. 2A) and the native protein in schistosomal extracts (Fig. 2B). A crude membrane fraction was prepared from cells expressing FLAG-tagged SmGPCR (Hamdan et al., 2002a) and then probed both with anti-SmGPCR and an anti-FLAG antibody. The anti-SmGPCR antibody consistently recognized two prominent bands, one corresponding to the size of the SmGPCR monomer (~ 65 kDa) and a larger band at approximately twice that size (~ 130 kDa). In addition, we detected a „„smear” of very high molecular weight (>180 kDa) species at the top of the gel and, in some samples, a small presumed proteolytic fragment. It must be emphasized that these bands are all SmGPCR specific, since they were also recognized by the anti- FLAG antibody and none could be seen in the „„mock”-transfected control probed with either antibody (Fig. 2A). The Western blot analysis of S. mansoni extracts produced a similar pattern of immunoreactive proteins (Fig. 2B). The results show two bands at about 65 and 130 kDa, as well as a larger species that migrated just above the 180 kDa markers.

To verify these results, we immunoprecipitated the native receptor from schistosome extracts, using covalently attached anti-SmGPCR antibody beads. The protein was eluted from the beads under acidic conditions, neutralized and then tested by Western blotting with anti- SmGPCR first test of the neutralized (pH ~7.4). IP eluate confirmed the existence of multiple SmGPCR species, the monomer (65 kDa) and the dimer (130 kDa) bands being particularly prominent (Fig. 2C). Further analysis of the IP eluate revealed that the 130 kDa species was resistant to treatment with 100 mM DTT, 1% Triton X-100 and, surprisingly, 4 M 79

urea (data not shown) but was sensitive to acidic pH (Fig. 2C). Analysis of the acidified eluate (pH ~ 3.0) showed only one immunoreactive band corresponding to the monomer. Increasing the pH from 3.0 to 7.4 in a stepwise fashion caused progressive appearance of the 130 kDa band and a concomitant decrease in the intensity of the monomer, suggesting the two forms are interconvertible. Though originally described as monomeric, there is increasing evidence that some GPCRs form dimers and even larger oligomeric species that resist denaturation on SDS– PAGE gels (Zhu et al., 2005; Zanna et al., 2008). Our results suggest that SmGPCR exists both as a monomer and an exceptionally stable dimer of about 130 kDa. The dimer is resistant to common denaturing and reducing agents but is sensitive to pH, suggesting it is non-covalent in nature. A larger >180 kDa species could also be detected but the intensity of this band was weaker and more variable. It is unknown if the larger species is produced by non-specific aggregation of SmGPCR during SDS–PAGE, a common problem in gel analysis of integral membrane proteins, or if it represents an oligomeric form of the receptor.

3.3. Confocal immunofluorescence analysis of SmGPCR in S. mansoni

To determine the tissue localization of SmGPCR in different life cycle stages of S. mansoni, we probed miracidia, sporocysts, schistosomula and adult worms with the polyclonal anti-SmGPCR IgG, followed by a FITC-labeled secondary antibody. Animals were also treated with rhodamine-conjugated phalloidin to label cytoskeletal elements and muscle (Mair et al., 1998, 2000, 2003). Two different types of fixation protocols were tested, one that used cold acetone as a fixative (Avarzed et al., 1998; Guedes et al., 2002) and the standard 4% PFA protocol (Mair et al., 2000, 2003). Acetone fixation was found to provide more detailed structural information but it disrupts phalloidin binding to actin (Bernard-Trifilo et al., 2006) and therefore only those samples treated with PFA were counterstained with phalloidin. An analysis of the early larval stages revealed virtually no SmGPCR in miracidia, whereas sporocysts exhibited distinct 80

green fluorescence localized particularly on and within the surface tegument and cells embedded in the parenchymal matrix. This was seen most clearly in 4-day old sporocysts (Fig. 3A and B) and could not be detected in any of the controls tested, including the pre- adsorbed antibody control (Fig. 3C and D). Schistosomulae exhibited strong and surprisingly widespread SmGPCR fluorescence staining in the tegument, acetabulum, anterior muscle cone, parenchyma and, less consistently, within the esophagus (Fig. 3E and G). To test whether SmGPCR was associated with muscle, we repeated the experiment in schistosomulae that were counterstained with TRITC-conjugated phalloidin. Intense phalloidin staining was clearly seen in the schistosomula mainly as striated muscle bands (Fig. 3I). An overlay of SmGPCR fluorescence (green) with phalloidin (red) produced regions of intense yellow fluorescence, suggesting that SmGPCR co-localizes with muscle. The strongest area of co-localization was in the musculature of the anterior cone, the acetabulum and the subtegumental musculature (Fig. 3I–K). Finally, in adult S. mansoni, SmGPCR was widely expressed in the tubercles of the male tegument (Fig. 4A, C and D). There was no comparable pattern of fluorescence in any of the male controls tested (Fig. 4B). Counterstaining with TRITC-conjugated phalloidin revealed a surface pattern of green fluorescent tubercles surrounded by red spines, with no apparent co- localization. Aside from the tubercles, significant SmGPCR fluorescence was detected in the subtegumental musculature, particularly in the anterior end and head region (Fig. 4E). Female worms exhibited strong fluorescence in the reproductive organs (not shown) but this was presumed to be an artifact caused by autofluorescence, since it could also be seen in the negative controls.

3.4. Quantitative RT-PCR analysis of SmGPCR mRNA expression

The confocal IFA analysis detected SmGPCR in all parasitic stages tested, including sporocysts, schistosomula and adults, but not miracidia. We also noted that the extent of immunolabeling varied among the parasitic stages, with the schistosomula showing widespread fluorescence, whereas in the adults the 81

receptor was more restricted to the tegument and subtegumental region. This led us to question whether SmGPCR expression might be developmentally regulated in S. mansoni. To address this question, we started by comparing the level of SmGPCR protein in different stages of S. mansoni by means of Western blot analyses. We detected the monomer and dimer in all the stages tested (cercariae, schistosomula and adults) but the analysis was not sufficiently quantitative to assess differences in expression among these stages. As an alternative strategy, we measured expression at the mRNA level, using real-time quantitative RT-PCR. Expression levels were compared first in S. mansoni cercariae, schistosomula and adult worms. In a separate experiment SmGPCR transcript levels were compared between miracidia and in vitro cultured sporocysts. The qPCR data were standardized relative to housekeeping genes (GAPDH or 18S rRNA) and the differences in expression were calculated, using standard curve and/or comparative Ct methods (Bustin, 2000; Cikos et al., 2007; Livak and Schmittgen, 2001), with similar results. Results of the analysis of the mammalian host stages suggest that SmGPCR is upregulated immediately after cercarial transformation (Fig. 5A). We detected a strong ~10-fold increase (P < 0.01) in SmGPCR expression in newly transformed schistosomulae (S0) compared to cercariae. SmGPCR levels increased further at 7 days post-transformation (P <0.0001) and then returned to S0 level in older (14-day) schistosomula and the adult worms. To test if SmGPCR is upregulated in the snail parasitic stage, we performed a second comparative RT-qPCR analysis of S. mansoni sporocysts and miracidia. The results showed that SmGPCR mRNA levels were increased 20- fold in 4-day old sporocysts (P < 0.01) and greater than 200-fold in 20-day old sporocysts (P < 0.001) compared to miracidia (Fig. 5B).

4. Discussion

Nearly 50 years after the first discovery of biogenic amines in schistosomes, very little is known about their mode of action. One of difficulties is the continuing lack of molecular information about amine receptors in any of the 82

parasitic flatworms. Aside from the prototype, rhodopsin (Hoffmann et al., 2001) and SmGPCR (Hamdan et al., 2002a), only one other member of this receptor superfamily has yet been cloned from these animals (Pearson et al., 2007). SmGPCR was previously shown to be activated by HA when expressed in mammalian cells (Hamdan et al., 2002a). The response to HA was dose- dependent and produced an intracellular calcium response, suggesting this receptor may be coupled to Gq and the Ca2+/phosphoinositol signaling pathway. Importantly, the receptor could not be activated by any of the other known biogenic amine transmitters, suggesting it is selective for HA. We cannot, however, rule out the possibility that there may be an unknown, structurally related amine in schistosomes that can activate the receptor in vivo. SmGPCR is unusual in that it shows about the same level of homology with all different types of biogenic amine receptors, including the histaminergic receptors, and so cannot be identified by sequence analysis. The most surprising feature of this receptor is that its predicted binding pocket lacks a highly conserved TM3 aspartate (Asp3.32), which is replaced with an asparagine. Asp3.32 is believed to be directly involved in the binding of biogenic amines and is present in every other aminergic GPCR cloned to date, both vertebrate and invertebrate (Roth and Kristiansen, 2004; Roth, 2006). In SmGPCR, however, the Asp Asn3.32 substitution does not hinder HA-induced activity (Hamdan et al., 2002a), suggesting the conformation of the binding pocket is quite different in this receptor. Following the completion of the S. mansoni genome project, we can see at least two closely related homologues of SmGPCR in the SchistoDB genome database, both of which have the same unique Asp Asn3.32 substitution in TM3. This raises the possibility that SmGPCR is part of a cluster of amine receptors that diverged early in evolution and may be unique to the parasites. It remains to be determined if these other SmGPCR-like sequences also encode functional receptors and if they are similarly activated by HA.

To further characterize this receptor, we began by raising polyclonal antibodies to a portion of the third intracellular loop (il3) of SmGPCR. This is the most divergent region among GPCRs and as such is often targeted for antibody production. A first evaluation based on ELISA, in situ immunofluorescence and Western blotting indicated that the antibody was of high titer and could recognize the full-length 65 kDa protein both in transfected HEK293 cells and schistosomal extracts. We noted, however, that the antibody also recognized a band of about 130 kDa as well as a mixture of high MW species that migrated at the top of the gel. To assess the specificity of the signal, we used a FLAG-tagged SmGPCR expressed in HEK293 cells and repeated the Western analysis with a different antibody targeting the FLAG epitope. The results showed virtually the same pattern of immunoreactive species with both anti-SmGPCR and anti-FLAG antibodies, whereas no signal could be detected in the mock-transfected cells, cells treated with pre-immune serum or the il3-preadsorbed antibody control. Thus we concluded that the multiple bands are all derived from SmGPCR and represented different forms or aggregates of the receptor. There are many reports of GPCRs forming dimers and higher order species that resist denaturation on SDS–PAGE gels (Bai et al., 1998; Bouvier, 2001; Romano et al., 1996; Romano et al., 2001; Balasubramanian et al., 2004; New et al., 2006). The higher MW species could be artifacts caused by membrane solubilization and aggregation of the receptor in the SDS-rich environment of the gel, a common problem in studies of membrane proteins. GPCR dimers, on the other hand, appear to be physiologically relevant. GPCR dimerization has been reported to play an important role in the regulation of ligand specificity, binding affinity and conformational activation (Bouvier, 2001). The exact chemical nature of these dimers is not known but there is increasing evidence they can involve intermolecular disulfide linkages, non-covalent interactions, or both (Hebert et al., 1996; Bai et al., 1998; Romano et al., 1996; Romano et al., 2001; Franco et al., 2007; Dalrymple et al., 2008). The 130 kDa species described here is most likely a non-covalent dimer of SmGPCR. This is suggested by the fact that it is resistant 84

to reducing agents such as DTT but can be made to dissociate at acidic pH. That it was seen consistently in preparations of recombinant and native receptor suggests the dimer is a biologically relevant species. The higher MW forms are harder to interpret at this point. Their large size, variable intensity and the fact they were more abundant in transfected cells, where the receptor is overexpressed, all suggest these are SDS-induced aggregates that formed during the gel analysis. We cannot rule out other explanations, however, including the possibility of receptor oligomerization and/or heavy glycosylation producing these larger forms of the receptor.

The anti-SmGPCR antibody was subsequently used to investigate the developmental stage-specific expression and tissue distribution of the receptor in S. mansoni. HA has been implicated in the control of neuromuscular function in these animals (see Ribeiro et al., 2005) and therefore we had expected SmGPCR to be associated with neuronal and/or muscle structures that are innervated by HA-containing neurons. It, therefore, was surprising to find that SmGPCR immunoreactivity was found predominantly in the „„parasitic” stages (sporocysts, schistosomulae and adults), compared to the free-swimming miracidial and cercarial larval forms. This finding was corroborated by SmGPCR gene expression data demonstrating significant receptor gene upregulation in parasitic vs. free swimming stages, suggesting a critical role of SmGPCR in the establishment and/or maintenance of parasitism within their hosts. To date, however, that role remains unclear. Histaminergic neurons have proven difficult to visualize in S. mansoni but in another trematode (H. cylindracea) they were shown to innervate the musculature of several tissues, including the body wall muscles and the acetabulum, as well as the cerebral ganglia and major nerve cords of the CNS (Eriksson et al., 1996). The pattern of HA innervation in the musculature correlates well with that of SmGPCR described here. The results showed significant co-localization of SmGPCR immunofluorescence and phalloidin in the subtegumental musculature, particularly the outer layer beneath

the tegument, both in schistosomula and adult worms. In the schistosomula, we also detected significant expression in the musculature of the suckers, in particular the acetabulum. These results are consistent with a role for SmGPCR in neuromuscular transmission or modulation. That the receptor is expressed in the body wall musculature further suggests that SmGPCR mediates at least some of the effects of HA on schistosome motility. One obvious difference between the tissue distribution of this receptor and the previously described pattern of HA innervation is in the CNS, which was shown to be rich in HA fibers (Eriksson et al., 1996) and yet did not express SmGPCR. If the same neuronal architecture exists in schistosomes, this suggests that there may be other HA receptors that mediate neuronal signaling within the CNS.

Aside from the musculature, the most conspicuous site of SmGPCR expression was the tegument. We detected consistent immunoreactivity in the tegument of all parasitic stages tested, including sporocysts, schistosomula and the adult worms. Adult males, in particular, showed very robust expression in the tubercles of the outer tegument. The discovery of a signal transducing receptor on the surface of schistosomes is surprising but not unprecedented. In recent years, researchers have identified an acetylcholine (nicotinic) receptor (Camacho et al., 1995; Bentley et al., 2004), receptor tyrosine kinases (Davies et al., 1998; Forrester et al., 2004; Osman et al., 2006) and at least one other GPCR of unknown function (Pearson et al., 2007) in the tegument of S. mansoni or S. hematobium. Being situated on the surface, these receptors are not likely to be part of an endogenous signaling system. Rather, they are believed to be activated by exogenous signals and to mediate some form of host–parasite communication. The presence of SmGPCR on the surface suggests that it too is activated by an exogenous substance, presumably HA or a HA-like substance that is present in host blood. There is evidence that the parasite stimulates HA production to facilitate passage through blood vessels during the initial migration (Catto et al., 1980; Boros, 1989; Rao et al., 2002). A tegumental HA receptor may be part of a

system that allows the parasite to respond to a change in environmental HA, for example by increasing motility or some other unknown behavior. Given that SmGPCR is clustered in the tubercles, which are enriched in sensory nerve endings (Gustafsson, 1987), this receptor could be acting through chemosensory circuits that originate at the parasite surface. The neuronal processes that supply the tubercles connect to peripheral elements and ultimately the CNS, so that any signaling through these circuits could have profound effects on worm behavior.

These results raise new questions about the role of HA in S. mansoni. Since it was first described nearly four decades ago, there have been conflicting reports about the biological relevance of HA in these parasites. On the one hand, the tissue level of HA in S. mansoni appears to be low, lower than that of other parasitic platyhelminths (Schwabe and Kilejian 1968; Perez-Keep and Payares 1978; Eriksson et al., 1996). This has made it difficult to visualize histaminergic neurons in situ. On the other hand, there is evidence that HA has effects on parasite motility (Ercoli et al., 1985; Ribeiro et al., 2005) and the existence of SmGPCR suggests these are receptor-mediated effects. The present results reinforce the notion that HA signaling is important in schistosomes. Based on the distribution of SmGPCR, we suggest there may be two HA systems operating in these animals, an endogenous system that controls primarily the musculature, and an exogenous one located on the tegument that is probably activated by host- derived amine. The data also suggest these systems are differentially expressed during the course of the life cycle. Sporocysts show robust SmGPCR expression both at the RNA and protein levels and the receptor appears to be localized mainly to the tegument and its surface. However, we could not detect evidence of association with muscle fibers at this stage. In contrast the schistosomula and adult worms showed a more widespread distribution that included the musculature and other tissues, in addition to the tegument. These observations reinforce the previous hypothesis of a dual system of HA signaling involving direct receptor communication through the tegument (perhaps with exogenous or environmental

HA) and a neuronal receptor-HA interactions involving regulation of motility, muscle activity and the like.

As mentioned previously, SmGPCR was found to be more widely expressed in the parasitic sporocyst and young schistosomula than other stages, including adult worms. At the RNA level, we found that SmGPCR was upregulated several fold in day 7 schistosomula compared to older larvae (14 days) or adults. The timing of this upregulation suggests that HA signaling could be particularly important during initial larval development and the lung stage, which occurs roughly at 7–8 days post-infection. The most striking upregulation was seen in sporocysts. SmGPCR expression levels increased >200-fold in 20-day old in vitro cultured sporocysts compared to miracidia. At this time, the sporocysts have increased in length approximately two- to threefold and contain numerous embryonic cell masses, which are destined to become the next generation of motile daughter sporocysts (Yoshino and Laursen, 1995). The coincidence of this increased growth and cellular differentiation capacity with SmGPCR expression strongly suggests a possible regulatory role of this receptor in sporocyst growth and development. HA in sporocysts may be obtained through endogenous synthesis or more likely, may be contributed by the snail host itself. The freshwater gastropod, Lymnaea stagnalis, possesses abundant central and peripheral histaminergic neurons (Hegedus et al., 2004) that could serve as sources of free HA in innervated tissues or fluid spaces, as has been suggested for other biogenic amines (e.g., 5HT, dopamine) found in B. glabrata, intermediate host of S. mansoni (Manger et al., 1996; Boyle and Yoshino, 2002). Unfortunately, initial attempts to silence SmGPCR expression by RNAi have not been successful either in sporocysts or schistosomula (data not shown), even under conditions known to effectively silence other schistosome targets (Boyle et al., 2003; Nabhan et al., 2007). Thus the biological relevance of this upregulation remains unclear. More research is needed to elucidate the function of HA signaling and SmGPCR in these parasites.

