MSc Chemistry
Molecular Sciences
Literature Thesis
Functional and molecular characterization of
G protein-coupled receptors in Schistosoma mansoni
Exploring the possibility of schistosome GPCRs as anthelmintic targets
by
Roxane Biersteker
10808272 (UvA) / 2565601 (VU)
July 2020
12 EC
Supervisor/Examiner:
Department Research Institute:
Prof. dr. R. Leurs
Medicinal chemistry (AIMMS)
2
Abstract
Schistosomiasis is one of the most devastating tropical diseases, affecting at least 230 million people. Currently, no vaccine exists to treat this disease and the only drug available is praziquantel. This drug is inactive against juvenile schistosomes and the first signs of resistance of S. mansoni to PZQ have been observed. Therefore, there is an urgent need to identify new drug targets. This paper has explored the possibility of schistosome GPCRs as anthelmintic targets. This paper showed that all major GPCR subfamilies and a flatworm specific GPCR family (PROF) are represented in the genome of S. mansoni. Analysis of GPCR transcription profiles revealed that GPCRs are involved in non-gonad, pairing-dependent processes as well as in gonad-specific functions. Furthermore, this paper has shown that schistosome GPCRs are expressed in various life-stages, and several receptors are upregulated in schistosomula. This paper has analyzed the characterization of all deorphanized GPCRs in S. mansoni. These included histamine, dopamine, acetylcholine, glutamate and serotonin receptors. Immunolocalization and RNAi studies suggest that these receptors play a role in worm motility and/or oogenesis. The majority of the deorphanized receptors have different pharmacological profiles than those of receptors in the mammalian host. This indicates that schistosome GPCRs have great potential for parasite-selective drug targeting. The first flatworm library screen of a flatworm GPCR has been described and indicated that structure-activity profiling can provide a deeper understanding of pharmacophores that are selective for parasite receptors. Schistosome GPCRs are promising antischistosomal drug targets because they are expressed in various life-stages, both sexes and various tissues, including the gonads. Therefore, compounds that target these receptors may be used to combat the acute as well as the chronic phase of schistosomiasis, and prevent the dissemination of schistosome eggs.
3
Index
Introduction 4
1. Schistosoma mansoni 6
1.1 Lifecycle and pathogenesis 6
1.2 Nervous system of S. mansoni 8
1.3 Summary 9
2. GPCR (sub)families in S. mansoni 10
2.1 All major GPCR families are present in S. mansoni 12
2.2 Rhodopsin family GPCRs 14
2.3 Platyhelminth-specific Rhodopsin-like orphan family 15
2.4 Adhesion/Secretin family GPCRs 18
2.5 Glutamate family GPCRs 18
2.6 Frizzled family GPCRs 20
2.7 Summary 20
3. Tissue-specific and pairing dependent GPCR expression in S. mansoni 22
3.1 Analysis of tissue-specific GPCR expression in paired and unpaired schistosomes 22
3.2 S. mansoni GPCRs contribute to non-gonad, pairing-dependent processes 23
3.3 S. mansoni GPCRs play a role in gonad-specific functions 25
3.4 Summary 27
4. GPCR expression in various S. mansoni life-cycle stages 28
4.1 GPCRs are low-abundantly expressed in adult schistosomes 28
4.2 Biogenic amine receptors are upregulated in parasitic stages 30
4.3 The larval nervous system undergoes broad changes during its development 32
4.4 Increased expression of PROF complements during juvenile life stages 33
4.5 Summary 35
5. Deorphanized S. mansoni GPCRs 36
5.1 Glutamate receptor SmGluR 39
5.2 Acetylcholine receptor SmGAR 42
5.3 Dopamine receptor SmD2 48
5.4 SmGPR-like receptors 52
5.5 Serotonin receptor Sm5HTR 75
5.6 Summary 80
6. Pharmacology of GPCRs in S. mansoni 81
6.1 Pharmacological profile of SmGluR 81
6.2 Pharmacological profile of SmGAR 83
6.3 Pharmacological profile of SmD2 85
6.4 Drug profiles of SmGPR-like receptors 87
6.5 Pharmacological profile of Sm5HTR 92
6.6 Repurpose promethazine as an antischistosomal drug 99
6.7 Summary 106
7. Discussion 108
4
Introduction
Schistosomiasis afflicts at least 230 million people in the (sub)tropics and is caused by flatworms of the genus Schistosoma.1 Acute schistosomiasis is characterized by abdominal pain, fatigue, fever, malaise and myalgia.2 Active and late chronic disease occurs because of immunopathological reactions against eggs that are retained in host tissue. These reactions result in an inflammatory and obstructive disease. The flatworm causes systemic morbidities that are associated with continuous inflammation due to multiple infections during the child’s early life. These include cognitive impairment, decreased aerobic capacity, anemia and growth stunting.1,3–6 Symptoms often persist after the infection has been terminated, especially in humans that have been infected in early life.1,7
Currently, no vaccine exists and the only drug available to cure this neglected parasitic disease is praziquantel (PZQ), which displays various disadvantages. For example, it is inactive against juvenile schistosomes and it needs to be administered multiple times in order to reach optimal cure rates.8,9,10 Chemical derivatives of PZQ all proved to be less effective and the lack of understanding of the mechanism of action hampers improvements in the efficacy.11 Moreover, the treatment of schistosomiasis will become increasingly difficult due to the emerging resistance of PZQ.12 For these reasons, there is an urgent need to identify new drug targets that are expressed throughout all life-stages of Schistosoma mansoni (S. mansoni).
Since G-protein coupled receptors (GPCRs) are the targets of 30–40% of currently marketed drugs for humans, they are an obvious target to explore for developing new anthelmintics.13,14 However, very little is known about platyhelminth GPCRs. In silico analyses showed that all major GPCR subfamilies are present in S. mansoni, including a rhodopsin subfamily specific to Platyhelminthes.15,16 Although only a limited number of schistosome GPCRs have been functionally characterized, the variety of GPCR genes suggests their involvement in a wide array of functions.17 GPCRs that play a role in reproduction are interesting potential drug targets because schistosome egg production is crucial for life cycle completion as well as for inducing the morbidity caused by schistosome infections.18,19 However, a deeper understanding of the roles of GPCR signaling in schistosome biology is necessary in order to identify candidate receptors.
5
The aim of this paper is to explore the possibility of schistosome GPCRs as anthelmintic targets. This study investigates the expression of schistosome GPCRs in various S. mansoni life-stages and tissues, and analyzes their functional characterization. Furthermore, this paper assesses the druggability potential of schistosome GPCRs by analyzing their pharmacological profiles and comparing them to those of mammalian GPCRs. In addition, this paper traces the development of a miniaturized screening assay and potential parasite-selective inhibitors.
This paper begins by discussing the schistosome lifecycle and induction of pathogenesis. It will then go on to highlight the potential of the schistosome nervous system for drug targeting. Chapter 2 analyzes the identification and classification of putative schistosome GPCRs at the family level. Chapter 3 discusses tissue-specific and pairing dependent GPCR expression in S. mansoni and chapter 4 analyzes schistosome GPCR expression in various life-cycle stages. Chapter 5 investigates the characterization of deorphanized GPCRs in S. mansoni. Lastly, chapter 6 analyzes the pharmacology of schistosome GPCRs.
