bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 Profiling G protein-coupled receptors of Fasciola hepatica
2 identifies orphan rhodopsins unique to phylum
3 Platyhelminthes
4
5 Short title: Profiling G protein-coupled receptors (GPCRs) in Fasciola hepatica
6
7 Paul McVeigh1*, Erin McCammick1, Paul McCusker1, Duncan Wells1, Jane
8 Hodgkinson2, Steve Paterson3, Angela Mousley1, Nikki J. Marks1, Aaron G. Maule1
9
10
11 1Parasitology & Pathogen Biology, The Institute for Global Food Security, School of
12 Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn
13 Road, Belfast, BT9 7BL, UK
14
15 2 Institute of Infection and Global Health, University of Liverpool, Liverpool, UK
16
17 3 Institute of Integrative Biology, University of Liverpool, Liverpool, UK
18
19 * Corresponding author
20 Email: [email protected]
21
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22 Abstract
23 G protein-coupled receptors (GPCRs) are established drug targets. Despite their
24 considerable appeal as targets for next-generation anthelmintics, poor understanding
25 of their diversity and function in parasitic helminths has thwarted progress towards
26 GPCR-targeted anti-parasite drugs. This study facilitates GPCR research in the liver
27 fluke, Fasciola hepatica, by generating the first profile of GPCRs from the F. hepatica
28 genome. Our dataset describes 146 high confidence GPCRs, representing the
29 largest cohort of GPCRs, and the most complete set of in silico ligand-receptor
30 predictions, yet reported in any parasitic helminth. All GPCRs fall within the
31 established GRAFS nomenclature; comprising three glutamate, 135 rhodopsin, two
32 adhesion, five frizzled and one smoothened GPCR. Stringent annotation pipelines
33 identified 18 highly diverged rhodopsins in F. hepatica that maintained core
34 rhodopsin signatures, but lacked significant similarity with non-flatworm sequences,
35 providing a new sub-group of potential flukicide targets. These facilitated
36 identification of a larger cohort of 76 related sequences from available flatworm
37 genomes, representing new members of existing groups of flatworm-specific
38 rhodopsins. These receptors imply flatworm specific GPCR functions, and/or co-
39 evolution with unique flatworm ligands, and could facilitate development of
40 exquisitely selective anthelminthics. Ligand binding domain sequence conservation
41 relative to deorphanised rhodopsins enabled high confidence ligand-receptor
42 matching of seventeen receptors activated by acetylcholine, neuropeptide F/Y,
43 octopamine or serotonin. RNA-Seq analyses showed expression of 101 GPCRs
44 across various developmental stages, with the majority expressed most highly in the
45 pathogenic intra-mammalian juvenile parasites. These data identify a broad
46 complement of GPCRs in F. hepatica, including rhodopsins likely to have key
47 functions in neuromuscular control and sensory perception, as well as frizzled and
48 adhesion families implicated, in other species, in growth, development and
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49 reproduction. This catalogue of liver fluke GPCRs provides a platform for new
50 avenues into our understanding of flatworm biology and anthelmintic discovery.
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51 Author Summary
52 Fasciola spp. liver fluke are important veterinary pathogens with impacts on human
53 and animal health, and food security, around the world. Liver fluke have developed
54 resistance to most of the drugs used to treat them (flukicides). Since no vaccines
55 exist, we need to develop new flukicides as a matter of urgency. Most anthelmintic
56 drugs used to treat parasitic worm infections operate by impeding the functioning of
57 their nerve and muscle. In flatworms, most nervous signals are received by a type of
58 receptor called a G protein-coupled receptor (GPCR). Since GPCRs control
59 important parasite functions (e.g. movement, egg-laying, feeding), they represent
60 appealing targets for new flukicides, but have not yet been targeted as such. This
61 work exploited the F. hepatica genome to determine the quantity and diversity of
62 GPCRs in liver fluke. We found more GPCRs in the Fasciola genome than have
63 been reported in any other parasitic worm. These findings provide a foundation that
64 for researchers to determine the functions of these receptors, and which
65 molecules/ligands they are activated by. These data will pave the way to exploring
66 the potential of F. hepatica GPCRs as targets for new flukicides.
67
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68 Introduction
69 Fasciola spp. liver fluke are pathogens of veterinary ruminants that threaten the
70 sustainability of global meat and dairy production. Infection with Fasciola
71 (fasciolosis/fascioliasis) inhibits animal productivity through liver condemnation,
72 reduced meat and milk yields, and reduced fertility (for recent impact surveys see [1-
73 4]. Fasciola spp. also infect humans, with fascioliasis considered a neglected
74 tropical disease [5]. Anthelmintic chemotherapy currently carries the burden of fluke
75 control, since there are no liver fluke vaccines [6]. Six flukicidal active compounds
76 are available for general use, with on-farm resistance reported for all except
77 oxyclozanide [7]. Resistance to the frontline flukicide, triclabendazole, also exists in
78 human F. hepatica infections [8,9]. Given the absence of alternative control
79 methods, new flukicides are essential for secure future treatment of veterinary and
80 medical liver fluke infections.
81
82 The helminth neuromuscular system is a prime source of molecular targets for new
83 anthelmintics [10-12], not least because many existing anthelmintics (dichlorvos,
84 levamisole, morantel, piperazine, pyrantel, macrocyclic lactones, paraherquamide,
85 amino acetonitrile derivatives) act upon receptors or enzymes associated with
86 classical neurotransmission in nematodes [11] The G protein-coupled receptors
87 (GPCRs) that transduce signals from both peptidergic and classical
88 neurotransmitters are of broad importance to helminth neuromuscular function.
89 Despite industry efforts to exploit helminth GPCRs in the context of anthelmintic
90 discovery [13], only a single current anthelmintic (emodepside) has been attributed
91 GPCR-directed activity as part of its mode of action [14-16]. GPCRs are druggable
92 targets, since 33% of human prescription medicines are attributed a GPCR-based
93 mode of action [17].
94
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95 Despite two F. hepatica genomes [18,19], no GPCR sequences have been reported
96 from F. hepatica. In contrast, GPCRs have been profiled in the genomes of
97 trematodes (Schistosoma mansoni and Schistosoma haematobium [20,21]),
98 cestodes (Echinococcus multilocularis, E. granulosus, Taenia solium and
99 Hymenolepis microstoma [22]), and planaria (Schmidtea mediterranea, Girardia
100 tigrina [21-24]). These datasets illustrated clear differences in the GPCR
101 complements of individual flatworm classes and species, with reduced complements
102 in parasitic flatworms compared to planarians.
103
104 This study profiles the GPCR complement of the temperate liver fluke F. hepatica for
105 the first time, permitting comparisons with previously characterised species that
106 inform evolutionarily and functionally conserved elements of flatworm GPCR
107 signalling. We have identified and classified 146 GPCRs by GRAFS family
108 (glutamate, rhodopsin, adhesion, frizzled, secretin) assignment [25], the majority of
109 which are expressed in Fasciola RNA-Seq datasets. These include clear
110 orthologues of GPCRs activated by known neurotransmitters, within which we
111 performed the deepest in silico ligand-receptor matching analyses to date for any
112 parasitic helminth. The latter predicted ligands for 17 F. hepatica GPCRs,
113 designating these as primary targets for deorphanisation. Intriguingly, the dataset
114 included a set of flatworm-expanded GPCRs lacking orthologues outside of phylum
115 Platyhelminthes. Evolution of such GPCRs across the parasitic flatworm classes
116 may have been driven by flatworm-specific functional requirements or co-evolution
117 with flatworm ligands, either of which could help support novel anthelmintic
118 discovery. This dataset provides the first description of GPCRs in liver fluke, laying a
119 foundation for future advances in GPCR-directed functional genomics and flukicide
120 discovery.
121
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122 Results and Discussion
123 A first look at GPCRs in the F. hepatica genome
124 This study represents the first description of the GPCR complement of the temperate
125 liver fluke, F. hepatica. Using HMM-led methods to examine available F. hepatica
126 genome datasets, we identified 166 GPCR-like sequences in F. hepatica (Figure 1,
127 S1 Table). Figure 1B shows that 49.7% contained 7 TM domains, with 88% of
128 sequences containing at least four TMs. The remainder of this manuscript focuses
129 on 146 sequences containing ≥4TM domains (S1 Table; S2 Text). Twenty
130 sequences containing ≤3 TMs were analysed no further (Figure 1).
131
132 Our ≥4TM dataset (146 sequences) was comprised of three glutamate, 135
133 rhodopsin, two adhesion, five frizzled, and one smoothened GPCR. Sequence
134 coverage was generally good in terms of TM and extracellular domain
135 representation, so we did not attempt to extend truncated sequences into full-length
136 receptors. The overall dataset contained excellent representation of seven TM
137 domains, while N-terminal extracellular LBDs and cysteine-rich domains (CRD) were
138 also detected (in glutamate, frizzled/smoothened, adhesion families). However, we
139 could not identify N-terminal secretory signal peptides in any sequence, suggesting
140 incomplete sequence coverage at extreme N-termini. Rhodopsins are designated by
141 ubiquitously conserved motifs on TMs 2, 3, 6 and 7. All rhodopsin sequences
142 contained at least one of these motifs (Figure 2, S3 Table), including in the highly
143 diverged flatworm-specific rhodopsins described below.
144
145 Table 1 compares the F. hepatica GPCR complement with other flatworms,
146 illustrating that F. hepatica has the largest GPCR complement reported from any
147 parasitic flatworm to date. The bulk of the expansion involves rhodopsins, while the
148 other GRAFS families are comparable between F. hepatica and other flatworm
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149 parasites. Secretin is the notable exception, at least one of which has been identified
150 in every other species studied, but which was absent from the datasets scrutinized
151 here.
152
153 Table 1. Comparison of the Fasciola hepatica G-protein coupled receptor 154 (GPCR) complement with those reported from other flatworms. Species 155 complements are shown in the context of GRAFS nomenclature [25]. * Saberi et al 156 [24] described 566 GPCRs in Schmidtea mediterrannea, of which 516 fall within 157 GRAFS nomenclature. 158
159
[21] [24]
[20] 160 [22]
[21] [20] 161
162
163 F. hepatica F. mansoni S. mansoni S. haematobium S. multilocularis E. mediterrannea S. mediterrannea S.
Glutamate 3 2 2 2 5 9 11164 Rhodopsin 135 105 59 53 48 418 461 Adhesion 2 3 - - 4 9 14165 Frizzled/Smoothened 6 5 4 4 5 11 10 Secretin 0 2 5 5 1 1 20166 Total 146 117 64 64 83 448 516* 167
168 Stringent annotation of flatworm-specific orphan rhodopsin GPCRs in F.
169 hepatica
170 Encompassing 135 sequences, the rhodopsin family is the largest of the GRAFS
171 classifications in F. hepatica. Rhodopsins comprise four subfamilies (α, β, γ and δ)
172 [26]; we identified members of both α and β groups, with nucleotide-activated (P2Y)
173 receptors (γ group), and olfactory (δ group) receptors absent from our dataset
174 (Figures 1, 2; S1 Table). The F. hepatica α subfamily contained 38 amine receptors
175 and three opsins, with the β subfamily comprised of at least 47 peptide receptors.
176 Homology-based annotations were supported by an ML phylogeny (Figure 2A),
177 which clearly delineated between amine and opsin α clades, and the peptide-
178 activated β-rhodopsin clades. Amine and peptide receptors were further delineated
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179 by additional phylogenetic and structural analyses, permitting high-confidence
180 assignment of putative ligands to 16 GPCRs (see below).
181
182 Six clades contain an additional 44 rhodopsin sequences with low scoring (median E
183 = 5.6e-5) similarity matches to a range of disparate α and β rhodopsins. Due to the
184 subsequent difficulty in designating these clades as amine, peptide or opsin, we
185 labelled them orphan rhodopsins (“R” clades in Figure 2A). Eighteen GPCRs within
186 the orphan clades displayed exceptionally low similarity scores relative to non-
187 flatworm sequences (Figure 2A,B). Seven returned no-significant hits in BLASTp
188 searches against non-flatworm members of the ncbi nr dataset (the most diverse
189 sequence dataset available to the research community), and the remaining eleven
190 scored E>0.01. Domain analysis (InterPro) identified rhodopsin domains
191 (IPR000276 or IPR019430) in thirteen of these (S1 Table, S3 Table), confirming their
192 identity as rhodopsin-like GPCRs. More troublesome to classify were five that, in
193 addition to lacking significant BLASTp identity to non-flatworm sequences, also
194 lacked any identifiable protein domains/motifs (with the exception of TM domains).
195 We annotated these as rhodopsins because: (i) They did not contain motifs/domains
196 representative of any other protein family; (ii) They displayed topological similarity to
197 GPCRs (ten had seven TM domains, seven had six TMs, one had five TM domains);
198 (iii) They contained at least two of the conserved rhodopsin motifs in TM domains 2,
199 3, 6 and 7 similar to those seen in the rest of the F. hepatica rhodopsins (Figure 2C;
200 S4 Table). As highly diverged rhodopsins with little or no sequence similarity versus
201 host species, these 18 F. hepatica receptors have obvious appeal as potential
202 targets for flukicidal compounds with exquisite selectivity for parasite receptors over
203 those of the host. This potential is contingent on future work demonstrating essential
204 functionality for these receptors; showing their wider expression across flatworm
205 parasites would enable consideration of anthelmintics with multi-species activity. To
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206 investigate the latter question, we used BLASTp to search the 18 F. hepatica
207 rhodopsins against other available genomes representing phylum Platyhelminthes.
208
209 An orphan family of lineage-expanded rhodopsins in flatworm genomes
210 Although lacking similarity against non-flatworm datasets, each of the 18 lineage-
211 expanded F. hepatica rhodopsins returned high-scoring hits in BLASTp searches
212 against the genomes of other flatworms (WormBase Parasite release WBPS9). All
213 returns were subsequently filtered through a stringent five-step pipeline (Figure 3A)
214 consisting of: (i) Removal of duplicate sequences; (ii) Exclusion of sequences
215 containing fewer than four TM domains; (iii) A requirement for reciprocal BLASTp
216 against the F. hepatica genome to return a top hit scoring E<0.001 to one of the
217 original 18 F. hepatica queries; (iv) A requirement for BLASTp against ncbi nr non
218 flatworm sequences to return a top hit scoring E>0.01; (v) Removal of sequences
219 lacking conservation of the ubiquitous rhodopsin motifs seen in the divergent F.