Acknowledgments

The authors thank Dr. Fred Lewis (Biomedical Research Institute, Bethesda, Maryland, USA) for the supply of Schistosoma mansoni-infected snails, Jacynthe Laliberte and Jaime Sanchez- Dardon for the confocal microscopy assistance and Dr. F. Hamdan (Université de Montréal, Quebec, Canada) for the gift of expression plasmid pFLAG-SmGPCR-EYFP. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to P.R. and NIH Grant AI061436 to T.P.Y.

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Figures

Figure 1: Production of an anti-SmGPCR polyclonal antibody. A portion of the third intracellular loop (il3) of SmGPCR (pos. 355–449) was expressed in E. coli as a recombinant His-tagged protein and purified to homogeneity prior to injecting into rabbits to produce anti-SmGPCR antibody (A) Schematic representation of SmGPCR showing a typical seven transmembrane topology and the il3 region targeted for antibody production. (B) Anti- SmGPCR antibody (lane I) or antibody that was pre-adsorbed with an excess of purified il3 antigen (Lane II) were tested against the purified il3 protein by Western blot analysis. The results show a strong, single immunoreactive band of the correct size in samples probed with anti- SmGPCR IgG but only a weak band in the pre-adsorbed control. (C) The antibody was tested first in HEK293E cells that were stably transfected with SmGPCR fused at the C-terminal end to EYFP. Cells were incubated with anti-SmGPCR antibody followed by a rhodamine-conjugated secondary antibody. An overlay of rhodamine (red) and EYFP (green) produced bright yellow fluorescence, indicating co-localization of the two signals. No rhodamine fluorescence could be detected in cells incubated with pre-immune serum (not shown) or anti-SmGPCR antibody that was pre-adsorbed with purified il3 antigen (pre-adsorbed control) Cells were counterstained with Dapi (blue) to visualize the nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Figure 2: Western blot and immunoprecipitation analyses of SmGPCR. (A) HEK293E cells were transfected with a FLAG-tagged SmGPCR expressing vector (+) or empty vector (_). Cells were homogenized and aliquots of a solubilized crude membrane fraction were immunoblotted either with anti-SmGPCR IgG or with a monoclonal anti-FLAG antibody. The sizes of the protein ladder are indicated. (B) A representative Western blot analysis of an adult S. mansoni extract probed with anti-SmGPCR antibody. (C) SmGPCR was immunoprecipitated from a crude extract of S. mansoni, using beads covalently coupled to anti-SmGPCR IgG and then immunoblotted with the same antibody. The receptor was eluted from the antibody beads under acidic conditions and immediately neutralized to pH 7.5. At neutral pH we see three bands corresponding to the monomer (65 kDa), dimer (130 kDa) and a faint high MW species (>180 kDa) but only the monomer can be detected when the sampled was acidified to pH 3 prior to immunoblotting (left panel). A stepwise increase in pH (pH 3–9) caused progressive dimerization of the receptor (right panel). IB, immunoblotted; IP, immunoprecipitated.

Figure 3: Localization of SmGPCR in larval stages of S. mansoni. Samples were fixed with 4% PFA or ice-cold acetone, permeabilized with 0.5% Triton X-100 and then probed with anti-SmGPCR IgG followed by a FITC-labeled 2ry antibody. Red TRITC- phalloidin was used as a counterstain to visualize the musculature. Sporocysts: SmGPCR immunofluorescence (green) was detected on the tegument (arrows) and within the parenchyma of 4-day-old in vitro cultured sporocysts incubated with anti-SmGPCR (A and B) but not the pre- adsorbed anti-SmGPCR IgG control (C and D). An overlay of the phalloidin (red) and SmGPCR (green) signals showed no apparent co-localization (B). Schistosomula: In vitro transformed schistosomula were cultured for 4 days (E) or 8 days (G) and then probed with anti-SmGPCR antibody. Fluorescence can be seen in the acetabulum, the tegumental region, the esophagus and parenchyma cells. A close-up of the posterior end of a 8-day animal shows a distinct pattern of SmGPCR fluorescence in the acetabulum (H). No significant immunoreactivity could be detected in the negative controls treated with il3-preadsorbed antiserum (F), pre-immune serum or when the primary antibody was omitted (not shown). 28-day-old schistosomula were probed with anti- SmGPCR antibody (green) and phalloidin (red) to test for possible colocalization of the receptor with the musculature. Red phalloidin labeling of the major longitudinal, circular and oblique body wall muscles is clearly visible (I). The overlay identified significant co-localization (yellow fluorescence) in the muscle cone of the head region (arrow), musculature of the acetabulum (arrow head) and the subtegumental musculature (box) (I and J). A close-up of the parasite body wall shows co-localization of SmGPCR and phalloidin in the outer layer of the musculature (yellow fluorescence) (K).

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Figure 4: Localization of SmGPCR in adult worms. Adult male S. mansoni were probed with anti-SmGPCR IgG or a pre-adsorbed anti-SmGPCR IgG control, followed by green FITC labeled secondary antibody. Red TRITC labeled phalloidin was used to label the muscles and the tegumental spines. Animals incubated with antiserum show strong immunoreactivity in the tubercles of the dorsal tegument (A and C), whereas only background fluorescence could be detected in the negative control (B). Co-labeling with anti- SmGPCR and phalloidin produced a distinctive pattern of green immunoreactive tubercles surrounded by red phalloidin–labeled tegumental spines (D). Co-localization of SmGPCR with phalloidin was detected in the subtegumental musculature, particularly near the anterior head region (E, yellow fluorescence).

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Figure 5: Developmental expression of SmGPCR in S. mansoni. Quantitative PCR was performed on reverse-transcribed RNA from S. mansoni cercariae, adult worms and in vitro transformed schistosomula harvested immediately after transformation (stage 0, S0) and at 7 days (S7) or 14 days (S14) post-transformation (A) and from miracidia and 4-day and 20-day cultured sporocysts (B). The qPCR data were standardized by simultaneous amplification of internal housekeeping controls (GAPDH or 18S rRNA) and differences in expression data were calculated according to the comparative DDCT method. The results are shown as the fold-change in SmGPCR expression relative to the cercariae (A) or miracidia (B) and are the means ± SEM of a minimum of three experiments, each in triplicates. S, schistosomula; spo, sporocyst.

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CONNECTING STATEMENT 1

In manuscript I, we described the subunit organization of SmGPCR, its mRNA expression and tissue distribution in different stages of S. mansoni. The histamine receptor was found to be preferentially expressed in the parasitic forms and was enriched in the tegument, subtegumental musculature and/or tubercles. In the next manuscript, we describe the cloning, functional analysis and immunolocalization of second S. mansoni receptor that is structurally related to SmGPCR. As discussed in the manuscript, the two receptors belong to a group of novel schistosome biogenic amine receptors that have no mammalian or invertebrate orthologues. Due to their novelty, we have named these receptors S. mansoni GPR (SmGPR) in keeping with the nomenclature used for human orphan GPCRs. Thus SmGPCR has been re-named SmGPR-1 and the second receptor is SmGPR-2. Similar to the prototype, SmGPR-2 was found to be activated by HA. The finding of a second HA receptor suggests this is an important biogenic amine in S. mansoni with potential multiple roles. We also investigated the distribution of SmGPR-2 and compared to that of histaminergic neurons in adult parasites.

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CHAPTER III (manuscript II)

Histamine signalling in Schistosoma mansoni: Immunolocalization and characterization of a new histamine receptor (SmGPR-2)

Fouad El-Shehabi and Paula Ribeiro*

Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9

* To whom correspondence should be addressed Tel: (514) 398-7607 Fax: (514) 398-7857

E-mail: [email protected]

in preparation for submission to International Journal for Parasitology

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Abstract

In parasitic platyhelminths, including Schistosoma mansoni, biogenic amines (BAs) play several important roles in the control of motility, metabolism, reproduction and survival. A bioinformatics analysis of the S. mansoni genome identified >15 G-protein coupled receptors (GPCRs) that share significant homology with aminergic receptors from other species. Six of these sequences are structurally related to SmGPCR, a previously described histamine (HA) receptor of S. mansoni and constitute a new clade of BA GPCRs. Here we report the cloning of a second member of this clade, named SmGPR-2 (Smp_043340). The full-length receptor cDNA was expressed in Saccharomyces cerevisiae and shown to be activated by histamine and 1-methylhistamine, whereas other common BA had no significant effect. Antagonist assays showed that SmGPR-2 was inhibited by classical BA antagonists but the pharmacological profile was unlike those of known mammalian histamine (H1-H4) receptors. Confocal immunolocalization studies identified SmGPR-2 in the subtegumental neuronal plexuses of the adult S. mansoni and larvae. HA was widely distributed in both the Central and peripheral nervous systems, including the subtegumental regions where the receptor is expressed. Finally, SmGPR-2 was shown to be developmentally regulated at the RNA level. Quantitative PCR studies showed it was upregulated in the parasitic stages compared to the cercaria and expressed at the highest level in young schistosomula. The widespread distribution of HA and the presence of at least two receptors in S. mansoni suggest that HA is an important neuroactive substance in schistosomes.

Keywords: Schistosoma mansoni, histamine, biogenic amines, GPCR, receptor, platyhelminth, neurotransmitter, immunolocalization.

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1. Introduction

Among the five bloodfluke species that infect more than 200 million people worldwide and cause schistosomiasis, Schistosoma mansoni (Platyhelminth, trematoda) is the causative agent of >90% of all infections. This trematode exists where its intermediate host, the freshwater snail Biomphalaria glabrata is available, notably in Africa, the Middle East, South America and the Caribbean (Olds and Dasarathy, 2001). Praziquantel (PZQ) 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 of the pharmaceutical drugs currently available on the market exert their effects by interacting with G-protein coupled receptors (GPCRs) (Wise et al., 2002; Eglen, 2005), in particular Family A (Rhodopsin-like) receptors, 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 characterized. 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 (NTs), hormones or modulators (Ribeiro et al., 2005; Maule et al., 2006). They include such ubiquitous substances as serotonin (5-hydroxytryptamine, 5-HT), catecholamines such as dopamine (DA) and noradrenaline (NA) and histamine (HA). In platyhelminths, especially the parasitic flatworms, BAs play many vital roles in metabolism, the control of motility, and survival within the host. The most widespread and best studied BA is 5-HT. Serotonergic neurons are abundantly distributed in the central nervous system (CNS) and peripheral nervous system (PNS) of every flatworm tested to date, including S. mansoni. Moreover, 5-HT is strongly myoexcitatory (Pax et al., 107

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 transport (Patocka and Ribeiro, 2007), all of which is indicative of an important neurotransmitter system. By comparison, less is known about the role of other BAs, particularly histamine (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 the flatworms is unclear but it most probably affects the musculature and the outcome is concentration-dependent. It was reported that HA significantly affects movement in the posterior region of the strobila in Hymenolepis diminuta (Sukhdeo et al., 1984) and also 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 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 SmGPCR. 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 and functional analysis of a member of this new clade, named SmGPR-2. We expressed the cDNA in a heterologous yeast system for activity

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assays and the results indicate that SmGPR-2 is a second histaminergic receptor of S. mansoni. Immunolocalization studies identified the receptor in the subtegumental neuronal plexuses of the adult worms and larvae. A HA monoclonal antibody revealed that HA is widely distributed in the adult worms in both the CNS and PNS. Finally, we found that SmGPR-2 receptor is developmentally regulated and it is expressed at the highest level in schistosomula.

2. Materials and methods

2.1. The parasite

Biomphalaria glabrata snails infected with a Puerto Rican (NMRI) strain of S. mansoni were kindly provided by Dr. Fred Lewis, Biomedical Research Institute, Rockville, Maryland, USA. S. mansoni cercaria were collected 35-45 days post-infection (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 37ºC and 5%

CO2 in OPTI-MEM I reduced serum medium (Invitrogen) supplemented with 10% fetal bovine serum, streptomycin 100µg/ml, penicillin 100U/ml and fungizone 0.25µg/ml (El-Shehabi et al., 2009). To obtain adult parasites, 28-day old female CD-1 mice were infected with 150 cercariae / animal by skin penetration. Adult S. mansoni worms were recovered 6-7 weeks post-infection by perfusion of the liver (Basch and Humbert, 1981), washed extensively and either flash-frozen in liquid nitrogen for subsequent RNA extraction or fixed in two changes (1+3hours) of 4% paraformaldehyde in phosphate buffer solution, PFA/PBS at room temperature for immuno-localization experiments.

2.2. Cloning of S. mansoni SmGPR-2

The full-length SmGPR-2 cDNA was cloned from adult S. mansoni based on a predicted coding sequence (Smp_043340) obtained from S. mansoni

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GeneDB (http://www.genedb.org/ genedb/smansoni/). Total RNA was purified from 25-30 adult S. mansoni worms (Qiagen RNeasy kit) and was oligo-dT reverse-transcribed with MMLV reverse transcriptase (Invitrogen), according to standard procedures. To clone SmGPR-2, we designed primers that targeted the beginning and end of the predicted coding sequence. The primer sequences were as follows: 5‟- ATGAAACAAGTGTTTTTAAATGACAACAG-3‟ (sense) and 5‟-TTATATATTCCTTCCAATATGTAATAAACG-3‟ (antisense). A proofreading Platinum Pfx DNA polymerase (Invitrogen) was used to amplify the cDNA in a standard PCR reaction (35 cycles of 94C/15s, 55.6C/30s and 68C/90s). The resulting amplicon (1656 bp) was gel excised, purified (QIAquick spin kit, Qiagen), ligated to pGEM-T Easy vector (Promega) and verified by DNA sequencing.

2.3. Yeast functional expression assays

The SmGPR-2 coding sequence was subcloned between the NcoI / XbaI restriction sites of the yeast expression vector Cp4258 (Wang et al., 2006), kindly provided by Dr J. Broach, Princeton University, NJ, USA) and the resulting construct was confirmed by DNA sequencing. The functional expression assay was adapted from the protocol of Wang et al, 2006 as described by (Kimber et al., 2009). The receptor was expressed in Saccharomyces cerevisiae strain YEX108

(MAT PFUS1-HIS3 PGPA1-Gq(41)-GPA1-Gaq(5) can1 far1 1442 his3 leu2 lys2 sst22 ste14::trp1::LYS2 ste186-3841 ste31156 tbt1-1 trp1 ura3; kindly provided by J. Broach, Princeton University). This strain expresses the HIS3 gene under the control of the FUS1 promoter (Stevenson et al., 1992) and contains an integrated copy of a chimeric G gene in which the first 31 and last 5 codons of native yeast G (GPA1) were replaced with those of human Gq. Strains carrying chimeras of GPA1 and human Gi2, G12, Go or Gs were also tested in preliminary experiments but were found to yield lower or no receptor activity compared to strain YEX108. The budding yeast S. cerevisiae were cultured in yeast YPD medium, according to standard conditions and transformation was 110

performed by the lithium acetate method (Gietz et al., 1995), using approximately 200 µl mid-log phase cells, 200 µg carrier ssDNA (Invitrogen) and 1 µg Cp4258- SmGPR-2 or empty plasmid as a negative control. Positive transformants were selected on synthetic complete (SC) 2% glucose solid medium lacking leucine (SC/leu-). For the agonist assay, single colonies of transformants carrying plasmid Cp4258-SmGPR-2 or vector alone (mock control) were cultured overnight in SC/leu- liquid medium at 250 rpm/30C. Next day, the cells were washed three times in SC 2% glucose liquid medium that lacked both leucine and histidine (SC/leu-/his-). Cells were finally resuspended in SC/leu-/his- medium supplemented with 50 mM MOPS, pH 6.8 and 1.5 mM 3-Amino-1, 2, 4-Triazole (3-AT) to an approximate density of 3000 cells/well of 96-well plate. 3- Aminotriazole inhibits the gene product of HIS3 and was used to reduce basal growth due to endogenous background signaling (Wang et al., 2006). Aliquots of cell culture containing about 3000 cells were added to each well of a 96-well plate containing test agonist or vehicle plus additional medium for a total reaction volume of 100 µl. The plates were incubated at 30C for 22-26 hours, after which 10 µl of Alamar blue (Invitrogen) was added to each well. The plates was returned to the 30C incubator until the blue color of Alamar blue began to shift to pink (approximately 1-4 hours) and the fluorescence (560 nm ex / 590nm em) was measured at 30C every 30 minutes for 3-4 hours using a plate fluorometer (FlexStation II, Molecular Devices). The antagonist assay was done in the same way except that each well contained 10-4 M agonist (HA or 1-mHA, as indicated) and the antagonist at the specified concentration. Data analyses and dose response curve fits were performed using Prism v5.0 (GraphPad software Inc.).

2.4. Quantitative PCR analyses

Total RNA was purified from S. mansoni cercariae, schistosomulae and adult worms using RNeasy mini or micro kits, as required (Qiagen, Mississauga, Ontario, Canada). The RNA was quantitated with a Nanodrop ND1000 spectrophotometer (Wilmington, USA) and equal amounts of RNA from the 111

various developmental stages were used for the RT. The RT was performed according to standard protocols in a 20 µl reaction volume containing purified total RNA (130–180 ng), 200 U M-MLV reverse transcriptase (Invitrogen), 40U

RNaseOUT ribonuclease inhibitor (Invitrogen), 0.5 µM oligo (dT)12–18, 0.5 mM dNTPs and 10 mM DTT in 1X first strand buffer (Invitrogen). The real-time qPCR was carried out with the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) in a final volume of 25 µl containing 2 µl of cDNA and 0.2 µM of each primer. The primers for qPCR were designed so as to amplify approximately 200 bp of either SmGPR-2 (Accession # GQ397114) or S. mansoni GAPDH (Accession # M92359), which was used as a housekeeping gene for data normalization. The SmGPR-2 primer pair was: 5‟-CGTATCAAGAGGTATCTC- 3‟ (sense) and 5‟-CATTCCACTCTGGTTGTAC-3‟ (antisense) and the GAPDH primers were: 5‟-GTTGATCTGACATGTAGGTTAG-3‟ (sense) and 5‟- ACTAATTTCACGAAGTTGTTG-3‟ (antisense). The reactions were performed in a Rotor-Gene RG3000 instrument (Corbbett Research, Australia) and the cycling conditions were as follows: 50C/2 min, 95C/2 min followed by 45 cycles of 94C/15 s; 53C/30 s; 72C/30 s. At the termination of all qPCR reactions, the generation of specific PCR products was confirmed by melting point dissociation curve analyses and DNA gel electrophoresis. Expression levels of the different parasite stages were normalized to the internal housekeeping genes GAPDH control. The normalized data were then calculated as the fold- change in expression relative to the cercarial stage and calculated by the comparative CT method (Livak and Schmittgen, 2001; Cikos et al., 2007). For graphic purposes, all qPCR data are represented as fold-difference relative to transcript levels found in the cercarial stage.