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1. Schistosoma mansoni
1.1 Schistosome eggs play a crucial role in schistosome lifecycle and induction of pathogenesis
In contrast to most viruses, protists and bacteria, schistosomes and various other helminths cause chronic infections where parasite and host both live for years. As a result, the parasite is able to maximize its opportunity for transmission and reproduction.20,21 In order to eliminate schistosomiasis, it is fundamental to understand the pathogenesis and life cycle of S. mansoni.
The life cycle of Schistosoma is complex (Figure 1).2 The schistosome eggs are secreted in faeces into the water, and after a motile miracidium has hatched from the egg, it searches for a specific water-snail host, depending on the species. Then, miracidia transform into sporocysts in an intermediate water-snail host.1,22 Subsequently, they reproduces asexually by developing mother and daughter sporocysts. Cercariae are then produced within daughter sporocysts. After approximately four to six weeks, the infectious cercariae leave the snail, move through the water and enter the skin of the human host. Consequently, they lose their tails and become schistosomula. In the human host, the maturing larvae transform into adult male and female schistosomes within the duration of five to seven weeks. The periods in humans and snails in which there is an infection but no release of eggs (humans) or cercariae (snails) can be detected, are called prepatent periods. The schistosomula travel via venous circulation to the lungs and are now called lung schistosomula.23 The schistosomula then travel to the left heart and into circulation. In the liver, the larvae mature and are now called adult schistosomes. Male and female schistosomes form a pair while passing through the liver. As a consequence, the female reproductive organs mature.22 In most species, the paired schistosomes migrate to the mesenteric veins of the gut. However, in S.
haematobium the final destination is the urinary venous plexus of the bladder. A matured female
can produce 300–3,000 eggs per day, depending on the species. Once the eggs are produced, half of them end up in the gut lumen (most schistosomes) or the bladder (S. haematobium), where they are excreted in faeces or along with the urine. The other half reach the spleen and liver via the blood system. Here they enter the tissues, which causes liver cirrhosis as well as severe inflammation and induces the morbidity. On average, schistosomes live three to ten years in their human hosts, but it is possible for them to live up to forty years.
7
Figure 1: Lifecycle of Schistosoma. The images represent Schistosoma mansoni.2
Schistosome egg production is not only crucial for life cycle completion but also for inducing the morbidity caused by schistosome infections.18,19 Schistosomiasis is predominantly caused by the immune response of the human host to the eggs of schistosomes.1,18 The eggs of S. mansoni schistosomes that are not excreted end up in the liver and there a granulomatous host immune response is induced. The granulomas contain egg proteolytic enzymes that cause the destruction of the eggs, thereby neutralizing pathogenic antigens. However, they also cause fibrogenesis in the infected tissues.24 The formation of granuloma in the host can be attributed to CD4+ T cell and B cell responses, which are regulated by a variety of cytokines, chemokines and cell populations.21,24– 26 Research has shown that the inherent propensity of antigens in the eggs of schistosomes is the primary reason for the induction of the Th2-type response.25
In summary, schistosome eggs play a fundamental role in schistosome life cycle as well as in the induction of pathogenesis. For this reason, it is crucial to identify and analyze schistosome genes that control reproductive development. Section 3.3 discusses GPCRs that display a gonad preferential expression.
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1.2 The nervous system of S. mansoni has great potential for drug targeting
As described in the previous section, S. mansoni has a complex life cycle. The nervous system of
S. mansoni plays a crucial role in this life cycle and thus in the survival of the parasite. It is involved
in attachment, migration, reproduction and feeding.27 During the migration phase, neuronal signaling enables S. mansoni to respond to host cues and is involved in navigating the migration and controlling the somatic muscles.28 The central nervous system enables the suckers of the worm to attach to the host. The nervous system is also used to control the muscle lining of the viscera, including digestive, excretory, and reproductive tracts. Moreover, the central nervous system is involved in the tight coupling of female and male schistosomes. When any of these activities is disrupted through pharmacological intervention, it could lead to interference of the normal life cycle. As a consequence, the worms may be eliminated from the host.
The flatworm nervous system is composed of a simple brain in the head region and multiple pairs of longitudinal nerve cords. The latter are cross-linked by transverse commissures, which results in an orthogonal pattern.29 The central nervous system is connected to a peripheral network of nerve plexuses and nerve cords that innervate the major structures of the worm’s body such as the somatic musculature, the suckers and the tegument. The subtegumental nerve plexuses that innervate the body wall musculature coordinate the activity of various types of fibers, which results in controlled movement. There are diagonal, circular and longitudinal muscle fibers in the body wall.29 These allow the worm to bend, lengthen and shorten.
Since schistosomes do not have a circulatory system, they lack the capability for classical endocrine signaling. Therefore, it is suggested that the nervous system is involved in the signal transduction via paracrine and synaptic mechanisms. Neuroactive compounds bind to their receptors, which results in a direct effect or an effect via second messenger cascades.30 The first class of receptors that are involved are the cysteine-loop ligand-gated ion channels, and the second class consists of GPCRs. These GPCRs have strong potential for drug targeting. In particular, those that are involved in the control of muscle function and movement.31
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1.3 Summary
In summary, the central nervous system of S. mansoni plays a crucial role in the survival of the parasite. In particular, it is involved in the control of muscle function and movement. Therefore, GPCRs that are expressed in the central nervous system of S. mansoni have strong potential for antischistosomal drug targeting. Furthermore, the pathology that is associated with schistosomiasis is caused almost entirely by schistosome eggs. Therefore, GPCRs that are involved in the reproductions system of S. mansoni are potential drug targets as well. The next chapter analyzes GPCR (sub)families in S. mansoni.
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2. GPCR (sub)families in Schistosoma mansoni
G protein-coupled receptors are well-established drug targets. All GPCRs have a seven-transmembrane (TM) α-helical structure with the N-terminus being extracellular and the C-terminus intracellular (Figure 2).13,3213 The receptor undergoes conformational changes when an agonist binds, which leads to heterotrimeric G proteins being coupled and activated (Figure 3).32 GPCRs play a role in a wide range of physiological processes. The primary sequence varies significantly among GPCRs. There are two GPCR classification systems that are predominantly used. Firstly, the GRAFS system divides GPCRs into protein families that share a common evolutionary origin.33 These families are Rhodopsin, Frizzled/taste2, Glutamate, Secretin and Adhesion. The second classification system is the A–F system. This system divides GPCRs in invertebrates as well as vertebrates and is primarily based on functional similarities and amino acid sequences.34,35 The GPCRs are divided into classes named class A (rhodopsin-like receptors), class B (secretin/adhesion receptors), class C (glutamate receptors) and class F (frizzled receptors). The two systems mostly differ in the further division of class B into the Adhesion family and the Secretin family in the GRAFS system. This is based on the finding that they do not share a common evolutionary origin. In addition to these families/classes, some organisms have lineage-specific receptors that are part of distinct GPCR families. Since GPCRs modulate a wide variety of critically important physiological and biochemical pathways, they have great potential as therapeutic targets for disrupting crictical proliferation and survival activaties of S. mansoni.15 Therefore, it is important to identify and analyze the GPCRs in S. mansoni. In this section, the GPCR (sub)families that are present in S. mansoni are analyzed.
