Fig 1.
Bioinformatic analyses of the three 5-HT1 serotonin receptors identified in this work (Eca-5-HT1a, Mvo-5-HT1a and Hmi-5-HT1a, GenBank accession numbers MW535743, MW535744 and MW535745 respectively).
A. The structural representation and prediction of residues of potential N-glycosylation of Eca-5-HT1a were obtained with the Protter program (http://www.enzim.hu/hmmtop/index.php). The intracellular and extracellular loops are indicated as ICL and ECL respectively. The third intracellular loop used for antibody generation is shaded in grey. Residues potentially involved in N-linked glycosylation were marked in green. B. The amino acid sequences of predicted serotonin receptors ortologues with best scores in blast searches with the cestode Eca-5-HT1a were aligned using the ClustalW method. The new 5-HT1 type cloned receptors’ names are marked in bold and aligned against sequences of orthologous receptors from Hydatigera taeniaeformis (Hta-5-HT1a, gene model number TTAC_0000125301), Taenia asiatica (Tas-5-HT1a, gene model number TASK_0000705401), Hymenolepis nana (Hna-5-HT1a, gene model number HNAJ_0000723201), Echinococcus granulosus (Egr-5-HT1a, gene model number EgrG_001050800.1), Taenia saginata (Tsa-5-HT1a, gene model number TSAs00002g00673m00001), Echinococcus multilocularis (Emu-5-HT1a, gene model number EmuJ_001050800.1), Taenia solium (Tso-5-HT1a, gene model number TsM_000928200), Hymenolepis diminuta (Hdi-5-HT1a, gene model number HDID_0000514801), Taenia multiceps (Tmu-5-HT1a, gene model number Tm1G003304), Schistocephalus solidus (Sso-5-HT1a, gene model number SSLN_0001996401), Dibothriocephalus latus (Dla-5-HT1a, gene model number DILT_0000437701), Spirometra erinaceieuropaei (Ser-5-HT1a, gene model number SPER_0002466701) and Gyrodactylus salaris (Gsa-5-HT1a, gene model number scf7180006953168). The transmembrane (TM), intracellular (ICL) and extracellular (ECL) domains are indicated above each alignment. For the sake of simplicity, the amino terminal end, the intracellular loop three and the carboxy terminal end were trimmed partially or completely. The position of residues involved in G-protein coupling are indicated with asterisks below each alignment. Residues present in the new predicted receptors that were not seen in other GPCRs are underlined. Critical residues involved in ligand binding and receptor function were indicated in bold below each alignment. Cysteine residues potentially involved in disulphide bond formation are marked as S-S between cysteines. The reader is referred to the S1 Table for the complete list of receptor names, species and identification numbers. C. The amino acid sequences of predicted cestode serotonin receptors were aligned with a repertoire of serotonin receptors cloned from Echinococcus canadensis (Eca-5-HT1b, gene model EcG7_00799; Eca-5-HT7a and Eca-5-HT7b, Genbank accession numbers MH707372 and MH707373 respectively), Mesocestoides vogae (Mvo-5-HT7a, Genbank accession numer MH707374), Dugesia japonica (Dja-5-HT1a to Dja-5-HT1e, Dja-5-HT2a to Dja-5-HT2c and Dja-5-HT7a to Dja-5-HT7h; PMID PMC4569474), Schistosoma mansoni (Sma-5-HT1 and Sma-5-HT7b, Genbank accession numbers XP_018645423 and KX150867 respectively), Caenorhabditis elegans (Cel-5-HT1, Cel-5-HT2 and Cel-5-HT7, Uniprot accession numbers G5EGH0, O17470 and Q22895 respectively) and Drosophila melanogaster (Dme-5-HT1a, Dme-5-HT1b, Dme-5-HT2a, Dme-5-HT2b and Dme-5-HT7, Genbank accession numbers CAA77570.1, CAA77571.1, CAA57429.1, NP_001262373.1 and NP_524599.1 respectively). The alignment included representative examples of the three major classes of serotonergic GPCRs from invertebrates. These include type 2 serotonin receptors (5-HT2, blue), type 7 (5-HT7, green) and type 1 receptors (5-HT1, red). The cestode GPCR sequences cloned and functionally expressed in this study (name labels shaded in gray: Eca-5-HT1a, Mvo-5-HT1a and Hmi-5-HT1a) as well as the putative receptor identified in databases (name label shaded in gray: Eca-5-HT1b) cluster within a clade of 5-HT1 like receptors. The FMRFaR neuropeptide receptor from D. melanogaster (DmeFMRFaR, Uniprot accession number Q9VZW5) was used as outgroup. The tree was tested by bootstrap analysis with 500 iterations. The length of the branches is proportional to the genetic distance between sequences (see scale bar).