2.5. Immunolocalization studies

A polyclonal anti-SmGPR-2 antibody was purchased from 21st Century Biochemicals, MA, USA. The antibody was raised in rabbits against two unique SmGPR-2 peptides, which were conjugated to ovalbumin as a carrier. The 112

peptides correspond to the first sixteen amino acids in the predicted extracellular N-terminal domain and positions 416-437 (of the 3rd intracellular loop region). Peptide sequences were BLASTed against the Schistosome Gene Database as well as the general protein database at NCBI to insure specificity. The antiserum was tested first by ELISA and shown to be of high titer. The IgG fraction was subsequently purified by protein A sepharose affinity chromatography (Sigma, Canada), dialyzed against PBS, pH 7.4, and the protein concentration was adjusted to 5 mg/ml. Confocal immunolocalization studies were performed both in adult worms and in vitro transformed schistosomula. The procedure is based on the protocols of D. Halton and colleagues (Mair et al., 2000; Mair et al., 2003) and El-Shehabi et al (2009). Adult worms and larvae were washed three times in PBS, pH 7.2 and were fixed in 4% PFA in PBS overnight at 4°C. The samples were washed 3-4 times in PBS and blocked overnight in PBS containing 5% goat serum and 0.5% TritonX-100 (PBST). Next day, the samples were incubated with purified anti SmGPR-2 primary antibody (1:150 dilution in PBST) for 3-4 days at 4°C with end-over-end rotation. Animals were washed 3 times in PBST and were incubated in the secondary antibody labelled either with FITC or rhodamine as required (1:300 dilution in PBST) for 3 days 4°C with rotation. In HA immunolocalization experiments, mouse anti-HA monoclonal antibody (Millipore) was used in 1:150 diluted in 5% goat serum in PBST for 3 days and the secondary antibody was rhodamine labeled goat anti mouse antibody (1:300), incubated for 3 days at 4C with gentle rotation. When phalloidin (0.2mg/ml) was used as a counter stain, 400 ng of tetramethylrhodamine B isothiocyanate (TRITC)-labeled phalloidin (Sigma, USA) was added during the last two days of incubation with secondary antibody. Following incubation, the worms were washed in PBST, mounted on a slide and examined using a BIO-RAD RADIANCE 2100 confocal laser scanning microscope equipped with Nikon E800 fluorescence microscope for confocal image acquisition and the LASERSHARP 2000 software package. The following controls were used routinely (a) omission of primary antibody (b) replacing the primary SmGPR-2 antibody with 113

preimmune serum or (c) using antibody that was preadsorbed with an excess of peptide antigen.

2.6. Other methods

Protein content was measured with a Lowry assay, using a commercial kit (BioRad). Indirect ELISA was performed in 96-well plates coated with individual or pooled SmGPR-2 peptides (50 - 500 ng/well) and incubated with a serial dilution of rabbit anti-SmGPR-2 antiserum or pre-immune serum (1:30,000 – 1:100), followed by incubation with a HRP-labeled secondary antibody (goat anti- rabbit IgG, 1:2000), according to standard protocols. Statistical comparisons of qPCR data were done with the Student t-test or a one-way ANOVA, followed by a Tukey pairwise comparison. A P 0.05 was considered statistically significant.

3. Results

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

A bioinformatics search of the S. mansoni genome database (http://www.genedb.org/ genedb/ smansoni/) identified a sequence (Smp_043340) that was structurally related to SmGPR-1 (formerly SmGPCR; accession # AF031196), 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) and was designated SmGPR-2. When compared to the sequences available in the current version (v.4.0) of the S. mansoni genome, we identified 18 possible biogenic amine GPCRs, of which 6 sequences (including the novel SmGPR-2) are closely related to SmGPR-1. The pairwise alignment analysis of SmGPR-2 with other related aminergic receptors, using the BLOSUM62 matrix of BioEdit Sequence Alignment Editor (V7.0.9.0) include Smp_043270 (62.4% homology), Smp_043300 (73.5% homology), Smp_145520 (55.6% homology), Smp_043290 (46.0% homology), Smp_043460 (38.0% homology) and the

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prototype, SmGPR-1 (43.0% homology). SmGPR-2 is also related to aminergic GPCRs from other species but the level of homology is lower, about 30%, and there is no clear relationship to any one particular type of amine receptor. A large dendogram analysis of amine GPCRs from schistosomes and other species shows that the SmGPR sequences cluster together and appear to have evolved from a common ancestor, suggesting this new clade of amine-like receptor (Fig. 1).

These receptors have the characteristic heptahelical topology and all of the signature motifs of class A GPCRs, including the DRY motif in TM3, GxxNS and NPxxY in TM7. We also identified several residues that have been implicated in biogenic amine binding and receptor activation, notably the aromatic cluster FxxCWxPFF of TM6 (Choudhary et al., 1993; Kristiansen et al., 2000; Ballesteros and Palczewski, 2001). The SmGPR receptors are unusual, however, in that they lack an important functional aspartate (D3.32) of TM3 (Fig. 2). This residue is conserved in every biogenic amine GPCR identified to date, both vertebrate and invertebrate, and it is considered to be essential for receptor activity (Shi and Javitch, 2002; Roth and Kristiansen, 2004; Roth, 2006). In modeling studies, this aspartate serves as an anchoring point for the different amines (Massotte and Kieffer, 2005). The other predicted amine receptors in the S. mansoni database also carry this conserved aspartate, whereas the majority of the SmGPR-1-like sequences, including the novel SmGPR-2, have an asparagine at this position (Fig. 2). Even conservative mutations of D3.32 are sufficient to abolish receptor activity in other species (Muntasir et al., 2006). Thus the Asn substitution marks a significant departure from current models of receptor structure. The data suggest that SmGPR-2 belongs to a new type of amine receptor that diverged early in evolution and may be schistosome-specific.

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3.2. Functional assays: SmGPR-2 is a second histamine receptor of S. mansoni To test for receptor activity, the full-length SmGPR-2 cDNA was ligated to a yeast expression plasmid and introduced into S. cerevisae. We used a histidine auxotrophic strain that expresses a HIS3 reporter gene under the control of the FUS1 promoter. Activation of a recombinant GPCR in this system in the presence of the appropriate ligand increases expression of the HIS3 reporter via the yeast‟s endogenous pheromone response, which in turn allows the cells to grow in histidine-deficient medium (Wang et al., 2006). Thus receptor activity was quantified based on measurements of yeast growth in the selective medium, using a fluorometric Alamar Blue assay. Cells transformed with SmGPR-2 or empty vector were initially tested with all different biogenic amines, each at 10-4 M (Fig. 3A). The results showed that SmGPR-2 was selectively activated by HA. The receptor exhibited constitutive activity in the absence of agonist but it was further activated by addition of HA, whereas other biogenic amines had no significant effect. Experiments were repeated with different concentrations of HA and the response was shown to be dose-dependent (Fig. 3B). Moreover, SmGPR-2 could be activated by 1-methylhistamine (1-methylHA), a common HA metabolite, and was strongly inhibited by the histaminergic antagonist, promethazine (Fig. 4A). 1- methylHA was a more powerful agonist than HA itself in two separate clones of SmGPR-2-expressing cells, causing 5-6.5 fold increase in growth compared to the untreated (no agonist) control and as much as 60-fold increase when compared to the mock-transfected control. As for promethazine, the addition of the drug at 10-4 M inhibited all receptor activity either in the presence of HA (10-4M) or 1-metHA (10-4M) and the effect was dose-dependent (Fig. 4B). Because the assay is based on cell growth, we questioned whether the inhibitory effect of promethazine was due to drug-induced toxicity leading to cell death. To test this possibility, we repeated the assay in medium supplemented with histidine (10-4M), which enables cell growth irrespective of receptor activation. The results showed normal growth in promethazine-treated cells in the presence of histidine (Fig. 4B), indicating that 116

the inhibitory effect of the drug was receptor-mediated and not the product of generalized toxicity. In addition to promethazine, we tested three classical (mammalian) HA antagonists (diphenhydramine, cimetidine and ranitidine) as well as a battery of drugs that normally target other BA receptors (Fig. 5). Among the histaminergics, only promethazine was able to inhibit HA-induced activity in three separate clones of the receptor. The other three drugs had no antagonist activity and produced, instead, a small increase in activity. Aside from promethazine, we observed significant inhibition by cyproheptadine, flupenthixol and, to a lesser extent, buspirone. Cyproheptadine has broad specificity and has been shown to target HA receptors as well 5HT receptors in vertebrates. Flupenthixol and buspirone are classical antagonists of DA and 5HT receptors, respectively, and are not known to have antihistaminic activity. As in the case of promethazine, these drugs did not inhibit normal cell growth in histidine- containing medium at the concentrations tested (data not shown) and therefore the inhibition is presumed to be specific. Mianserin, a mixed adrenergic/5HT antagonist had no effect on SmGPR-2, nor did sulpiride, a classical DA antagonist.

3.3. SmGPR-2 expression is upregulated in schistosomula

We have previously shown that SmGPR-1 is markedly upregulated at the RNA level in young schistosomula compared either to cercaria or adult worms (El-Shehabi et al., 2009). Because SmGPR-2 is structurally related, and to address whether its expression is developmentally regulated, we compared mRNA levels in different developmental stages of S. mansoni by real-time qPCR. The data were calculated according to the comparativeCt method, using the housekeeping gene GAPDH as an internal control and the cercarial stage as the calibrator reference. The results show that the receptor mRNA is expressed in all stages tested but the level of expression is developmentally regulated. SmGPR-2 expression increased 4-5 fold immediately after transformation from cercaria to stage 0 schistosomula (S0) and the expression level continued to increase up to 117

about 60-fold within the first week of culture (Fig. 6). As the animals aged beyond 7 days, SmGPR-2 levels were downregulated first in the 14-day schistosomula and more so in the adult worms, where the level of expression is comparable to that of the newly transformed S0 larvae. This developmental pattern is similar to that of SmGPR-1 (El-Shehabi et al., 2009) and suggests that HA receptors are particularly important during early larval development.

3.4. In situ localization of histamine (HA) in S. mansoni

HA is present in schistosomes (Perez-Keep and Payares, 1978; Ercoli et al., 1985) but its tissue distribution is unknown. Here we used a commercial monoclonal anti-HA antibody to localize the amine in S. mansoni (Fig. 7). The results revealed surprisingly abundant and widespread HA immunoreactivity throughout the nervous system of the parasite. HA was identified in the main longitudinal nerve cords and transverse commissures throughout the length of the body of both male and female worms (Fig. 7A-C). The pattern of histaminergic neurons in the transverse commissures has the characteristic “orthogonal” organization of the platyhelminth CNS (Fig. 7B). Some of these processes are seen to innervate other tissues, especially the reproductive tract in the females (Fig 7C) and also excretory ducts (not shown). HA immunoreactive nerve fibers are prominent in the musculature of both the oral and the ventral suckers of the adult male (Fig 7D, E) and to a lesser extent in the smaller female suckers. This innervation is varicose in appearance and runs deeply into the musculature of the suckers. Another distinctive area of HA immunoreactivity is the subtegumental region, where HA-containing cell bodies and processes are clearly visible in the subtegumental nerve plexuses that supply the somatic musculature (Fig. 7F). These occur along the length of the body but are particularly enriched in the tail end of both genders (Fig. 7G). The HA detected in this study was associated mainly with neuronal elements. We observed some fluorescence in the female reproductive tract, notably the vitellaria but this is presumed to be non-specific since it was also present in the negative controls. 118

3.5. Confocal immunofluorescence analysis of SmGPR-2 in S. mansoni

The tissue localization of the receptor SmGPR-2 was examined in adult and larval S. mansoni. We selected in vitro cultured 7 day- old schistosomula for these studies because they were shown in the qPCR analysis to have the highest SmGPR-2 expression level. The larvae and adult worms were probed with rabbit polyclonal anti-SmGPR-2 IgG, followed by a FITC-labeled goat anti-rabbit secondary antibody. Some animals were also treated with TRITC-conjugated phalloidin to label cytoskeletal elements and muscle (Mair et al., 2000; Mair et al., 2003). The results showed strong SmGPR-2 green fluorescence in the subtegumental region of the larvae (Fig.8A, B). The signal could be seen along the entire length of the body and was associated with the subtegumental innervation rather than the musculature. We could not detect co-localization of SmGPR-2 (green) and muscle (red) in larvae that were counterstained with TRITC-conjugated phalloidin (Fig 8C). SmGPR-2 immunoreactivity in the adults was generally weaker than in the larvae but the distribution pattern was similar. Most of the expression was restricted to the peripheral neuronal plexuses of the subtegumental region (Fig. 8D & E). Importantly, we observed that the localization of the receptor in this area closely resembles that of HA itself. Animals probed with the two antibodies showed distinctive anti-SmGPR-2 immunofluorescence (green) in close proximity to anti-HA immunofluorescence (red) in the subtegumental plexus (Fig. 8F). No co-localization could be seen, however, indicating that the transmitter and its receptor are present on different cells. Aside from this region, we observed weak expression of the receptor in the oral and ventral suckers (not shown) but not in the CNS.

4. Discussion

Previously, our lab described the first HA-responsive receptor of S. mansoni, named SmGPR-1 (formerly SmGPCR; Hamdan et al., 2002). In the present study, we report the cloning and expression of a structurally related

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receptor, which we have named SmGPR-2. The bioinformatics analysis identified a S. japonicum EST (SJCHGC07612) and a total of six orphan receptors in the genome of S. mansoni that share high sequence homology with SmGPR-1 and SmGPR-2. These sequences do not align within the known clades of the biogenic amine GPCR tree and thus appear to constitute a new type of amine receptor. The SmGPR homologues are characterized in part by the absence of the highly conserved aspartate D3.32 of TM3, which is replaced with an asparagine in all but one of these receptors (Smp_043290; SmGPR-3, accession # GQ259333). As mentioned earlier, D3.32 is a critical residue in the ligand-binding pocket of BA receptors (Shi and Javitch, 2002; Roth and Kristiansen, 2004; Roth, 2006). The side-chain carboxylate of D3.32 is believed to form direct contact with the protonated amino moiety of the different BAs via a salt bridge interaction (Strader et al., 1987; Strader et al., 1991; Mansour et al., 1992; Boess et al., 1998; Shi and Javitch, 2002; Jongejan et al., 2008). Studies have shown that mutagenesis of D3.32 can decrease binding affinity, decrease specificity or even abolish ligand binding activity all together. For instance, D3.32N or D3.32A single-point mutations were shown to abrogate ligand binding to 5HT2C and histaminergic (H4) receptors (Muntasir et al., 2006) and to reduce binding affinity of muscarinic and H1 receptors (Ohta et al., 1994; Nonaka et al., 1998). Thus the D3.32N substitution of the schistosome sequences is surprising and suggests a fundamental difference in the organization of the binding pocket. The three dimensional models of both SmGPR-1 and SmGPR-2 do not show any obvious acidic residues on the TM3 helix that could substitute for D3.32. There is, however, a unique glutamate (SmGPR-2, position 128, E128) in the first extracellular loop region (EL1) near the boundary of TM3. This glutamate residue is present in all SmGPR-receptors but is not conserved in other aminergic receptors (Fig. 2). If this residue contributes to the binding pocket, it could be a schistosome-specific substitution that compensates for the absence of D3.32.

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SmGPR-2 was tested for activity by expressing the cDNA in yeast. The system used in this study is designed for functional expression of GPCRs (Dowell and Brown, 2002) and offers many advantages over other heterologous expression systems, particularly for receptor deorphanization. Besides low cost of growth and maintenance, yeast cells have robust translational and folding mechanisms for expression of foreign eukaryotic proteins and they can be easily adapted to high- throughput activity assays (Dowell and Brown, 2002; Ladds et al., 2005). Many GPCRs have been successfully expressed in yeast, including helminth receptors (Kimber et al., 2009; Taman and Ribeiro, 2009).

SmGPR-2 expressed in yeast was selectively activated by HA. The receptor showed some intrinsic activity in the absence of ligand. However, in the presence of HA or a methylated derivative, that activity was several fold greater and the stimulation was dose-dependent. Other common BAs had no effect on this receptor, indicating the response was specific. The EC50 for HA was in the micromolar range, a value higher than that of mammalian HA receptors. This difference could be due to the aforementioned D3.32N substitution, which might lower binding affinity, or it could be an artefact caused by heterologous expression in yeast. Although the yeast system has many advantages for GPCR expression, the cell wall hinders access of ligands to the receptor, with the result that more ligand is needed for activation. Agonist potency is often greatly reduced in yeast GPCR expression systems compared to mammalian cells (Ladds et al., 2005), in some instances by more than 100-fold (Taman and Ribeiro, 2009) .

One distinctive feature of this receptor is its high constitutive activity in the yeast cell. Cells expressing SmGPR-2 exhibited significant activity compared to the mock control, even in the absence of HA. It is not uncommon for GPCRs to show some spontaneous (ligand-independent) activity when they are expressed in a heterologous environment, possibly due to protein overexpression in the foreign cell. Some receptors, however, have a natural propensity towards spontaneous activation and the resulting basal activity is biologically relevant in vivo.

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Mammalian receptors like the H3 histamine receptor, 5HT4, 5HT2C, β1- adrenoceptor and the parathyroid hormone (PTH) receptor all show spontaneous activation in vivo, which, in some instances, has been linked to disease (Bond and Ijzerman, 2006). Viral GPCRs are constitutively activated and this is thought to contribute to the infection mechanism (Leurs et al., 2003; Vischer et al., 2006; Cannon, 2007). The cause of high basal activity has been linked to single nucleotide polymorphisms (SNPs), splicing and/or RNA editing events that disrupt the normal constraints on GPCR activation (Huang and Chen, 2005). Many of the amino acids implicated in these constraints are conserved in SmGPR- 2 (e.g. D3.49, R3.50, E6.30, T6.34) but there may be additional interactions among neighbouring residues that destabilize the inactive conformation, allowing the receptor to spontaneously activate. Whether this basal activity is relevant to the parasite in vivo or simply a function of heterologous expression remains to be determined. In the case of SmGPR-2, the constitutive activity may be due in part to the absence of D3.32. Single point mutations of D3.32 in some GPCRs such as

-opiod and 1B-AR receptors were shown to increase agonist-independent activity (Porter et al., 1996; Befort et al., 1999; Huang and Chen, 2005).