11
Figure 2: Structure and topology of GPCRs. A) GPCRs have seven transmembrane helices (gray), three intracellular loops
(ICLs), a carboxyl terminus (purple) and three extracellular loops (ECLs) and an amino terminus (orange). The transmembrane domain consists of the transmembrane helices, as well as the extracellular and intracellular loops. B) Cartoon representation of the
β2 adrenergic receptor in which the transmembrane helices (TM), loops, and terminal tails are highlighted.32
Figure 3: GPCR activation and signaling. A) An orthosteric ligand (orange) binds an inactive GPCR (example: the β2 adrenergic
receptor). B) Subsequently, the GPCR, which is bound to a ligand, undergoes a conformational change to its active state. C) The active GPCR is able to bind a G protein, which then promotes nucleotide release from, and the activation of, the G protein
12
2.1 All major GPCR families are present in S. mansoni
In order to find anthelmintic drug targets, a deeper understanding of the biology of parasites is crucial. Whole genome sequence data aids in the search for potential receptors. Analysis of the genome of S. mansoni showed that it encodes a minimal of 11,809 genes.36 In 2011, an updated genome of S. mansoni was described.37 More than 45% of the predicted genes were modified and the total number of genes was reduced to 10,852. The first draft of the genome of S. mansoni provided a starting point for the identification and analysis of GPCRs in S. mansoni. Phylogenetic analysis of putative receptors was performed to divide GPCRs in S. mansoni into GRAFS families.15 Various bioinformatic methods were used, including family-specific profile hidden Markov models (HMMs), motif-driven queries and homology-based searching (BLAST).38 A machine-learning approach was used for ligand-based (sub)classification of full-length Rhodopsin GPCRs. The results of the phylogenetic analysis showed that at least 117 GPCR genes and all major GPCR families are present in S. mansoni (Table 1).15 In addition, it revealed a Rhodopsin-like orphan family (PROF1) that is specific to Platyhelminthes.
A more recent study presented an updated phylogenetic analysis of GPCRs in the genome of S.
mansoni.17 Every receptor that was used in this study is connected to a gene model that was validated by previously conducted whole transcriptome RNA sequencing (RNA-seq) experiments.37 This updated study found 115 putative GPCRs with at least three predicted transmembrane domains (TMs). 105 of these GPCRs had more than four TMs and were analyzed phylogenetically (Figure 4).17 Subclassifications shown in this figure each correspond to a highly supported node. Each gene ID has a color depending on transcriptomic enrichment. A note of caution is due here since gene IDs can differ per study. For example, they differ between the study conducted by Lu et al. 2016,22 and Hahnel et al. 2018.17 For a comparison between the gene IDs in these two studies, consult supplementary table 1 provided by Hahnel et al. 2018.17 The main difference between the findings in Table 1 and Figure 4 is that the updated study (Figure 4) found less class A GPCRs and a single receptor more in class B as well as in class C. The number of class F receptors is the same in both studies. Figure 4 shows that class A aminergic receptors, including opsins, biogenic amines and orphan amines share a common peptide receptor-like ancestor.
13
This study phylogenetically analyzed 105 putative GPCR genes that had more than four TMs. In total, 67 seven-transmembrane receptors were identified. All GPCRs that have been deorphanized so far (Chapter 5), contain seven TMs. This raises the question whether putative S. mansoni GPCRs with less than seven TMs are functional. In the following section, the GPCR families that are present in S. mansoni are analyzed. The first group that will be discussed is the Rhodopsin family.
Table 1: Comparison of GPCR repertoires in H. sapiens and S. mansoni based on the GRAFS system.15
H. sapiens S. mansoni R AMIN (α) 44 24 MEC (α) 22 0 MTN (α) 3 0 OPN (α) 11 4 PTGER (α) 13 0 PEP (β) 43 36 CHEM () 43 0 MCHR () 1 0 SOG () 10 0 LGR( ) 7 0 MRG () 7 0 OLF () 494 0 PUR () 35 0 PROF1 0 19 Unclassified 20 22 F FZD/SMO 11 5 TAS2 13 0 G GLR 24 2 A/S ADH 27 3 SEC 20 2 PARF1 0 0
14
Figure 4: Phylogenetically analyzed S. mansoni GPCR genes. Putative GPCRs were analyzed with MrBayes3.2 to infer a
Bayesian tree.39 It is rooted between class A and classes, F, B, and C. Subclassifications are shown and they each correspond to a
highly supported node. Each gene ID has a color depending on transcriptomic enrichment. sF (pairing-inexperienced females), bF (pairing-experienced females), bM (pairing-experienced males), sM (pairing-inexperienced males), bT (testes from bM), sT (testes
from sM). PROF (Platyhelminth-Specific Rhodopsin-like Orphan-Family), FLPR (FMRFamide-like Peptide GPCRs).17
2.2 Two main groups of the Rhodopsin family GPCRs are present in S. mansoni
The four main groups of the Rhodopsin family (α, β, , ) are subdivided into thirteen subclasses in mammalian genomes.40 Group α and β are present in S. mansoni while and are absent (Table
1).15 The α group includes opsin-like, amine and melatonin receptors. The amine subclass is largest for both humans and S. mansoni, with a minimum of 44 and 24 putative aminergic receptors, respectively (Table 1).15 Biogenic amines such as histamine, dopamine and serotonin regulate core activities like metabolism, transport and movement in flatworms.41 These activities are crucial for the survival of the organism, and therefore the corresponding receptors are interesting potential drug targets. The other subclass that is present in S. mansoni are the melanopsin-like receptors. Four potential receptors belonging to this subclass were identified. Remarkably, no melatonin-like receptors were identified in S. mansoni, while 9 of such receptors are present in S. mediterranea.
15
Melatonin has been suggested to have regeneration effect in planaria.42 Not all receptors could be classified.15 Many of these 22 unclassified receptors in S. mansoni are likely to be specific to Platyhelminthes.
The β subgroup houses the greater part of peptide and peptide hormone GPCRs. Multiple studies have shown that neuropeptidergic signaling plays an important role in feeding, locomotion, reproduction, regeneration and host-finding.43 36 putative peptide receptors were identified in S.
mansoni (Table 1).15 Most of these receptors have no assigned ligand. However, a part of the receptors showed moderate homology to previously characterized FMRFamide-like Peptide (FLP) receptors and neuropeptide F-like (NPF) receptors.44,45 The putative peptidergic receptors are divided into three clades with high posterior probabilities (Figure 4).17 The first clade includes GPCRs that show similarity to Neuropeptide FF (NPFF), Neuropeptide F (NPF) and Neuropeptide Y (NPY). The second clade houses GPCRs that are similar to FMRFamide-like Peptide GPCRs (FLPRs). The third clade is platyhelminth-specific and contains receptors that are members of the Platyhelminth-specific Rhodopsin-like Orphan-Family (PROF). This clade is discussed in the next section.
