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Figure 1. Asterias rubens luqin-type precursor (ArLQP) and luqin-type neuropeptide (ArLQ). (A) Amino acid sequence of ArLQP, with the predicted signal peptide shown in blue, the predicted luqin-type neuropeptide shown in red and a potential dibasic cleavage site shown in green. Shown in purple is a region of the precursor near to the C-terminus containing two cysteine residues (underlined), which is a conserved feature of luqin-type precursors (see alignment in (C). (B) Mass spectrometric identification of ArLQ (EEKTRFPKFMRW-NH2) from an acetic acid extract of radial nerve cords from A. rubens. Annotated MS/MS spectrum indicated in red, with ions matched to the sequence used for Mascot scoring. A triply charged peptide was identified with a Mascot ions score of 45, expect value of 0.00018 based on UniProt/TrEMBL protein database filtered for taxon identifier 7586 (Echinodermata, 70,885 sequences) with a precursor mass error of 2.7 ppm. (C) Alignment of the N-terminal neuropeptide-containing region and C-terminal region of ArLQP with corresponding regions of other luqin/RYamide-type precursor proteins. Conserved residues are highlighted in black or grey. The C-terminal residues of the luqin/RYamide-type peptides and species names are highlighted in phylum-specific colours: light blue (Echinodermata), dark blue (Hemichordata), pink (Annelida), red (Mollusca), yellow (Priapulida), green (Arthropoda) and purple (Nematoda). Species names are as follows: Arub (Asterias rubens), Ovic (Ophionotus victoriae), Ajap (Apostichopus japonicus), Spur (Strongylocentrotus purpuratus), Skow (Saccoglossus kowalevskii), Ctel (Capitella teleta), Obim (Octopus bimaculoides), Cgig (Crassostrea gigas), Acal (Aplysia californica), Aful (Achatina fulica), Iobs (Ilyanasa obsoleta), Bgla (Biomphalaria glabrata), Pcau (Priapulus caudatus), Tcas (Tribolium castaneum), Dmel (Drosophila melanogaster), Aaeg (Aedes aegypti), Tsui (Trichuris suis), Cele (Caenorhabditis elegans). The accession numbers of the sequences included in this alignment are listed in supplementary Table 2.
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Figure 2. Phylogenetic tree showing luqin/RYamide-type receptors from bilaterians, including the starfish A. rubens, and other closely related neuropeptide receptors. The tree, which was generated in PHYML 3.0 42 using the Maximum likelihood method47,48, comprises three distinct receptor clades – luqin/RYamide-type receptors, tachykinin-type receptors, neuropeptide-Y-type receptors, with TRH-type receptors as an outgroup. Taxa are colour-coded and bootstrap support (1000 replicates49) for clades is represented with coloured stars, as explained in the key. Species in which the peptide ligands that activate luqin/RYamide-type receptors have been identified experimentally are shown with blue lettering. Species names are as follows: Aaeg (Aedes aegypti), Acal (Aplysia californica), Apis (Acyrthosiphon pisum), Arub (Asterias rubens), Cele (Caenorhabditis elegans), Cint (Ciona intestinalis), Ctel (Capitella teleta), Dmel (Drosophila melanogaster), Dpul (Daphnia pulex), Hsap (Homo sapiens), Lgig (Lottia gigantea), Lsta (Lymnaea stagnalis), Obim (Octopus bimaculoides), Ovul (Octopus vulgaris), Pcau (Priapulus caudatus), Pdum (Platynereis dumerilii), Skow (Saccoglossus kowalevskii), Spur (Strongylocentrotus purpuratus), Tcas (Tribolium castaneum), Tsui (Trichuris suis), Uuni (Urechis unicinctus). The accession numbers of the sequences included in this phylogenetic tree are listed in the supplementary Table 3.