The yeast antagonist assays suggest that SmGPR-2 has a very distinctive pharmacological profile, which is quite different from those of mammalian HA receptors and may be unique to the parasite. The HA or 1-metHA activated receptor was strongly inhibited by promethazine, a classical (mammalian) H1 antagonist and, to a lesser degree, by cyproheptadine, a mixed antagonist that has both serotonergic and histaminergic activity. SmGPR-2 was also inhibited by drugs that are not known to interact with HA receptors like flupenthixol and buspirone. In contrast, classical antihistamines such as diphenhydramine, cimetidine and ranitidine had no effect on receptor activity. The novelty of this pharmacological profile reinforces the notion that SmGPR-2 belongs to a new type of BA receptor, one that is activated by HA but does not conform to any known histaminergic receptor, either at the structural or pharmacological level.

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Importantly, some of the drugs that interact with SmGPR-2 in vitro are known to have adverse effects on schistosomes. Promethazine was previously shown to cause rapid paralysis of S. mansoni adults and larvae in culture (Ercoli et al, 1985; Ribeiro et al, 2005) and we have observed a similar effect following treatment with flupenthixol and buspirone (unpublished results). These observations highlight the potential of SmGPR-2 for drug targeting and the development of new antischistosomal drugs.

To explore the biological role of this receptor, we began by examining its developmental expression at the RNA level. SmGPR-2 mRNA levels were measured by RT-qPCR in the free-living cercarial stage, adult worms and at different points during schistosomula growth. The comparative analysis shows that SmGPR-2 mRNA expression is developmentally regulated and its pattern of expression is similar to that of SmGPR-1 (El-Shehabi et al, 2009). The expression patterns are alike in two ways: First, the receptors are both upregulated in the parasitic stages compared to the free-living cercaria; expression levels in the cercaria were consistently lower than in the schistomula or adult worms. Second, the receptors achieve their highest expression levels during the first week of schistosomula development and peak at around day 7. The fact that both HA- activated receptors are so strongly upregulated in schistosomula suggests that HA is particularly important during early parasite development. It has been suggested that young schistosomula exploit the host‟s HA system to increase vascular permeability, which in turn facilitates passage through blood vessels during early larval migration (Catto et al., 1980; Gerken et al, 1984). The upregulation of the parasite‟s own HA system may be linked to this response, perhaps to stimulate movement in the bloodstream or some other unknown behaviour.

The discovery of a second HA-activated receptor in schistosomes suggests this amine is more important to the parasite than is presently believed. Though HA has been detected in S. mansoni extracts by biochemical methods the distribution of HA neurons was largely unknown (Ercoli et al., 1985). We have

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examined this neuronal system by confocal immunofluorescence, using an anti- histamine monoclonal antibody. HA-containing neurons are abundantly distributed throughout the nervous system of S. mansoni. Histaminergic fibers could be seen both in the CNS and PNS, and were prominent in the innervation of the suckers and the subtegumental neural plexuses. Our HA-immunolocalization results are similar to what was described earlier about the distribution of this amine in parasitic helminths. For example, histamine was identified in the nervous system, the suckers and the musculature of the pharynx in the frog lung trematode Haplometra cylindracea (Eriksson et al., 1996), where HA is considered to be a major neurotransmitter and in the nervous system of the fish tapeworm D. dendriticum (Wikgren et al., 1990). These observations provide new clues into the mode of action of HA in parasitic flatworms.

The confocal microscopy-IFA experiments demonstrated that SmGPR-2 receptor expression was stronger in the schistosomula than in the adult stage, consistent with the findings of the qPCR analysis. Both the receptor and HA are located in the neuronal plexuses of the subtegumental layer but they do not colocalize, suggesting they are expressed in different neurons. Their presence in this region reveals a possible role in the control of the somatic musculature and parasite motility. HA is believed to stimulate motor activity in schistosomes (Ercoli et al., 1985), in part by acting through SmGPCR1, which is expressed directly on the parasite musculature (El-Shehabi et al., 2009). If SmGPR-2 also contributes to this regulation, its mode of action is probably indirect. The receptor could exert effects on the musculature by modulating the activity of neurons that in turn control motor function. The subtegumental plexuses are rich in serotonergic, peptidergic and cholinergic neurons, any one of which could be regulated in this manner. Alternatively, SmGPR-2 may be targeting a different tissue and its function is unrelated to parasite motility. It should be emphasized that histamine is present in sites where the SmGPR-2 signal is weak, such as the suckers. However, these regions are rich in SmGPR-1, which was previously

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reported to be present in the tegument, musculature, suckers, mainly the acetabulum and the tubercles (El-Shehabi et al., 2009). Thus, the bloodfluke expresses different SmGPCR-like receptors in different locations, which presumably allows for more than one HA function in these animals. For example, HA is potentially involved in host attachment when it interacts with SmGPR-1 (and not SmGPR-2) in the suckers, especially the holdfast acetabulum. The expression of SmGPR-1 in the tegument and within the tubercles of the adult worm suggests host-parasite interaction and sensory function since the tubercles were determined to be enriched in sensory nerve endings (Gustafsson, 1987). It is uncertain why the parasite would use two HA signalling systems unless HA has more than one function. Preliminary RNA interference experiments to knock down SmGPR-1 expression did not show any measurable phenotypic changes, although the corresponding mRNA was silenced by ~ 60% after five day of siRNA transfection (unpublished, appendix).

Given the importance of HA in S. mansoni, one important question that needs to be answered is whether the parasite synthesizes its own HA, or whether it is obtained from the host. In the case of serotonin (5-HT), the amine is synthesized endogenously (Hamdan and Ribeiro, 1999) but it can also be derived from the host, through a specific transporter system (Patocka and Ribeiro, 2007). HA can be synthesized by some parasites (Mettrick and Telford, 1963; Eriksson et al., 1996), whereas in other worms it is taken up by simple diffusion (Yonge and Webb, 1992). Bioinformatics analyses of the S. mansoni genome identified a potential histidine decarboxylase, the enzyme responsible for histamine biosynthesis but this has yet to be cloned and characterized enzymatically. Additional research is needed to fully characterize the HA system of schistosomes and possibly to identify additional receptors. Efforts are underway to examine if other members of the SmGPR clade are also activated by HA or other BAs.

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Acknowledgements

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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Figures

Figure 1: Dendogram analysis of biogenic amine GPCRs. A radial tree of 55 invertebrate and vertebrate biogenic amine (BA) receptors was constructed from a ClustalW sequence alignment, using MEGA 4 (Tamura et al., 2007). Bootstrap analyses were conducted with 1000 iterations, according to the neighbor-joining method. Fifteen predicted Schistosoma BA GPCR sequences were analyzed, of which eight clustered together into a separate clade (shown in red). These receptors share sequence homology with SmGPR-1 (), a previously described histamine-activated receptor, and also include SmGPR-2 (●), the receptor described in this paper. The remaining S. mansoni receptor sequences in the alignment are marked ().

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Figure 2: SmGPR-1-like receptors lack the conserved aspartate (D 3.32) of transmembrane domain 3. A ClustalW protein sequence alignment was performed from 57 biogenic amine GPCR receptors, including the “SmGPR-1-like” receptors described above (horizontal box) and the Class A GPCR prototype, rhodopsin. TM regions were predicted by the TMpred server at http://www.ch.embnet.org/software/TMPRED_form.htm. Shown is a portion of the alignment representing transmembrane (TM) domains II and III. The invariant residue in each TM segment (D2.50 and R3.50) are identified by asterisks. Many conserved residues of Class A GPCRs, including the DRY/F motif at the boundary of TMIII are present in the SmGPR-1-like sequences (see the conservation histogram below the alignment). However, the SmGPR-1-like receptors lack a highly conserved aspartate (D3.32) of TMIII. D3.32 is present in all vertebrate and invertebrate BA receptors cloned to date but is replaced with an asparagine in all but one of the SmGPR-1-like receptors (vertical right box, red). A glutamate (E) in the loop region near the extracellular boundary of TMIII (E128 in SmGPR-2 sequence), is present in the SmGPR-1-like receptors but absent in the other BA receptors examined, including other schistosome receptors (vertical left box, blue). Sequences are identified by their accession numbers and the species names are abbreviated as follows: D.m. (Drosophila melanogaster), A.m. (Apis mellifera), H.s. (Homo sapiens), R.n. (Rattus norvegicus), M.m. (Mus musculus), C.e. (Caenorhabditis elegans), D.r. (Danio rerio), P.x. (Papilio xuthus), B.t. (Bos taurus), S.j. (Schistosoma japonicum). Predicted S. mansoni coding sequences are identified by their “Smp” designation obtained from the S. mansoni Genome database (S. mansoni GeneDB; www.genedb.org/genedb/smansoni/). SmGPR-2 is marked by an arrow and SmGPR-1 by a triangle. DA (dopamine); 5-HT (5-hydroxytryptamine, serotonin); OA (octapamine); TA (tyramine); H (histamine); Ach M (acetylcholine, muscarinic type).

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Figure 3: Functional expression studies of SmGPR-2 (Smp_043340) in yeast. (A) The full-length SmGPR-2 cDNA was expressed in Saccharomyces cerevisae strain YEX108 and grown in selective leu/histidine-deficient (leu-/his-) medium containing 10-4 M test agonist or vehicle (no drug control, ND). Yeast cells transformed with empty plasmid were used as a negative control (mock). Receptor activation was quantified from measurements of yeast growth in relative fluorescence units (RFU), using an alamar blue fluorescence assay. The results are the means ± SEM of three individual experiments, each performed in triplicate. SmGPR-2 exhibits constitutive activity in the absence of ligand, but is further activated by histamine (HA). Other common biogenic amines tested had no effect, including DA (dopamine), 5-HT (5- hydroxytryptamine, serotonin), OA (octapamine), TA (tyramine), A (adrenaline) and NA (noradrenaline). (B) Functional assays were repeated in the same yeast strain, using variable concentrations of histamine (HA). The data are the means ± SEM of two experiments, in triplicates.

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Figure 4: Pharmacological studies of SmGPR-2.

(A) SmGPR-2 expressed in yeast strain YEX108, is activated by 100 µM of either histamine (HA) or its metabolite 1-methylhistamine (1-metHA). Two independent clones were tested with similar results. Measurements of receptor activity were obtained from yeast growth assays in restrictive leu-/his- medium, as described in Fig. 3 and the results were normalized relative to the untreated (ND) control. The results are the means ± SEM of minimum of three individual experiments, each performed in triplicate. (B) Dose-dependent inhibition of SmGPR-2 activity by the antihistaminic drug, promethazine. Two independent clones of SmGPR-2 expressed in yeast were treated with 100 µM agonist (HA or 1-metHA) and increasing concentrations of promethazine or vehicle (- ).The data were normalized relative to the untreated control (ND) that lacked both agonist and promethazine. To test for drug induced toxicity, assays were repeated in the presence of 100 µM HA and 100 µM promethazine in histidine-supplemented (his+) medium, which enables the cell to grow irrespective of receptor activation (+ve control; see text for details). The results are the means ± SEM of three individual experiments, each performed in triplicate.

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Figure 5: SmGPR-2 has an atypical drug profile.

Three independent clones of SmGPR-2 were tested for activity in the presence of 100 µM histamine (HA) and a test antagonist or vehicle. Drugs were used at 100µM except for Flupenthixol (FLP), which was tested at 10µM. The data are shown as the percentage of a control sample that contained HA but no antagonist (control, hatched line). Error bars are derived from the means  SEM values of three individual experiments, each in triplicates. Tested drugs are Promethazine (PMZ), Diphenhydramine (DPH), Cimetidine (CMT), Ranitidine (RNT), Cyproheptadine (CPH), Buspirone (BUS), Flupenthixol (FLP), Mianserin (MNS) and Sulpiride (SLP).

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Figure 6: Developmental expression of SmGPR-2 in S. mansoni.

Quantitative PCR was performed on reverse-transcribed RNA from S. mansoni cercariae (C), adult worms (A) and freshly transformed schistosomula (S) at stage 0 (S0), 3 days (S3), 7 days (S7) or 14 days (S14) post-transformation.The qPCR data were standardized by simultaneous amplification of internal housekeeping controls (GAPDH) and differences in expression data were calculated according to the comparative CT method. The results are shown as the fold-change in SmGPR-2 expression relative to the cercariae and error bars are the means ± SEM of a minimum of three experiments, each in triplicates.

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Figure 7: Localization of histamine (HA) in adult S. mansoni.

Adult worms were probed with an anti-HA monoclonal antibody (mouse) and followed by rhodamine-labeled goat anti-mouse secondary antibody. HA immunoreactivity was seen in both genders and occurs along the nerve cords (arrows) and transverse nerve commissures (arrow heads) (panels A-C). Histaminergic neurons are abundant in the male oral sucker (D) and the acetabulum (E). HA-containing cell bodies and fibers can be seen in the subtegumental neuroplexuses (F) and the tip of the tail in both sexes (G). Autofluorescence occurs in the female reproductive tract (asterisks, A and C).

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Figure 8: Localization of SmGPR-2 in S. mansoni.

7-day old schistosomula (A-C) and adult females (D-F) were probed with rabbit anti SmGPR-2 polyclonal IgG, followed by FITC-labeled goat anti-rabbit seconday antibody (green). In the larval stage, SmGPR-2 is enriched in the subtegumental layer (panels A and B). When a counterstain TRITC-labeled phalloidin (red) is used to probe the musculature of the larva, the expressed receptor was detected in neurons with no co-localization in the muscles (C). Panels D and E show two typical adult female specimens probed with anti-SmGPR-2 antibody (green). SmGPCR2 immunofluorescence is weaker in the adult stage than the larvae and it is restricted to the neuroplexuses of the subtegumental layer. When adult females were simultaneously probed with anti SmGPR-2 (green) and an anti-histamine antibody (red) we observed closely juxtaposed signals in the subtegumental plexuses with no apparent co-localization (panel F).

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CONNECTING STATEMENT 2

In the previous manuscript, we demonstrated that HA neurons are widely distributed in S. mansoni and we characterized a second HA receptor in this parasite, SmGPR-2. It was found that SmGPR-2 is an atypical HA receptor, which can be stimulated by 1-methyl histamine and does not recognize many of the inhibitors of mammalian histaminergic receptors. These findings encouraged us to investigate another SmGPR homologue, SmGPR-3. This receptor differs from other members of the clade in that it carries the conserved aspartate (D3.32) of TM 3. We questioned whether SmGPR-3 would also be activated by HA and whether its pharmacological profile was similar to that SmGPR-2. In the next manuscript, we describe the cloning and functional analysis of SmGPR-3. We further analyze the effects of several SmGPR-3 agonists and antagonists on schistosome motility in vitro.

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CHAPTER IV (manuscript III)

Characterization of a novel catecholamine receptor (SmGPR-3) in the bloodfluke Schistosoma mansoni

Fouad El-Shehabi and Paula Ribeiro*

Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9

* To whom correspondence should be addressed Tel: (514) 398-7607 Fax: (514) 398-7857

E-mail: [email protected]

Manuscript in preparation

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Abstract

A novel G-protein coupled receptor (SmGPR-3) was cloned from adult Schistosoma mansoni. Based on sequence homology, SmGPR-3 shares little sequence homology with other biogenic amine (BA) receptors and SmGPR-3 belongs to a new clade of schistosome-specific BA receptors that was characterized recently. The full length SmGPR-3 cDNA was expressed in the yeast Saccharomyces cerevisiae and the functional assay revealed that it was activated by catecholamines. The strongest response was obtained with dopamine and was dose-dependent. The receptor also recognized noradrenaline, adrenaline and two closely related metabolites, epinine and metanephrine, suggesting broad specificity for catechol derivatives. The pharmacological profile of SmGPR-3 does not conform to that of any mammalian receptors. Haloperidol and flupenthixol (dopamine antagonists) and promethazine (histamine antagonist) are the most potent inhibitors of SmGPR-3 as well as the non-selective inhibitor, cyproheptadine. In contrast, SmGPR-3 activity is not affected by other common anti-dopaminergic drugs (clozapine and spiperone). As a first investigation of SmGPR-3 function in the intact parasite, we tested the effects of various SmGPR- 3 ligands on schistosome motility in vitro. The results show that dopamine, adrenaline and epinine all caused a significant decrease in motility, whereas metanephrine was strongly myoexcitatory. The results suggest that SmGPR-3 contributes to the control of motor function and is probably one of several receptors involved in catecholamine-mediated motility in schistosomes.

Keywords : Schistosoma mansoni, dopamine, biogenic amines, GPCR, receptor, platyhelminth, neurotransmitter, catecholamines.