2.3 Platyhelminth-specific Rhodopsin-like orphan family
GPCRs are a major area of interest within the field of helminth neurochemistry and identification of novel targets for antischistosomal drugs.15,28 However, high conservation of GPCRs between the human hosts and the parasites can be a source of selectivity problems. Molecules that target multiple GPCRs may cause toxic side effects.46 A possible solution to this problem is focusing on phylum-specific proteins, in particular orphan proteins. Recently, a highly-diverged Platyhelminth-specific Rhodopsin-like Orphan Family was discovered.15 Platyhelminth Rhodopsin Orphan Family 1 (PROF1) is the largest subfamily because of an expansion in trematodes.47 Other subfamilies are called PROF2 PROF3a PROF3b. No readily identifiably similarity to non-flatworm GPCRs was found.15 It is predicted that PROF1 receptors are solely derived from intronless genes and have a 7 TM domain with an extracellular N-terminus.15 Although the receptors show remnants of ubiquitous Rhodopsin family motifs, no homology was found to previously characterized GPCRs. In S. mansoni, 19 PROF1 receptors were found (Table 1) and in platyhelminth S. mediterranea, 47 receptors were identified. Maximum parsimony analysis of
16
PROF1 GPCRs made it possible to subdivide the receptors into three subfamilies named PROF1 I, II and III (Figure 5).15 Group I contains the most receptors, including 29 members from S.
mediterranea and 13 members from S. mansoni. There are no clear orthologs between species,
which suggest that contraction or expansion of the receptors happened after the splitting of trematodes and planaria in the flatworm lineage. Group II houses 12 S. mediterranea and 6 S.
mansoni sequences. Group III includes 6 S. mediterranea sequences.
To study transcript expression for putative PROF1 receptors, RT-PCR was utilized.15 Planarian or schistosome tissues were used to extract total RNA from. The putative receptors that were selected are marked with an asterisk in the maximum parsimony tree (Figure 5).15 The two schistosome PROF1 sequences that were used are Smp_084270 (group I) and Smp_041880 (group II). The results confirmed transcript expression of the two schistosome and thirteen S. mediterranea targets in the adult stage. Life stage-specific expression data from S. mansoni was utilized to create expression profiles for PROF genes.37,47 The analysis of these profiles is discussed in section 4.4. Another study discovered that Smp_041880 is predominantly described in the ovary of S. mansoni (section 3.3).17 Since it is grouped into the PROF1 family, it is suggested that natural ligands of this GPCR are flatworm-specific molecules.17 Together, these findings highlight that this receptor is an interesting potential parasite-selective drug target. Drugs that target this receptor could potentially disrupt reproduction processes in S. mansoni.
Research to date has not yet assigned putative ligands or functions to the PROF1 family. It is suggested that members of this family may respond to phylum-specific peptide ligands. Due to the fact that the receptors are highly diverged, determining receptor function will prove challenging. However, the receptors form one of the largest Rhodopsin-like subfamilies that are conserved between the monophyletic species S. mansoni and S. mediterranea. Therefore, the receptors are suggested to play a prominent biological role in both species.
17
Figure 5: Phylogenetically analyzed PROF1 GPCR genes. A maximum parsimony tree was inferred for all PROF1 receptors
that are identified. TM domains I-IV were included in an alignment block, which was bootstrapped 1000 times for parsimony analysis. Three subfamilies were identified for the PROF1 family (I, II, III). Sequences from Schmidtea (blue) and S. mansoni (green) were analyzed. The parsimony tree is rooted with S. mansoni opsin-like GPCR (AAF73286.1). The putative receptors that
were selected for RT-PCR analysis are marked (*).15
HMMs that were trained against the originally published S. mediterranea and S. mansoni GPCR complements, were used to identify GPCRs in the genome of F. hepatica.17 In total, six putative PROF1 receptors were identified. This result further supports the notion that the PROF1 receptors are expanded within flatworm lineages. Some studies suggested that the PROF family is similar to the nematode Srw family, which is an ancient chemoreceptor family.48,49 However, while the Caenorhabditis elegans Srw family is primarily (90%) located on one chromosome,50 the S.
18
The Platyhelminth-specific Rhodopsin subfamily (PROF) is a lineage specific novel receptor grouping with large numerical representation. Further research into this family may reveal functions or ligands that are specific to Platyhelminthes. Moreover, these receptors are interesting to investigate as selective antischistosomal drug targets. In the next section, the Adhesion and Secretin GPCR Families in S. mansoni are analyzed.
2.4 Six Adhesion/Secretin GPCRs are identified in S. mansoni
Although adhesion and secretin GPCRs have similar sequences in their 7 TM domains, they differ in their N-terminal ectodomains.15 Usually, Adhesion receptors contain a long serine and threonine-rich N-terminus that has a variety of functional domains. Deorphanization of a couple of adhesion receptors linked them to a wide variety of ligands such as FMRFamide-like neuropeptides, transmembrane glycoproteins, collagen and complement proteins.51,52 It is hypothesized that vertebrate adhesion GPCRs’ functions vary widely. Mammalian adhesion GPCRs are implicated in controlling cellular migration. In the vertebrate lineage this family is subdivided into eight subfamilies. In total, three adhesion receptors were identified in S. mansoni.15 One of these receptors (Smp_099670) contains a Somatomedin B domain and another adhesion-like receptor was found (Smp_058380) that contains an N-terminal GPS domain.15
An archetypical secretin GPCR contains a hormone-binding domain (HBD) in the N-terminus that interacts with peptide hormones.53Two GPCRs of this family were found in S. mansoni. One of these receptors showed homology to diuretic hormone receptors and is likely to be involved in homeostatic regulation (SMP125420). The other secretin receptor was found to have a hormone receptor domain (HRM) domain and showed sequence similarity to parathyroid hormone receptors (SMP170560). The aforementioned updated study,17 identified an additional adhesion/secretin (class B) receptor (Smp_125420).
2.5 S. mansoni houses three putative glutamate-like GPCRs
Glutamate GPCRs respond to a wide variety of endogenous agonists such as γ-aminobutyric acid (GABA), glutamate, odorants and calcium ions.15 There are eight mammalian metabotropic glutamate receptors (mGluRs) types that are split into three subgroups based on G-protein coupling properties, agonist pharmacology and amino acid sequence similarity.54 They all contain a large extracellular domain that has a ligand binding domain (LBD).
19
Two glutamate-like receptors were found in S. mansoni. The previously mentioned updated study,17 identified an additional glutamate (class C) receptor. One glutamate-like receptor (GSmp_052660) showed homology with a Drosophila mGluRlike receptor (DmGluRA).15 However, the second receptor (GSmp_128940) seemed to be significantly diverged from mGluR as well as from GABAB receptors. The characterization of this receptor is discussed in section 5.1.
Figure 6 shows a comparison between conserved mouse mGluR3 LBD residues that take part in
glutamate binding with Glutamate-like receptors GSmp_128940 (S. mansoni) and GSMD004608 (Schmidtea mediterranea).15 This analysis showed that GSMD004608 has retained key residues that interact with glutamate α-amino and α-carboxylic groups. Furthermore, it shows that GSmp_128940 has an unusual LBD with only one putative glutamate-interacting residue. This receptor displays overall divergence with the canonical glutamate binding pocket and most likely have atypical pharmacology or responds to different amino acid-derived ligands. The pharmacological profile of Smp_128940 is analyzed in section 6.1.
Figure 6: Comparison between conserved mouse mGluR3 LBD residues that take part in glutamate binding (underlined) with Glutamate-like receptor GSmp128940/SmGluR (S. mansoni) and GSmd004608 (Schmidtea mediterranea). The residue
20
2.6 Five S. mansoni GPCRs are identified that are part of the Frizzled Family
GPCRs in the Frizzled family usually contain a cysteine-rich ligand-binding domain and seven hydrophobic domains.55 The endogenous ligands are, among others, secreted Wnt proteins. The receptors play a critical role in developmental processes such as cell motility, synaptic organization, cell polarity and cell fate determination. Moreover, in planarian flatworms the canonical Wnt/b-catenin pathway is involved in regeneration polarity.56,57 Four Frizzled sequences and one Smoothend-like sequence were identified in S. mansoni.15 In humans, ten Frizzled GPCRs are phylogenetically grouped into four clusters. A part of the S. mansoni GPCRs could also be grouped into these clusters. A single Frizzled GPCR in S. mansoni grouped in cluster IV (FSmp_118970), which shares ~38% amino acid identity with human Fzd6 and ~45% with Drosophila Fzd1. Two receptors grouped in cluster II (FSmp_155340 and FSmp_139180). The remaining receptors could not be grouped.
