Ruthenium Ben

To search for transcripts encoding putative neuropeptide or peptide hormone precursor proteins in A. rubens, the sequences of neuropeptide or peptide hormone precursors previously identified in the sea urchin S. purpuratus [5 (link),6 (link),11 (link),16 (link),17 (link),37 (link),38 (link)], the sea cucumber A. japonicus [10 (link)] and the starfish species Asterina pectinifera [39 (link)] were submitted individually as queries in tBLASTn searches of the contig database with the BLAST parameter e-value set to 1000. Contigs identified as encoding putative precursors were analysed after translation of their full-length DNA sequence into protein sequence using the ExPASy Translate tool (http://web.expasy.org/translate/). Proteins were assessed as potential precursors of secreted bioactive peptides by investigating: (i) the presence of a putative N-terminal signal peptide sequence, using the SignalP v. 3.0 online server [40 (link)], (ii) the presence of putative monobasic or dibasic cleavage sites N-terminal and C-terminal to the putative bioactive peptide(s), with reference to known consensus cleavage motifs [41 (link)–43 (link)], and (iii) the presence, in some cases, of a C-terminal glycine residue that is a potential substrate for amidation.

Specimens of starfish (Asterias rubens) with a diameter > 4 cm were collected at low tide from the Thanet Coast (Kent, UK) or were obtained from a fisherman based at Whitstable (Kent, UK). The starfish were maintained in an aquarium with circulating seawater (salinity of 32 ‰) under a 12 h–12 h light–dark cycle (lights on at 8 a.m.) at a temperature of ~12 °C, located in the School of Biological & Behavioural Sciences at Queen Mary University of London. Animals were fed on mussels (Mytilus edulis) that were collected at low tide near Margate (Kent, UK). Additionally, juvenile specimens of A. rubens (diameter 0.5–1.5 cm) were collected from the University of Gothenburg Sven Lovén Centre for Marine Infrastructure (Kristineberg, Sweden).

Peptides corresponding to the C-terminal region of the A. rubens asterotocin precursor (ArASTP; KERLLDALLRQP; Fig. 1a), corazonin-type precursor (ArCRZP; KLLDNVRLPQTERK; Fig. 1b) and luqin-type precursor (ArLQP; KGKVPATA; Fig. 1c) were custom synthesized by Peptide Protein Research Ltd (Fareham, UK). Naturally occurring lysine residues at the N-terminus of ArASTP and ArCRZP peptide antigens and an introduced lysine at the N-terminus of the ArLQP peptide antigen, replacing a naturally occurring cysteine residue (Fig. 1c), facilitated glutaraldehyde-mediated coupling to porcine thyroglobulin (Sigma-Aldrich, Gillingham, UK) as a carrier protein, using 5% glutaraldehyde (Sigma-Aldrich, Gillingham, UK) in phosphate buffer (0.1 M; pH 7.2). Then, antigen peptide-thyroglobulin conjugates were used for the immunisation of one rabbit per antigen peptide (70-day protocol; Charles River Biologics, Romans, France). Conjugates were emulsified in Freund’s complete adjuvant for primary immunisations (~100 nmol antigen peptide) and in Freund’s incomplete adjuvant for three booster immunisations (~50 nmol antigen peptide). The presence of antibodies to the antigen peptides in post-immunisation serum samples was assessed using an enzyme-linked immunosorbent assay (ELISA; see below), in comparison with pre-immune serum.Fig. 1

Amino acid sequences of a vasopressin/oxytocin-type (asterotocin) precursor (ArASTP), b corazonin-type precursor (ArCRZP) and c luqin-type precursor (ArLQP) in Asterias rubens. Predicted signal peptides are shown in blue, neuropeptides are shown in red but with C-terminal glycine residues that are substrates for amidation shown in orange, dibasic cleavage sites are shown in green and the neurophysin domain of ArASTP is shown in pink. The cysteine residues in ArASTP that form a disulphide bridge in the mature neuropeptide are underlined. The sequences of the C-terminal peptides that were used as antigens for antibody production are shown in bold yellow. Note, however, that the underlined cysteine residue in ArLQP was replaced with a lysine residue at the N-terminus of the antigen peptide to provide reactive sites for glutaraldehyde-mediated coupling to a carrier protein (thyroglobulin). GenBank Accession numbers: a ALJ99953.1 (Semmens et al. 2016 (link); Odekunle et al. 2019 (link)); b ALJ99955.1 (Semmens et al. 2016 (link); Tian et al. 2017 (link), 2016 (link)); c ALJ99961.1 (Semmens et al. 2016 (link); Yañez-Guerra et al. 2018 (link))