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1. Introduction

The bloodfluke Schistosoma mansoni is of three species of schistosomes that cause significant disease in humans. Approximately 210 million people are infected and another 600 million are at risk of infection. Over 90% of all human schistosomiasis is due to S. mansoni. This species exists in Africa, the Middle East, South America and the Caribbean, in regions where the intermediate snail host, Biomphalaria glabrata, is also present (Olds and Dasarathy, 2001). There is no vaccine available for schistosomiasis and the arsenal of drugs available for treatment is limited. Praziquantel (PZQ) is the drug of choice but the emergence of PZQ resistant strains and the drug‟s inability to prevent re-infection urge researchers to search for alternative antischistosomal drugs (Fallon and Doenhoff, 1994; Ismail et al., 1994; William et al., 2001; Doenhoff and Pica-Mattoccia, 2006). Since approximately 30%-40% of marketed drugs exert their effects through interaction with G-protein coupled receptors (GPCRs) (Sautel and Milligan, 2000; Wise et al., 2002; Eglen, 2005), schistosome GPCRs are promising targets for antischistosomal drug development. These receptors are integral plasma membrane glycoproteins that span the cell surface seven times to form seven hydrophobic transmembrane domains. They are also known as 7-TM receptors. GPCRs play key roles in cell responses to external stimuli and are expressed in all eukaryotes. Based on protein homology, GPCRs can be divided into three main families A-C. Family A comprises rhodopsin and the vast majority (> 90%) of non-odorant (transmitter-activated) receptors (Massotte and Kieffer, 2005; Jacoby et al., 2006). The external stimuli or ligands for Family A GPCRs are very diverse and include small (classical) neurotransmitters, peptides, hormones, nucleotides, amino acids, ions and even photons. Although very few GPCRs have been cloned from schistosomes (Hoffmann et al., 2001; Hamdan et al., 2002; Pearson et al., 2007; Taman and Ribeiro, 2009; El-Shehabi and Ribeiro, 2009 submitted), the recently published S. mansoni genome encodes a total of 92 putative GPCR

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sequences (Berriman et al., 2009), many of which are novel sequences that have yet to be characterized. These GPCRs are potentially good targets for new antischistosomal drugs and thus are worthy of further investigation. In this study we focus our attention of GPCRs that are activated by biogenic amines (BAs). BAs are naturally occurring derivatives of aromatic amino acids or histidine and serve as neurotransmitters (NTs) and modulators in a wide range of animal phyla. The most common BAs among invertebrates are serotonin (5- hydroxytryptamine: 5HT), histamine, the phenolamines (octopamine and tyramine) and the catecholamines, a collection of catechol derivativates that includes dopamine (DA), noradrenaline (NA) and adrenaline (A). BAs are abundantly distributed in flatworms and play many roles that are critical for parasite survival. Serotonin, in particular, helps to regulate parasite muscle contraction and motility, metabolic activity and reproduction (Ribeiro et al., 2005; Maule et al., 2006). Catecholamines are also present in the flatworm nervous system but less is known about their function. Immunoassays and biochemical analyses detected the presence of DA and NA in several planarian species (Joffe and Kotikova, 1991), trematodes such as S. mansoni, S. japonicum and Fasciola hepatica(Chou et al., 1972; Bennett and Gianutsos, 1977; Gianutsos and Bennett, 1977) and the cestodes, Hymenolepis diminuta and Diphyllobothrium dendriticum (Ribeiro and Webb, 1983; Gustafsson and Eriksson, 1991). The rat tapeworm H. diminuta is capable of synthesizing DA from its tyrosine precursor (Ribeiro and Webb, 1983) and the same is true of schistosomes, since the rate-limiting biosynthetic enzyme, tyrosine hydroxylase (SmTH) was previously cloned from S. mansoni (Hamdan and Ribeiro, 1998). Catecholamines are thought to have a neuromuscular function in flatworms but there is no consensus on their mode of action. For example, it was reported that DA inhibits motor activity in S. mansoni (Pax et al., 1984) but it is stimulatory in the monogenean Diclidophora merlangi (Maule et al., 1989) as well as in F. hepatica (Holmes and Fairweather, 1984). Another study reported that DA has no direct effect on the motility of schistosomes and acts instead as a modulator of myoexcitatory substances, such 145

as serotonin (Willcockson and Hillman, 1984). With respect to NA, it was shown to have an inhibitory effect in the liverfluke (Holmes and Fairweather, 1984), whereas in S. mansoni neither NA nor adrenaline had a measurable effect on muscle tone or contractile activity (Pax et al., 1984). Based on these conflicting results, it has been suggested that the effects of DA and other catecholamines on flatworm motor function are species-dependent (Maule et al., 2006). Recently, a DA receptor was cloned from S. mansoni and shown to be abundantly expressed in the bodywall musculature (Taman and Ribeiro, 2009). This new evidence adds to the notion that DA is an important regulator of motor activity in this parasite. A bioinformatics analysis of putative GPCRs encoded in the S. mansoni genome identified at least 18 potential BA or cholinergic receptors (Berriman et al, 2009), some of which have no identifiable mammalian or invertebrate orthologues. Among these novel receptors are seven structurally related GPCRs that have been designated as SmGPR1-like due to their similarity to a previously described receptor of S. mansoni (SmGPR1, formerly known as SmGPCR) (Hamdan et al, 2002). The SmGPR1-like receptors resemble BA GPCRs but they constitute a separate clade within the BA receptor tree and are believed to represent a new structural type of aminergic receptor (El-Shehabi and Ribeiro, submitted). Two members of this clade (SmGPR-1 and SmGPR-2) were previously cloned and characterized in vitro (Hamdan et al, 2002; El-Shehabi and Ribeiro, submitted). In the present study, we report the functional analysis of a third SmGPR-1 homologue, SmGPR-3. The results show that SmGPR-3 is activated by catecholamines, in particular DA and closely related catechol derivatives but the pharmacological profile is unlike that of mammalian dopaminergic receptors. Moreover, we show that some of the ligands that interact with SmGPR-3 also have pronounced effects on worm motility. Together, the evidence suggests that SmGPR-3 as an important new catecholamine receptor of S. mansoni and a possible neuromuscular drug target.

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2. Materials and methods

2.1 The parasite

Fresh water Biomphalaria glabrata snails infected with a Puerto Rican (NMRI) strain of S. mansoni miracidia were kindly provided by Dr. Fred Lewis (Biomedical Research Institute, Rockville, Maryland, USA). S. mansoni cercaria were collected 35-45 days post-infection (Lewis et al., 1986) and were mechanically transformed to produce schistosomula. In vitro transformed schistosomula were cultured at 37ºC and 5% CO2 in OPTI-MEM I reduced serum medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), streptomycin 100µg/ml, penicillin 100U/ml and fungizone 0.25µg/ml (El-Shehabi et al., 2009). To obtain adult parasites, 28-day old female CD-1 mice were infected with 150 cercaria / animal by skin penetration. Adult S. mansoni worms were recovered 6-7 weeks post-infection by perfusion of the liver (Basch and Humbert, 1981), washed extensively and either flash-frozen in liquid nitrogen for subsequent RNA extraction.

2.2 Cloning of S. mansoni SmGPR-3

The full-length SmGPR-3 cDNA was cloned from adult S. mansoni based on a predicted coding sequence (Smp_043290) obtained from S. mansoni GeneDB (http://www.genedb.org/ genedb/smansoni/). Total RNA was purified from 25-30 adult S. mansoni worms (Qiagen RNeasy kit) and was oligo-dT reverse-transcribed with MMLV reverse transcriptase (Invitrogen), according to standard procedures. To clone SmGPR-3, we designed primers that targeted the beginning and end of the predicted coding sequence. The primer sequences were as follows: 5‟-ATGAATTTCATAAGAAACAAAACCAATTATTC-3‟ (sense) and 5‟-CTATCTACATCCTTTCAAAAGTACAATATG-3‟ (antisense). A proofreading Platinum Pfx DNA polymerase (Invitrogen) was used to amplify the cDNA in a standard PCR reaction (35 cycles of 94C/15s, 53.1 C/30s and 68 C/90s). The resulting amplicon (1494 bp) was gel excised, purified (QIAquick 147

spin kit, Qiagen), ligated to pGEM-T Easy vector (Promega) and verified by DNA sequencing. The verified sequence was submitted online and given the name SmGPR-3 with the nucleotide accession # GQ259333 and the protein accession # ACT36165.

2.3 Yeast functional expression assays

The SmGPR-3 coding sequence was subcloned between the NcoI / XbaI restriction sites of the yeast expression vector Cp4258 (Wang et al., 2006) ; kindly provided by Dr J. Broach (Princeton University, NJ, USA) and the resulting construct was confirmed by DNA sequencing. The functional expression assay was adapted from the protocol of Wang et al, 2006 and was recently described (Kimber et al., 2009). The receptor was expressed in the budding yeast

Saccharomyces cerevisiae strain YEX108 [MAT PFUS1-HIS3 PGPA1-Gq(41)- GPA1-Gaq(5) can1 far11442 his3 leu2 lys2 sst22 ste14::trp1::LYS2 ste186- 3841 ste31156 tbt1-1 trp1 ura3] kindly provided by J. Broach, Princeton University. This strain expresses the HIS3 gene under the control of the FUS1 promoter (Stevenson et al., 1992) and contains an integrated copy of a chimeric G gene in which the first 31 and last 5 amino acids of native yeast G (GPA1) were replaced with those of human Gq. Strains carrying chimeras of GPA1 and human Gi2, G12, Go or Gs were also tested in preliminary experiments but were found to yield lower or no receptor activity compared to strain YEX108. S. cerevisiae were cultured in yeast YPD medium, according to standard conditions and transformation was performed by the lithium acetate method (Gietz et al., 1995), using approximately 200µl mid-log phase cells, 200µg carrier ssDNA (Invitrogen) and 1 µg Cp4258-SmGPR-3 or empty plasmid as a negative control. Positive transformed colonies were selected on synthetic complete (SC) + 2% glucose and 2% agar solid medium lacking leucine (SC/leu-). For the agonist assay, positive isolated colonies of transformed yeast carrying plasmid Cp4258- SmGPR-3 or vector alone (mock control) were subcultured overnight in SC/leu- liquid medium at 250 rpm/30 C. Next day, the cells were washed three times in 148

SC 2% glucose liquid medium that lacked both leucine and histidine (SC/leu-/ his-). Finally, cells were resuspended in SC/leu-/his- medium supplemented with 50mM MOPS, pH 6.8 and 1.5mM 3-Amino-1, 2, 4-Triazole (3-AT) and the 7 density was measured spectrophotometry (OD600nm 0.3= 1x10 cells/ml). Aliquots of diluted cell culture containing 3000 cells/90µl of MOPS and 3AT containing medium were added in individual wells of a 96-well plate plus 10 µl of tested agonist (10X of the desired concentration). The plates were incubated at 30C for 22-26 hours, after which 10 µl of Alamar blue (Invitrogen) was added to each well. The plates was returned to the 30C incubator until the blue color of Alamar blue began to shift to pink (approximately 1-4 hours) and the fluorescence (560 nm ex / 590nm em) was measured at 30 C for 3-4hours using a plate fluorometer (FlexStation II, Molecular Devices). The antagonist assay was done in the similar way except that each well contained 100 µM DA (agonist) and the antagonist at the specified concentration. Data analyses, statistic manipulations and dose response curve fits were performed using Prism v5.0 (GraphPad software Inc.).

2.4 Measurements of motor activity

3-day old in vitro transformed and cultured schistosomula were placed in individual wells of a 24-well plate (30-40 animals/well) in 300 l of OPTI-MEM + 10% dialyzed serum. Following an adaptation period of 15 min at room temperature, test substances were added at a final concentration of 500M or as indicated. Animals were monitored 5 min after drug addition by placing the 24- well plate on a compound microscope (Nikon, SMZ1500) equipped with a digital video camera (QICAM Fast 1394, mono 12 bit, Qimaging) and SimplePCI version 5.2 (Compix Inc.) for image acquisition. Images were obtained at a rate of ≈ 3 frames / s for a period of 1 minute and the data were analyzed with ImageJ software (version 1.41, NIH, USA). Cultured schistosomula display complex motor behaviours that are dominated by repeated changes in length, both shortening and elongation. To quantify this type of movement, we used the “Fit- Ellipse” command of ImageJ to draw best-fit ellipses of individual animals in 149

each recorded frame. An estimate of body length was obtained by measuring the principal (“major”) axis of each ellipse, using calibrated units (µm), and the frequency of length changes during the observation period was calculated. Any change representing > 10% of body length, whether an increase or decrease, was included in the calculation; changes ≤ 10% were disregarded. Between 12-15 animals were monitored per well and the experiment was repeated 4-5 times. To monitor for possible drug induced toxicity, viability tests were performed routinely using the methylene blue dye exclusion assay described elsewhere (Gold, 1997).

3. Results

3.1 SmGPR-3 belongs to a new clade of BA receptors

A bioinformatics analysis of the S. mansoni genome database (http://www.genedb.org/ genedb/ smansoni/) identified a sequence (Smp_043290) that was structurally related to SmGPR-1 (formerly called SmGPCR, accession # AF031196) and SmGPR-2 (Accession # GQ397114), two previously described HA receptors of S. mansoni (Hamdan et al., 2002; El-Shehabi and Ribeiro, 2009, submitted). The new predicted receptor cDNA was cloned from adult S. mansoni by RT-PCR, verified by DNA sequencing and was designated SmGPR-3 (Accession # GQ259333). When compared to the sequences available in the current version (v.4.0) of the S. mansoni genome, we identified at least 16 possible biogenic amine GPCRs, of which 6 sequences (including SmGPR-2 and SmGPR-3) are closely related to the prototype SmGPR-1. The SmGPR1-like receptors are more related to each other than to other vertebrate or invertebrate BA receptors. A dendogram analysis of nearly 54 BA receptors from various vertebrates and invertebrates, including other putative schistosome BA receptors, suggests that the SmGPR1-like sequences form a separate clade in the BA GPCR tree (Fig. 1). Further analysis within the clade indicates that SmGPR-3 is most closely related to the prototype, SmGPR-1 (53.8% similarity; 36.3% identity)

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followed by SmGPR-2 (46.2% similarity and 29.6% identity (Tables 1 and 2). The SmGPR receptors have the characteristic heptahelical organization of the GPCR superfamily and exhibit many of the structural motifs of BA receptors (Choudhary et al., 1993; Kristiansen et al., 2000; Ballesteros and Palczewski, 2001). These include the DRY motif at the cytosolic interface of TM3, FxxCWxPF in TM6 and NPxxY in TM7 (Fig. 2). The newly cloned SmGPR-3 receptor also has the highly conserved TM3 aspartate, D3.32, a well-established ligand binding residue of BA receptors (Shi and Javitch, 2002; Roth and Kristiansen, 2004; Roth, 2006). This residue is surprisingly absent in all the other SmGPR-1-like receptors, which have an asparagine at this position, but it is conserved in SmGPR-3 (Fig. 2).

3.2 Functional assays: SmGPR-3 is a catecholamine receptor of S. mansoni Functional expression assays were performed in transfected mammalian cells and yeast. Preliminary experiments failed to detect receptor expression in two mammalian cell lines (HEK293 and COS-7 cells) despite considerable optimization of transfection and cell growth conditions (data not shown). Thus we used yeast as a heterologous expression system throughout the study. The full- length SmGPR-3 cDNA was ligated to a yeast expression plasmid Cp4258 and introduced into S. cerevisae to test for activity. Histidine auxotrophic yeast strains that express a HIS3 reporter gene under the control of the FUS1 promoter were used. In the presence of the appropriate ligand, activation of recombinant SmGPR-3 in this system increases expression of the HIS3 reporter via the yeast‟s endogenous pheromone response, which in turn allows the cells to grow in histidine-deficient medium (Wang et al., 2006). Thus, the assay quantifies receptor activity indirectly based on measurements of yeast growth in the selective medium, using a fluorometric Alamar Blue assay. Cells transformed with SmGPR-3 or empty vector were initially tested with common biogenic amines, each at 200 µM (Fig. 3A). Two to four independent clones were tested

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and the results showed consistent, strong activation by DA compared to the mock- transformed control. Other catecholamines, (NA) and (A) also stimulated receptor activity but not to the same extent as DA. This receptor did not exhibit constitutive activity in the absence of agonist (no drug control), unlike the SmGPR-2 homologue, which was partially activated (El-Shehabi and Ribeiro, submitted). Experiments were repeated with different concentrations of DA and the response was shown to be dose-dependent (EC50 = 13 µM, Fig. 3B). In subsequent experiments, we tested two naturally occurring catecholamine metabolites, deoxyepinephrine (Epinine, EPN) and 3-O-methyl-epinephrine (Metanephrine, MTN) and found they both activated SmGPR-3 almost to the same extent as DA (Fig 3A).

3.3 Antagonist assays: SmGPR-3 has a novel pharmacological profile

We further studied the effect of classical (mammalian) antagonists on the activity of SmGPR-3. Drugs were tested initially at a single concentration of 100 µM (10 µM in the case of flupenthixol) in the presence of 100 M DA. The drug effects revealed an unusual pharmacological profile, which does not resemble any of the dopaminergic (D1-D5) or adrenergic receptors of mammals (Fig. 4 A-F).

Surprisingly, spiperone, a selective D2 receptor antagonist enhanced the activity of SmGPR-3 nearly 2-fold, thus behaving more as an agonist than a receptor blocker (Fig. 4A). Propranolol, a β-adrenoceptor antagonist had no effect on this receptor, while the remaining drugs tested showed various degrees of effectiveness. The most effective antagonists were haloperidol and flupenthixol, two classical DA antagonists, followed by promethazine, an antihistaminic drug not known to interact with dopaminergic receptors. Mianserin, a mixed adrenergic/5HT antagonist effectively inhibited 67% of receptor activity while cyproheptadine, the H1 receptor and 5-HT receptor antagonist caused >70% decrease of SmGPR-3 activity. Finally the remaining drugs tested caused modest inhibition at 100 µM. Clozapine, a selective antagonist for D4-dopamine receptor caused 40% inhibition and buspirone, a 5-HT1A serotonin receptor agonist caused 152

55% inhibition (Fig. 4A). Because the assay is based on cell growth, we questioned whether the strong inhibitory effects of promethazine, flupenthixol and haloperidol were due to drug-induced toxicity leading to cell death. To test this possibility, we repeated the assay in medium supplemented with histidine (100 µM), which enables cell growth irrespective of receptor activation. The results showed normal or nearly normal growth in the drug-treated cells in the presence of histidine, indicating that the inhibitory effect of the drug was receptor-mediated and not the product of generalized toxicity (Fig. 4A). Moreover, we studied the dose-dependent effects of five antagonists on SmGPR-3 and the IC50 values were measured (Fig. 4B-F). Based on these results, the order of drug potency for SmGPR-3 is Haloperidol > Flupenthixol > Promethazine > Cyproheptadine > Mianserin > Buspirone > Clozapine > Propranolol > Spiperone.