2.7 Summary
This chapter analyzed the identification and classification of putative schistosome GPCRs at the family level. The most recent study based on whole genome sequencing data has described the S.
mansoni genome with 10,852 genes.37 Based on these data, 115 putative GPCRs were found with a minimum of three predicted TMs. 105 of these GPCRs had more than four TMs and were analyzed phylogenetically. A previously conducted phylogenetic analysis described 117 GPCR genes that belonged to five major GPCR families.15 These include the Glutamate family (2 GPCRs), Rhodopsin family (105 GPCRs), Adhesion family (3 GPCRs), Frizzled family (5 GPCRs) and the Secretin family (2 GPCRs). The updated study reduced the number of GPCRs in the Rhodopsin family (class A), added one receptor to the Secretin/Adhesion family (class B) and one to the Glutamate family (class C).37 The majority of the GPCRs that were identified belonged to the α or β Rhodopsin subgroup. The identification of these receptors may help identify biogenic amine and neuropeptide-like ligands in S. mansoni. The most striking result is the identification of a new and highly-diverged receptor family named Platyhelminth Rhodopsin Orphan Family 1. The members of this family (19 GPCRs) share little sequence similarity with any previously discovered GPCR. For this reason, they have enormous appeal as potential drug targets for antischistosomal drugs with high selectivity for S. mansoni GPCRs over those of the mammalian host.
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The identification of S. mansoni GPCRs provides opportunities to identify receptors that are fundamental for parasitic platyhelminth life and survival. These are potential drug targets in schistosomes. The identified GPCRs are potential candidates for future functional and biological studies. The next chapter analyzes the tissue-specific and pairing dependent GPCR expression in
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3. Tissue-specific and pairing dependent GPCR expression in adult S. mansoni
3.1 Analysis of tissue-specific GPCR expression in paired and unpaired schistosomes
The reproductive biology of schistosomes is unique, as they are the only one to have evolved separate sexes among trematodes.58 The reproduction of schistosomes involves a high degree of interdependence between females and males. Adult females and males live paired, which is necessary female gonad development.59 Pairing-inexperienced females (sF) exhibit an immature reproductive system. The vitellaria is underdeveloped and the ovary is small and contains stem cell-like precursor oocytes. The maturation of the vitellarium and ovary is induced when the female pairs with the male schistosome, leading to a sexually mature female (bF). Contrary to females, pairing-inexperienced males (sM) have testes with differentiated spermatocytes and are not morphologically different from pairing-experienced males (bM).60,61 Nonetheless, sM and bM gene expression differ as a result of pairing.22,62 In this chapter, the tissue-specific and pairing dependent GPCR expression in S. mansoni is analyzed. This can provide new insights into GPCR functions. Moreover, the data can provide a selection of GPCR candidates for functional studies.
Figure 7: Female and male worm paired.63
Whole-organ isolation can be used to analyze specific tissues in S. mansoni.64,65 Based on this method, a tissue-specific comparative RNA-seq analysis was conducted on complete testes and ovaries of males and females from paired and unpaired schistosomes.22 In adult schistosomes, most GPCRsdetected in the RNA-seq analysis exhibited tissue-specific and/or sex-/pairing-dependent transcription. For this reason, GPCRs can be categorized into functional groups (Figure 8).17
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Figure 8: Hierarchical clustered GPCR genes that are expressed in adult S. mansoni. A light color indicates a relative low
expression level, whereas a darker color indicates a relative high expression (Color Key). bF (pairing-experienced females), sF (pairing-inexperienced females), sM (pairing-inexperienced males), bM (pairing-experienced males), bT (testes from bM), sT
(testes from sM), bO (ovaries from bF), sO (ovaries from sF).17
3.2 S. mansoni GPCRs contribute to non-gonad, pairing-dependent processes
The majority of GPCRs that were found during the RNA-seq analysis are predominantly expressed in non-gonad tissues of both male and female schistosomes.17 For example, the dopamine-responsive GPCR SmD2 (Smp_127310) is located in the acetabulum and the subtegumental somatic musculature of all larval stages.66 In adult schistosomes, the receptor was mainly present in the somatic muscles and in a lower amount in the muscular lining of the caecum. The results of functional assays showed that that SmD2 responded dose-dependently to dopamine (section 5.3).
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It is suggested that this receptor plays an important role in the neuromuscular system of S. mansoni. A second receptor that is mostly expressed in non-gonad tissues is the serotonin receptor Sm5HTR (Smp_126730). Immunolocalization of the receptor showed that it is widely expressed in the nervous system of adult worms and schistosomula.31 Specifically, it is present in the main nerve cords and the cerebral ganglia. Furthermore, the receptor is expressed in the tegument and the peripheral innervation of the body wall muscles. In section 5.5,a functional analysis of Sm5HTR is described.
Confocal immunolocalization studies showed that glutamate receptor SmGluR (Smp_128940,
section 5.1) is widely expressed in the central nervous system of both adult schistosomes and larvae.67 No expression was measured in the vitellarium and ovary. In adult schistosomes, the receptor is located in the cerebral commissures and the main longitudinal nerve cords. Moreover, it is expressed in the peripheral nerve plexuses and fibers that innervate the body wall musculature and acetabulum. In female schistosomes, SmGluR was located at the uterus, ootype and oviduct. Since SmGluR is expressed in the main nerve cords, it may be involved in interneuronal signaling. In other organisms, mGluRs are also often involved in signaling within the central nervous system. In vertebrates, they are located presynaptically where they modulate transmitter release, or post-synaptically to mediate excitatory responses. The authors note that distribution of SmGluR resembles that of acetylcholine and the peptidergic system in central nervous system of schistosomes. This suggests that the receptor could play a role in the control of one or more of these circuits.29
Interestingly, most of the GPCRs that are predominantly expressed in non-gonad tissue exhibited a pairing-dependent transcription. This is in agreement with recent studies that revealed that neuronal processes are important in S. mansoni male-female interaction.22,68 Furthermore, most of the non-gonad tissue GPCRs belong to the sM-bM-sF group (Figure 8).17 This is consistent with the finding that the sF transcriptome is more similar to the male than to the bF.22 This observation suggest that biological processes such as partner attraction and locomotion are equally important in sF, sM and bM. About 30% of the receptors within this group showed a bias towards one of the three subgroups. Receptors that showed a bias towards sM may play a role in female attraction or locomotion. Similarly, GPCRs that showed a bias towards sF may be involved in male attraction
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or repression of sexual maturation. Previous microarray studies showed that transcript levels of genes that are involved in egg production increase in bF compared to sF.22,62 In addition, transcription levels of genes that are associated with motility are higher in sF than in bF. In contrast, these levels are increased in bM compared to sM. This also supports the suggestion that the transcription levels of GPCRs are increased in bM to increase muscle activity, which is necessary to successfully transport the female.17 In addition, GPCRs that are preferentially transcribed in bM might be associated with clasping and pairing processes.
