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Figure 1.
A. rubens relaxinâ€like gonadâ€stimulating peptide (AruRGP) precursor and comparison with RGP precursors from other starfish species. A: The cDNA sequence (lowercase, 560 bases) encoding the AruRGP precursor protein (uppercase, 109 amino acid residues) is shown. The predicted signal peptide is shown in blue and predicted dibasic cleavage sites are shown in green. Relaxinâ€type peptides (with cysteine [C] residues underlined) are shown in red, with the A and B chains highlighted in green and blue, respectively. Nucleotides and amino acids that differ from the previously reported AruRGP precursor cDNA and protein sequence that was assembled from transcriptome sequence data (GenBank accession number KT601728; Semmens et al., 2016) are highlighted in gray. Nucleotide sequences that were used as primers for cDNA cloning are shown in red and the asterisk shows the position of the stop codon. B: Alignment of the AruRGP precursor with RGP precursors from A. amurensis (AamRGP, GenBank accession number LC040882), A. japonica (AjaRGP, GenBank accession number LC104980), and P. pectinifera (PpeRGP, GenBank accession number AB496611). Regions of the precursors corresponding to the A and B chains are labeled (green and blue, respectively) and amino acid residues that are identical in all four precursors are highlighted in yellow.
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Figure 2. Neighbor joining tree showing the relationships of starfish relaxinâ€like gonadâ€stimulating peptide precursors with precursors of other members of the relaxin/insulin/insulinâ€like growth factor (IGF) peptide family. The A. rubens RGP (AruRGP) precursor (blue arrow) and other starfish RGP precursors form a distinct clade within the relaxin/insulinâ€like precursor family, which is highlighted in pink to distinguish it from the insulin/IGF precursor family that is highlighted in purple. A second A. rubens relaxinâ€type precursor (AruRLP2; green arrow) is a paralog of the AruRGP precursor that is also positioned within the relaxin/insulinâ€like clade of precursors. The full names and accession numbers of the 36 protein sequences included in the tree are as follows: AruRGP, relaxinâ€like gonadâ€stimulating peptide (ALJ99970.1, Asterias rubens); AamRGP, relaxinâ€like gonadâ€stimulating peptide precursor (BAR40315.1, Asterias amurensis); AjaRGP, relaxinâ€like gonadâ€stimulating peptide precursor (BAU20369.1, Aphelasterias japonica); PpeRGP, relaxinâ€like gonadâ€stimulating peptide precursor (BAI44654.1, Patiria pectinifera); AruRLP2, relaxinâ€like peptide precursor 2 (ALJ99971.1, Asterias rubens); AplRGP, relaxinâ€like gonadâ€stimulating peptide precursor (LC033566.1, Acanthaster planci); PmiRGP, relaxinâ€like gonadâ€stimulating peptide precursor (LC057656.1, Patiria miniata); RLN1 Human, relaxin 1 precursor (NP_008842.1, Homo sapiens); RLN2 Human, relaxin 2 precursor (NP_604390.1, Homo sapiens); RLN3 Human, relaxin 3 precursor (NP_543140.1, Homo sapiens); RLN1 Mouse, relaxin 1 precursor (NP_035402.2, Mus musculus); RLN3 Mouse, relaxin 3 precursor (NP_775276.1, Mus musculus); RLN3 Alligator, relaxin 3 precursor (XP_006023546.1, Alligator sinensis); INSL3 Alligator, insulinâ€like 3 (XP_006017481.1, Alligator sinensis); RLN3a Zebrafish, relaxin 3a precursor (NP_001032892.1, Danio rerio); RLN3b Zebrafish, relaxin 3b precursor (NP_001108535.1, Danio rerio); RLN3c Zebrafish, relaxin 3c precursor (NP_001108525.2, Danio rerio); INS Human, insulin precursor (NP_000198.1, Homo sapiens); INSL3 Human, insulinâ€like peptide 3 precursor, (NP_005534.2, Homo sapiens); INSL4 Human, insulinâ€like peptide 4 precursor (NP_002186.1, Homo sapiens); INSL5 Human, insulinâ€like peptide 5 precursor (NP_005469.2, Homo sapiens); INSL6 Human, insulinâ€like peptide 6 precursor (NP_009110.2, Homo sapiens); INSL3 Mouse, insulinâ€like 3 precursor (NP_038592.3, Mus musculus); INSL5 Mouse, insulinâ€like peptide precursor (NP_035961.1, Mus musculus); INSL6 Mouse, insulinâ€like peptide precursor (NP_038782.1, Mus musculus); INSL5 Zebrafish, insulinâ€like 5 precursor (NP_001122028.1, Danio rerio); INSL3 Deer, relaxinâ€like peptide (AAR25542.1, Capreolus capreolus); IGF1a Human, insulinâ€like growth factor 1 precursor (NP_000609.1, Homo sapiens); IGF2 Human, insulinâ€like growth factor 2 precursor (NP_000603.1, Homo sapiens); IGF1 Mouse, insulinâ€like growth factor 1 precursor (NP_001104745.1, Mus musculus); IGF2 Mouse, insulinâ€like growth factor 2 precursor (NP_034644.2, Mus musculus); INS1 Mouse, insulinâ€1 precursor (NP_032412.3, Mus musculus); INS2 Mouse, insulinâ€2 precursor (NP_032413.1, Mus musculus); RLNL BRAFL, relaxinâ€like peptide (EEA41967.1, Branchiostoma floridae); ILPl BRAFL, insulinâ€like peptide 1 precursor ((Mita et al., 2009b), Branchiostoma floridae); ILP2 BRAFL, insulinâ€like peptide 2 precursor ((Mita et al., 2009b), Branchiostoma floridae). Bootstrap values for selected nodes are shown.