3.4 In vitro motility assays

As a first step towards the elucidation of SmGPR-3 function, we tested the effects of several agonists and antagonists on the motor activity of intact schistosomula in culture. The goal of these studies was to determine whether substances that interact with the receptor also influence worm movement, which would suggest a possible role for SmGPR-3 in motor control. Measuring schistosome movement is challenging, in part because the worms do not travel (i.e. swim) in culture and there are no locomotory behaviours that can be easily quantified. To obtain an index of motor activity, we monitored individual schistosomula in the presence and absence of test substances, using a microscope equipped with a video camera and imaging software. Approximately 180 consecutive frames were recorded for each animal over 1 min of observation and the approximate length of the animal in each frame was measured in calibrated units (µm). A typical recording of a control (untreated) animal is shown in Fig 5A. When cultured in vitro, schistosomula exhibit repeated cycles of elongation and contraction, which cause the animal to increase or decrease its body length by as much as 20% in either direction. Treatment with catecholamines or catechol 153

derivatives significantly altered this behaviour. Among the SmGPR-3 agonists tested, DA, epinine and adrenaline all caused inhibition of motor activity when added at a concentration of 500 µM. The DA-treated animals were unable to elongate and both the amplitude and frequency of contractions were substantially reduced (Fig. 5B). Epinine (Fig. 5C) and adrenaline (not shown) caused a more pronounced inhibition than DA, leading to virtual paralysis of all animals tested. In contrast, metanephrine, also an agonist of SmGPR-3 (Fig 3A), produced significant hyperactivity with repeated bursts of elongation and contraction (Fig. 5D). The amplitude of length changes was substantially increased in metanephrine-treated animals compared to the control. To quantify the level of motor activity under the various conditions, we calculated the frequency of length changes, both decrease and increase, for each individual animal during the 1 min of observation. Mean values were obtained from the average of 12-15 animals/ experiment and each experiment was repeated a minimum of 3 times. The data (Fig. 6A) confirmed the inhibitory effect of catecholamines in these animals. Compared to the no-drug control, DA reduced the frequency of expansions/contractions (EC) from 14.5 ± 2.9 / min to 4.0 ± 1.5 /min, a decrease of about 72.4%, and adrenaline caused a further decrease to 2.2 ± 0.8 /min. Epinine was the strongest inhibitor of motor activity (1.4 ± 1.0 EC/ min) whereas metanephrine increased the frequency to 33.8 ± 6.12 EC/ min, nearly 2.5 times the control level. Though all are catechol derivatives, metanephrine differs from the other test agonists in that its catechol group is methylated (Fig. 6B).

In addition to agonists, assays were performed with several antagonists of SmGPR-3. We tested three drugs that caused strong inhibition of SmGPR-3 activity in vitro (haloperidol, flupenthixol, promethazine) and one that did not (clozapine). The results do not correlate well with the drug profile seen in the yeast cells. Aside from haloperidol, all the drugs tested caused significant inhibition of motility, whether they were tested individually (Fig. 6) or in combination with DA (not shown). Haloperidol, the most potent of the SmGPR-3

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antagonists in vitro, had no significant effect on the frequency of expansions/ contractions (14.6 ± 3.8 EC / min) compared to the untreated control sample (14.4 ± 2.9 EC / min) (Fig. 6). Drug-treated animals were routinely assayed for viability, using a methylene blue dye exclusion assay (Gold, 1997). There were no measurable effects on viability at the drug concentrations tested.

4. Discussion

The genome of S. mansoni encodes 92 putative GPCRs, of which 18 are predicted to be aminergic or cholinergic (Berriman et al., 2009). Among these receptors, we identified a new clade of six GPCRs that are structurally related to the SmGPR-1 (SmGPCR) receptor of S. mansoni (Hamdan et al, 2002). A distinctive feature of these receptors is the absence of a highly conserved TM3 aspartate (D3.32), which is normally involved in BA binding (Strader et al., 1988; Kristiansen et al., 2000) but is replaced with an asparagine in the majority of the SmGPR-1 homologues. The receptors are also characterized by the presence of unique glutamate in the first extracellular loop near the TM3 boundary, which may compensate for the absence of D3.32.Two of the receptors carrying the D3.32N substitution (SmGPR-1 and SmGPR-2) were previously shown to be activated by HA in vitro (Hamdan et al, 2002; El-Shehabi and Ribeiro, 2009 submitted). Given the structural similarity among these receptors, we had predicted that SmGPR-3 might also be activated by HA. Instead, the results presented here indicate that SmGPR-3 is specific for catecholamines and is particularly responsive to DA. SmGPR-3 is the only member of this clade to carry the conserved D3.32 in the TM3 helix.

The functional expression analysis of SmGPR-3 was conducted in yeast cells. Yeast presents a very robust expression system for recombinant, hard to express genes. SmGPR-3 could not be expressed in HEK293E or COS-7 cells but it produced a functional receptor when expressed in the budding yeast S. cerevisiae. As noted, the activity assays revealed that SmGPR-3 was

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preferentially activated by catecholamines. The strongest response was obtained with DA but the receptor also recognized NA, A and two closely related metabolites, EPN and MTN, suggesting broad specificity for catechol derivatives. When tested against classical dopaminergic and other BA antagonists, SmGPR-3 exhibited a mixed pharmacological profile that did not conform to any known mammalian receptor. The most potent inhibitors of SmGPR-3 included two classical DA antagonists (haloperidol, flupenthixol), an anti-histaminic drug (promethazine) and a relatively nonselective inhibitor (cyproheptadine) that normally targets serotonin and histaminergic receptors. In contrast, anti- dopaminergics such as clozapine and spiperone either had little inhibitory effect or exhibited agonist activity. The unusual drug profile of SmGPR-3 is not surprising, given the novelty of the structure and the fact that the drugs tested are all designed for mammalian receptors. We note that three of the top inhibitors of SmGPR-3 (promethazine, flupenthixol and cyproheptadine) are also powerful inhibitors of SmGPR-2 (El-Shehabi and Ribeiro, submitted), suggesting common pharmacological properties among members of this GPCR clade, even if the amine ligands are different. More research is needed to elucidate the structure- function relationships of these schistosome receptors and to identify new, more selective inhibitors.

Several studies have shown that DA plays an important role in flatworm motility, especially in the free-living turbellarians. In planarians, DA has been implicated in two different types of hyperkinesias, screw-like (SL) and curling- like (CL) postures, which are thought to be mediated by distinct D1-like and D2- like receptors (Venturini et al., 1989; Passarelli et al., 1999). Disruption of DA biosynthesis by RNA interference (RNAi) abrogated both types of hyperkinesias (Nishimura et al., 2007), thus confirming that DA was required for the locomotory response. Opposite to DA motility mediated roles in the planarians, there are few articles available on the mode of action of DA and other catecholamines in trematodes. Not only are these articles old, but there are discrepancies amongst

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them. The discovery of SmGPR-3 raises questions about the function of DA and other catecholamines in parasitic flatworms. As mentioned earlier, there have been conflicting observations on the mode of action of DA and NA, some studies reporting no effect, whereas others describe inhibition of motor activity or increased motility, depending on the species. In the case of schistosomes, researchers have reported a lengthening effect (Tomosky et al, 1974; Mellin et al, 1983) and pronounced relaxation of the bodywall musculature (Pax et al, 1984). More recently, a dopamine receptor (SmD2) was identified in the somatic muscles of both adult and larval forms of S. mansoni (Taman and Ribeiro, 2009). Based on the signalling mechanism of this receptor, the authors predicted an inhibitory mode of action (Taman and Ribeiro, 2009) thus adding to the notion that DA causes muscle relaxation in this parasite. To reassess this question, and to investigate the role of SmGPR-3, we tested the effects of DA and several SmGPR-3 agonists/antagonists on schistosome motility in vitro. As an experimental system, we used cultured S. mansoni schistosomula instead of adult worms because the larvae are more suitable for quantitative measurements of motor activity. The results support the notion that DA has inhibitory neuromuscular effects in schistosomes; DA-treated larvae were significantly less motile than the controls. An examination of individual motility patterns showed a much diminished frequency and amplitude of body wall contractions and virtually no elongations during the observation period. This is consistent with earlier reports that DA causes relaxation of both circular and longitudinal muscle in schistosomes but the effect is stronger on the circular musculature (Pax et al, 1984), which normally drives elongation of the body. We did not see the lengthening effect of DA described in some earlier studies (Tomosky et al., 1974; Mellin et al., 1983). The reason for this apparent discrepancy is unknown. It may be due to a difference in developmental stage, since the previous studies were conducted on adult worms, or possibly a difference in experimental protocol. The lengthening described in the earlier studies was measured at the end of treatment by sucking the worms into a calibrated pipet or tubing (Tomosky et al, 1974; 157

Mellin et al, 1983), whereas in this study measurements were taken continuously during treatment with imaging software.

Is the inhibitory effect of DA on motor activity mediated by SmGPR-3? To address this question we tested the effects of several SmGPR-3 agonists and antagonists on schistosomula motility using the same imaging assay. Virtually every drug tested had strong effects on motor activity but the nature of these effects did not correlate well with the pharmacological profile of SmGPR-3. Notably, not all agonists of SmGPR-3 produced the same effects on motility, as would be expected if they were working solely through this receptor. Whereas DA, epinine and adrenaline inhibited motor activity, metanephrine was strongly myoexcitatory. The different effects of these substances can be due to different reasons. First, it is possible that the ligands bind to SmGPR-3 in the parasite but produce different conformational states, leading to different responses. Second and a more likely explanation, however, is that the drugs are interacting with other receptors in addition to SmGPR-3. It is expected that schistosomes have multiple DA receptors since this is the case in most vertebrates and invertebrates. A probable candidate is the aforementioned SmD2 receptor but there may be others as well. Thus SmGPR-3 is probably one of several receptors involved in catecholamine-mediated motility in these animals. In addition, none of the SmGPR-3 blockers tested behaved as a DA antagonist in the motility assay. Haloperidol had no significant effect and the remaining drugs produced near complete paralysis, whether tested individually or in combination with DA. A probable explanation for these results is that the drugs are interacting with more than one receptor in the intact parasite, for example the aforementioned SmD2 receptor, a SmGPR homologue, or others that have yet to be identified. The paralysis caused by promethazine and flupenthixol could be due to interactions with SmGPR-2, which is also strongly inhibited by these drugs (El-Shehabi and Ribeiro, 2009 submitted). Another consideration is the possibility of general (receptor-independent) toxicity effects. The drugs used in this study did not affect

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parasite viability but we cannot rule out other, more subtle effects that might have hindered motility and were not detected in this study. Thus the function of SmGPR-3 remains unresolved. The fact that so many of the receptor‟s ligands have strong effects on motility suggests a probable role in motor control but the mechanism is unknown.

The strong effects of epinine and metanephrine on larval motility are surprising and raise interesting questions about the role of these substances in the parasite. In mammals, epinine is a naturally occurring but relatively minor byproduct of DA metabolism. It is produced from DA by the activity of phenylethanolamine N-methyltransferase (PNMT) and can be further metabolized to adrenaline, though this reaction rarely occurs in mammals (Laduron et al., 1974). Metanephrine is one of the major metabolites of adrenaline in humans; it is synthesized through the action of catechol-O-methyl transferase (COMT) and it is normally excreted in the urine. Although they are considered to be inactive byproducts in higher organisms, epinine and metanephrine are biologically active in some protozoa and invertebrates. For instance, epinine replaces NA as the major substrate for adrenaline biosynthesis in the unicellular protozoan Tetrahymena pyriformi (Takeda and Sugiyama, 1993). In the cnidarian, sea pansy Renilla koellikeri, epinine, metanephrine and another related metabolite, normetanephrine, are all present at high levels and are believed to function as neuroactive substances (Pani and Anctil, 1994). It is unknown if these substances also occur in flatworms. We note, however, that at least some of the enzymes required for endogenous biosynthesis of these substances are present in schistosomes. The S. mansoni genome encodes a putative O-methyltransferase (CAZ32787, Smp_052470) that shares significant homology with COMTs from other species, in particular Taeniopygia guttata (XP_002192673, 54% similarity) and Gallus gallus (XP_421605, 54% similarity). If these metabolites are produced by the parasite, their activation of SmGPR-3 in vitro and strong effects on larval motility could prove to be biologically important. As noted earlier, epinine and

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metanephrine have opposite effects on motility that cannot be explained in terms of SmGPR-3 activation alone. More research is needed to identify other receptors involved in this response and to elucidate the mechanism of action.

Acknowledgements

The authors would like to thank Dr J. Broach (Princeton University, NJ, USA), who kindly provided us with the yeast pheromone GPCR mediated strains. We also thank Dr. Fred Lewis (Biomedical Research Institute, Rockville, Maryland, 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.

References

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Tables

Table 1: Pairwise alignments of SmGPR receptors.

The predicted protein sequences of six SmGPR-1-like sequences were aligned with ClustalW and compared using similarity matrix (BLOSUM 62) algorithm. The % identity and (% similarity) among these sequences are shown. The (accession #) for the sequences listed are as follows: SmGPR-1 (AAF21638); SmGPR-2 (GQ397114); SmGPR-3 (ACT36165); Smp_043270 (CAZ31902); Smp_043300 (CAZ31905); Smp_145520 (CAZ31903).

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Figures

Figure 1: Phylogenetic tree of biogenic amine GPCRs.

The tree was generated from a ClustalW alignment of 55 invertebrate and vertebrate BA receptors and was constructed with MEGA4, version 4.0 (Tamura et al., 2007). Bootstrap analyses were conducted with 1000 iterations, according to the neighbor-joining method. Fourteen predicted Schistosoma BA GPCRs were analyzed, of which eight receptors constitute a separate clade (red). These receptors share sequence homology with SmGPR-1 (▲), histamine activated receptor of S. mansoni (formerly called SmGPCR). Accordingly, the receptors within that group are designated as SmGPR1-like receptors. The receptor described in this study, SmGPR-3 is identified by an arrow. Seven other S. mansoni receptors that do not belong to the SmGPR1-like clade but they share sequence homology with other common BA clades are labeled (♦). The protein accession# of each receptor is indicated and is followed by the putative ligand and the species. Species abbreviations are: A.g. Anopheles gambiae, D.m. Drosophila melanogaster, H.s. Homo sapiens, M.m. Mus musculus, R.n. Rattus norvegicus, C.e. Caenorhabditis elegans, Smp Schistosoma mansoni protein, SJ S. japonicum, A.m. Apis mellifera, M.p.f. Mustela putorius furo, E.c. Equus caballus, B.t. Bos taurus. Ligand abbreviations: H: histamine, DA: dopamine, 5-HT: 5- hydroxytryptamine or serotonin, mACh: muscarinic acetylcholine, OA: Octopamine, TA: Tyramine.

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Figure 2: SmGPR-3 is a class A GPCR.

The protein sequence of SmGPR-3 (arrow) is aligned with four other SmGPR1-like receptors and several representatives of DA (D1-D5) receptor subtypes. The seven transmembrane domains (TMI-TMVII) are marked by horizontal lines and the most conserved residue within each TM is identified by an asterisk (Ballesteros and Weinstein, 1995). Other conserved residues of interest are identified by solid circles. Signature motifs are boxed. According to the Ballesteros and Weinstein system of nomenclature for class A GPCRs, the most conserved residue within each TM domain is assigned the number 50, preceded by the number of the TM. Thus, 1.50 is the most conserved amino acid residue of the first TM domain and it is an asparagine (N1.50). The other amino acids in the TM region are numbered accordingly. The positions of a predicted binding site of TM3 (D3.32) and a glutamate of the first extracellular loop (position E105 in SmGPR-3) are shown (see text for details). The ClustalW protein alignment was visualized with CLC sequence viewer (version 6.0.2); dark color represents identical residues and grey tones are conservative substitutions. The protein accession numbers are as described in Fig. 1.

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Figure 3: Functional expression studies of SmGPR-3 in yeast.

A. Yeast strain YEX108 expressing SmGPR-3 was tested for activity with all common biogenic amines and two catecholamine metabolites, epinine (EPN) and metanephrine (MTN) (all at 200 µM) or vehicle alone (no drug, ND). Assays were conducted in restrictive histidine-deficient (his-) medium and receptor activity was estimated from measurements of yeast growth, using a fluorescence Alamar blue assay. The results are the means and SEM of 3-4 experiments, each performed in triplicate. Four independent clones of SmGPR-3 were tested (named P2, P4, B2, B4) and were compared to control yeast transformed with empty plasmid (mock). Statistical comparisons were performed with one-way ANOVA, followed by a Tukey pairwise comparison with values of P<0.05 (*) and P<0.0001 (***). B. Dopamine stimulates SmGPR-3 in a dose- -5 dependent manner (EC50= 1.34x10 M). Drug abbreviations: 5-HT: 5-hydroxytryptamine (serotonin), TA: tyramine, HA: histamine, OA: octapamine, A: adrenaline (epinephrine), NA: noradrenaline (norepinephrine), DA: dopamine, EPN: deoxyepinephrine (epinine), MTN: metanephrine.

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Figure 4: Antagonist effects on SmGPR-3 activity.

A. Yeast YEX108 auxotrophic his strain expressing SmGPR-3, was incubated with the agonist (DA, 100 M) and test antagonist or vehicle. Drugs were tested at 100M except for flupenthixol, which was used at 10M. The data were normalized relative to the control sample that contained 100 µM DA but no antagonist. To test for drug induced toxicity, assays were repeated in the presence of 100 µM test antagonist in histidine-supplemented (his+) medium, which enables the cell to grow irrespective of receptor activation (His +ve control; see text for details). B-F. Dose- dependent inhibiton by haloperidol (IC50= 1.35 M), flupenthixol (IC50= 4.0 M), promethazine

(IC50= 30.4 M), mianserin (IC50= 52.0 M) clozapine (IC50 > 100µM). The error bars are the means ± SEM for 3-4 experiments and at least 2 clones (in triplicates).

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Figure 5: Effects of SmGPR-3 agonists on intact schistosomula.

In vitro transformed 3-day-old larvae were incubated with test drug (5x 10-4 M), dopamine (DA), epinine (EPN), metanephrine (MTN) or vehicle (control). Animals were treated for 5 min at room temperature, after which they were examined with a compound microscope equipped with a digital video camera and SimplePCI (Compix Inc.) for image acquisition. Images were recorded for 1 minute (~3 frames /second) and an estimate of body length in µm was obtained for each animal in every frame. Each tracing shown is of an individual animal and is representative of 12-15 larvae per experiment and 3-4 independent experiments per treatment.

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Figure 6: In vitro drug effects on the motor activity of S. mansoni schistosomula: (A) In vitro transformed 3 day-old schistosomula were treated with test substances or vehicle (CT) and then monitored under a microscope, as described in Fig. 5. The approximate body length of each animal was measured at every frame over 1 min of observation (180 frames at 3 frames / s) and the frequency of length changes (increase or decrease) per minute was calculated. The data are the means and SEM of a minimum of 3 experiments each with 12-15 animals. Epinine (EPN), promethazine (PMZ) and flupenthixol (FLP) were tested at a final concentration of 50 µM. The remaining drugs were tested at 500 µM (DA, dopamine; EPN, epinine; A, adrenaline; MTN, metanephrine; CLZ, clozapine; FLP, flupenthixol; PMZ, promethazine; HLP, haloperidol). Statistical comparisons were made relative to the no drug control (CT) with unpaired t-student tests. P<0.05 was considered significant (*). (B) Chemical structures of catecholamines and related metabolites used in the current study.