220 hepatica rhodopsins (Figures 2C, 3C). The latter motifs were largely absent from
221 cestode rhodopsins (with the exception of a single sequence from Diphyllobothrium
222 latum, and three sequences from Schistocephalus solidus), and present in only two
223 sequences from a single monogenean (Protopolystoma xenopodis). This left our
224 final dataset consisting of 76 “flatworm-specific” rhodopsins (fwRhods; Figure 3B,
225 Table S4) in phylum Platyhelminthes, heavily biased towards trematodes (70
226 sequences). Nineteen sequences from nine species of cestode were omitted from
227 the final dataset despite meeting the inclusion criteria in most respects, because they
228 lacked conservation of ubiquitous rhodopsin motifs (filtering step (v)). Although their
229 further characterisation was beyond the scope of this study, they warrant more
230 detailed examination in future studies as potential cestode-specific rhodopsins. Note
231 that our filtering pipeline also excluded initial hits from Gyrodactylus salaris
232 (Monogenea), and the Turbellarians Macrostomum lignano and S. mediterranea.
233 Individual species complements of fwRhods showed some consistency (Figure 3B);
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234 the trematodes F. hepatica and Echinostoma caproni (both phylum Platyhelminthes
235 Order Echinostomida) bore 18 and 19 sequences, respectively, most species of
236 family Schistosomatidae contained 3-4 sequences each. The inclusion of two
237 cestode species and a single monogenean may be an indication of the existence of
238 distantly related rhodopsins in those lineages, rather than a true measure of the
239 extent of cestode and monogenean fwRhod diversity. Again, proper classification of
240 these groups will require further more focused study that was beyond the scope of
241 the current work.
242
243 Our method for identification of fwRhods is supported by a similar BLAST-driven
244 approach used to identify highly diverged “hidden orthologues” in flatworms [27]. It
245 should be noted that the existence of sequences lacking sequence similarity to
246 genes of other species is not a new finding. “Taxonomically-restricted genes”
247 comprise 10-20% of every sequenced eukaryote genome, and may be essential for
248 phylum-specific morphological and molecular diversity [28]. How do our fwRhods
249 compare to previously reported groups of flatworm restricted GPCRs in S. mansoni,
250 S. mediterrannea and E. granulosus [21,22,24]? Phylogenetic comparisons of these
251 groups (Figure 3D) demonstrated that the previously described Schmidtea Srfb
252 cluster [24] and the PROF1 clade (E. multilocularis, Schmidtea, S. mansoni [21]) are
253 equivalent, and likely represent a single group. Our phylogeny added 23 fwRhods to
254 this clade, including three from F. hepatica (BN1106_s6156B000040, D915_03083,
255 D915_13002). Figure 3D designated the remaining fwRhods within additional pre-
256 existing groups [24], placing 34 within Rho-L (including eight from F. hepatica), nine
257 in Srfc (one from F. hepatica), four in Rho-R (one from F hepatica) and two in Srfa
258 (one from F. hepatica). Four fwRhod sequences were omitted from this tree due to
259 poor alignment.
260
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261 Our approach to classifying flatworm-restricted rhodopsins was to err towards
262 stringency, and this may have resulted in erroneous exclusion of some sequences
263 from the dataset. There is no set definition for lineage specificity in the literature, but
264 ours is the most stringent yet applied to flatworm GPCRs. Applying our BLASTp
265 E≥0.01 cutoff (modified from Pearson [29]) to the previously described groups of
266 flatworm-specific rhodopsins [21,22,24], excludes 57 of the 62 PROF1s described
267 from S. mansoni and S. mediterranea and 287 of the 318 RhoL/R and Srfa/b/c
268 flatworm-specific clusters in S. mediterranea. Further pursuit of the extent of lineage
269 restricted GPCRs in the wider phylum was beyond the scope of this study, but we
270 are currently trawling for taxonomically restricted flatworm GPCRs on a phylum wide
271 scale.
272
273 We have established the existence of a group of rhodopsin GPCRs that appear
274 restricted to, and expanded in, phylum Platyhelminthes. By definition these
275 receptors are orphan (i.e. their native ligands are unknown), so key experiments
276 must focus on identifying their ligands and functions. Such experiments can exploit
277 the expanding molecular toolbox for flatworm parasites, which in F. hepatica includes
278 RNA interference (RNAi) [30-33], interfaced with enhanced in vitro maintenance
279 methods, and motility, growth/development and survival assays [31,34,35]. Our
280 phylogeny (Figure 2A) suggests that fwRhods are more similar to peptide than amine
281 receptors. If their heterologous expression can be achieved, one approach to
282 characterisation would be to screen them with the growing canon of peptide ligands
283 from flatworms [36-38], as well as from other genera, in a receptor activation assay.
284 Subsequent localisation of their spatial expression patterns would provide additional
285 data that would inform function.
286
287 Predicting ligands for F. hepatica rhodopsin GPCRs
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288 In addition to the flatworm-specific fwRhod sequences described above, for which
289 the ligands and functions remain cryptic, we also identified many rhodopsins with
290 clear similarity to previously annotated GPCRs. Figure 2A shows the phylogenetic
291 delineation of these sequences into amine-, opsin- and peptide-like receptors,
292 distinctions that are supported by BLASTp comparisons with general (ncbi nr) and
293 lineage-specific (superphylum level) datasets, as well as by gross domain structure
294 (InterProScan) (S1 Table). These data provided a foundation for deeper
295 classification of putative ligand-receptor matches.
296
297 The structure and function of GPCR LBDs can be studied using molecular modelling
298 to predict interactions with receptor-bound ligands. These predictions can then be
299 validated by targeted mutagenesis of residues within the LBD, measuring impacts
300 with downstream signalling assays. Such experiments have been performed in
301 model vertebrates and invertebrates, enabling identification of evolutionarily
302 conserved binding residues/motifs. These data inform assignment of putative
303 ligands to newly discovered receptors. Since mutagenesis experiments have not yet
304 been performed in flatworm GPCRs, we employed a comparative approach to
305 identify 17 F. hepatica rhodopsins with LBD motifs diagnostic of receptors for NPF/Y,
306 5-HT, octopamine (Oct) or acetylcholine (ACh) (Figure 4; S4 Table), thus enabling in
307 silico ligand-receptor matching of these GPCRs.
308
309 Comparison of F. hepatica rhodopsins by structural alignment with LBD residues
310 conserved across vertebrate NPY and dipteran NPF receptors [39-44] identified
311 three peptide receptors with more than 75% identity across 9 ligand-interacting
312 positions (Figure 4A). The two highest scoring GPCRs (BN1106_s3169B000088
313 and D915_05685) are also found, in our phylogenetic analysis (S5 Figure) in the
314 same clade as the deorphanized NPF/Y receptors of human (HsNPYR2), Glossina
315 mortisans (Glomo-NPFR) and S. mediterranea (SmedNPYR1). These data
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316 designate these three F. hepatica GPCRs as prime candidates for further work to
317 deorphanize and confirm these receptors as NPF/Y-activated, and to probe the
318 biology of NPF/Y receptors in parasitic flatworms. A single NPF/Y receptor has been
319 functionally characterised in S. mediterranea, displaying a role in the maintenance of
320 sexual maturity [24]. If related functions are conserved in liver fluke NPF/Y receptors
321 they could have appeal as therapeutic targets in adult fluke that could interrupt
322 parasite transmission, although their utility for the control of acute fasciolosis, caused
323 by migrating juveniles, would be open to question.
324
325 Broad phylogenetic comparison of our peptide receptor set with a comprehensive
326 collection of deorphanized bilaterian rhodopsin GPCRs (S5 Figure), identified F.
327 hepatica receptors similar to those for myomodulin, FLP, luqin and Neuropeptide KY
328 (NKY). These ligands have all been predicted or demonstrated in previous
329 biochemical or in silico studies of flatworm neuropeptides [36-38]. We also
330 uncovered F. hepatica GPCRs with phylogenetic similarity to allatotropin, allatostatin,
331 thyrotropin-releasing hormone and sex peptide receptors. These ligands have not
332 yet been reported in flatworms, although the existence of allatostatin-like receptors in
333 flatworms is supported by the inter-phyla activity of arthoropod allatostatins in
334 helminth (including flatworm) neuromuscular assays [45].
335
336 No F. hepatica neuropeptide sequences have been published yet, but our
337 unpublished data suggest the presence of at least 36 neuropeptide genes in the F.
338 hepatica genome (Duncan Wells, Queen’s University Belfast, personal
339 communication). These ligands would facilitate deorphanisation of heterologously-
340 expressed peptide GPCRs (S1 Table). This is essential work, since although two
341 planarian peptide receptors have been deorphanised [23,24], no flatworm parasite
342 peptide GPCRs have been ligand matched yet. Receptor deorphanisation provides
343 a starting point for drug discovery, by enabling development of agonists or
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344 antagonists that modulate the interaction of a GPCR with its cognate ligand. Such
345 compounds could form the basis of ligand series for screening pipelines that would
346 lead to new flukicides [46,47].
347
348 Serotonin (5-hydroxytryptamine, 5-HT) is abundant throughout flatworm nervous
349 systems, and is considered the primary flatworm excitatory neurotransmitter [48].
350 Deorphanized GPCRs activated by 5-HT have been described in turbellarians and
351 trematodes, with an S. mansoni 5-HT receptor (Sm5HTR) involved in neuromuscular
352 control [49-51]. Five F. hepatica rhodopsins (Figure 4B) bore appreciable (≥80%)
353 positional identity in amino acids shown to be key ligand-interacting residues in the
354 human 5HT1A LBD [52,53]. Notably, these residues were also conserved in the
355 deorphanized S. mansoni 5-HT receptor (Sm5HTR, Smp_126730) [51]. Three of the
356 sequences (BN1106_s362B000177, BN1106_s81B000700 and
357 BN1106_s10B000515) also resembled Sm5HTR in our phylogenetic analysis,
358 identifying them as likely 5-HT receptors. The remaining two (D915_00277 and
359 BN1106_s1436B000114) appeared phylogenetically more similar to an S. mansoni
360 dopamine receptor (Smp_127310) [54]. These annotations provide rational starting
361 points for receptor deorphanization using functional genomic and/or heterologous
362 expression tools. We found that F. hepatica dopamine-like receptors, identified by
363 phylogeny (S5 Figure), displayed poor conservation (max 56% overall identity) to the
364 human D2 LBD [55]. Due to this lack of selectivity, we did not annotate any F.
365 hepatica GPCRs as dopamine receptors.
366
367 Although common in other invertebrates, octopamine has not yet been directly
368 demonstrated as a neurotransmitter in flatworms. Evidence for its presence is
369 indirect, based on tyramine β-hydroxylase (octopamine’s biosynthetic enzyme)
370 activity in cestodes and planaria [56,57]. Three rhodopsins (Figure 4C) showed
371 100% conservation of the arthropod octopamine LBD, as determined from
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372 Periplaneta americana and Bombyx mori [58,59], with an additional four showing
373 88% conservation. Of these seven rhodopsins, four resolved in close phylogenetic
374 proximity to Drosophila mushroom body octopamine receptors (D915_02972),
375 Drosophila octopamine beta-receptors (D915_08505 and BN1106_s1016B000108)
376 (S5 Figure) or a Drosophila tyramine receptor (D915_05578), denoting these as
377 high-confidence octopamine receptors. These data provide further evidence in
378 support of a functional role for this enigmatic classical neurotransmitter in flatworms.
379
380 Acetylcholine has species-specific impacts on flatworm neuromuscular preparations
381 in vitro, with myoinhibitory effects in Fasciola [60]. Two putative muscarinic
382 acetylcholine receptors (mAChRs), shared highest LBD identity with a Rat M3 ACh
383 receptor (Figure 4D) [61]. Although these were only 67% identical to the rat
384 sequence, the five ligand-interacting residues within their LBDs were 100% identical
385 to those of a deorphanised S. mansoni mAChR, known to be involved in
386 neuromuscular coordination (SmGAR) [62]. These receptors (D915_00814 and
387 BN1106_s1913B000092) were also the most similar to SmGAR in our phylogeny (S5
388 Figure) so we consider them amongst our high confidence candidates for
389 deorphanization.
390
391 F. hepatica glutamate receptors bear divergent glutamate binding domains
392 At least three glutamate-like GPCRs exist in F. hepatica (Figure 5A, S1 Table). All
393 three are defined by significant BLASTp similarity (median E=2.3e-34) to metabotropic
394 glutamate receptors (mGluRs), and/or by the presence of InterPro GPCR family 3
395 (Class C) domains IPR017978, IPR000162 or IPR000337. Phylogenetic analysis of
396 these GPCRs was performed alongside receptors representative of the various Class
397 C subgroups (Figure 5) [63], including Ca2+-sensing receptors, γ-aminobutyric acid
398 type B (GABAB) receptors, metabotropic glutamate (mGluR) receptors, and
399 vertebrate taste receptors; for reference we also included the two previously reported
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400 mGluRs from S. mansoni [21]. One F. hepatica GPCR (BN1106_s2924B000081)
401 resolved alongside Smp_052680, which has previously been described as an S.
402 mansoni mGluR; these receptors form a close outgroup from the mGluR clade,
403 supporting their designation as mGluRs. A second F. hepatica glutamate receptor
404 (BN1106_s1717B000113) also has a close S. mansoni ortholog (Smp_128940), both
405 of which reside in an orphan outgroup that is of uncertain provenance. The third F.
406 hepatica glutamate receptor resides within another orphan group, close to human
407 GPR158 and GPR179, two closely related class C GPCRs expressed respectively in
408 the human brain and retina [64]. Although these receptors have been linked with
409 specific disease states [65,66], their ligands remain unknown.