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Figure 3. Mass spectrometric identification of AruRGP A chain and B chain in extracts of A. rubens radial nerve cords. A: Predicted dimeric structure of AruRGP, showing the sequences of the A chain and B chain. The positions of disulfide bridges are shown with red lines and tryptic cleavage sites are marked with arrowheads. B,C: MS/MS data for the A chain and B chain, respectively, from reduced and alkylated samples of radial nerve extract without tryptic digestion. The b series of peptide fragment ions are shown in red, the y series in blue and additional identified peptide fragment ions in green. The amino acid sequence identified in the mass spectrum is highlighted at the top of the figures. C+57 represents cysteine modified by carbamidomethylation and M+16 represents oxidized methionine. The observed m/z of the precursor ion for the A chain (PETYVGMGSYCCLVGCTRDQLSQVC; B) is 980.75 with a charge state 3 + and an error of 0.41 ppm between the experimentally determined and predicted values (Mascot score = 57). The observed m/z of the precursor ion for the B chain (AEKYCDEDFHMAVYRTCTEH; C) is 860.02 with a charge state of 3 + and an error of 0.65 ppm between the experimentally determined and predicted values (Mascot score = 31). D,E: MS/MS data for the complete sequences of fragments of the A chain and B chains, respectively, derived from reduced and alkylated samples of radial nerve extract subjected to tryptic digestion, with annotations in the same format as in B and C. The observed m/z of the precursor ion for the A chain fragment (PETYVGMGSYCCLVGCTR; D) is 1055.44 with a charge state of 2 + and an error of –4.7 ppm between the experimentally determined and predicted values (Mascot score = 98). The observed m/z of the precursor ion for the B chain fragment (YCDEDFHMAVYR; E) is 541.22 with a charge state of 3 + and an error of –0.83 ppm between the experimentally determined value and predicted value (Mascot score = 45).
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Figure 4. Mass spectrometric identification of a dimeric fragment of AruRGP in an extract of A. rubens radial nerve cords. The mass spectrum of a disulfide bridge linked dimeric peptide comprising DQLSQVC from the AruRGP A chain and TCTEH from the AruRGP B chain is shown. This dimeric peptide was detected in samples of radial nerve extract that were subjected to tryptic digestion without reduction. Peptide fragments from the A chain are shown in green and peptide fragments from the B chain are shown in blue. The observed m/z of the precursor ion is 690.28 with a charge state 2 + and an error of –0.73 ppm between the experimentally determined value and predicted value (Stavrox score = 145).
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Figure 5. Comparison of the in vitro bioactivity of AruRGP and PpeRGP as inducers of spawning in A. rubens. A: Isolated ovary from A. rubens. B: AruRGPâ€induced spawning of an ovary fragment from A. rubens. C: Graph showing the doseâ€dependent effects of AruRGP (•) and PpeRGP (â–´) in causing spawning of ovarian fragments. + + + denotes spawning occurred and most of oocytes were matured, + + denotes about 50% oocytes were matured, + denotes a few oocytes were matured, and – denotes no spawning occurred. Means ± SEM for five separate assays using ovarian tissue from different animals are shown. The median effective concentration (EC50) of AruRGP required to induce spawning (1.33 ± 0.09 nM) is approximately 10â€fold lower than for PpeRGP (14 ± 1 nM).