This section analyzed the expression of GPCRs in non-gonad tissue of female and male schistosomes. The results revealed that GPCRs are involved in non-gonad, pairing-dependent processes in S. mansoni. These potentially mediate functions that are not related to the gonads during the schistosome male-female interaction. The next section analyzes GPCR expression in the gonads of schistosomes.
3.3 S. mansoni GPCRs play a role in gonad-specific functions
As discussed in section 1.1, schistosome eggs play a fundamental role in schistosome life cycle and in the induction of pathogenesis.22 Therefore, understanding the processes that control egg production and gonad development is essential for discovering strategies to eliminate schistosomiasis.69 In this section, GPCRs that are preferentially transcribed in the gonads of S.
mansoni are discussed.
The schistosome female undergoes sexual maturation upon pairing, which is marked by a change in gene expression. Remarkably, only a few GPCRs exhibited gonad-biased transcription (Figure
8).17 A previous study has demonstrated that transcription levels of genes that are associated with egg production are increased in bF compared to sF.22,62 Four S. mansoni GPCRs are preferentially transcribed in bF (Smp_041880, Smp_049330, Smp_170560 and Smp_145240). All of these are functionally uncharacterized. These receptors are abundantly expressed in the vitellarium, since this is the most prevalent tissue in this group.17,59,70 The vitellarium plays an important part in the synthesis of composite eggs. Drugs that target these receptors may disturb the reproduction process of S. mansoni. Smp_041880 is the only one that is preferentially transcribed in the ovary.17 This putative receptor is an ortholog of a potential allatostatin receptor, which is involved in the
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reproductive development in female S. japonicum.71 Allatostatin family neuropeptides are crustacean and insect hormones that play a role in feeding, reproduction and the generation of juvenile hormone.72 However, Smp_041880 is grouped into the PROF1 family (section 2.3), which suggests that natural ligands of this GPCR are flatworm-specific molecules.17 Smp_145240 was phylogenetically grouped in the FLPR clade, while Smp_170560 belonged to the Adhesion/Secretin family (class B) (Figure 4).17 The last receptor in the bF group could not be classified (Smp_049330). Smp_174350 is also preferentially expressed in the female gonads. It is a frizzled ortholog and its expression levels are increased in the ovary of sF (Figure 8).17 It is hypothesized that it is involved in a Wnt signaling pathway and preserving the immature state of the oocytes.59,61
A few GPCRs are preferentially transcribed in the male reproductive organs. Seven GPCRs are preferentially transcribed in the testis (Figure 8; sT-bT group).17 These include Smp_178420 and Smp_127720, which are phylogenetically classified as class A orphan amine and biogenic amine receptors, respectively (Figure 4).17 Smp_244240 and Smp_242910 are also part of the sT-bT group and are connected to neuropeptide signaling. Interestingly, a recent study showed that neuropeptides play an important role in planarian germline development.44 The remaining GPCRs in the sT-bT group are classified as class B (secretin/adhesion) GPCRs (Smp_099670, Smp_176820) and as an opsin receptor (Smp_104210). Notably, the transcription levels of four GPCRs in the sT-bT group increased in the testis upon pairing. In accordance with this result, previous studies have demonstrated that changes occur pairing-dependently in the schistosome testis.22,65,68 Possibly, one of these changes is the increase of sperm production upon pairing.
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3.4 Summary
In summary, the results in this chapter showed that there are GPCRs that are preferentially transcribed in the gonads of S. mansoni. These GPCRs may contribute to embryogenesis, vitellogenesis and/or gametogenesis. Furthermore, most GPCRs have a non-gonad-preferential, pairing-dependent transcript profile. These GPCRs may play a role in the interaction between male and female schistosomes. Sinceschistosome eggs play a critical role in schistosome life-cycle and in the induction of pathogenesis, identifying receptors that mediate egg production and gonad development will help in the identification of new drug targets in S. mansoni. Similarly, the male-female interaction is crucial for the induction of germ-cell differentiation. GPCRs that play a role in this interaction are interesting potential candidates for antiparasitic drug targeting. The following chapter analyzes GPCR expression in various S. mansoni life-cycle stages.
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4. GPCR expression in various S. mansoni life-cycle stages
As described in section 1.1, S. mansoni goes through many different and complex life stages. Therefore, it is expected that the expression of the GPCRome differs per life stage. Since juvenile schistosomes are refractory to the currently available drug (PZQ) to treat Schistosomiasis,8 it is important to develop drugs that are effective against this life stage. The molecular target of PZQ remains unknown. However, it is known that downstream factors play a role in PZQ action. Similar to adult S. mansoni, schistosomula that are refractory to PZQ experience Ca2+- dependent paralysis and contraction.73 However, whereas adults worms do not survive, juvenile worms recover and live. This indicates that the initial target is probably similar, but adaptive responses that result in the survival of the animals are activated in juvenile, and not adult, worms.9 This example illustrates that gene expression patterns differ per S. mansoni life stage. Understanding the link between the
S. mansoni GPCRome and the various life stages will help to find new drug targets. This chapter
examines the differences in GPCR expression in various S. mansoni developmental stages. Firstly, GPCR expression in adult schistosomes compared to that of other life-stages is described. Secondly, the developmental expression pattern of biogenic amine receptors (1, SmGPR-2) is analyzed. Then, this developmental expression pattern is compared to that of glutamate receptor SmGluR in order to gain insight into the changes in the larval nervous system during its development. Lastly, the expression of PROF complements during various life stages is analyzed.
4.1 GPCRs are low-abundantly expressed in adult schistosomes
A tissue-specific comparative RNA-seq analysis was conducted by Lu et al. on complete testes and ovaries of males and females from paired and unpaired schistosomes.22 Data obtained from this study showed that, compared to the GPCRome described by Hahnel et al.,17 60% of the GPCR genes were expressed in adult schistosomes. Moreover, it revealed that GPCRs of all classes were present. A possible explanation for the fact that not all GPCR genes are expressed, or are weakly expressed, in adult schistosomes is that the missing GPCRs could be predominantly important for earlier life stages. For example, the need for miracidia to quickly find a host without losing their infectivity illustrates that miracidia need highly adapted sensory mechanisms.74 A previous RNA-seq study that investigated GPCR genes in miracidia showed that Smp_180030 was present in light-responsive miracidia.74 This study suggested that this gene together with Smp_104210 is involved in photokinesis and plays a fundamental role in host-finding. Both were predicted as
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opsin-like GPCRs. Interestingly, only Smp_104210 was detected in adult schistosomes and exhibited a testis-biased transcription.17 Seven other genes were found to be expressed in free-living miracidia, each sequence containing 7 TM domains. These include Smp_173010, which has been described as a PROF receptor.75
Analysis of the data from a previously conducted RNA-seq study,37 showed that the majority of the 47 putative S. mansoni GPCRs that were missing in the Lu et al. dataset are less abundantly transcribed in adult schistosomes in comparison to other life stages (Figure 9).17 This explains their absence in the Lu et al. dataset. Furthermore, this dataset did not include other GPCRs that were previously characterized, but were transcribed below threshold.22 These included the amine receptors SmGPR-1 (Smp_043260), SmGPR-2 (Smp_043340) and SmGPR-3 (Smp_043290). The next section discusses the gene expression patterns of two of these biogenic amine GPCRs.