410
411 Divergence within the LBD can inform the ligand selectivity of Class C receptors
412 [21,67]. To further classify the two orphan glutamate GPCRs described above, we
413 generated multiple sequence alignments to analyse the conservation of established
414 agonist-interacting residues between mammalian mGluR and GABAB receptors and
415 our F. hepatica GPCRs. These analyses identified no significant conservation of
416 either mGluR or GABAB LBD residues (Figure 6B). Figure 6B also includes the
417 previously reported S. mansoni glutamate receptors [21], where Smp_052660
418 contained a relatively well-conserved LBD with Smp_062660 appearing more
419 atypical. Since all three F. hepatica glutamate GPCRs bear atypical LBDs with
420 respect to both GABAB and mGluR, it remains difficult to unequivocally define their
421 ligand selectivity on the basis of conserved motifs. Nevertheless, the lack of in silico
422 evidence for F. hepatica GABAB GPCRs reflects the dominance of GABAA-like
423 pharmacology, which suggests that flatworm GABA signal transduction is probably
424 entirely mediated by ionotropic receptors [48,68].
425
426 The Wnt binding domain is conserved in F. hepatica frizzled/smoothened
427 receptors
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428 Ten frizzled (fzd) GPCRs and a single smoothened (smo) GPCR are recognised in
429 the human genome. In F. hepatica we identified five fzd-like sequences and one
430 smo-like sequence (Figure 6; Table S1; Table S7). All of these show high scoring
431 similarity to annotated sequences in the ncbi nr dataset (median E=3.8e-83), and all
432 five fzd contain InterPro domain IPR000539, with the single smo containing domain
433 IPR026544 (Table S1). Phylogenetic analysis of these alongside vertebrate and
434 invertebrate receptors placed all in close proximity to existing fzd/smo groups (Figure
435 6A). Four F. hepatica fzd had individual direct orthologs with the four known S.
436 mansoni fzd [21].
437
438 Frizzled receptors are activated by cysteine-rich glycoprotein ligands known as Wnts
439 (Wingless and Int-1), and are involved in developmental signalling through at least
440 three different signalling pathways [69]. Crystallography of mouse fz8, docked with
441 Xenopus wnt8, identified 14 amino acids within the fz8 CRD that make contact with
442 the Wnt8 ligand [69]. Positional conservation of these residues is apparent when fz8
443 is aligned with the five F. hepatica fzd sequences (Figure 6B; S6 Table), suggesting
444 conservation of the wnt-frizzled interaction between liver fluke and vertebrates.
445
446 Two Wnt ligands have been described in S. mansoni [70,71] ; our BLAST searches
447 identified at least three Wnt-like sequences in the F. hepatica genome
448 (BN1106_s198B000330.mRNA-1, BN1106_s1256B000163.mRNA-1,
449 BN1106_s737B000430.mRNA-1; Figure 6C). These showed conservation of the 23
450 conserved cysteine residues that are diagnostic of Wnt glycoproteins [72]. Norrin, a
451 non-Wnt protein ligand, can also activate Fz4, and the canonical β-catenin pathway.
452 The amino acids involved in norrin binding to the fz4 CRD have also been
453 determined [73], but we did not observe conservation of these in any of the F.
454 hepatica fzd. Similarly, BLASTp searches of human norrin (Uniprot Q00604) against
455 the F. hepatica genome did not return significant hits, suggesting that the norrin-fz
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456 signaling axis may not function in liver fluke. Smoothened receptors are structurally
457 similar to frizzleds, but operate in a ligand-independent fashion within hedgehog
458 signaling pathways that control several developmental processes [74]. Model
459 organism genomes typically contain only one smoothened gene (SMO); this was the
460 case in S. mansoni and S. mediterranea [21], and here we have identified a single F.
461 hepatica smoothened (BN1106_s1509B000194 ; Figure 6; Table S1).
462
463 Fzd/smo GPCRs are involved broadly in the control of cellular development. Our
464 discovery of fzd/smo GPCRs, and their Wnt ligands, in F. hepatica opens avenues
465 towards probing molecular aspects of development and differentiation in the putative
466 stem cells/neoblasts of liver fluke [35]. Neoblasts are the cells that impart the
467 regenerative capacity of free-living turbellarian flatworms [75], and neoblast-like cells
468 also represent the only proliferating cells in several parasitic species [76-78].
469 Therefore, these cells are important in understanding fundamental fluke biology and
470 represent potential repositories of unique anthelmintic targets, capable of inhibiting
471 worm growth or development. The presence of both receptor and ligand sequences
472 will permit functional genomic dissection of Wnt-Frizzled ligand-receptor signalling
473 networks, aimed at elucidating their roles in the development and differentiation of
474 liver fluke neoblast- like cells. These FhGPCRs will enable comparisons between
475 the biology of parasitic and free-living flatworms, where Wnt signaling is known to be
476 essential for anterior-posterior polarity in regenerating planaria [79,80].
477
478 Class B (Adhesion and Secretin) receptors
479 Class B receptors incorporate both adhesions and secretins. Adhesions are
480 characterised by a long N-terminal extracellular domain (ECD) that includes several
481 functional motifs. These ECDs are auto-proteolytically cleaved into two subunits that
482 subsequently reassemble into a functional dimer [26]. We identified two Class B
483 sequences in the F. hepatica genome (S7 Figure, S1 Table), both of which
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484 (scaffold181_78723-79604, and BN1106_s537B000355) contained GPCR class B
485 InterPro domain IPR000832 and displayed closest BLASTp similarity (E=5.6e-7) to
486 latrophilin-like receptors. These data suggest that both are adhesions, rather than
487 secretins. Phylogenetic analysis of these GPCRs alongside human Class B
488 receptors supports the definition of scaffold181_78723-79604 as an adhesion,
489 alongside two previously reported S. mansoni adhesions (Smp_176830,
490 Smp_099670) [21], while the other receptor appears more divergent.
491 BN1106_s536B000355 pairs with another known S. mansoni adhesion
492 (Smp_058380) [21], although both sit in closer proximity to human secretins than
493 adhesions.
494
495 Deorphanization of a handful of adhesions matches them with a complex assortment
496 of ligands including collagen, transmembrane glycoproteins, complement proteins
497 and FMRFamide-like neuropeptides [81]. This assortment of potential ligands, and
498 their expression in almost every organ system has led to the proposal of a diverse
499 range of functions for vertebrate adhesions. The F. hepatica adhesion complement
500 of two GPCRs is greatly reduced compared to the 33 receptors known in humans; in
501 other flatworms 14, 4 and 1 adhesions have been described in S. mediterranea, E.
502 multilocularis and S. mansoni, respectively [21,22,24]. Functional characterisation
503 will be a challenging task given the wide range of possible functions to be assayed;
504 an appealing starting point would be to investigate roles in neoblast motility prior to
505 differentiation, given that mammalian adhesion GPCRs are involved in the control of
506 cellular migration [81].
507
508 Developmental expression
509 Using RNA-Seq methods, we were able to confirm expression of 101 GPCRs across
510 libraries representing several F. hepatica life-stages. These datasets included
511 publically available reads from individual developmental stages [18], and a
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512 transcriptome that we generated in-house for 21-day liver stage ex-vivo juveniles
513 (juv2). Since these datasets were generated independently and clearly display
514 distinct sequence diversities, we avoided any further direct comparisons between
515 Cwiklinski juv1 and our juv2 datasets. Each dataset is analysed separately, below.
516
517 Figure 7A illustrates detection of 83 GPCRs across Cwiklinski’s developmentally
518 staged RNA-Seq datasets. These comprised four FZD, thirteen aminergic
519 rhodopsins, two opsins, 41 peptidergic rhodopsins, and 23 orphan rhodopsins. The
520 latter included nine fwRhods. Clustering within Figure 7A’s expression heatmap
521 shows clear developmental regulation of GPCR expression, outlining nine GPCRs
522 with relatively higher expression in adults, two with higher expression in 21d
523 juveniles, 64 GPCRs preferentially expressed in either 1h, 3h or 24h NEJs, and six
524 receptors expressed most highly in eggs. GPCR classes appear to be randomly
525 distributed across these expression clusters, giving little opportunity to infer function
526 from expression. Adult-expressed GPCRs include five orphan fwRhods, three
527 peptide receptors including a putative NPF/Y receptor, and a predicted octopamine-
528 gated aminergic rhodopsin. The majority of expressed GPCRs occurred in the NEJ-
529 focused expression cluster. Given data implicating GPCRs in motility,
530 growth/development and sensory perception [11], it is no surprise to find high levels
531 of GPCR expression in the NEJs, which must navigate and burrow their way from the
532 gut lumen into the liver parenchyma, while also sustaining rapid growth from the start
533 of the infection process. The high expression in these stages, of receptors that we
534 predict to be activated by myomodulators such as ACh, FMRFamide, GYIRFamide,
535 myomodulin, myosuppressin and 5-HT, provide tentative support for these
536 predictions. The focused expression of six GPCRs in eggs suggests potential roles
537 in the control of cellular proliferation and fate determination processes that occur
538 during embryonation of liver fluke eggs. This complement did not include frizzled or
539 adhesion GPCRs that are traditionally implicated in the control of development,
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540 instead consisting of rhodopsins (including an angiotensin-like peptide receptor, two
541 octopamine-like amine receptors, one opsin receptor and one fwRhod receptor).
542
543 Focusing on the pathogenic 21-day juvenile stage, we detected 76 GPCRs in our
544 juv2 datasets, and 29 in the corresponding juv1 samples from Cwiklinski’s dataset
545 (Figure 7B). Our juv2 dataset included three glutamate, one adhesion, four frizzled,
546 one smoothened, and 67 rhodopsins. The identity of the receptors expressed here
547 again attest to the key role of neuromuscular co-ordination in this highly motile life
548 stage, which must penetrate and migrate through the liver parenchyma en route to
549 the bile ducts. Amongst the receptors expressed in this stage and thought to have a
550 role in neuromuscular function are several activated by classical neurotransmitters
551 including ACh, dopamine and 5-HT. The peptide receptors include some with
552 phylogenetic similarity to receptors for myoactive flatworm peptides (FMRFamide,
553 GYIRFamide, NPF) [11], as well as receptors from other invertebrates activated by
554 peptide ligands known to have excitatory effects on flatworms (allatostatin A,
555 myomodulin, proctolin) [82]. The presence of highly expressed GPCRs with
556 probable neuromuscular functions in liver stage juveniles, points to the importance of
557 studying these receptors with a view to flukicide discovery. The damage caused by
558 migrating juvenile fluke requires that new flukicides are effective against this stage.
559 The neuromuscular GPCRs expressed in migrating juveniles provide compelling
560 targets for new drugs.
561
562 Conclusions
563 GPCRs are targets for 33% of human pharmaceuticals [17], illustrating the appeal of
564 GPCRs as putative anthelmintic targets. This study provides the first description of
565 the F. hepatica GPCR complement permitting consideration of a GPCR target-based
566 screening approach to flukicide discovery. To facilitate the deorphanization
567 experiments that will precede compound screening efforts, we have described a set
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568 of high confidence rhodopsin ligand-receptor pairs. We identified these GPCRs,
569 including receptors for ACh, octopamine, 5HT and NPF/Y, through phylogenetic
570 comparison with existing deorphanised receptors and positional conservation of
571 ligand-interacting residues within ligand binding domains. Our additional descriptions
572 of flatworm-specific rhodopsins support the potential for synthetic ligands to be
573 parasite-selective anthelmintics.
574
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575 Materials and Methods
576 Liver fluke sequence databases
577 We exploited two F. hepatica genome assemblies available from WormBase
578 ParaSite [83], generated by Liverpool University
579 (http://parasite.wormbase.org/Fasciola_hepatica_prjeb6687/Info/Index/; [18], and
580 Washington University, St Louis
581 (http://parasite.wormbase.org/Fasciola_hepatica_prjna179522/Info/Index/; [19].
582
583 Identification of GPCR-like sequences from F. hepatica
584 Figure 1 summarises our GPCR discovery methodology, which employed Hidden
585 Markov Models (HMMs) constructed from protein multiple sequence alignments
586 (MSAs) of previously described S. mansoni and S. mediterranea GPCR sequences
587 [21]. Individual HMMs were constructed for each GRAFS family [25]. Alignments
588 were generated in Mega v7 (www.megasoftware.net) [84] using the Muscle algorithm
589 with default parameters. HMMER v3 (http://.hmmer.org) was employed to construct
590 family-specific HMMs (hmmbuild) from alignments and these were searched
591 (hmmsearch) against a predicted protein dataset from F. hepatica genome
592 PRJEB6687 consisting of 33,454 sequences [18]. Returned sequences were filtered
593 for duplicates and ordered relative to the hmmsearch scoring system, enabling the
594 classification of hits according to the GRAFS family to which they showed most
595 similarity (i.e. highest score, lowest E value). All remaining returns were then used
596 as BLAST queries (BLASTp and tBLASTn) to identify matching, or additional,
597 sequences originating from the PRJEB6687 and PRJNA179522 genomes (Figure 1).
598 Where sequences appeared in both genomes, we kept the longest annotated
599 sequence (S1 Table).
600
601 GPCR annotation
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602 Sequences resulting from HMM searches were filtered by transmembrane (TM)
603 domain composition, using hmmtop (http://www.sacs.ucsf.edu/cgi-bin/hmmtop.py)
604 [85,86]. Sequences containing ≥4 TMs were analysed as described below.
605
606 Homology analyses
607 All GPCRs were used as BLASTp [87] queries, to identify their closest (highest
608 scoring) match in the ncbi non-redundant (nr) protein sequence dataset
609 (https://blast.ncbi.nlm.nih.gov/Blast.cgi), with default settings and the “Organism” field
610 set to exclude Platyhelminthes (taxid: 6157). All GPCRs were additionally searched
611 against more phylogenetically limited datasets, by using the “Organism” field to limit
612 the BLASTp searches to: (i) Basal phyla, Ctenophora (taxid:10197), Porifera
613 (taxid:6040), Placozoa (taxid:10226), Cnidaria (taxid:6073); (ii) Superphylum
614 Lophotrochozoa (taxid: 1206795), excluding phylum Platyhelminthes (taxid: 6157);
615 (iii) Superphylum Ecdysozoa (taxid: 1206794); (iv) Superphylum Deuterostomia
616 (taxid: 33511). For BLASTp searches against other flatworms, we performed local
617 BLAST+ [88] on the WBPS9 release of WormBase Parasite, which included
618 predicted protein datasets from 30 flatworm species. In all cases, we recorded the
619 single highest scoring hit, or recorded “no significant similarity found” in cases where
620 no hits were returned (Table S1); sequences generating both GPCR hits and “no
621 hits” were retained. Where the top hit was not to a GPCR, that sequence was
622 removed from the dataset.