Table 1.
Important amino acid residues in ligand binding and receptor activation of the cloned cestode GPCRs and homologous invertebrate GPCRs.
Table 2.
Residues potentially involved in receptor and G-protein coupling.
Table 3.
Prediction of coupling specificity for the 5-HT1 type of cestode receptors.
Fig 2.
Structural comparative analysis of cestode 5-HT1 type serotonergic receptors.
(A-E) Structural similarities and differences among the 5-HT1 type serotonergic receptors of Homo sapiens (Hsa-5-HT1b) and cestodes. (A) Superposition of crystallized Hsa-5-HT1b (orange; PDB 4IAR) and the homology models of Eca-5-HT1a (blue; Echinococcus canadensis G7), Mvo-5-HT1a (green; Mesocestoides vogae), and Hmi-5-HT1a (red; Hymenolepis microstoma). (B-E) Close-up views of the residues involved in the putative ergotamine interaction site of (B) Hsa-5-HT1b, (C) Eca-5-HT1a, (D), Mvo-5-HT1a, and (E) Hmi-5-HT1a, in the transmembrane domains III (labeled III) and V (labeled V). (F-Q) Comparative analysis of residues involved in the ligand binding and G protein coupling sites between cestode 5-HT1 and 5-HT7 type serotonergic receptors. (F, G, J, K, N and O) Close-up views of residues involved in the ligand binding site of (F) Eca-5-HT1a, (G) Eca-5-HT7a, (J), Mvo-5-HT1a, (K), Mvo-5-HT7a, (N), Hmi-5-HT1a, and (O) Hmi-5-HT7a, in the transmembrane domains III (labeled III) and V (labeled V). Note that threonines at positions 3.40 and 5.39 and serine at 5.42 (in bold) in 5-HT1a receptors are replaced by isoleucine, glutamine, and alanine (in red); respectively, in 5-HT7a receptors. (H, I, L, M, P and Q) Close-up views of residues involved in the G protein coupling site of (H) Eca-5-HT1a, (I) Eca-5-HT7a, (L), Mvo-5-HT1a, (M), Mvo-5-HT7a, (P), Hmi-5-HT1a, and (Q) Hmi-5-HT7a, in the transmembrane domains V (labeled V) and VII (labeled VII). Note that histidine, isoleucine, serine, and lysine at the positions 5.68, 5.69, 7.57, and 7.58 (in bold); respectively, in 5-HT1a receptors are replaced by methionine, alanine, asparagines, and arginine residues (in red); respectively, in 5-HT7a receptors. In all the plots, protein structures were represented as cartoons and the residue side chains involved in the putative ergotamine interaction, ligand binding, and G protein coupling sites were represented as sticks.
Fig 3.
Heterologous expression of Eca-5-HT1a receptor.