Figure 9: Analysis of the expression of the 47 putative GPCRs that were missing in the Lu et al. dataset per S. mansoni
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4.2 Biogenic amine receptors are upregulated in parasitic stages
Biogenic amine receptors SmGPR-1 (Smp_043260), SmGPR-2 (Smp_043340) and SmGPR-3 (Smp_043290) are expressed in the nervous system of adult schistosomes (section 5.4).76–78 SmGPR-1 and SmGPR-2 were both shown to be activated by histamine (section 5.4.3 & 5.4.6),76,79 whereas SmGPR-3 responds to dopamine (section 5.4.8).80 A study focused on the relative transcription levels of SmGPR-1 in various life stages of S. mansoni.77 Quantitative RT-PCR was used to measure expression of SmGPR-1 at the mRNA level. First, the expression levels of adult schistosomes, cercariae and schistosomula were compared (Figure 10A).77 The results suggested that the receptor is upregulated after cercarial transformation. Compared to cercariae, a 10-fold increase (P < 0.01) in expression was measured in transformed schistosomula (S0). These levels were measured again seven days later (S7), which showed that they increased even further (P < 0.0001). Remarkably, the expression levels went down again after 14-days and in adults down to S0 level. Since this upregulation in schistosomula peaks at day 7, it could be hypothesized that histamine signaling plays a role in the initial development of larvae and during the lung-stage, which occurs approximately on 7-8 days post-infection.23,77
A second experiment was performed in which mRNA levels of SmGPR-1 were measured in S.
mansoni miracidia and sporocysts (Figure 10B).77 Compared to miracidia, a 20-fold (P < 0.01) increase in mRNA levels was observed in 4-day old sporocysts. Remarkably, a more than 200-fold increase in 20-day old sporocysts (P < 0.001) was observed compared to miracidia. Possibly, the receptor plays a role in the regulation of the growth and development of sporocysts. This significant SmGPR-1 gene upregulation in parasitic stages (sporocysts, schistosomula and adults) in comparison to free swimming stages (miracidia and cercariae) suggests that SmGPR-1 has a fundamental role in the establishment and/or maintenance of parasitism within the hosts.
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Figure 10: Developmental expression of SmGPR-1. A) Expression of SmGPR-1 relative to cercariae. Quantitative RT-PCR
was performed on reverse-transcribed RNA obtained from S. mansoni cercariae, schistosomula up to 14 days after transformation (S14) and adult schistosomes. B) Expression of SmGPR-1 relative to miracidia. Data obtained from qPCR on reverse-transcribed
RNA obtained from miracidia and sporocysts (4-day and 20-day).77
Structurally related to SmGPR-1 is SmGPR-2 (Smp_043340).76 To establish whether the expression of SmGPR-2 is also developmentally regulated, real-time qPCR was used to compare mRNA levels in various S. mansoni developmental stages. Similar to SmGPR-1, an increase in expression level was observed immediately after cercariae transformed to stage 0 schistosomula (S0) (Figure 11). This level increased up to 60-fold at day 7 (P < 0.001), after which the expression levels decreased. In adult schistosomes, the expression level is similar to S0 level. The SmGPR-1 and SmGPR-2 developmental patterns are highly comparable,77 which suggest that these kind of receptors are especially important during the development of early schistosomula.
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Figure 11: Developmental expression of SmGPR-2 in S. mansoni. Expression of SmGPR-2 relative to cercariae. qPCR was
performed on reverse-transcribed RNA from cercariae (C), schistosomula up to 14 days after transformation (S14), and adult
schistosomes (A).76
The mRNA levels of SmGPR-1 and SmGPR-2 are lower in adults compared to other life stages. This might be an explanation for that these receptors were missing, due to transcription levels below threshold, in the transcriptome data obtained by the study conducted by Lu et al.22 The described expression pattern could be a result of general upregulation of neuronal genes in young schistosomula compared to other life stages. Another explanation could be that the upregulation is linked to some part of histamine signaling that happens early in development. Previous research has suggested that young schistosomula increase vascular permeability by using the host’s histamine system.81–83 This facilitates passage through blood vessels during larval migration. It might be the case that the upregulation of S. mansoni’s own histamine system is involved in this.
4.3 The larval nervous system undergoes broad changes during its development
Similar to SmGPR-1 and SmGPR-2, SmGluR (Smp_128940) is widely expressed in the nervous system of S. mansoni (section 3.2).67 SmGluR was shown to be activated by glutamate (section
5.1). Remarkably, the expression pattern of up- and down-regulation that was observed for
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quantitative RT-PCR was used to determine the expression of SmGluR at mRNA level in various life stages of S. mansoni. The results indicated that expression levels of the receptor are similar for adult schistosomes and cercariae. Remarkably, a significant 8-fold upregulation of SmGluR was found in 8-day old schistosomula compared to cercariae. These findings suggest that the receptor is upregulated during the development of early schistosomula, followed by down-regulation during the transformation from larvae into adult worms. The expression pattern of up- and down-regulation for these three S. mansoni neurotransmitter receptors, SmGPR-1, SmGPR-2 and SmGluR,suggests that the larval nervous system undergoes broad changes during its development. The next section discusses the developmental expression profiles for PROF genes.
4.4 Increased expression of PROF complements during juvenile S. mansoni life stages
As explained in in section 2.3, PROF1 is a new highly diverged platyhelminth specific GPCR subfamily.15 The receptors that within this family are interesting because they are exclusively expressed in flatworms, and could possibly function as specific targets for anthelmintics. Since PZQ lacks efficacy against juvenile S. mansoni, it is desirable that new antischistosomal drugs are effective against various life stages.8 Therefore, this section analyzes the expression of PROF complements during various life stages. Life stage-specific expression data from S. mansoni was utilized to create expression profiles for PROF genes (Figure 12).37,47 The results were normalized, allowing stage-to-stage comparison. The results showed that there is a general pattern of increased expression in the juvenile schistosomula life stage. Remarkably, this is upregulation in schistosomula was also observed for SmGPR-1, SmGPR2 and SmGluR (section 4.2 & 4.3).
The expression of PROF receptors in parasitic stages and the restriction of these receptors to flatworms make them interesting potential antischistosomal drug targets. To develop a full picture of PROF receptors, additional studies need to investigate their biological function and endogenous ligands.
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4.5 Summary
Taken together, the results in this chapter showed that GPCRs in adult schistosomes are generally expressed at low levels.17 In accordance with earlier observations in other organisms that suggested that GPCR transcription levels are lower compared to other genes.84 Biogenic amine receptors SmGPR-1 and SmGPR-2 are upregulated in parasitic stages, which makes them interesting drug targets. These receptors and SmGluR, which are all neurotransmitter receptors, seem to be upregulated during the development of early schistosomula. This is followed by down-regulation during the transformation into adult schistosomes. These results shows that the larval nervous system undergoes broad changes during its development. Furthermore, analysis of expression data from S. mansoni showed that PROF genes are upregulated in schistosomula as well. This is notable, since it is desired that the next antischistosomal drug is effective against schistosomula. Therefore, SmGPR-1, SmGPR-2 and SmGluR are interesting potential drug targets. In the next chapter, deorphanized S. mansoni GPCRs will be discussed.