623
624 Domain composition
625 GPCR identities were confirmed using InterProScan Sequence Search
626 (www.ebi.ac.uk/interpro/search/sequence-search) [89] and/or HMMER HMMScan
627 (www.ebi.ac.uk/Tools/hmmer/search/hmmscan) [90]. Again, sequences returning
628 non-GPCR domains were omitted from the dataset, with all others retained.
629
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630 Motif identification
631 As an additional measure of confidence in our identifications, we analysed the
632 presence/absence of key motifs diagnostic of receptor families and subfamilies.
633 These analyses were performed for rhodopsins generally, the ligand binding domains
634 (LBDs) of rhodopsin receptors for acetylcholine (ACh), neuropeptide F/Y (NPF/Y),
635 octopamine and serotonin (5-hydroxytryptamine, 5HT), and for the LBDs of
636 glutamate and frizzled/smoothened families. Motifs were identified via protein
637 multiple sequence alignment (MSA) of GPCRs, performed in MAFFT
638 (www.mafft.cbrc.jp/alignment/server) [91]. Only identical amino acids were accepted
639 at each site, with conservation expressed as % identity across all sites. Motif
640 illustrations (Figures 3 & 7) were generated using WebLogo 3
641 (http://weblogo.threeplusone.com) [92].
642
643 Phylogenetic reconstruction
644 Maximum likelihood (ML) phylogenetic trees were constructed using PhyML
645 (http://www.phylogeny.fr) [93], from protein MSA generated in MAFFT
646 (www.mafft.cbrc.jp/alignment/server/) [91]. Alignments were manually edited (in
647 Mega v7) to include only TM domains, by removing extramembrane blocks aligned
648 with human glutamate, rhodopsin, adhesion or frizzled proteins. Trees were
649 constructed from these TM-focused alignments in PhyML using default parameters,
650 with branch support assessment using the approximate likelihood ratio test (aLRT),
651 under “SH-like” parameters. Trees, exported from PhyML in newick format were
652 drawn and annotated in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).
653
654 RNA-Seq analyses
655 Expression of F. hepatica GPCRs was investigated in publically available and in-
656 house generated RNA-Seq datasets. These included developmentally staged
657 Illumina transcriptome reads associated with one of the F. hepatica genome projects
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658 [18] (reads accessed from the European Nucleotide Archive at
659 http://www.ebi.ac.uk/ena/data/search?query=PRJEB6904). These samples
660 originated from distinct developmental stages of US Pacific Northwest Wild Strain F.
661 hepatica (Baldwin Aquatics), including egg (n=2), metacercariae (met; n=4), in vitro
662 NEJs 1h post-excystment (NEJ1h; n=1), in vitro NEJs 3h post-excystment (NEJ3h;
663 n=2), in vitro NEJs 24h post-excystment (NEJ24h; n=2), ex-vivo liver-stage juveniles
664 (juv1; n=1) and ex-vivo adult parasites (Ad; n=3). Our in-house datasets were
665 generated from ex vivo liver stage F. hepatica juveniles (Italian strain, Ridgeway
666 Research Ltd, UK), recovered from rat (Sprague Dawley) hosts at 21 days following
667 oral administration of metacercariae (juv2; n=3). Total RNA, extracted with Trizol
668 (ThermoFisher Scientific) from each of the 3 independent biological replicates, was
669 quantified and quality checked on an Agilent Bioanalyzer, converted into paired-end
670 sequencing libraries and sequenced on an Illumina HiSeq2000 by the Centre for
671 Genomic Research at the University of Liverpool, UK. RNA samples were spiked
672 prior to library construction with the ERCC RNA Spike-In Mix (ThermoFisher
673 Scientific) [94]. All read samples were analysed using the TopHat and Cufflinks
674 pipeline [95-100], with mapping against PRJEB6687 genome sequence and
675 annotation files (accessed from WormBase Parasite;
676 http://parasite.wormbase.org/ftp.html). Data were expressed as number of fragments
677 mapped per million mapped reads per kilobase of exon model (FPKM). In juv2
678 datasets we discarded GPCRs represented by fewer than 0.5 FPKM (the minimum
679 linear sensitivity that we detected with our ERCC spike in); for the staged datasets,
680 we included only receptors represented by ≥0.5 FPKM in at least one life stage.
681 Heatmaps were generated with heatmapper (http://www.heatmapper.ca/) [101] set
682 for Average Linkage, and Pearson Distance Measurement.
683
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1087 glutamate, rhodopsin, adhesion, frizzled/smoothened and secretin families, and two
1088 rhodopsin subfamilies, were built from protein multiple sequence alignments of
1089 Schistosoma mansoni and Schmidtea mediterranea GPCRs [21]. HMMs were built
1090 and searched respectively using the hmmbuild and hmmsearch modules of HMMER
1091 v3.0. Searches were performed against two publically available F. hepatica
1092 genomes using hmmsearch and basic local alignment search tool (BLAST) tools.
1093 Each putative FhGPCR sequence was assessed for transmembrane (TM) domain
1094 composition with hmmtop before classification using tools including BLASTp,
1095 Interproscan and CLANS. (B) The largest proportion (49%) of FhGPCRs carried the
1096 full complement of 7 TMs, with 88% of sequences bearing at least 4 TMs. (C)
1097 GRAFS composition of 146 FhGPCRs carrying ≥4 TMs. (D) Rhodopsins were
1098 subject to further classification, including BLASTp vs datasets representing major
1099 non-flatworm animal phyla and superphyla. These rhodopsin homology
1100 classifications fed back into phylogenetic analyses versus deorphanised bilaterian
1101 GPCRs to confirm their putative ligand selectivity, with a final analysis of ligand
1102 binding domain composition comparing conservation of ligand interacting residues
1103 for characterised GPCRs reported in the literature with our F. hepatica assignments.
1104
1105 Fig 2: Phylogenetic classification of Fasciola hepatica rhodopsin G protein-
1106 coupled receptors. (A) Maximum-likelihood cladogram of F. hepatica rhodopsins.
1107 Phylogeny delineated clades containing rhodopsins with distinct homologies (RA,
1108 amine; RP, peptide; RO, opsin: R, orphan rhodopsin). The orphan clades contained
1109 sequences with generally low BLASTp similarity to their closest non-flatworm
1110 BLASTp hit, but concentrated within them were 18 sequences with exceptionally low
1111 (E>0.01) BLASTp similarity to non-flatworm sequences (fwRhods). The tree was
43 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1112 midpoint rooted and was generated from a multiple protein sequence alignment
1113 trimmed to TM domains I-VII. Numbers at nodes indicate statistical support from
1114 approximate likelihood ratio test (aLRT). Tip colours are coded according to the E-
1115 value scale (as indicated) of that GPCR’s closest BLASTp match in the ncbi nr
1116 database, excluding phylum Platyhelminthes. (B) Summary of sequence similarity
1117 comparisons between GPCRs within each rhodopsin clade, and their closest
1118 BLASTp hits in major phylogenetic groups (Basal: Cnidaria, Ctenophora, Porifera,
1119 Placozoa; superphylum Lophotrochozoa, omitting Platyhelminthes; Superphylum
1120 Ecdysozoa; superphylum Deuterostomia; phylum Platyhelminthes). BLASTp E-value
1121 (median) is summarised in each case, colour coded as a heat map on the same
1122 colour scale as (A). The number of GPCRs comprising each F. hepatica clade (n) is
1123 also indicated. (C) Sequence diversity within ubiquitous rhodopsin motifs of the
1124 majority (117) of the F. hepatica rhodopsins (upper panel), compared to those motifs
1125 in 18 F. hepatica fwRhods (lower panel). The mammalian consensus motifs are
1126 illustrated above the top panel, along with an illustration of the location of each motif
1127 within the rhodopsin 7TM domain structure. Some variability is visible within the TM2
1128 and TM6 motifs, but TM3 and TM7 motifs are well conserved.
1129
1130 Fig 3: Identification of flatworm-specific rhodopsins (fwRhods) in genomes
1131 from phylum Platyhelminthes. (A) The 18 Fasciola hepatica GPCRs in our dataset
1132 that had poor BLASTp similarity (E>0.01) to non-flatworm sequences in the ncbi nr
1133 dataset (lsGPCRs), were used as queries in BLASTp searches of flatworm genomes
1134 in WormBase Parasite (release WBPS9). All hits scoring E<0.01 were back-
1135 searched by BLASTp against our F. hepatica GPCR dataset. Sequences scoring
1136 E<0.01 against one of the original F. hepatica GPCRs were retained as matches.
1137 These sequences were then filtered to identify those lacking matches in ncbi nr,
1138 lacking non-GPCR protein domains, possessing at least 4 transmembrane (TM)
1139 domains, and containing rhodopsin motifs consistent with those seen in the majority
44 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1140 of F. hepatica rhodopsins (see C). (B) This process identified 76 fwRhods in phylum
1141 Platyhelminthes, the majority (70) of which were from class Trematoda. Small
1142 numbers were returned from classes Cestoda and Monogenea. Note that no
1143 fwRhods fitting these criteria were identified in class Turbellaria. (C) Sequence
1144 diversity within ubiquitous rhodopsin motifs of 18 F. hepatica fwRhods (upper panel),
1145 compared to those motifs in the 58 fwRhods identified in the wider phylum (lower
1146 panel); motifs are broadly similar between F. hepatica and the rest of the phylum.
1147 (D) Maximum likelihood phylogeny of 76 fwRhods, alongside flatworm-specific
1148 rhodopsins described previously (70 platyhelminth rhodopsin orphan family 1
1149 (PROF1) [21,22], and 245 S. mediterranea G protein coupled receptor [GCRs,
1150 comprising RhoL, RhoR, Srfa, Srfb and Srfc families, reported as lacking non-
1151 flatworm homologues [24]) with branches coloured to indicate Family (dark blue,
1152 PROF1; mid blue, Srfa; cyan, Rho-L; green, Rho-R; orange, Srfb; purple, Srfc; red,
1153 fwRhod). Tree was rooted to a human rhodopsin (P08100) and was generated from
1154 an alignment trimmed to transmembrane domains I-VII. Numbers at nodes indicate
1155 statistical support from approximate likelihood ratio test (aLRT).
1156
1157 Fig 4: Conservation of ligand-interacting residues between 17 Fasciola
1158 hepatica G protein-coupled receptors (GPCRs) and structurally characterized
1159 homologues from other species. (A) Neuropeptide F/Y receptor ligand binding
1160 residues as characterised by mutagenesis in human neuropeptide Y receptor NPY1R
1161 [39-43], and conserved in Anopheles gambiae (Ag) and Drosophila melanogaster
1162 (Dm) neuropeptide F receptors (NPFR) [44]. Numbering relative to HsNPY1R. (B)
1163 Serotonin (5-hydroxytryptamine; 5HT) receptor ligand binding residues as
1164 characterised by mutagenesis in human 5HT receptor (Hs5HT1A) [102], and
1165 conserved in Schistosoma mansoni 5HTR [51]. Numbering relative to Hs5HT1A.
1166 (C) Octopamine receptor (OaR) ligand binding residues as characterised by
1167 homology modelling of the Periplaneta americana (Pa) [58], and mutational analysis
45 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1168 of the Bombyx mori (Bm) [59] octopamine receptor ligand binding domain.
1169 Numbering relative to PaOAR, except for Y412 which is shown relative to BmOAR.
1170 (D) Acetylcholine receptor ligand binding residues as characterised by homology
1171 modelling of the S. mansoni G protein-coupled acetylcholine receptor (SmGAR) [62];
1172 numbering relative to SmGAR. In each case, only F. hepatica sequences displaying
1173 at least 75% identity across the stated ligand binding residues are shown. Relative
1174 positions of residues across seven transmembrane domains (TM1-7) are shown. TM
1175 diagrams are not to scale.
1176
1177 Fig 5: Fasciola hepatica glutamate G-protein coupled receptors (GPCRs)
1178 display divergent phylogeny and ligand binding domain (LBD) composition.
1179 (A) Maximum likelihood phylogeny containing three F. hepatica glutamate receptors,
1180 alongside representative receptors from the various recognised GPCR Class C
1181 subgroups (subclasses indicated by blue boxes: Ca2+, Ca2+-sensing receptor;
1182 GABAB, γ-aminobutyric acid type B receptors; mGluR, metabotropic glutamate
1183 receptors; Orphan, receptors with no known ligand; Taste, vertebrate taste
1184 receptors). Two previously reported Schistosoma mansoni glutamate receptors are
1185 also included; F. hepatica sequences are coloured red, S. mansoni are coloured
1186 blue, all others are black. Node numbers indicate statistical support as determined
1187 by approximate likelihood ratio test (aLRT). Tree was midpoint rooted. (B)
1188 Conservation of ligand-interacting residues between vertebrate GABAB and
1189 metabotropic glutamate receptors (mGluR), and F. hepatica class C GPCRs.
1190 Agonist-interacting residues were identified by multiple protein sequence alignment
1191 of F. hepatica glutamate receptors against mutationally-identified ligand interacting
1192 residues (those causing a significant reduction in receptor signalling activity), from
1193 mouse GABAB receptor (top panel), or selected human mGluR subtypes (lower
1194 panel). Identical amino acids in F. hepatica/S. mansoni GPCRs are represented by
1195 white text on black background, functionally conserved amino acids by black text on
46 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1196 grey background. In lower panel, mutations causing a significant reduction in mGluR
1197 receptor activity are bold and numbered, with the region of the glutamate molecule
1198 bound by each residue indicated (COOH, C-terminus; NH2, N-terminus). For
1199 references see [63,103,104].