A) Time resolved measurements of Ca2+ accumulation (measured as raw fluorescence units, RFU) in cells expressing the cestode Eca-5-HT1a before and after addition of different concentrations of 5-HT (arrow, concentrations indicated in legend in μM). B) Concentration-response curve measuring peak amplitude of 5-HT evoked fluorescence change (measured as a percentage) in cells expressing Eca-5-HT1a (green closed squares), or untransfected HEK293-GNA15 cell line (open circles). C) Bar graph showing normalized peak responses in cells transfected with the new receptor to indicated neurotransmitters (10 μM). D) Concentration-response curve measuring 5-HT evoked fluorescence change (measured as raw fluorescence units, RFU) in cells expressing wild type Eca-5-HT1a (red solid dot), or the following mutants: D118A3.32 (blue triangle), C122A3.36 (green inverted triangle), T123A3.37 (purple rhombus) and W542A6.48 (brown triangle). Black solid squares represent the untransfected control (HEK293-GNA15 cell line). E) molecular model of the orthosteric binding pocket in Eca-5-HT1a, showing the role of the mutated residues in serotonin binding. The molecule shown in yellow represent 5-HT. The gray sticks represent the R groups from the amino acids mutated here. In all cases, the amine groups are shown in blue, thiol group is shown in yellow and hydroxyl groups in red. Hypothetical interactions between the 5-HT molecule and receptor mutated residues were marked by dotted lines. Abbreviations are: 5-HT, serotonin; Tyra, tyramine; Octo, octopamine; Ach, acetylcholine; His, histidine; Dopa, dopamine. For panels A) and B), each point represents the average of three biological experiments with three technical replicates each. For panel C), each point represents the average of two biological experiments with three technical replicates each. Error bars corresponds to the standard error of the mean.
Fig 4.
Effect of various agonists and antagonist on Eca-5-HT1a in HEK293-GNA15 cells.
(A) Concentration-dependent activation of Eca-5-HT1a with 5-HT (black box) or the 5-HT receptor agonists α-methylserotonin (α-MTS, red ball) and 5-methoxytryptamine (5-MTT, blue triangle), shown as the percentage of activation achieved with 5-HT (maximum response = 100%). (B) Concentration-dependent inhibition of Eca-5-HT1a obtained with methysergide on 5-HT (0.03 μM), α-methylserotonin (10 μM) and 5-methoxytryptamine (1 μM) mediated activation. In all cases, data represent the mean of three independent measurements (each performed in triplicate).
Fig 5.
Inmunolocalization of Eca-5-HT1a in protoscoleces of Echinococcus canadensis.
Protoscoleces were probed with anti-Eca-5-HT1a antibody (red) and anti-tropomyosin antibody or anti-serotonin antibody (green) and visualized by confocal microscopy. A) Phase contrast view of the protoscolex shown in B. B) Fluorescent image of the same protoscolex co-labelled with anti-Eca-5-HT1a antibody and anti-tropomyosin antibody. No evidence of proximity between the two signals were found. Intense signal was found in the cerebral ganglia (cg) and the main longitudinal nerve cords (lnc). C) Phase contrast view of the protoscolex shown in D. D) Fluorescent image of protoscolex co-labelled with anti-Eca-5-HT1a antibody and anti-tropomyosin antibody. The intense green signal shows the complex pattern of muscle fibers (mf). The strong red signal was observed in the cerebral ganglia (cg), the longitudinal nerve cord (lnc) and transverse commissure (tc). E) Phase contrast of the protoscolex shown in F. F) Fluorescent image of protoscolex labelled with preimmune serum obtained from the same mice which were later inoculated with Eca-5-HT1a. G) Phase contrast view of the protoscolex shown in H. H) Fluorescent image of the same protoscolex labelled with anti-Eca-5-HT1a antibody (red). Strong Eca-5-HT1a signal was found in the cerebral ganglia (cg) and the main longitudinal nerve cords (lnc). Some weak signal (arrowheads) could be seen in the surface region of the scolex. I) and J) Two different stack integrations and picture magnifications of the fluorescent image of the protoscolex shown in H. Alternating regions of receptor and serotonin immunoreactivity can be seen (arrowheads) in the scolex region. K) Phase contrast of the protoscolex shown in L. L) Fluorescent image of protoscolex incubated without primary antibody. M) Phase contrast view of the protoscolex shown in N. N) Fluorescent image of the same protoscolex co-labelled with anti-Eca-5-HT1a antibody and anti-serotonin antibody. Strong signal was observed in the cerebral ganglia (cg), the lateral nerve cord (lnc) and the cerebral commissure (co). For the phase contrast images, the tegument (te), body (bo), scolex region (sc) with sucker (su) and the rostellum (ro) were marked. Scale bar 20 μm.