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CHAPTER V

Discussion and Conclusions

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This thesis presents work on three novel and structurally related 7-TM receptors, which were cloned and characterized from S. mansoni. These GPCRs possess many of the conserved residues and signature motifs of aminergic receptors. However, none of them has identifiable mammalian or invertebrate orthologues. They share about the same level of sequence homology with all different types of biogenic amine GPCRs. In a BA receptor dendogram, these sequences constitute a separate clade, suggesting they represent a new type of amine receptor. Among the three receptors discussed in this thesis, SmGPR-1 was the first to be discovered and remains the prototype for this new clade. This receptor was initially named SmGPCR (Hamdan et al., 2002a) and that was also the name used in the first manuscript of the thesis. The name was changed to SmGPR-1, however, to better reflect the novelty of the structure. GPR is the designation assigned to orphan GPCRs encoded in the human genome and I used the same nomenclature here to describe these schistosome receptors.

The main distinctive feature of these receptors, including the prototype SmGPCR/SmGPR-1, is the absence of Asp3.32, a key functional residue of the BA binding pocket. That acidic Asp3.32 is replaced with an uncharged but polar residue, Asn3.32, in six of the seven receptors. The exception is SmGPR-3, which has the conserved aspartate. There is a large body of evidence pointing to Asp3.32 as the principal binding residue of BA GPCRs; even conservative mutations of Asp3.32 are sufficient to decrease or eliminate activity in a large number of receptors. Thus the Asn3.32 substitution of the schistosome sequences marks a significant departure from the predicted structure. Although the mechanism of ligand-binding to SmGPR1-like receptors is still unresolved, it is likely they evolved a new way of interacting with their ligands. This is suggested for two reasons. First, the loss of Asp3.32 is not a random event; the majority of receptors in the clade lack this acidic residue. Second, Asp3.32 is consistently replaced with an asparagine, suggesting the substitution is under some form of selective pressure. If that were not the case, we would expect different amino acid

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substitutions occurring, at least in some of the sequences. As previously shown, a reverse Asn Asp3.32 mutation does not alter HA binding in SmGPR-1 (Hamdan et al., 2002a) and therefore the native Asn3.32 is unlikely to be directly involved in ligand binding. However, it may have other functions that have not been identified, perhaps a structural role or by contributing to the activation of the receptor. If position 3.32 is not directly involved in binding, how do BAs interact with these receptors? The sequence analysis identified a unique glutamate present in the first extracellular loop, near the boundary of TM3 in these receptors. Although its role has yet to be characterized, the glutamate is consistently present in all the SmGPR1-like receptors but absent in other aminergic GPCRs. This specific glutamate may play an important functional role in the SmGPR1-like receptors, perhaps to compensate for the absence of Asp3.32.

In manuscript I, I followed up the characterization of SmGPR-1 (SmGPCR), which had been previously cloned and shown to be activated, by HA (Hamdan et al., 2002a). We asked questions about the pattern of gene expression (both at RNA and protein levels), the tissue distribution of the receptor in different developmental stages of the parasite and how this could be correlated with function. SmGPR-1 was shown to form a stable, non-covalent dimer that resisted common denaturation conditions (boiling, SDS, 4M urea) and disulfide reagents (mercaptoethanol and DTT) but was acid-sensitive. Dimerization was detected both in native and recombinant forms of SmGPR-1 suggesting this was likely to be physiologically relevant. We do not know, however, if the protein forms homo- or heterodimers, if there are GPCR-interacting proteins (GIP) associated with the receptor, and how dimerization impacts on receptor function. Not all S. mansoni BA receptors form dimers. The dopaminergic SmD2 receptor that was cloned recently did not (Taman and Ribeiro, 2009). Further studies are needed to determine if dimerization occurs in other SmGPR1-like receptors.

Continuing our investigation of SmGPR-1 (Manuscript I), we found that the receptor was expressed at different levels during the course of the life cycle. 179

Moreover, the pattern of developmental expression was virtually identical to that of SmGPR-2, the second HA receptor described in this study (Manuscript II). They were both upregulated in the parasitic stages compared to the free-living cercariae: schistosomula had the highest expression level, followed by adults and then cercariae. During schistosomula development, both receptors showed the highest level of transcript expression in 7 day-old larva, which is roughly equivalent to the lung stage of schistosomula in a normal infection. The expressions of SmGPR-1 and SmGPR-2 were quickly upregulated within hours of cercarial transformation and continued to rise in the developing schistosomula, reaching the maximal level at day 7. This suggests that HA-receptors could play an important role in the early stages of infection. The significance of this expression pattern is unclear but it may be important for morphological and other developmental changes that occur during the first week of infection. The upregulation may also be important for the tissue migration of the young larvae. Compared to the skin stage, the lung stage schistosomula tend to be elongated so they can easily pass through the narrow pulmonary capillary beds within the lung tissue (Forrester and Pearce, 2006). If HA contributes to neuromuscular function and movement, as suggested previously (Ercoli et al., 1985), the upregulation of the receptors at this stage may be linked to these changes in body shape and increased movement through blood vessels.

In manuscript II, we described the cloning and functional analysis of SmGPR-2 by expressing the receptor in yeast. The results showed that the receptor had some intrinsic activity but was further activated by HA in a dose- dependent manner. Other common biogenic amines had no effect. Interestingly, SmGPR-2 was also activated by the HA byproduct, 1-methylhistamine (1- metHA). In humans and other mammals, HA is enzymatically methylated to terminate the signal; 1-methyl-HA binds weakly to HA receptors and is considered to be an inactive product (Stanovnik and Erjavec, 1982). In contrast, SmGPR-2 was more responsive to 1-methyl-HA than HA itself. We do not know

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if the parasite can synthesize HA or 1-methyl-HA endogenously. A bioinformatics analysis of the S. mansoni genome identified a putative histidine decarboxylase (HDC) gene, the biosynthetic enzyme of HA, but it is unknown if this gene encodes a functional enzyme. With respect to methyl-HA, we did not find an orthologue of histamine-N-methyltransferase, the enzyme that converts HA to 1- methyl HA (also known as N-methylHA; see Fig. 5 of the literature review). There are, however, many putative N-methyltransferases encoded in the S. mansoni genome that have not been identified. It is possible one of these new enzymes is capable of methylating HA. Alternatively, the parasite could obtain HA and/or 1-methyl-HA from the host since both products are present in serum. If that is the case, the import could occur by diffusion across the tegument or a transporter-mediated process. The last possibility is that the parasite combines both mechanisms (i.e. endogenous synthesis and import), as is the case for other BAs such as serotonin (Hamdan and Ribeiro, 1999; Patocka and Ribeiro, 2007). The monoclonal anti-histamine antibody that we used to identify HA immunoreactivity in these worms can also recognize methyl-HA if present at high levels (according to the manufacturer). Thus, it is possible that some of that immunoreactivity was due to a methylated form of HA rather than HA itself. If this is the case, methyl-HA could be an important agonist of SmGPR-2 in vivo. These results raise the interesting question of whether schistosomes can use a host by-product (e.g. 1-methyl-HA) for their own benefit.

The yeast antagonist assays suggest that SmGPR-2 has a distinctive pharmacological profile, which is different from those of mammalian HA receptors and may be unique to the parasite (Manuscript II). Although promethazine, the anti-H1 drug can inhibit SmGPR-2 efficiently and in a dose- dependent manner, other common histamine antagonists (Diphenhydramine, Cimetidine, Ranitidine) do not. Moreover, the activated receptor can be inhibited by some non-histaminic drugs, including adrenergics and dopaminergics. The novelty of this pharmacological profile reinforces the notion that SmGPR-2

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belongs to a new type of BA receptor, one that is activated by HA but does not conform to any known histaminergic receptor, either at the structural or pharmacological level.

We identified widespread distribution of HA-containing neurons in the CNS and PNS, including suckers and the subtegumental layer in S. mansoni adult worms. This was a surprising result because HA was not believed to be present at high levels in schistosomes. HA is a major neurotransmitter in some amphibian trematodes, such as Haplometra cylindracea and Mesocoelium monodi (Mettrick and Telford, 1963; Eriksson et al., 1996) but the tissue level of HA in S. mansoni was reported to be low and it was unknown if the amine was present in neurons. (Perez-Keep and Payares, 1978; Ercoli et al., 1985). The immunoreactivity described in this study is presumed to be specific for HA (or a closely related metabolite) since the monoclonal antibody that does not crossreact with other common biogenic amines or the HA precursor, histidine. Thus we conclude that S. mansoni has a well-developed histaminergic system, similar to that of H. cyclindracea, where HA neurons are also widely distributed (Eriksson et al., 1996).

To investigate the function of HA in schistosomes, we studied the tissue distributions of the two HA-activated receptors, SmGPR-1 and SmGPR-2 in adult and larval stages of the parasite (Manuscripts I and II). The results suggest that S. mansoni may have two HA signaling systems, one located on the tegument that involves only SmGPR-1, while the second is endogenous and consists of both receptors, SmGPR-1 and SmGPR-2. Expressed on the surface, the tegumental SmGPR-1 is likely interacting with host HA and mediates some form of host- parasite communication. This receptor may have a sensory function since it is enriched in the male tubercles, where sensory nerve endings are also present (Gustafsson, 1987). The endogenous system has SmGPR-1, which is expressed in the subtegumental musculature and suckers, and SmGPR-2, which is mainly associated with the neuroplexuses of the subtegument. In both cases, the receptors

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are expressed in areas that are also innervated by histaminergic neurons and thus could be activated by neuronal HA released endogenously. Given its location in the somatic musculature, SmGPR-1 is probably involved in the previously described effects of HA on parasite motility (Ercoli et al, 1985) and is presumed to control muscle function. SmGPR-1 may also play a role in host attachment through the holdfast acetabulum. SmGPR-2, in turn, is neuronal and is closely positioned to HA neurons in the subtegumental neuroplexuses. This suggests that HA is acting through SmGPR-2 to modulate the activity of other neurons in the subtegumental region, possibly neurons that control motor activity, or a different unknown function. We note that HA is abundant in locations where neither SmGPR-1 nor SmGPR-2 is expressed (e.g. longitudinal and transverse nerves of the CNS), suggesting there may be other HA receptors in schistosomes that have not been identified.

In manuscript III, we cloned and expressed a third SmGPR1-related receptor, SmGPR-3. Structurally, SmGPR-3 is most closely related to the prototype SmGPR-1 (54% homology) than to SmGPR-2 (43% homology). Given the similarity between theses sequences, SmGPR-3 was predicted to be another HA-receptor of S. mansoni. However, the functional expression analysis of SmGPR-3 revealed that it was a catecholamine receptor instead. When expressed in yeast, SmGPR-3 was activated most effectively by dopamine and its metabolites, epinine and metanephrine. To a lesser extent, the receptor was also responsive to noradrenaline and adrenaline, suggesting a broad specificity for catecholamines and related metabolites. It is unknown why SmGPR-3 has a different amine preference compared to the other two receptors. One possible explanation is the presence of D3.32, SmGPR-3 is the only member of the clade that has this aspartate. In addition, SmGPR-3 has several predicted catecholamine binding residues, in particular S5.42 and S5.43 of TM5. Although these residues are also present in SmGPR-1, it is possible that the combination of the TM5 serines and TM3 aspartate in SmGPR-3 confers a different ligand specificity.

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Mutagenesis studies are needed to test the role of these residues in the different receptors.

SmGPR-3 could not be expressed in any of the mammalian cell lines tested. This was also true of SmGPR-1, which had to be codon-optimized for mammalian cell expression (Hamdan et al., 2002b). In the case of SmGPR-3, we circumvented the problem by using a yeast system. Yeast cells possesses a robust translational machinery to express recombinant proteins. The particular strain of yeast we used was genetically modified, not only to produce “hard to express” GPCRs, but also to identify their activating ligands, via the endogenous pheromone signaling pathway. This yeast assay has been widely used in mammalian GPCR studies but it was only recently applied to studies of helminth receptors (Kimber et al., 2009; Taman and Ribeiro, 2009). Our results show that yeast can be used for functional expression studies of schistosome GPCRs. Aside from having strong translational mechanisms, yeast are easily adapted to high- throughput activity assays, which are especially useful to express and deorphanize novel GPCRs (Dowell and Brown, 2002; Ladds et al., 2005).The yeast system can also be used in drug screens to search for selective receptor blockers.

Like SmGPR-2, the pharmacological profile of SmGPR-3 is distinctive from mammalian BA receptors. The most potent inhibitors of SmGPR-3 included anti-histaminics, adrenergics as well as dopaminergic drugs. Many of these drugs also inhibited SmGPR-2, suggesting the receptors have similar antagonist preferences, despite being activated by different amines. The most interesting characteristic of SmGPR-3 was the activation by catecholamine metabolites (epinine and metanephrine) that are considered to be inactive in mammals. Metanephrine, in particular, is a major breakdown product of adrenaline in humans (see section 3.1 of the Literature Review) and is not known to interact with mammalian dopaminergic (or adrenergic) receptors. In contrast, SmGPR-3 was activated by these substances to about the same extent as dopamine itself. As in the case of SmGPR-2 and 1-methyl-histamine (see above), this is another

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example of a schistosome receptor responding to metabolites that are inactive in the host. The significance of these results is unknown at present. It is possible these BA derivatives are normally active in lower phyla but lost their function through evolution. Epinine and metanephrine are biologically active in some lower invertebrates (e.g. sea pansy) and protozoa (Tetrahymena pyriformi) and the same could be true in flatworms. Alternatively, schistosomes may have evolved strategies to use inactive host metabolites for their own advantage. BA breakdown products can accumulate in the bloodstream and thus could be obtained by the parasite without harmful effects to the host. It is possible the SmGPR receptors have broad specificity so they can be activated by endogenously produced BAs, as well as BA metabolites that are taken from the host.

Dopamine and its metabolites were shown to have strong effects on parasite motility. The nature of these effects is complex and suggests the involvement of more than one receptor. DA and epinine caused inhibition whereas metanephrine stimulated movement. The effect of metanephrine, in particular, was very strong, stronger than that of serotonin (data not shown), which is known to be myoexcitatory. Thus if these metabolites are present in schistosomes, as discussed above, they could be important modulators of motor activity. Some of the motility effects of DA and metabolites are presumed to be mediated by SmGPR-3, since these substances all activate the receptor in vitro. However, there must be other dopamine receptor(s) involved to explain the opposite effects on movement. One possibility is SmD2, a recently described dopamine receptor of S. mansoni (Taman and Ribeiro, 2009). SmD2 is strongly expressed in the somatic musculature and is believed to cause muscle relaxation. SmD2 does not respond to adrenaline or noradrenaline (Taman and Ribeiro, 2009) but it has not been tested for activity with epinine or metanephrine. If metanephrine blocks the normal activity of SmD2, this could explain the increase in motility observed in the present study. Alternatively, these substances may be interacting with other receptors, possibly one of the remaining SmGPR receptors that have not been

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characterized. The mode of action of these myoactive catechol derivatives deserves further investigation.

In conclusion, we have identified a group of novel aminergic receptors with predicted roles on neuromuscular function, parasite motility and, in the case of SmGPR-1, host-parasite interaction. The selectivity of these receptors for histamine- (SmGPR-1, SmGPR-2) and catecholamine-related substances (SmGPR-3) show that these are important neurotransmitters in schistosomes. Finally, the receptors have atypical pharmacological profiles and thus are potential targets for the developmental of anti-schistosomal drugs.

Reference

Dowell, S.J., Brown, A.J., 2002. Yeast assays for G-protein-coupled receptors. Receptors Channels 8, 343-352. Ercoli, N., Payares, G., Nunez, D., 1985. Schistosoma mansoni: neurotransmitters and the mobility of cercariae and schistosomules. Exp Parasitol 59, 204- 216. Eriksson, K.S., Johnston, R.N., Shaw, C., Halton, D.W., Panula, P.A., 1996. Widespread distribution of histamine in the nervous system of a trematode flatworm. J Comp Neurol 373, 220-227. Forrester, S.G., Pearce, E.J., 2006. Immunobiology of Schistosomes (Chapter 8). In: Maule A. G. and Marks N. J. (Ed) Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Physiology. CAB International 2006. pp:174-185. Gustafsson, M.K., 1987. Immunocytochemical demonstration of neuropeptides and serotonin in the nervous systems of adult Schistosoma mansoni. Parasitol Res 74, 168-174. Hamdan, F.F., Ribeiro, P., 1999. Characterization of a stable form of tryptophan hydroxylase from the human parasite Schistosoma mansoni. J Biol Chem 274, 21746-21754. Hamdan, F.F., Abramovitz, M., Mousa, A., Xie, J., Durocher, Y., Ribeiro, P., 2002a. A novel Schistosoma mansoni G protein-coupled receptor is responsive to histamine. Mol Biochem Parasitol 119, 75-86. Hamdan, F.F., Mousa, A., Ribeiro, P., 2002b. Codon optimization improves heterologous expression of a Schistosoma mansoni cDNA in HEK293 cells. Parasitol Res 88, 583-586.

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Kimber, M.J., Sayegh, L., El-Shehabi, F., Song, C., Zamanian, M., Woods, D.J., Day, T.A., Ribeiro, P., 2009. Identification of an Ascaris G protein- coupled acetylcholine receptor with atypical muscarinic pharmacology. Int J Parasitol. Ladds, G., Goddard, A., Davey, J., 2005. Functional analysis of heterologous GPCR signalling pathways in yeast. Trends Biotechnol 23, 367-373. Mettrick, D.F., Telford, J.M., 1963. Histamine in the Phylum Platyhelminthes. J Parasitol 49, 653-656. Patocka, N., Ribeiro, P., 2007. Characterization of a serotonin transporter in the parasitic flatworm, Schistosoma mansoni: cloning, expression and functional analysis. Mol Biochem Parasitol 154, 125-133. Perez-Keep, O., Payares, G., 1978. Histoquimica de la cercaria de Schistosoma mansoni. Determinaci6n de la histamina e histaminoxidasa. Acta Cientifica Venezolana 29 (Suppl. l), 147. Stanovnik, L., Erjavec, F., 1982. Analysis of the dose-response relationship of histamine and N tau-methylhistamine. Agents Actions 12, 162-165. Taman, A., Ribeiro, P., 2009. Investigation of a dopamine receptor in Schistosoma mansoni: Functional studies and immunolocalization. Mol Biochem Parasitol.