1200
1201 Fig 6: Frizzled/smoothened seven transmembrane receptors and wnt ligands in
1202 Fasciola hepatica. (A) Maximum likelihood phylogeny containing six F. hepatica
1203 frizzled/smoothened receptors, alongside those from Schistosoma mansoni,
1204 Drosophila melanogaster, Caenorhabditis elegans and Homo sapiens (identified by
1205 FSMP, d, c and h, respectively). F. hepatica sequences are coloured red, S.
1206 mansoni are coloured blue, all other species are coloured black. Radial labels
1207 indicate human frizzled clusters (hClust) I-IV, and the smoothened clade. Node
1208 numbers indicate statistical support as determined by approximate likelihood ratio
1209 test (aLRT). Tree was rooted against a Dictyostelium frizzled sequence (dicty-fslJ-1).
1210 Tree composition adapted from [21]. (B) WebLogo comparison of ligand interacting
1211 residues between mouse fz1-10 (top panel) and F. hepatica frizzled receptors.
1212 Numbering in top panel x-axis is relative to mouse fz8 [69]. (C) Three wnt-like
1213 sequences exist in F. hepatica. Shading indicates positions of 22 characteristic Cys
1214 residues, positions numbered relative to D. melanogaster wnt-1 (Dro-wnt-1).
1215
1216 Fig 7: Expression profiling of 101 G protein-coupled receptors (GPCRs) in
1217 Fasciola hepatica life stages. (A) Expression heatmap generated from log2 FPKM
1218 values of 83 GPCRs identified from developmentally staged RNA-seq libraries. Life
1219 stages are represented in columns (Egg; Met, metacercariae; NEJ_1h, newly-
1220 excysted juvenile collected 1h post excystment; NEJ_3h, NEJ collected 3h post-
1221 excystment; NEJ_24h, NEJ collected 24h post-excystment; Juv_21d, liver stage
1222 juvenile parasites collected from murine livers 21 days following oral administration of
1223 metacercariae; Adult, adult parasites collected from the bile ducts of bovine livers).
47 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1224 Rows indicate individual GPCRs, as denoted by the ID and phylogeny columns. The
1225 latter indicates receptor classification and predicted ligand where available (see S1
1226 Table). Expression cluster column indicates clusters of GPCRs with highest
1227 expression focused in particular life stages. (B) Detection of 76 GPCRs in Illumina
1228 RNA-Seq libraries generated from F. hepatica 21 day liver-stage juveniles, recovered
1229 ex vivo from rat infections. Data show expression of three glutamate (G), one
1230 adhesion (A), four frizzled (F), one smoothened (S) and 67 rhodopsin (R) GPCRs.
1231 The rhodopsins include representatives of amine (RA1, RA3), opsin (RO), peptide
1232 (RP1-7), and orphan (R2,3,4,6). Data points (each at n=3) represent mean log2
1233 FPKM ± 95% confidence intervals, as calculated by cuffdiff. In both panels, flatworm
1234 rhodopsins (fwRhods) are marked in red text. ACh, acetylcholine; AstA. Allatostatin
1235 A; Dop, dopamine; FMRFa, FMRFamide; GHS, growth hormone secretagogue;
1236 GYIRFa, GYIRFamide; Myom, myomodulin; Myos, myosuppressin; NPF/Y,
1237 neuropeptide F/Y; Oct, octopamine; Pkt, prokineticin; Tyr, tyramine; 5HT, 5-
1238 hydroxytryptamine.
1239
48 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1240 Supporting Information Captions
1241 S1 Table. Fasciola hepatica G protein coupled receptor (GPCR) dataset
1242 summary. Table describes sequences containing 4-9 transmembrane (TM)
1243 domains. Each sequence is defined in terms of GRAFS family, and annotated for
1244 TM composition, sequence length, phylogeny, domain composition and homology
1245 relative to various datasets.
1246
1247 S2 Text. Fasciola hepatica G protein-coupled receptor (GPCR) protein
1248 sequence dataset.
1249
1250 S3 Table. Flatworm-specific rhodopsins (fwRhods) in Fasciola hepatica and
1251 other flatworms. Species and genome IDs of sequences that have ≥4
1252 transmembrane (TM) domains, and lack high-scoring orthologues in non-flatworms
1253 (BLASTp score E≥0.01 vs ncbi nr excluding Platyhelminthes), and show
1254 conservation of at least two of the four ubiquitous rhodopsin motifs. Each sequence
1255 is annotated for protein domains where present (Pfam HMMScan). Accessions refer
1256 to WormBase ParaSite.
1257
1258 S4 Table. Rhodopsin ubiquitous motifs and ligand binding domains for ACh,
1259 NPF/Y 5-HT, octopamine. Note data are in individual tabs. Rhodopsin: Sequence
1260 motifs extracted from alignment of F. hepatica rhodopsins, corresponding to
1261 ubiquitous rhodopsin motifs of TMs 2, 3, 6 and 7; Acetylcholine, NPF, 5-HT,
1262 Octopamine: Amino acids extracted from alignment of F. hepatica rhodopsins with
1263 mutationally or structurally-characterised GPCRs (comparators). Summary: Percent
1264 identity of ACh, NPF, 5-HT and Oct receptor LBDs, indicating most conserved LBD
1265 sequences showing at least 75% identity.
1266
49 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1267 S5 Fig. Phylogenetic comparison of Fasciola hepatica GPCRs with
1268 deorphanised bilaterian GPCRs. (A) Peptide receptors (F. hepatica black or
1269 magenta as described in Fig x); (B) Amine receptors (F. hepatica dark blue); In all
1270 cases, non-flatworm receptors are coloured light blue. In (A) outer labels indicate
1271 positions of receptors for neuropeptide families previously reported in flatworms
1272 (McVeigh et al., 2009; Collins et al., 2010; Koziol et al., 2016); in (B), outer labels
1273 represent major groups containing phylogenetically similar F. hepatica sequences.
1274 Trees were midpoint rooted, maximum likelihood phylogenies of transmembrane
1275 domains I-VII. Numbers at nodes indicate statistical support from approximate
1276 likelihood ratio test (aLRT). Scale bars at the centre of each tree indicate number of
1277 substitutions per site. Abbreviations: ACh, acetylcholine; CCAP, crustacean
1278 cardioactive peptide; Dop, dopamine; FLP, FMRFamide-like peptide; GrH,
1279 gonadotropin-releasing hormone; Luq, luqin; Mmd, myomodulin; NKY, neuropeptide
1280 KY; NPF/Y, neuropeptide F/Y; Oct, octopamine; PK, pyrokinin; SIFa, SIFamide; Tyr,
1281 tyramine; SmGPR, schistosome GPCRs; 5HT, 5-hydroxytryptamine.
1282
1283 S6 Table. Frizzled receptor ligand binding domain motifs. Amino acids
1284 extracted from alignment of F. hepatica frizzled and smoothened GPCRs with
1285 mutationally characterised mouse fz8, also showing positionally-conserved residues
1286 in mouse fz1-10 (top panel) and F. hepatica frizzled receptors. Numbering in top row
1287 relative to mouse fz8 (Janda et al., 2012). Green boxes indicate identical residues in
1288 F. hepatica vs mammalian Fzd.
1289
1290 S7 Figure: Adhesion receptor phylogeny. Maximum likelihood phylogeny of
1291 Fasciola hepatica adhesion/secretin-like GPCRs alongside class B GPCRs from
1292 human and Schistosoma mansoni. Tree was a midpoint rooted, maximum likelihood
1293 phylogeny of transmembrane domains I-VII. Numbers at nodes indicate statistical
50 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1294 support from approximate likelihood ratio test (aLRT). Scale bars at the centre of
1295 each tree indicate number of substitutions per site.
1296
51 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which Schistosomawas not certified mansoni by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A availableHMMER under a CC-BY-NC-ND3.0 4.0 International license. Schmidtea mediterrannea hmmbuild > Glutamate > Rhodopsin F. hepatica genome > Adhesion PRJEB6687 > Frizzled/Smoothened HMMER 3.0 Proteins FhGPCRs v1 > Secretin hmmsearch Genome scaffolds tBLASTn
≤3TM FhGPCRs v2 F. hepatica genome hmmtop 20 seqs 166 seqs PRJNA179522 Proteins BLASTp
≥4TM Genome scaffolds tBLASTn 146 seqs
Classification Classification - BLASTp vs - Interproscan ncbi nr - BLASTp vs ncbi nr -Interproscan
9 B C 4-9TM dataset summary 8 146 GPCRs 7 Glutamate = 3 6 Adhesion = 2 5 Frizzled = 5 4 Smoothened = 1 Rhodopsin = 135
#TM domains #TM 3 2 1 01020304050 % sequences D > FhRhodopsins BLASTp vs ncbi nr: - Non-flatworms Basal Phyla (Cnidaria, Ctenophora, Placozoa, Porifera) Rhodopsin subclassification (Figure 2) - Lophotrochozoa RP:E<0.01 non-FW peptide GPCRs - Ecdysozoa RA:E<0.01 non-FW amine GPCRs - Deuterostomia RO:E<0.01 non-FWopsin GPCRs - Phylum Platyhelminthes R: E≥0.01 nonFW sequences > Maximum Likelihood Phylogeny - vs Deorphanised Bilaterian Rhodopsins
>Ubiquitous rhodopsin motif conservation - TMs 2,3,6,7
> Ligand binding domain conservation - vs structurally characterised GPCRs bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Peptide RP1 A Opsin B RO Orphan Amine Rhodopsin 0.83 R1 BasalLophotrochozoaEcdysozoaDeuterostomiaPlatyhelminthes RA3 RA1 n=21 Amine RA2 n=15 RA2 Orphan RA3 n=2 0.96 Rhodopsin RO n=3 1.00 0.81 R2 RP1 n=18 0.80 RP2 n=10 0.92 0.84 0.92 Amine RP3 n=2 0.50 0.87 0.76 0.59 RA1 Orphan RP4 n=2 0.81 0.89 Rhodopsin RP5 n=5
0.82 R3 RP6 n=8 0.49 RP7 n=2 0.75 R1 n=14 1.00 3.0 R2 n=8 1.00 Orphan R3 n=6 Rhodopsin R4 n=13 R6 R5 n=3 0.70 R6 n=3 Orphan 1.00 Rhodopsin