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Figure 6. Localization of AruRGP precursor mRNA in the radial nerve cord and circumoral nerve ring of A. rubens using in situ hybridization. A,B: Transverse sections of radial nerve cord incubated with antisense probes (main panels of A and B) showing a bilaterally symmetrical group of 2–3 stained cells (arrowheads) in the epithelium of the ectoneural region of the nerve cord. Panel B shows a highâ€magnification view of the rectangular region highlighted in panel A. The inset of panel A shows the absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. C,D: Longitudinal parasagittal sections of the radial nerve cords incubated with antisense probes showing groups of cells interspersed along the length of the nerve cord in the ectoneural epithelium. Panel D shows a highâ€magnification view of the rectangular region highlighted in panel C. E,F: Transverse section of the disk region in A. rubens incubated with antisense probes, showing the circumoral nerve ring and tube feet. Stained cells can be seen in the ectoneural epithelium of the nerve cord, highlighted by the rectangle in E and shown at higher magnification in F. CONR, circumoral nerve ring; Ec, ectoneural region of radial nerve cord; Hy, hyponeural region of radial nerve cord; TF, tube foot. Scale bars = 50 μm in A and A inset; 10 μm in B,D; 25 μm in C; 200 μm in E; 20 μm in F.
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Figure 7. Localization of AruRGP precursor mRNA in tube feet of A. rubens using in situ hybridization. A: Longitudinal section of a tube foot showing three stained cells (arrowheads and rectangle) in the subepithelial layer of the podium. B: The region highlighted with a rectangle in A is shown here at higher magnification, with a stained cell located between the external epithelium and connective tissue layer. C: Stained cells (arrowheads) located in the subepithelial layer near to the base of adjacent tube feet. D: A group of stained cells (see rectangle) in the tube foot subepithelial layer just above the sucker. E: The region highlighted with a rectangle in D is shown here at higher magnification. CL, connective tissue layer; Ep, epithelium; ML, muscle layer; Su, sucker TF: tube foot. Scale bars = 100 μm in A; 10 μm in B; 25 μm in C; 50 μm in D; 10 μm in E.
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Figure 8. Localization of AruRGP precursor mRNA in the arm tips of A. rubens using in situ hybridization. A: Photograph of a living specimen of A. rubens showing the arm tip region viewed from the underside (oral) of the animal, taken using a Leica DFC420 C camera linked to a Leica S8 APO microscope. The most prominent feature is the pigmented optic cushion, which is located at the base of the terminal tentacle. The terminal tentacle and optic cushion are bounded on each side by spines and rows of tube feet can be seen adjacent to the optic cushion. B: Section of the arm tip showing the pigmented optic cushion and terminal tentacle. Stained cells expressing AruRGP precursor transcripts (arrowheads) can be seen in the body wall epithelium lining a cavity that surrounds the terminal tentacle and optic cushion. C: Section of an arm tip showing the terminal tentacle cut obliquely. Stained cells can be seen in the terminal tentacle (rectangle and arrowheads) and in the body wall epithelium at the base of the spines that surround the terminal tentacle (arrowheads). D: Detail of the region highlighted with a rectangle in panel C, showing stained cells (arrowhead) in the subepithelial layer of the terminal tentacle. E: Section through the distal region of the arm tip beyond the terminal tentacle, showing stained cells (arrowheads and rectangle) in the body wall epithelium at the base of two adjacent spines; the region highlighted with a rectangle is shown in panel F. The inset shows absence of staining (arrowhead) in a section of the arm tip adjacent to the section shown in the main panel and which was incubated with sense probes instead of the antisense probes used in the main panel E. F: Detail of the region highlighted with a rectangle in panel E, showing stained cells with processes (arrowheads) at high magnification. CL, connective tissue layer of terminal tentacle; Ep, epithelium of body wall; ML, muscle layer of terminal tentacle; OC, optic cushion; TF, tube foot; Sp, spine; TT, terminal tentacle. Scale bars = 400 μm in A; 100 μm in B, E inset; 50 μm in C,E; 10 μm in D,F.
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Figure 9. Neuronâ€like characteristics of cells expressing AruRGP in the arm tips of A. rubens. A: Transverse section of A. rubens arm tip showing two cells in the body wall epithelium that express the AruRGP precursor, as revealed by mRNA in situ hybridization, and that have stained axonâ€like processes (arrowheads). B: Longitudinal section of A. rubens arm tip showing a cell in the body wall epithelium that expresses the AruRGP precursor, as revealed by mRNA in situ hybridization, and that has a stained axonâ€like process (arrowhead). C,D: Transverse sections of A. rubens arm tip showing cells expressing AruRGP precursor transcripts, as revealed by mRNA in situ hybridization, in the body wall epithelium lining the cavity that contains the terminal tentacle (TT). E,F: Transverse sections of A. rubens arm tip adjacent to the sections shown in panels C and D, respectively, showing that the unstained region in panels C and D underlying the AruRGP expressing cells (see asterisks in panels C and D) is immunoreactive with monoclonal antibodies (1E11) to the axonal protein synaptotagmin B. This provides supporting evidence that the AruRGP expressing cells in the arm tip epithelium are neurons. Scale bars = 5 μm in A,B; 20 μm in C,E; 10 μm in D,F.
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