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APPENDIX

RNA interference (RNAi) studies of SmGPR receptors in cultured Schistosoma mansoni schistosomula

In this appendix, I describe a method of RNA interference (RNAi) that was used to target several genes in cultured schistosomula. Portions of this work, including the description of the method and optimization conditions appeared in the following publication:

Nabhan, J., El-Shehabi, F., Patocka, N. and Ribeiro, P. (2007) The 26S proteasome in Schistosoma mansoni: Bioinformatics analysis, developmental expression and RNA interference (RNAi) studies. Exp. Parasitol. 117: 337- 347.

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1. Introduction

RNA interference (RNAi) is a powerful tool by which gene-specific double- stranded RNA (dsRNA) is used to trigger degradation of a homologous mRNA transcript. First discovered in the free-living nematode, Caenorhabditis elegans (Fire et al., 1998), RNAi has since been identified in many eukaryotic organisms of diverse phylogeny (Fire et al., 1998; Grishok and Mello, 2002). However, some unicellular eukaryotes, including the protozoan parasites Trypanosoma cruzi and Leishmania major as well as the budding yeast, Saccharomyces cerevisiae were reported to lack the RNAi machinery (Robinson and Beverley, 2003; DaRocha et al., 2004; Ullu et al., 2004). In the phylum platyhelminths, RNAi has been used to silence various genes in free-living planarians and parasitic flatworms Schistosoma mansoni and Fasciola hepatica (Sanchez Alvarado and Newmark, 1999; Salo and Baguna, 2002; Sanchez Alvarado et al., 2002; Takano et al., 2007). In S. mansoni, the first genes to be silenced were cathepsin B (Skelly et al., 2003), glyceraldehyde-3-phosphate dehydrogenase GAPDH and glucose transporter SGTP1 (Boyle et al., 2003) in schistosomula and sporocyst larval stages. This was followed by multiple reports of RNAi targeting a variety of the bloodfluke genes in different life cycle stages (Cheng et al., 2005; Correnti et al., 2005; Nabhan et al., 2007; Ndegwa et al., 2007; Morales et al., 2008; Pereira et al., 2008; de Moraes Mourao et al., 2009; Rinaldi et al., 2009). Recently, the dsRNA ribonuclease, dicer, was cloned from S. mansoni (SmDicer) and its expression was detected in all the developmental stages but mainly in the egg and schistosomula (Krautz-Peterson and Skelly, 2008). Successful gene silencing by RNAi relies on several factors, including the design of dsRNA (or short interfering siRNAs), the gene itself, and the method of dsRNA (or siRNA) delivery and length of treatment. Although many of these factors can be optimized, it is not always easy to identify RNAi phenotypes in worms due to the lack of behavioural assays. Another important consideration is the strong possibility of non-specific effects in dsRNA-treated animals. It is important to use

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appropriate controls in these assays, including an irrelevant dsRNA negative control, and to verify the silencing at the RNA level with a quantitative PCR method. To date, most studies of RNAi in schistosomes have used soaking methods, particle bombardment or electroporation (Bezanilla et al., 2003; Krautz- Peterson et al., 2007; Ndegwa et al., 2007; Ah-Fong et al., 2008) to deliver the dsRNAs. Here we describe a simple RNAi protocol based on the use of transfection reagents to deliver in vitro produced siRNAs in cultured schistosomula. The results show that transfection efficiency is superior to soaking methods alone, under the conditions described, and can be used to produce effective RNAi in this parasite.

2. Materials and Methods

2.1. dsRNA synthesis and production of siRNA

The Silencer® siRNA Cocktail Kit (Ambion, cat # 1625) was used to synthesize double-stranded RNA (dsRNA) for the different genes of interest to this study. Primers were designed so as to amplify a short amplicon (≈ 200-350 bp) of SmGPR-1 (SmGPCR), SmGPR-2, SmGPR-3 and GAPDH, which was used as an internal reference for data normalization (Table 1). We targeted the highly variable third intracellular loop of the different GPCRs for dsRNA production and verified specificity by BLAST analyses against the S. mansoni genome database. The primers were designed to include a T7 promoter sequence at the 5‟-end and were used to amplify the target by conventional (endpoint) PCR. We tested both PCR strategies shown on Fig. 1 and found there was no significant difference in the yield of PCR product; thus, we followed the simpler strategy in which the T7 promoter sequence was added both the forward and reverse primer. The conditions for the endpoint PCR were as recommended by the kit protocol. The resulting PCR amplicons were first gel purified and then in vitro transcribed to produce dsRNA, using 300ng of the template, T7 RNA polymerase and nucleotides (ATP, CTP, GTP and UTP) provided with the Silencer® siRNA

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cocktail kit in a total volume of 20 µl. The reaction was performed at 37 C for two hours, after which the product was treated with DNaseI/ RNase A at 37 C for one hour to remove any DNA or ssRNA contaminants. The dsRNA was purified on a Transcription Reaction Filter Cartridge provided by the kit and the purified dsRNA was quantified using a Nanodrop ND1000 spectrophotometer (Wilmington, USA). Aliquots of dsRNA (12 g-15 g) were subsequently digested with E.coli Ribonuclease III (RNase III; EC 3.1.24) at 37° C for 90 minutes, according to the kit protocol to produce a cocktail of short interfering RNA (siRNA). The resulting siRNAs were isolated with the siRNA Purification Unit provided by the kit. The quality of the siRNA was analyzed on 14% polyacrylamide gels (PAGE) and quantified by using a Nanodrop ND1000 spectrophotometer.

2.2 Labelling of siRNA

To optimize transfection efficiency, some of prepared duplex siRNA was labeled with the fluorescence dye FAM (exc. 492 nm, emt. 518 nm) using Silencer® siRNA Labeling Kit - FAM™ (cat #AM1634, Ambion). siRNA was phenol chloroform-extracted and ethanol-precipitated prior to labeling, as recommended by the manufacturer. Approximately 5g of siRNA was labeled in 50 l reaction at 37 C for 90minutes in the dark. The reaction was stopped by addition of 5 M NaCl (0.1 volume) and cold 100% ethanol (2.5 volumes) on ice for one minute. The labeled siRNA was collected by centrifugation, dried for 5 minutes and resuspended in nuclease-free water. The labeled siRNA was finally evaluated on a 14% PAGE gel stained with ethidium bromide, as the labeled siRNA runs slower than unlabeled siRNA (data not shown).

2.3. Transfection with siRNA

RNAi studies were performed in cultured, in vitro transformed schistosomula. Biomphalaria glabrata snails infected with S. mansoni miracidia of a Puerto Rican (NMRI) strain were obtained from the Biomedical Research 191

Institute, Rockville, Maryland, USA (Lewis et al., 1986). Cercariae were shed 45- days post infection and schistosomula were obtained in vitro by mechanical transformation. The conditions for cercarial transformation and culturing of schistosomula were described previously (El-Shehabi et al., 2009). Transfections were performed immediately after cercarial transformation. Approximately 50 schistosomula were added per well of a 24-well plate containing 250 µl OPTI- MEM reduced serum medium (Invitrogen). A 50 µl mix consisting of test siRNA, scrambled siRNA control (Ambion) or no siRNA, 2 l siPORT lipid transfection reagent (Ambion) and Opti-MEM reduced serum (Invitrogen) was applied to each well and left for up to 5 days before harvesting. The concentration of siRNA was optimized within a range of 1-100 nM. Other transfection conditions were tested (siPORT amine transfection reagent (Ambion) and no transfection reagent) but siPORT lipid was found to give the best results. Schistosomula cultures were maintained at 37°C / 5% CO2 in a humidified incubator. Cultures were supplemented with 5% fetal bovine serum (FBS) 2-3 h after transfection and fresh OPTI-MEM medium/serum was added every 2-3 days thereafter. To monitor transfection efficiency, experiments were repeated with 80 nM FAM-labeled siRNA and the larvae were examined by fluorescence microscopy, starting from the second day post-transfection.

2.4. Quantitative PCR analyses

Total RNA was purified from S. mansoni schistosomulae using RNeasy micro kit (Qiagen, Mississauga, Ontario, Canada). The RNA was quantitated with a Nanodrop ND1000 spectrophotometer and equal amounts of RNA from the various test and control samples were used for reverse-transcription (RT). The RT was performed according to standard protocols in a 20 µl reaction volume containing purified total RNA (130–180 ng), 200U M-MLV reverse transcriptase (Invitrogen), 40U RNaseOUT ribonuclease inhibitor (Invitrogen), 0.5 µM oligo (dT)12–18, 0.5 mM dNTPs and 10 mM DTT in 1X first strand buffer (Invitrogen). The real-time qPCR was carried out with the Platinum SYBR 192

Green qPCR SuperMix-UDG kit (Invitrogen) in a final volume of 25 µl containing 2 µl of cDNA and 0.2 µM of each primer. Primer sequences used for qPCR are listed in Table 1. The reactions were performed in a Rotor-Gene RG3000 instrument (Corbbett Research, Australia) and the cycling conditions were as follows: 50 C/2 min, 95 C/2 min followed by 45 cycles of 94 C/15 s; 53 C/30 s; 72 C/30 s. At the termination of all qPCR reactions, the generation of specific PCR products was confirmed by melting point dissociation curve analyses and DNA sequencing. Expression levels were determined according to the standard curve method (Bustin, 2000) and were normalized to the housekeeping gene, GAPDH.

3. Results

In this study, we describe a protocol for siRNA in cultured schistosomula, using a lipid-based transfection reagent. siRNAs targeting the three receptors of interest (SmGPR-1, SmGPR-2, SmGPR-3) were prepared from larger dsRNAs, which were digested with E. coli RNaseIII (Dicer) in vitro and then purified. A typical gel analysis of the SmGPR-1 dsRNA and resulting siRNAs after digestion is shown in Fig. 2. To test whether schistosomula could be transfected with siRNAs, we performed preliminary experiments in which animals were treated with fluorescent FAM-labelled siRNA in the absence and presence of different transfection reagents (siPORT lipid or siPORT amine, Ambion) and transfection was monitored by fluorescence microscopy. Very little labelled siRNA was seen inside the animals when no transfection agent was used. In contrast, animals treated with transfection reagents showed visibly higher levels of fluorescence (Fig. 3). This was true of both transfection reagents but siPORT lipid was more effective and thus was selected for subsequent experiments. The siPORT lipid- treated larvae showed strong widespread fluorescence, suggesting that the siRNAs could penetrate the tegument and also spread throughout the body using this protocol.

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For gene silencing experiments, we varied the siRNA concentration between 1 and 100 nM. A lower concentration (< 25 nM) is usually recommended because it minimizes the risk of non-specific downregulation (see Ambion‟s recommendations). To assess specificity, we conducted parallel experiments with the same concentration of a scrambled siRNA control (Ambion) or no siRNA and then measured silencing at the RNA level by quantitative RT-PCR. Fig. 4 shows a typical analysis of one the receptors targeted, SmGPR-1. Among the siRNA concentrations tested, 5nM gave the best combination of silencing and specificity. 1nM was ineffective and 10nM produced non-specific silencing of unrelated genes (not shown). Real-time qPCR analyses were conducted after 2 and 5 days of siRNA treatment and the data were normalized relative to the no siRNA negative control. The results show no effect after the first 2 days of treatment. However, by day 5, the expression level of SmGPR-1 was significantly decreased by 57.3% (Fig. 4). This is presumed to be specific since there was no change in the control samples treated with scrambled siRNA at the same concentration and for the same length of time.

To search for siRNA phenotypes, animals were monitored visually for changes in motor activity, color and morphology, as well as loss of viability, using a methylene blue dye exclusion assay (Gold, 1997). The analysis was repeated with siRNAs targeting all three receptors of interest (SmGPR-1, SmGPR-2 and SmGPR-3), each tested at 5nM, and compared to the scrambled siRNA control. No gross phenotypic differences could be seen under these conditions up to 8 days of treatment. These studies are being repeated in the presence of various biogenic amines and with a quantitative imaging assay to search for more subtle changes in motor activity, which might not have been detected by visual inspection.

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4. Discussion

Most RNAi studies of flatworms have used soaking methods or electroporation to deliver dsRNAs. (Sanchez Alvarado and Newmark, 1999; Salo and Baguna, 2002; Sanchez Alvarado et al., 2002; Boyle et al., 2003; Skelly et al., 2003; Correnti et al., 2005; Osman et al., 2006; Freitas et al., 2007; Ndegwa et al., 2007; Takano et al., 2007; Rinaldi et al., 2008; Rinaldi et al., 2009). The protocol described here provides an alternative, relatively simple transfection method that can be used with siRNA or longer dsRNA in cultured schistosomula. During optimization, we found that “soaking” with siRNA in the absence of transfection reagent was insufficient to transfect the larvae. This is contrary to the findings of a recent study, where soaking methods were reported to be as efficient as liposome – based transfection (Ndegwa et al, 2007). The reason for the discrepancy is unknown; it may be a function of the siRNA sequence itself or a difference in experimental conditions. We noticed that transfection agents can give different results, depending on the chemistry of the reagent (e.g. lipid-based versus amine- based), the concentration and more importantly, the manufacturer. Some transfection reagents tested were found to be toxic to the parasite (personal observation). We were able to knockdown the expression of SmGPR-1 by approximately 60% but did not observe a major phenotype. This is likely due to the target gene itself rather than the RNAi method. The same protocol described here produced > 80% silencing and a lethal phenotype when applied to a different target (Nabhan et al, 2007). In a recent study of S. mansoni sporocysts, only 11 out of 32 genes silenced by RNAi produced a measurable phenotype (de Moraes Mourao et al., 2009). This may be because the genes encode long-lasting (i.e. low turnover) proteins and thus the protein is still present, even when the transcript is reduced. Another explanation for the absence of a RNAi phenotype is gene redundancy, which can occur when there are multiple genes that share the same function. If one gene is silenced, the others are still functional and may substitute for the

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missing transcript. In the case of neuronal targets, such as the SmGPR receptors these problems are compounded by the difficulty in delivering siRNA to the nervous system and the lack of good behavioural assays to measure phenotypic changes. It is unknown if any of these factors contributed to the apparent lack of phenotype in the SmGPR RNAi experiments. Research is continuing to elucidate the role of these receptors in S. mansoni.

References

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Schistosoma mansoni, using RNA Interference. PLoS Negl Trop Dis 3, e502. El-Shehabi, F., Vermeire, J., Timothy, P., Yoshino, T., P., R., 2009. Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni. Exp Parasitol 122, 17-27. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. Freitas, T.C., Jung, E., Pearce, E.J., 2007. TGF-beta signaling controls embryo development in the parasitic flatworm Schistosoma mansoni. PLoS Pathog 3, e52. Gold, D., 1997. Assessment of the viability of Schistosoma mansoni schistosomula by comparative uptake of various vital dyes. Parasitol Res 83, 163-169. Grishok, A., Mello, C.C., 2002. RNAi (Nematodes: Caenorhabditis elegans). Adv Genet 46, 339-360. Krautz-Peterson, G., Radwanska, M., Ndegwa, D., Shoemaker, C.B., Skelly, P.J., 2007. Optimizing gene suppression in schistosomes using RNA interference. Mol Biochem Parasitol 153, 194-202. Krautz-Peterson, G., Skelly, P.J., 2008. Schistosoma mansoni: the dicer gene and its expression. Exp Parasitol 118, 122-128. Lewis, F.A., Stirewalt, M.A., Souza, C.P., Gazzinelli, G., 1986. Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations. J Parasitol 72, 813-829. Morales, M.E., Rinaldi, G., Gobert, G.N., Kines, K.J., Tort, J.F., Brindley, P.J., 2008. RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade. Mol Biochem Parasitol 157, 160-168. Nabhan, J.F., El-Shehabi, F., Patocka, N., Ribeiro, P., 2007. The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies. Exp Parasitol 117, 337- 347. Ndegwa, D., Krautz-Peterson, G., Skelly, P.J., 2007. Protocols for gene silencing in schistosomes. Exp Parasitol 117, 284-291.

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Table 1: Primers used in dsRNA/siRNA production and real-time quantative PCR (qPCR) analysis.

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Figures

Figure 1: Two strategies for PCR mediated in vitro transcription of

dsRNA (modified from Ambion). T7 promoter sequence was added at the 5‟ end of gene specific primers, which were selected to amplify a 200-400 base pair region of the gene of interest. (A) Four primers needed, two forward (with and without T7 promoter sequence) and two reverse (with and without T7 promoter sequence) and two PCR reactions are required. After transcription, equimolar amounts of each complimentary ssRNA were mixed and heated to 75°C for 5 min, then cool to room temperature to produce dsRNA. (B) Only two primers needed, each is designed to contain a T7 promoter sequence at the 5‟end and a single PCR reaction is required.

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Figure 2: Gel analysis of dsRNA and ssRNA.

Double-stranded RNA was produced byin vitro transcription of T7 promoter sequence linked DNA and was analyzed by electrophoresis, using 14% polyacrylamide gels stained with ethidium bromide. Some of the synthesized dsRNA (400 bp, upper arrow) was digested by E. coli RNase III (i.e. Dicer-like) to produce double-stranded short interfering RNA (siRNA) of 12-15 bp (the bottom arrow).

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Figure 3: Intake of siRNA by cultured schistosomula.

Freshly transformed schistosomula were incubated with in vitro transcribed and fluorescent labelled-siRNA in the absence (left panel) or presence of two transfection agents. The labelled- siRNA was seen inside the larvae after two days and up to 9 days post-transfection. The lipid- based transfection agent (right panel) was more effective than the siPORT amine-based transfection agent (middle panel).

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Figure 4: SmGPCR silencing in the cultured schistosomula.

SmGPR-1 specific siRNA (5nM) was added to freshly transformed schistosomula, using siPORT lipid transfection agent. The level of SmGPR-1 silencing was assessed 2 and 5 days post- transfection by RT-qPCR. As controls, experiments were repeated with an irrelevant scrambled siRNA sample and no siRNA vehicle (i.e. + transfection agent). In the three different samples, SmGPR-1 was normalized to an internal (GAPDH) control and compared to the untreated (i.e. no siRNA) sample. The error bars are for the mean  SEM of 3-5 independent experiments, each one in triplicates.

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