R5 0.30 0.64 Peptide Orphan BLASTp vs ncbi nr RP7 Rhodopsin (excluding Platyhelminthes) 3.0 R4 >0.01 0.77 Peptide 0.84 0.01-0.001 -5 RP6 0.001-1E -5 -10 Peptide 0.76 1E - 1E 0.01 -10 -20 RP5 Peptide 1E - 1E -20 RP4 Peptide Peptide <1E RP3 RP2 C TM1 TM2 TM3 TM4 TM5 TM6 TM7 Ubiquitous rhodopsin motifs LAxxD (D/E)RY FxxxWxP NPxxY 1.0 A1.0 1.0 I 1.0 F F. hepatica VS Y ICWL I A D L LLSTA V rhodopsins 0.5 V0.5 F0.5 RF0.5 LSIC E A V LQ P L VTET CLT F V P I Y Y N C V D L L probability probability probability probability Y F FAFH E n=117 Q A FD V Q S M A S FN F F I YHAY F Y E F L N W H I G GL G F N A F LS CA L N R WI M CI H DEADGSC MATI I CEA IS N LPFM M GGSQHV T P YSD C V A S LH T K GTGGP R G H Q HTMMQVT KAG NCVKVMCQ IQQ VARFP KGPPT LWTPS R TSCRIWG VKTYDYYYTWYSEVYTRYWRV 0.0 0.0 0.0 0.0 1.0 1.05 1.0 5 1.0 5 WebLogoI 3.5.0 WebLogo 3.5.0 AWebLogoV 3.5.0 WebLogo 3.5.0 F. hepatica YVW I MFP F L Y II AL 0.5 A0.5 0.5 F C 0.5A ILY fwRhods C C F MTI E Q D F EN V L L IF V FPCVM T G VV L MT G N n=18 probability I probability probability A L S probability TI LA S Q C G EL LMIQ H LATYEDEL LFP IA YTPFS T F E MFPVDNFTM QSCVTFDWTGT 0.0 YVWY0.0STRY0.0SLWYQYY0.0QGYWTW 5 5 5 WebLogo 3.5.0 WebLogo 3.5.0 WebLogo 3.5.0 WebLogo 3.5.0 bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. F. hepatica BLASTp ncbi nr E>0.01 A lineage-expanded BLASTp excl Platyhelminthes GPCRs (18) HMMScan No domains WormBase Parasite domain search 76 fwRhods F. hepatica WBPS9 Phylum Reciprocal GPCRs Platyhelminthes >3TM Platyhelminthes BLASTp HMMTop EmuJ_000996550.1_PROF1
SMDC22227.1_PROF1
≥4 TMs SMDC2150.1_PROF1
SMDC2514.1_PROF1
SMDGB6_PROF1
SMDC2955.1_PROF1 SMDGB1_PROF1 SMDGB3_PROF1 SMDC907.1A_PROF1 SMDGB2_PROF1 SMP041880_PROF1 SMDC907.1B_PROF1
GCR262 GCR315 GCR122 GCR157 GCR243 GCR330 Matches GCR324 GCR392 GCR244
GCR307 0.623 GCR577 0.999 0.997
SMDC2411.1_PROF1 GCR302 SMDGB4_PROF1 SMP134960_PROF1 EmuJ_000954500.1_PROF1 GCR419 schistosoma_mansoni_prjea36577_Smp_023710.1 0.024 schistosoma_rodhaini_prjeb526_SROB_0001125301-mRNA-1 schistosoma_margrebowiei_prjeb522_SMRZ_0000602501-mRNA-1 schistosoma_curassoni_prjeb519_SCUD_0000835601-mRNA-1 GCR301 schistosoma_mansoni_prjea36577_Smp_117340.1 0.975 Rhodopsinschistosoma_margrebowiei_prjeb522_SMRZ_0001802201-mRNA-1 motifs GCR254 0.999 Fhep_D915_03083
1 Fhep_BN1106_s6156B000040.mRNA-1 1 GCR572 1 schistosoma_margrebowiei_prjeb522_SMRZ_0000541701-mRNA-1 1 schistocephalus_solidus_prjeb527_SSLN_0000511401-mRNA-1 GCR104 schistocephalus_solidus_prjeb527_SSLN_0001437101-mRNA-1
1 echinostoma_caproni_prjeb1207_ECPE_0000405901-mRNA-1 GCR395 0.896 0.472 schistosoma_haematobium_prjna78265_A_05638 0.998 0.783 GCR574 GCR472 0.873 SMDC6858.4_PROF1 0.499 1 SMDC8572.2_PROF1 GCR312 1 SMDC7587.4_PROF1 GCR437 0.995 SMDGB7_PROF1 GCR410 0.984 0.911 GCR450 1 SMDC14497.4_PROF1 GCR387 1 GCR506 0.898 0.48 0.992 SMDC15273.1_PROF1 schistosoma_haematobium_prjna78265_A_07947 GCR342 0.509 SMDC6858.1_PROF1 GCR196 0.913 0.098 0.806 GCR539 GCR195 1 1 SMDC15168.2_PROF1 0.833 1 GCR519 GCR346 0.994 0.561 SMDC21116.2_PROF1 GCR079 0.877 0.839 0.956 0.731 SMDC12781.1_PROF1 GCR047 0.757 SMDC18000.2_PROF1 1 GCR526 GCR308 0.893 0.997 GCR048 0.993 0.201 SMDGB5_PROF1 0.084 0.999 1 SMDC14736.1_PROF1 GCR436 0.048 GCR501 GCR359 0.905 1 SMDC19969.1_PROF1 0.939 0.955 GCR314 0.582 SMDC4350.2_PROF1 GCR445 0.916 0.187 0 GCR117 0.82 0.902 SMDC423.2_PROF1 GCR355 GCR100 GCR388 0.339 0.871 0.814 0.757 0.999 SMDC5598.1A_PROF1 Platyhelminthes (76) GCR240 0.999 0.653 0.894 0.769 SMDC1825.1_PROF1 0.726 Fhep_D915_03393GCR241 0.841 0.683 SMDC1749.3A_PROF1 GCR460 0.958 0.973 GCR468 GCR191 SMDC1749.3B_PROF1 0.921 0.221 GCR149 GCR521 0.997 SMDC5598.1B_PROF1 GCR179 0.84 0.4920.444 1 SMDC4570.1_PROF1 0.875 0.95 0.998 SMPSC63_PROF1 0.403 SMPSC331A_PROF1 GCR458 0.99 0.699 SMPSC331B_PROF1 B 0.998 1 0.812 SMP083940_PROF1 GCR379 C Monogenea0.771 (2) SMPSC1003_PROF1 GCR583 0.749 0.72 1 SMPSC433_PROF1 GCR200 0.784 schistosoma_japonicum_prjea34885_Sjp_0056590 GCR181 0.996 0.385 1 0.87 SMP084290_PROF1 GCR257 0.906 0.99 schistosoma_curassoni_prjeb519_SCUD_0001875201-mRNA-1 GCR1030.784 0.879 schistosoma_margrebowiei_prjeb522_SMRZ_0001830401-mRNA-1 P.0.86 xenopodis (2) TM1 TM2 TM3 schistosoma_haematobium_prjna78265_A_02855TM4 TM5 TM6 TM7 GCR235 0.82 0.816 Trematoda GCR5710.586 0.79trichobilharzia_regenti_prjeb4662_TRE_0001051301-mRNA-1 GCR373 0.963 0.909 SMP084270_PROF1 0.997 0.9690.755 SMP084280_PROF1 GCR405 1 0.501 schistosoma_margrebowiei_prjeb522_SMRZ_0001830201-mRNA-1 GCR5680.97 schistosoma_mattheei_prjeb523_SMTD_0000474701-mRNA-1 Monogenea GCR321 0.811 0.977 1 0 schistosoma_haematobium_prjna78265_A_04157 GCR370 0.3560.994 SMP167870_PROF1 GCR3050.541 0.9990.9930.839 SMPSC12A_PROF1 GCR3930.9710.639 0.571 clonorchis_sinensis_prjda72781_csin102640 Cestoda GCR389 0.957Fhep_D915_13002 GCR042 0.849 0.814 0.924 0.816 SMP091950_PROF1 GCR055 0.822 EmuJ_000633100.1_PROF1 0.963 0.467 GCR171 GCR512 1 0.979 SMDC1889.2_PROF1 GCR524 0.77 0.761 0.872 0.621 0.998SMDC1026.2_PROF1 GCR499 0.17 GCR550 GCR560 0.973 0.985SMDC8510.3B_PROF1 GCR520 0.722 SMDC15385.3_PROF1 GCR5510.9770.79 0.997 GCR517 GCR118 0.657 0.966 SMDC27521.1_PROF1 0.663Cestoda (4) 0 GCR466 0.707 0.891 0.675 SMDC14380.2_PROF1 GCR564 0 0.593 0 1.0 0.0331.0 0 1.0 SMDC8510.3A_PROF1 1.0 GCR269 0.871 0.999 0.993 SMDC12554.3_PROF1 GCR5330.874 0.62 0.995GCR473 GCR523 0.244 0.987D.latum (1) 0 SMDC8510.2_PROF1 GCR510 I 0.408 SMDC27911.2_PROF1 0.938 GCR532 0.771 1 GCR208 AV 0.751 0.811SMDC1917.2_PROF1 GCR2390.905 0.756 GCR198 P GCR496 0.918 0.7371 F S.solidus0.989 (3) F. hepatica W ISMDC6472.1_PROF1 Trematoda (70) GCR465 YV 0.65 GCR483 0.986 MF A GCR475 Y 1ISMDC504.1_PROF1I GCR382 L HsRhodopsin L A C I GCR217 F GCR176 A Y 0.993 0.5 0.5 0.5 0.5 L GCR4540.742 0.9 fwRhods C C F0 GCR290 T E 0.907 I GCR371 0.952 0.916GCR218 M Q D F GCR335 0.963 GCR059 EN E. caproni (19) V L GCR067V L GCR189 IF 0.996 C M GCR481 FP V F. hepatica (18) T G V0.989V L MT G N probability I probability probability A probability I EmuJ_001085520.1_PROF1 0.727 GCR530L S T GCR397 0.956 n=18 S Q C0.4060.891GCR253G E LM Q GCR3900.9030.734 LAL FGCR093 L IS GCR565 H ATYEDEL 0 LGCR543P IA YTPF 0.022 T F E GCR411 0.682 0.886 M PVNFTM GCR434QSCVTFDWTGT GCR036 0.978 F D0.74 GCR078 0.175 0.919 YVWYSTYS WYQYYQGYWTW GCR0560.031 0.997 0.765 L0.972 GCR242 0.0 0.0 R0.0 GCR401 0.0 GCR110 0.741 0.989 Trematoda GCR075 0.915 0.993 GCR252 GCR3180.963 0.89 Phylum 1.0 1.00.1855 1.0 0.986 GCR457 5 1.0 5 GCR325 0.999 0.42 0.581GCR398 0.77 WebLogo 3.5.0 WebLogo 3.5.0 GCR237 WebLogo 3.5.0 WebLogo 3.5.0 S. japonicum (1)0.79 1 0.995 protopolystoma_xenopodis_prjeb1201_PXEA_0002666801-mRNA-1C F Fhep_D915_13937 A 0.417 I 0 trichobilharzia_regenti_prjeb4662_TRE_0000301801-mRNA-1 O. viverrini (1) 0.9050.835 0.904 Platyhelminthes 0.745schistosoma_rodhaini_prjeb526_SROB_0001137201-mRNA-1L GCR156 0.79 0.937Fschistosoma_mansoni_prjea36577_Smp_137980.1 0 Fhep_BN1106_s1984B000285.mRNA-1 0.994 0.857 I GCR515 G 0.965 schistosoma_curassoni_prjeb519_SCUD_0001747201-mRNA-1I 0.879 Y W 0 S. mattheei (2)GCR300 YV F schistosoma_haematobium_prjna78265_A_03895I GCR158 0.664 0.951 schistosoma_mattheei_prjeb523_SMTD_0000955601-mRNA-1 E GCR215 0.92 schistosoma_margrebowiei_prjeb522_SMRZ_0000949101-mRNA-1I ML 0.696 L 0.943 Q 0.928 F GCR219 GCR343 T fwRhods A 0.87 T S.margrebowiei (6) 0.5 0.5 0.5 GCR375 P0.5 0.99 P 0.606 T GCR566 0.952 0.891 0.871 V T. regenti (3) 0.774 L schistosoma_curassoni_prjeb519_SCUD_0001180801-mRNA-1 0.849 C C GCR222 T V GCR538 0.93 N trichobilharzia_regenti_prjeb4662_TRE_0000048201-mRNA-1schistosoma_mansoni_prjea36577_Smp_162980.1 0 GCR184Y GCR561 0.747 Q D 0.903 F GCR493 V GCR5140.1820.875 0.998 A N C 0.98 F 0.706GCR417A 0.996 V I G GCR489 0.861 0.912 GCR385 F S. rodhaini (3) 0.992 V L GCR5360.731 T n=58 0.614 GCR369 S E G 0.505 L probability probability probability 0.945I T probability E LC S. haematobium (5) GCR556 D0.734 YGCR255 MG L 0.43 0.932 DF GCR508 0.6010.847 S E 0.929GCR429 E MF WI 0.921 LA S Q T S. mansoni (4) GCR4760.636 0.025 0.901 GCR420C Q IL L N A VS T II H H A EFRGCR360 L Y S Y GCR152 Y A C YY I F M AA C C. sinensis (4) 0.312YGCR439 V MHL L AP 0.981 A T IG M GCR522 0.976 0.869 M PVNYH GCNP MA S. curassoni (4) 0.948 0.763 F LKGCR451 IGPQ DRSPFP GCR557 0.624Q1LLNPSVYSFTTGCQ DS GCR049 GQ YVWYS0.873 NTPWTYV VWSMV GCR142 0.528 0.913 0.992GCR344 GCR5590.974 0.969 0.989 0.0 0.0 GCR101 0.0 0.0 GCR545 0.907 0.698 GCR376 0.219 0.996GCR427 GCR164 0.55 0.793 0.773 GCR5050.995 0.844 5 GCR426 5 5 GCR463 0.978 0.8 1 GCR226 0.98 0.922 0.789 GCR363 GCR210 0.743 WebLogo0 3.5.0 opisthorchis_viverrini_prjna222628_T265_11077WebLogo 3.5.0 WebLogo 3.5.0 WebLogo 3.5.0 GCR582 0.09 0.026 clonorchis_sinensis_prjda72781_csin109678 GCR485 0.51 0 0.997 Fhep_BN1106_s2144B000269.mRNA-1 GCR207 0.985 GCR123 0.782 0.907 GCR130 GCR575 1 0.999 GCR127 GCR580 clonorchis_sinensis_prjda72781_csin110930 0.911 0.189 GCR570 0.804 echinostoma_caproni_prjeb1207_ECPE_0001431101-mRNA-1 GCR442 0.975 Fhep_scaffold3014_121504-122538 GCR081 0.747 schistocephalus_solidus_prjeb527_SSLN_0002053201-mRNA-1 0.598 protopolystoma_xenopodis_prjeb1201_PXEA_0002576201-mRNA-1 GCR500 0.158 GCR193