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Figure 3. ArLQ acts as a ligand for two A. rubens G-protein coupled receptors, ArLQR1 and ArLQR2. The graphs show that ArLQ causes dose-dependent activation of ArLQR1 (A, red) and ArLQR2 (B, blue) expressed in CHO-K1 cells expressing the promiscuous Gα16 protein and a calcium-sensitive bioluminescent GFP-aequorin fusion protein (G5A). Each point represents mean values (± S.E.M.) from at least four independent experiments, with each experiment performed in triplicate. Control experiments where cells were transfected with an empty pcDNA 3.1(+) vector are shown in black. Luminescence is expressed as a percentage of the maximal response observed in each experiment. The EC50 values for activation of ArLQR1 and ArLQR2 with ArLQ are 2.4 × 10−8 M and 7.8 × 10−10 M, respectively.
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Figure 4. Localisation of ArLQP expression in the nervous system of A. rubens using mRNA in situ hybridisation. (A) Schematic showing the anatomy of the starfish arm as seen from a transverse section. (B) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. (C) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels (D) and (E). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. (F). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel (G). (H) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I. am, apical muscle; conr, circumoral nerve ring; cut, cuticle; ec, ectoneural region; g, gonads; hy, hyponeural region; mn, marginal nerve; pc, pyloric caeca; pm, peristomial membrane; rhs, radial hemal sinus; rnc, radial nerve cord. Scale bars: 50 μm in C, C inset, F, H; 10 μm in D, E, G, I.
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Figure 5. Localisation of ArLQP expression in the tube feet and stomach of A. rubens using mRNA in situ hybridisation. (A) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. (B) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. (C) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. (D) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in (E), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in (F). a, anus; amp, ampullae; bnr, basal nerve ring; conr, circumoral nerve ring; cs, cardiac stomach; g, gonad; gcc, general coelomic cavity; l, lumen; m, mouth; md, madreporite; o, ossicle; oa, organ axial; p, papillae; pc, pyloric caecum; pd, pyloric duct; pm, peristomial membrane; ps, pyloric stomach; rc, rectal caecum; rca, ring canal; rn, radial nerve; rw, radial water vascular canal; sa, sinus of axial organ; sc, stone canal; tb, Tiedemann’s bodies; tf, tube foot; tfd, tube foot disc. Scale bars: 50 μm in B, D; 20 μm in C; 10 μm in E and F.
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Figure 6. ArLQ causes relaxation of in vitro preparations of tube feet from A. rubens. (A) Representative recording of an experiment where ArLQ (1 µM) causes partial reversal of acetylcholine (ACh; 10 µM) induced contraction of an in vitro preparation of a tube foot from A. rubens. (B). Graphs showing the dose-dependent relaxing effect of ArLQ (red) on tube foot preparations in comparison with a known tube foot relaxant, the SALMFamide neuropeptide S2 (blue). Each point represents the mean ± S.E.M. from at least 6 different experiments, with the effect calculated as the percentage reversal of contraction induced by 10 μM ACh.
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Figure 7. Phylogenetic diagram showing the occurrence and characteristics luqin-type neuropeptide signalling in the Bilateria. The phylogenetic tree shows relationships of selected bilaterian phyla. Phyla in which luqin-type precursors and luqin-type receptors have been identified are labelled with purple-filled boxes. The number in the precursor box indicates how many mature luqin-like neuropeptides are derived from the precursor, with a hashtag indicating mixed features. The inclusion of an asterisk in the receptor boxes indicates that the peptide ligand that activates the receptor has been determined experimentally. Note that the starfish Asterias rubens is the first and only deuterostome in which the neuropeptide ligand for luqin-type receptors has been identified. Note also the loss of the luqin-type signalling system in the chordate lineage, which is signified by the X and the white-filled boxes. C-terminally aligned peptides that are predicted/proven ligands for luqin-type receptors in the species listed are shown on the right side of the figure, illustrating that peptides with a C-terminal RWamide motif occur in ambulacrarians, peptides with a C-terminal RFamide motif occur in lophotrochozoans and peptides with a C-terminal RYamide motif occur in ecdysozoans. Species names are as follows: Arub (Asterias rubens), Skow (Saccoglossus kowalevskii), Pdum (Platynereis dumerilii), Lsta (Lymnaea stagnalis), Pcau (Priapulus caudatus), Dmel (Drosophila melanogaster), Cele (Caenorhabditis elegans). Silhouettes of representative animals from each phylum were created by Maria Eugenia Guerra.
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