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5. Deorphanized S. mansoni GPCRs
Most GPCRs in S. mansoni remain to be deorphanized. The identification of endogenous ligands for schistosome GPCRs is a necessary step towards the functional characterization of GPCRs and helps unravelling the biological meaning of neuronal signaling via GPCRs.85 This, in turn, advances the understanding of the biology of schistosomes and their complex interplay between the sexes. Furthermore, deorphanization of schistosome GPCRs reveals potential candidates for antischistosomal drug targeting. Cloning and functional expression studies are necessary to deorphanize receptors.28 To deorphanize a receptor, the recombinant protein is expressed in an appropriate cell environment, after which various ligands are tested in order to find a selective agonist (Figure 13).28 This can be a challenge for S. mansoni GPCRs, since there are no flatworm cell lines that can be used for transfection. Moreover, it can be problematic to express schistosome cDNAs in heterologous environments. More specifically, it can be challenging to target the GPCR to the cell membrane at sufficient levels so that the activity can be measured. Furthermore, it is often difficult or not possible to predict which ligands are effective based on sequence homology. As a result, a wide variety of ligands and metabolites at various concentrations needs to be tested. Therefore, a highly adaptable functional expression assay is necessary. A recent pilot study described a novel approach to deorphanize S. mansoni receptors.85 This study used the Membrane-Anchored Ligand And Receptor yeast two-hybrid system (MALAR-Y2H) to detect potential neuropeptide ligands for schistosome GPCRs. The results indicated that this system is suitable to detect S. mansoni GPCR–ligand interactions. An advantage of this method is that the ligands and the receptor are co-expressed at the membrane. This approach may help in the deorphanization of schistosome GPCRs. So far, seven S. mansoni GPCRs have been deorphanized (Figure 14).86 These include SmGluR (section 5.1), SmGAR (section 5.2), SmD2 (section 5.3), SmGPR-1, SmGPR-2 and SmGPR-3 (section 5.4) as well as Sm5HTR (section 5.5). This chapter investigates the characterization of these deorphanized GPCRs in S. mansoni.
37
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Figure 14: Deorphanized S. mansoni GPCRs. All receptors have been deorphanized by RNA interference or in heterologous
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5.1 Glutamate receptor SmGluR
Glutamate is a well-established neurotransmitter in helminths, both flatworms and nematodes.67 Research has shown that adult S. mansoni muscle fibers contract in response to glutamate in a dose-dependent manner.87 Furthermore, a study provided evidence for the presence of glutamatergic receptors by showing that S. mansoni worms underwent hyperkinesias and body wall contractions after treatment with a glutamate agonist.88 These results suggest that glutamate has neuromuscular activity in S. mansoni and show that glutamate receptors are potential drug targets. The first metabotropic glutamate receptor that was identified in S. mansoni is SmGluR ((G)Smp_128940).67 As discussed in section 4.3,the expression levels of SmGluR in S. mansoni are similar for adults and cercariae. Furthermore, this receptor is upregulated during the development of early schistosomula. This section discusses the functional analysis of SmGluR.
5.1.1 SmGluR is selectively activated by glutamate
Analysis of the protein sequence of SmGluR revealed that the receptor has 1026 amino acid residues.67 SmGluR has conserved features of glutamate receptors, but is only distantly related to mGluRs from other phyla.SmGluR cannot be grouped in any subtypes of mGluRs (Groups I, II and III) and therefore seems to be a novel type of glutamate receptor. To functionally analyze the receptor, SmGluR was heterologously expressed in mammalian HEK-293 cells.67 The full-length cDNA was modified using PCR in order to introduce a Kozak sequence to increase translational efficiency in mammalian cells followed by a FLAG fusion tag at N-terminal end of the receptor. Since most mGluRs inhibit adenylyl cyclase and decrease cAMP levels,89 receptor activity was tested by measuring changes in the level of intracellular cAMP. Important to mention is that the cloned SmGluR cDNA that was used in this study differs slightly from the new annotation (Smp_128940) at the 5’ end. The researchers assumed the sequence to be correct because the length of the SmGluR cDNA is in accordance with the size of the SmGluR protein in the Western blot analysis and it was verified in multiple clones. The authors also suggest that there could be multiple forms of SmGluR in S. mansoni.
Protein expression was monitored by probing the clones with an anti-FLAG m2 antibody, after which the cells were treated with a secondary FITC-conjugated anti-mouse antibody. Green fluorescence was observed in the positive clones (Figure 15A), which were picked for functional
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analysis.67 The results revealed that glutamate activated the receptor and stimulated the cAMP production dose-dependently with an EC50 of ±100 µM (Figure 15C).67 This suggests that the receptor is coupled to a Gs type protein, which stimulates adenylate cyclase. In contrast, mammalian mGluRs usually couple to Gi/o and decrease the cAMP level or bind to Gq/11 and increase intracellular Ca2+. The unusual signaling of SmGluR could be a result of the heterologous system. Since SmGluR did not respond to GABA or aspartate, it is selectively activated by glutamate (Figure 15B).67
Although SmGluR has conserved features of family C GPCRs, it is distantly related to glutamate receptors from other phyla and therefore it cannot be classified according to the mammalian system. The receptor shares low level of sequence homology (25–30%) with all types of mGluRs (mGluRs I, II, III).67 The receptor has residues in its binding domain that are associated with glutamate binding. To illustrate, residues Arg-323, Tyr-236, Asp-208, and Ser-165 are thought to undergo an interaction with glutamate in the binding pocket of mGluRs.90 These residues are all conserved in the sequence of SmGluR. However, the schistosome GPCR has a lysine in the position of Arg-78. A previous study showed that this arginine interacts with the carboxylate group in the side chain of glutamate, and is crucial for binding of glutamate to mGluR1 with high affinity.91 It may be the case that lysine has a similar function as arginine, or a different site is involved in the interaction between glutamate and SmGluR.
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Figure 15: Functional analysis of SmGluR. SmGluR was expressed heterologously in HEK-293 cells. A) Indirect
immunofluorescence assays (IFA) were performed by probing the clones with an anti-FLAG m2 antibody, after which the cells were treated with a secondary FITC-conjugated anti mouse antibody. Green fluorescence was observed in the positive clones. B)
Positive clones, expressing SmGluR, were treated with either GABA, l-aspartate or l-glutamate (10-4 M or vehicle -). A competitive
immunoassay was performed to test for cAMP. Mock transfected cells were used as controls and to normalize the data. C)
Experiments were repeated with various concentrations of glutamate. SmGluR is dose-dependently activated by glutamate.67
5.1.2 SmGluR may influence muscle function indirectly
As described in section 3.2, SmGluR is widely expressed in the nervous system of S. mansoni as well as in the female reproductive tract. A previous study suggested that glutamate has neuromuscular activity in S. mansoni.88 It is possible that SmGluR is involved in this effect. However, another study suggested that glutamate receptors are not present in isolated muscle fibers of S. mansoni.87 Hence, SmGluR could modulate neuronal circuits and by doing so influence muscle function indirectly. It is also possible that other glutamate receptors are responsible for the observed effect of glutamate on the musculature. In situ immunolocalization analysis also revealed that SmGluR is associated with the epithelium of the reproductive tract or the oocytes, similar to what has been observed for certain mammalian mGluRs.92,93 Glutamate has been suggested to be important for the regulation of egg production and release in mammals. If this is also the case in S.
mansoni, glutamate GPCRs could be potential targets for drugs aimed at inhibition of oogenesis.