GCR089 0.571 echinostoma_caproni_prjeb1207_ECPE_0000038901-mRNA-1 GCR563 0.954 echinostoma_caproni_prjeb1207_ECPE_0000941201-mRNA-1 0.922 0.254 0.814 0.796 echinostoma_caproni_prjeb1207_ECPE_0000977101-mRNA-1 GCR452 0.952 0.691 0.73 echinostoma_caproni_prjeb1207_ECPE_0000508001-mRNA-1 GCR259 0.795 0.246 echinostoma_caproni_prjeb1207_ECPE_0000818801-mRNA-1 0.912 GCR425 0.961 echinostoma_caproni_prjeb1207_ECPE_0000725501-mRNA-1 GCR261 0.868 0.351 echinostoma_caproni_prjeb1207_ECPE_0001599201-mRNA-1 0.96 1 echinostoma_caproni_prjeb1207_ECPE_0000834701-mRNA-1 GCR547 0.988 0.783 echinostoma_caproni_prjeb1207_ECPE_0000304501-mRNA-1 echinostoma_caproni_prjeb1207_ECPE_0001229101-mRNA-1 0.684 GCR509 0.982 fb 0.973 0.844 Fhep_BN1106_s3601B000055.mRNA-1 GCR204 0.914 r 0.968 Fhep_scaffold163_426785-427753 0.93 echinostoma_caproni_prjeb1207_ECPE_0000956301-mRNA-1 GCR146 S 0.404 echinostoma_caproni_prjeb1207_ECPE_0001207601-mRNA-1 GCR498 0.839 clonorchis_sinensis_prjda72781_csin110082 echinostoma_caproni_prjeb1207_ECPE_0000263901-mRNA-1 GCR488 0.154 1 Fhep_scaffold1723_86054-86959 0.907 0.997 echinostoma_caproni_prjeb1207_ECPE_0000410101-mRNA-1 GCR546 0.675 echinostoma_caproni_prjeb1207_ECPE_0000523001-mRNA-1 Fhep_scaffold37_374515-375306 1 GCR527 0.879 Fhep_scaffold5322_2710-3585 0.987 Fhep_scaffold2512_57274-58011 GCR148 Fhep_scaffold4204_44433-44774 Fhep_BN1106_s4918B000028.mRNA-1 Fhep_scaffold323_27651-28547 GCR544 0.925 Fhep_scaffold7452_19555-20052 F 0.486 diphyllobothrium_latum_prjeb1206_DILT_0000517801-mRNA-1 GCR548 GCR211 GCR540 0.998 GCR513 GCR562 GCR504 GCR534 GCR175 0.167 GCR531 O GCR518 D GCR511 0.999 GCR251 GCR470 0.975
R 0.633 GCR209 GCR478 GCR216 GCR431 P GCR160 GCR525 GCR145 GCR491 GCR507 GCR497 GCR487 GCR528 GCR541 GCR495 GCR260 GCR537 GCR490 GCR554
GCR464 GCR516
EmuJ_000996550.1_PROF1
SMDC22227.1_PROF1
SMDC2150.1_PROF1
SMDC2514.1_PROF1
SMDGB6_PROF1
SMDC2955.1_PROF1 SMDGB1_PROF1 SMDGB3_PROF1 SMDC907.1A_PROF1 SMDGB2_PROF1 SMP041880_PROF1 SMDC907.1B_PROF1
GCR262 GCR315 GCR122 GCR157 GCR243 GCR330 GCR324 GCR392 GCR244
GCR307 0.623 GCR577 0.999 0.997
SMDC2411.1_PROF1 GCR302 SMDGB4_PROF1 SMP134960_PROF1 EmuJ_000954500.1_PROF1 GCR419 schistosoma_mansoni_prjea36577_Smp_023710.1 0.024 schistosoma_rodhaini_prjeb526_SROB_0001125301-mRNA-1 schistosoma_margrebowiei_prjeb522_SMRZ_0000602501-mRNA-1 schistosoma_curassoni_prjeb519_SCUD_0000835601-mRNA-1 GCR301 schistosoma_mansoni_prjea36577_Smp_117340.1 0.975 schistosoma_margrebowiei_prjeb522_SMRZ_0001802201-mRNA-1 GCR254 0.999 Fhep_D915_03083
1 Fhep_BN1106_s6156B000040.mRNA-1 1 GCR572 1 schistosoma_margrebowiei_prjeb522_SMRZ_0000541701-mRNA-1 1 schistocephalus_solidus_prjeb527_SSLN_0000511401-mRNA-1 GCR104 schistocephalus_solidus_prjeb527_SSLN_0001437101-mRNA-1
1 echinostoma_caproni_prjeb1207_ECPE_0000405901-mRNA-1 GCR395 0.896 0.472 schistosoma_haematobium_prjna78265_A_05638 0.998 0.783 GCR574 GCR472 0.873 SMDC6858.4_PROF1 0.499 1 SMDC8572.2_PROF1 GCR312 1 SMDC7587.4_PROF1 GCR437 0.995 SMDGB7_PROF1 GCR410 0.984 0.911 GCR450 1 SMDC14497.4_PROF1 GCR387 1 GCR506 0.898 0.48 0.992 SMDC15273.1_PROF1 schistosoma_haematobium_prjna78265_A_07947 GCR342 0.509 SMDC6858.1_PROF1 GCR196 0.913 0.098 0.806 GCR539 GCR195 1 1 SMDC15168.2_PROF1 0.833 1 GCR519 GCR346 0.994 0.561 SMDC21116.2_PROF1 GCR079 0.877 0.839 0.956 0.731 SMDC12781.1_PROF1 GCR047 0.757 SMDC18000.2_PROF1 1 GCR526 GCR308 0.893 0.997 GCR048 0.993 0.201 SMDGB5_PROF1 0.084 0.999 1 SMDC14736.1_PROF1 GCR436 0.048 GCR501 GCR359 0.905 1 SMDC19969.1_PROF1 0.939 0.955 GCR314 0.582 SMDC4350.2_PROF1 GCR445 0.916 0.187 0 GCR117 0.82 0.902 SMDC423.2_PROF1 GCR355 GCR100 GCR388 0.339 0.871 0.814 0.757 0.999 SMDC5598.1A_PROF1 0.999 0.769 GCR240 0.653 0.894 0.726 SMDC1825.1_PROF1 Fhep_D915_03393GCR241 0.841 0.683 SMDC1749.3A_PROF1 GCR460 0.958 0.973 GCR468 GCR191 SMDC1749.3B_PROF1 0.921 0.221 GCR149 GCR521 0.997 SMDC5598.1B_PROF1 GCR179 0.84 0.4920.444 1 SMDC4570.1_PROF1 0.875 0.95 0.998 SMPSC63_PROF1 0.403 SMPSC331A_PROF1 GCR458 0.99 0.699 SMPSC331B_PROF1 0.998 1 0.812 SMP083940_PROF1 GCR379 0.771 SMPSC1003_PROF1 GCR583 0.749 0.72 1 SMPSC433_PROF1 GCR200 0.784 schistosoma_japonicum_prjea34885_Sjp_0056590 GCR181 0.996 0.385 1 0.87 SMP084290_PROF1 GCR257 0.906 0.99 schistosoma_curassoni_prjeb519_SCUD_0001875201-mRNA-1 GCR1030.784 0.879 schistosoma_margrebowiei_prjeb522_SMRZ_0001830401-mRNA-1 0.86 schistosoma_haematobium_prjna78265_A_02855 GCR235 0.82 0.816 GCR5710.586 0.79trichobilharzia_regenti_prjeb4662_TRE_0001051301-mRNA-1 GCR373 0.963 0.909 SMP084270_PROF1 0.997 0.9690.755 SMP084280_PROF1 GCR405 1 0.501 schistosoma_margrebowiei_prjeb522_SMRZ_0001830201-mRNA-1 GCR5680.97 schistosoma_mattheei_prjeb523_SMTD_0000474701-mRNA-1 GCR321 0.811 0.977 1 0 schistosoma_haematobium_prjna78265_A_04157 GCR370 0.3560.994 SMP167870_PROF1 GCR3050.541 0.9990.9930.839 SMPSC12A_PROF1 GCR3930.9710.639 0.571 clonorchis_sinensis_prjda72781_csin102640 GCR389 0.957Fhep_D915_13002 GCR042 0.849 0.814 0.924 0.816 SMP091950_PROF1 GCR055 0.822 EmuJ_000633100.1_PROF1 0.963 0.467 GCR171 GCR512 1 0.979 SMDC1889.2_PROF1 GCR524 0.77 0.761 0.872 0.621 0.998SMDC1026.2_PROF1 GCR499 0.17 GCR550 GCR560 0.973 0.985SMDC8510.3B_PROF1 GCR520 0.722 SMDC15385.3_PROF1 GCR5510.9770.79 0.997 GCR517 GCR118 0.657 0.966 SMDC27521.1_PROF1 0.663 0 GCR466 0.707 0.891 0.675 SMDC14380.2_PROF1 GCR564 0 0.593 0 0.033 0 SMDC8510.3A_PROF1 GCR269 0.871 0.999 0.993 SMDC12554.3_PROF1 GCR5330.874 0.62 0.995GCR473 GCR523 0.244 0.987 0 SMDC8510.2_PROF1 GCR510 0.938 0.408 SMDC27911.2_PROF1 GCR532 0.771 1 GCR208 0.9050.751 0.811SMDC1917.2_PROF1 GCR239 0.756 0.737 GCR198 GCR496 0.9180.989 3.0 1 SMDC6472.1_PROF1 GCR465 0.65 GCR483 GCR4750.986 1 SMDC504.1_PROF1 GCR382 HsRhodopsin GCR217 GCR176 S a 0.993 GCR4540.742 0.9 0 0.907GCR290 f GCR371 0.952 0.916GCR218
GCR335 0.963 GCR059 r r GCR189 0.996 GCR067
GCR481 f 0.989 GCR530
EmuJ_001085520.1_PROF1GCR397 0.956 0.727GCR253 a
S GCR3900.903 0.4060.891GCR093 GCR5650.734 GCR543 GCR411 0.022 0 0.682 0.886 GCR434 GCR036 0.9780.175 0.919 0.74 GCR078 GCR0560.031 0.997 0.765 0.972 GCR242 GCR110 0.741 0.989GCR401 GCR075 0.915 0.993 GCR252 GCR3180.963 0.89 0.185 0.986 GCR457 GCR325 0.999 0.42 0.581GCR398 0.77 GCR237 0.79 1 0.995 protopolystoma_xenopodis_prjeb1201_PXEA_0002666801-mRNA-1
0.417 Fhep_D915_13937 0 trichobilharzia_regenti_prjeb4662_TRE_0000301801-mRNA-1 S 0.9050.835 0.904 0.745schistosoma_rodhaini_prjeb526_SROB_0001137201-mRNA-1 GCR156 0.79 0 0.937 schistosoma_mansoni_prjea36577_Smp_137980.1
Fhep_BN1106_s1984B000285.mRNA-1GCR515 0.965 0.994 0.879 0.857 schistosoma_curassoni_prjeb519_SCUD_0001747201-mRNA-1 GCR300 0 schistosoma_haematobium_prjna78265_A_03895 r
GCR158 0.664 0.951 schistosoma_mattheei_prjeb523_SMTD_0000955601-mRNA-1
GCR2150.696 0.92 0.943 schistosoma_margrebowiei_prjeb522_SMRZ_0000949101-mRNA-1 f 0.928 GCR343 GCR219 c 0.99 0.606 0.87 GCR375 GCR566 0.952 0.891 0.871 schistosoma_curassoni_prjeb519_SCUD_0001180801-mRNA-1 0.774 schistosoma_mansoni_prjea36577_Smp_162980.1GCR538 0.849 0.93 GCR222 trichobilharzia_regenti_prjeb4662_TRE_0000048201-mRNA-1 GCR561 0.903 0 GCR184 0.875 0.747 0.998 GCR493 GCR5140.182 0.98 0.706GCR417 GCR489 0.861 0.912 0.996 GCR385 GCR5360.731 0.992 0.6140.505 GCR369 GCR556 0.945 0.734 GCR255 0.43 0.932 GCR508 0.6010.847 0.921 0.929GCR429 GCR4760.636 0.025 0.901 GCR420 GCR360 GCR152 0.312GCR439 GCR522 0.981 0.976 0.869 GCR5570.948 0.763 0.624 1 GCR451 0.873 GCR049 GCR142 0.528 0.913 0.992GCR344 GCR5590.974 0.969 0.989 GCR101 GCR545 0.907 0.698 GCR376 0.219 0.996GCR427 GCR164 0.55 0.793 0.773 GCR5050.995 0.844 GCR426 GCR463 0.978 0.8 1 GCR226 0.98 0.922 0.789 GCR363 GCR210 0.743 0 opisthorchis_viverrini_prjna222628_T265_11077 GCR582 0.09 0.026 clonorchis_sinensis_prjda72781_csin109678 GCR485 0.51 0 0.997 Fhep_BN1106_s2144B000269.mRNA-1 GCR207 0.985 GCR123 0.782 0.907 GCR130 GCR575 1 0.999 GCR127 GCR580 clonorchis_sinensis_prjda72781_csin110930 0.911 0.189 GCR570 0.804 echinostoma_caproni_prjeb1207_ECPE_0001431101-mRNA-1 GCR442 0.975 Fhep_scaffold3014_121504-122538 GCR081 0.747 schistocephalus_solidus_prjeb527_SSLN_0002053201-mRNA-1 0.598 protopolystoma_xenopodis_prjeb1201_PXEA_0002576201-mRNA-1 GCR500 0.158 GCR193
GCR089 0.571 echinostoma_caproni_prjeb1207_ECPE_0000038901-mRNA-1 GCR563 0.954 echinostoma_caproni_prjeb1207_ECPE_0000941201-mRNA-1 0.922 0.254 0.814 0.796 echinostoma_caproni_prjeb1207_ECPE_0000977101-mRNA-1 GCR452 0.952 0.691 0.73 echinostoma_caproni_prjeb1207_ECPE_0000508001-mRNA-1 GCR259 0.795 0.246 echinostoma_caproni_prjeb1207_ECPE_0000818801-mRNA-1 0.912 GCR425 0.961 echinostoma_caproni_prjeb1207_ECPE_0000725501-mRNA-1 GCR261 0.868 0.351 echinostoma_caproni_prjeb1207_ECPE_0001599201-mRNA-1 0.96 1 echinostoma_caproni_prjeb1207_ECPE_0000834701-mRNA-1 GCR547 0.988 0.783 echinostoma_caproni_prjeb1207_ECPE_0000304501-mRNA-1 echinostoma_caproni_prjeb1207_ECPE_0001229101-mRNA-1 0.684 GCR509 0.982 0.973 0.844 Fhep_BN1106_s3601B000055.mRNA-1 GCR204 0.914 0.968 Fhep_scaffold163_426785-427753 0.93 echinostoma_caproni_prjeb1207_ECPE_0000956301-mRNA-1 GCR146 0.404 echinostoma_caproni_prjeb1207_ECPE_0001207601-mRNA-1 GCR498 0.839 clonorchis_sinensis_prjda72781_csin110082 echinostoma_caproni_prjeb1207_ECPE_0000263901-mRNA-1 GCR488 0.154 1 Fhep_scaffold1723_86054-86959 0.907 0.997 echinostoma_caproni_prjeb1207_ECPE_0000410101-mRNA-1 GCR546 0.675 echinostoma_caproni_prjeb1207_ECPE_0000523001-mRNA-1 Fhep_scaffold37_374515-375306 GCR527 0.879 Fhep_scaffold5322_2710-3585 0.987 Fhep_scaffold2512_57274-58011 GCR148 Fhep_scaffold4204_44433-44774 Fhep_BN1106_s4918B000028.mRNA-1 Fhep_scaffold323_27651-28547 GCR544 0.925 Fhep_scaffold7452_19555-20052 0.486 diphyllobothrium_latum_prjeb1206_DILT_0000517801-mRNA-1 GCR548 GCR211 GCR540 0.998 GCR513 GCR562 GCR504 GCR534 GCR175 0.167 GCR531 GCR518 GCR511 0.999 GCR251 GCR470 0.975
GCR209 0.633 GCR478 GCR216
GCR431 GCR160
GCR525 GCR145
GCR491 GCR507 GCR497 GCR487
GCR528
GCR541 GCR495 GCR260 GCR537 GCR490 GCR554
GCR464 GCR516 R
-
o
h
R R
h
o
- L bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aCC-BY-NC-ND 4.0 International license.
HsNPY1R (AAA59920) Y100W106C113Q120 V126 W163 Q219 N283D287 AgNPFR (AAT81602) E W C Q V W Q N D DmNPFR (AAK50050) E W C Q V W Q N D TM1 TM2 TM3 TM4 TM5 TM6 TM7 BN1106_s3169B000088 L W C Q V W Q N D D915_05685 W W C Q V W Q N E BN1106_s19B000334 E W C Q V W Q Y G B
Hs5HT1A (NP_000515) D82 C109 D116 D133R134Y135 C187S199T200 N386 Sm5HTR (Smp_126730) D C D D R Y C A T Q TM1 TM2 TM3 TM4 TM5 TM6 TM7 BN1106_s1436B000114 D C D D R Y C S T - BN1106_s10B000515 D C D D R Y C A T L BN1106_s362B000177 D C D D R Y C A T I BN1106_81B000700 D C D D R Y C A T T D915_00277 D C D D R Y C S S T C PaOaR (AY333178.1)
BmOaR (AB255163.1)** D105V106T110 I156 S213 W504Y412**F507 TM1 TM2 TM3 TM4 TM5 TM6 TM7 BN1106_s1016B000108 D V T I S W Y F D915_02972 D V T I S W Y F D915_08505 D V T I S W Y F BN1106_s2834B000232 D V T I S A W F D915_01850 D V T I S N W F D915_05578 D I T I S Y W F D915_06018 D V T I S N W F D SmGAR (Smp_145540) D230Y231 Y869H870 Y898 TM1 TM2 TM3 TM4 TM5 TM6 TM7 D915_00814 D Y Y H Y BN1106_s1913B000092 D F Y H Y bioRxiv preprint doi: https://doi.org/10.1101/207316; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A BN1106_s1859B000140.mRNA-1 0.999 HsGP179 Orphan 0.502 0.925 HsGP158 (Galvez et al., 2000; JBC 275, 41166-41174).HsGABR2 0.927 GABAB HsGABR1
BN1106_s1717B000113.mRNA-1 0.742 Orphan Smp_128940
HsRAI3 0.929 HsGPC5D Orphan Geng et al (2013) Nature 504(7479) 10.1038/nature127251 HsGPC5B 0.925 0.866 Taste1R3 0.282 Taste1R2 Taste 0.904 0.985 Taste1R1 HsCASR Ca2+ 0.673 0.748 HsGPC6A Orphan
Table x. Conservation of ligand-interactingHs_mGR5 residues between vertebrate GABAB and metabotropic 0.996 glutamate receptors (mGluR),Hs_mGR1 and Fasciola hepatica class C GPCRs (FhGlu1-3). Amino acids of FhGlu1-3 with primary sequence orthology to agonist-interacting residues identified by protein alignment Hs_mGR3 FhGlu1-3 against0.908 mutation-identified0.986 human and rat GABAB receptors (A), or selected human mGluR Hs_mGR2 1 subtypes (B). A, Mutationally-identified agonist-interacting residues of ( )human GABAB receptor 1B and (2) rat GABAB receptor 1a. IdenticalHs_mGR4 amino acids in FhGlu1-3 are represented by white text on black 0.657 mGluR background, functionally conserved0.991 Hs_mGR7 amino acids by black text on grey background. B, Mutationally- 0.681 identified agonist-interacting residuesHs_mGR8 of human mGluR1-4, 7, 8. Mutations causing a significant reductio 0.811 in mGluR receptor activity0.915 are bold andHs_mGR6 numbered. FhGlu1-3 residues identical to any of these are represented by white text on black background, functionally-conserved residues by black text on grey 1 2 Smp_052660 3 background. ( Galvez et al., 2000;1 Geng et al., 2013; Wellendorph & Bräuner-Osborne, 2009) BN1106_s2924B000081.mRNA-1 BWellendorph & Brauner-Osborne (2009) Brit J Pharmacol 156, 869-884. A 0.3
MmGABAB1 S246 S269 S270 E465 D471 BN1106_s2924B000081 - - - - - BN1106_s1717B000113 S D A D N BN1106_s1859B000140 F L N C Y Smp_128940 S A S D N Smp_052660 S T T E D
B Glu binding region: COOH αNH2 αNH2 αNH2 αNH2 αNH2 COOH mGluR1 Y74 R78 S S165 T188 D208 Y236 E292 G293 D318 D K409 mGluR2 R57 R Y S145 T168 D188 Y216 R S D295 R K377 mGluR3 R64 R68 Y150 S151 T174 D194 Y222 R277 S D301 Q306 K mGluR4 K74 R78 G S159 T182 D Y N E D K K mGluR7 N R G S T D Y N D D K K mGluR8 K71 R75 A S T D Y N E D309 K K BN1106_s2924B000081 ------BN1106_s1717B000113 I F I S D A Y S S N D A BN1106_s1859B000140 - L - - - - A D V N - P Smp_128940 N L Q S A I H T S N T S Smp_052660 R R Y S T D Y G M D E K .658 N1106_s2849B000110.mRNA-10
0.9 0.994 hFzd4 bioRxiv preprint doi: https://doi.org/10.1101/2073160.924; this version posted0.876 October 23, 2017. The copyright holder for this preprint (which cFzd4 was not certified by peer review) is the author/funder, who has granted bioRxiv0.938 a license to display the preprint in perpetuity. It is made hFzd7
cFzd7 available under aCC-BY-NC-ND0.727 4.0 International license. 0.916 hFzd2 cFzd1
0.982
BN1106_s1665B000273.mRNA-1FSMP118970 hFzd1 A cFzd2 BN1106_s905B000183.mRNA-1
S m o
cFzd2a o cFzd2b t BN1106_s2220B000114.mRNA-1 FSMP139810 h e n cLin-17 BN1106_s33B000335.mRNA-1 e 1 0.988 0.827 cMig-1 d dFzd3 dFzd4 0.883 dSmoothened 0.368 0.963 hSmoothened FSMP155340 0.702 0.205 0.778 BN1106_s1509B000194.mRNA-1
0.913 0.949
0.855 FSMP125160 dFzd2
0.445 I I dicty-fslJ-1
t hFzd50.894
s
u 0.957 0.865 hFzd10 l 0.961
C hFzd8 h cFzd10 0.774 2.0 hFzd3 1 hFzd9 0.798 0.99
0.921
V hFzd6 FSMP174350
I 1
0.388
t
0.658 h s BN1106_s2849B000110.mRNA-1
dFzd1 0.437 C
u
l 0.99 0.994 hFzd4
0.876 l
u
C 0.924
cFzd4 s
h 0.938
hFzd7 t
cFzd7 I 0.727
0.916 hFzd2 I cFzd1 I
0.982
FSMP118970 hFzd1
cFzd2
BN1106_s905B000183.mRNA-1
I h
t C s l u 4.0 B 3.0 Ligand-contacting amino acids 2.0
M. musculus fz1-10, bits numbering relative to fz8 D Q V R 1.0 E I G H T E D E L I IQP FF E 4.0 N TAT PHLMYLYV S 0.0 M 2.0 F P 58 H59 64 68 71 72 74 75 78 Aligned positions 3.0 5 10122 125 127 130 132 BN1106_s2220B000114 WebLogo 3.4 BN1106_s1665B000273 2.0 bits BN1106_s2894B000110 1.0 L A E D I F R BN1106s33B000335 G Q D Q Q F V MG E P Y N BN1106_s905B000183 0.0 E V E R T Y A L Y LSFPQ 5 10 WebLogo 3.4 C Amino acid position relative to Dro-wnt-1 427 450
93 104 146 154156 185 233235 242 247 397 413 423 428443445 451 455 458 467 Dro-Wnt-1 ECQ NCS ACS CTC GCS CKC SCT TCW FCE QCN CGLMCC CAC CCEVKCKLC TCL BN1106_s737B000430 ------ACS CDC GCS CKC ACS TCW YCR LCN CNRLCC CDC CCEVRCKVC RCK BN1106_s198B000330 ------ACS CGC GCS CKC SCE TCW FCH RCV CEYLCC CNC CCKVICQTC TCN BN1106_s1256B000163 ECQ NCT SCK CGC GCG CRP L-- --- YCQ ECL CVHLCC CRC CCRVECQTC VCN Log (FPKM) − Row Z 02 20 2 B B A -1 0 1 2 3 − was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint Score G A − Row Z 02 20 F
Egg − egg_FPKM Score S
ACh doi:
Dop
Met egg_FPKM https://doi.org/10.1101/207316 RA1 met_FPKM
Oct
5HT NEJ_1h met_FPKM
Tyr NEJ1h_FPKM RA3 available undera NEJ1h_FPKM RO NEJ_3h
GPCR families NEJ3h_FPKM
FMRFa
Luqin
GYIRFa NEJ3h_FPKM
NPF/Y NEJ_24h ;
RP1 this versionpostedOctober23,2017.
Myomodulin
NEJ24h_FPKM CC-BY-NC-ND 4.0Internationallicense
Ast A NEJ24h_FPKM
GHS
Pkt Juv_21d R juv_FPKMAngiotensin
Endothelin
RP2 FMRFa juv_FPKM
NPF/Y
Proctolin
Adult RP3 adult_FPKM adult_FPKM RP4 FMRFa
Myom/Myos GPCR ID BN1106_s2144B000269.mRNA-1 BN1106_s1984B000285.mRNA-1 BN1106_s3601B000055.mRNA-1 BN1106_s6156B000040.mRNA-1 BN1106_s6156B000040.mRNA-1 D915_05591 D915_02221 BN1106_s51B000259.mRNA-1 D915_02595 BN1106_s1703B000178.mRNA-1 BN1106_s2967B000104.mRNA-1 BN1106_s1280B000188.mRNA-1 BN1106_s545B000172.mRNA-1 BN1106_s2220B000114.mRNA-1 BN1106_s1085B000091.mRNA-1 D915_01187 BN1106_s3137B000066.mRNA-1 BN1106_s3169B000088.mRNA-1 D915_00019 BN1106_s19B000334.mRNA-1 D915_06456 BN1106_s802B000072.mRNA-1 BN1106_s4244B000034.mRNA-1 D915_00383 BN1106_s2145B000022.mRNA-1 BN1106_s1984B000285.mRNA-1 BN1106_s81B000700.mRNA-1 BN1106_s2017B000126.mRNA-1 BN1106_s2188B000064.mRNA-1 BN1106_s258B000277.mRNA-1 BN1106_s1016B000108.mRNA-1 BN1106_s817B000144.mRNA-1 BN1106_s494B000132.mRNA-2 D915_06018 BN1106_s161B000174.mRNA-1 D915_03535 BN1106_s46B000392.mRNA-1 D915_00814 BN1106_s4451B000132.mRNA-1 BN1106_s3332B000065.mRNA-1 BN1106_s6153B000107.mRNA-1 BN1106_s1665B000273.mRNA-1 BN1106_s1362B000183.mRNA-1 D915_01727 D915_08505 BN1106_s1566B000115.mRNA-1 D915_05571 scaffold37:374515-375306 BN1106_s1385B000135.mRNA-1 scaffold3014:121504-122538 BN1106_s1037B000170.mRNA-1 BN1106_s2144B000269.mRNA-1 BN1106_s905B000183.mRNA-1 D915_10580 BN1106_s2017B000123.mRNA-1 D915_01691 scaffold5322:2710-3585 BN1106_s504B000398.mRNA-1 D915_02972 D915_01954 BN1106_s4129B000138.mRNA-1 BN1106_s2849B000110.mRNA-1 BN1106_s2682B000166.mRNA-1 scaffold4074:42842-43729 BN1106_s3778B000020.mRNA-1 BN1106_s3332B000064.mRNA-1 D915_12026 BN1106_s1278B000107.mRNA-1 D915_06722 D915_06590 D915_05685 D915_01953 D915_13002 D915_05265 BN1106_s3204B000090.mRNA-1 D915_06342 D915_01126 BN1106_s92B000566.mRNA-1 D915_05109 scaffold5738:2490-3377 BN1106_s6141B000027.mRNA-1 BN1106_s316B000427.mRNA-1 D915_01850 D915_15811 BN1106_s923B000146.mRNA-1 BN1106_s3601B000055.mRNA-1 D915_05097 scaffold37:374515-375306 scaffold3014:121504-122538 49 48 46 47 45 43 44 42 41 39 40 37 38 36 35 34 32 33 30 31 29 27 28 25 26 24 22 23 20 21 18 19 17 16 14 15 12 13 11 9 10 8 7 5 6 3 4 1 2 83 82 81 80 78 79 77 76 74 75 72 73 71 69 70 68 67 66 65 63 64 62 61 59 60 57 58 56 54 55 52 53 50 51 RP5 83 82 81 80 78 79 77 76 74 75 72 73 71 69 70 68 67 66 65 63 64 62 61 59 60 57 58 56 54 55 52 53 50 51 49 48 46 47 45 43 44 42 41 39 40 37 38 36 35 34 32 33 30 31 29 27 28 25 26 24 22 23 20 21 18 19 17 16 14 15 12 13 11 9 10 8 7 5 6 3 4 1 2
Proctolin The copyrightholderforthispreprint(which RP6 Sex peptide
Prostacyclin RP7 .
R2 BN1106_s1984B000285_fwRhod Classification R3 RO RA3 R1 RA1_Octopamine RA1_Octopamine RP6_Angiotensin R6 FZD Hormone RP1_Thyrotropin-releasing R1 RP1_FMRFamide R3 R4 RA1_Octopamine RP3 RP6 Y RP1_Neuropeptide RP1 R3 RP4_Myosuppressin/Myomodulin RP6_Sex Peptide RP4_Angiotensin RP2_Angiotensin RP1 RP6_Proctolin R1 R3 RP5_Myosuppressin/Myomodulin RA1 R4 RP6_Prostacyclin R4 RP6_Sex peptide R6 RP1_Prokineticin RP6 RP5_Myosuppressin/Myomodulin R4 A RP1_Allatostatin RP1_GYIRFamide RP2_Proctolin RP1 FZD RP1_GYIRFamide R2 RA1_Tyramine Y RP1_Neuropeptide RP2_Proctolin RP1_Luqin RP1_Myomodulin R2 R6 RP2_Proctolin RP5_Myosuppressin/Myomodulin R2 RA1_5-HT RO Hormone Secretagogue RP1_Growth RP2_FMRFamide RA1_Octopamine RP1 RP2 RA1 RP7 RA1_Octopamine RP2_proctolin RA1_Acetylcholine RP1 R3 R4 FZD RA3 RP2_Proctolin RA1_Octopamine Y RP2_Neuropeptide R3 R1 RP6 R2 R2 R5 FZD RP2_endothelin R4 R6 BN1106_s6156B000040_fwRhod Expression cluster Egg NEJ Adult 21d_juv