Antibodies to the antigen peptides were purified from the final bleed antiserum by affinity purification using the AminoLink Plus Immobilization Kit (Thermo Fisher Scientific, Waltham, MA), with bound antibodies eluted using glycine elution buffer (6.3 ml of 100 mM glycine [VWR Chemicals, Leicestershire, UK] and 0.7 ml of Tris [1 M, pH = 7.0]) and trimethylamine (TEA) elution buffer (6.3 ml of TEA [Sigma-Aldrich, Gillingham, UK] and 0.7 ml of Tris [1 M, pH = 7.0]). Eluates were dialysed and sodium azide (0.1%) was added for long-term storage of the affinity-purified antibodies at 4 °C. ArASTP, ArCRZP and ArLQP antibodies eluted with TEA were diluted in 5% normal goat serum (NGS; Sigma-Aldrich, Gillingham, UK)/PBST (phosphate-buffered saline containing 0.1% Tween-20) at 1:20, 1:15 and 1:15, respectively, and then used for immunohistochemistry (see below). The rabbit antisera to ArASTP, ArCRZP and ArLQP have been assigned the RRID numbers RRID:AB_2922389, RRID:AB_2922390 and RRID:AB_2922391, respectively.

Specimens of A. rubens were fixed by immersion in seawater Bouin’s fluid (75% saturated picric acid [Sigma-Aldrich, Gillingham, UK] in seawater, 25% formaldehyde, 5% acetic acid) for 3–4 days at 4 °C and then were decalcified for a week using a 2% ascorbic acid/0.3 M sodium chloride solution. Following dehydration and embedding in paraffin wax, sections of arms and the central disk region (8–10 μm; transverse or horizontal) were cut using a microtome (RM 2145, Leica Microsystems [UK], Milton Keynes, UK) and mounted on chrome alum/gelatin-coated microscope slides. Paraffin wax was removed by immersion of slides in xylene, and then slides were immersed in 100% ethanol. Endogenous peroxidase activity was quenched using a 0.3% hydrogen peroxide (VWT Chemicals, Leicestershire, UK)/methanol solution for 30 min. Subsequently, the slides were rehydrated through a graded ethanol series (90%, 70%, and 50%) and distilled water, blocked in 5% NGS made up in PBST.
Following preliminary tests in which antisera were tested at a range of concentrations, the specificity of immunostaining was assessed by testing antisera alongside antisera pre-absorbed with the corresponding antigen peptide. For these experiments, the ArASTP, ArCRZP and ArLQP antisera were tested at dilutions of 1:4000, 1:1000 and 1:4000 in PBS, respectively. For pre-absorption, the ArASTP, ArCRZP and ArLQP antisera were first prepared at dilutions of 1:400, 1:100 and 1:400 in PBS, respectively, and then were incubated with the corresponding antigen peptide at a concentration of 200 µM for 2 h at room temperature. Then, the pre-absorbed antisera were further diluted 1:10 in 5% NGS/PBST so that they were tested on starfish sections at the same final concentrations as the antisera without pre-absorption. After overnight incubation of slides with antisera or pre-absorbed antisera followed by a series of washes in PBST, indirect immunohistochemical detection was carried out using Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H + L) conjugated to Horseradish Peroxidase (RRID: AB_2313567; Jackson ImmunoResearch, West Grove, PA) diluted 1:1000 in 2% NGS/PBST. Bound antibodies were revealed using a solution containing 0.015% hydrogen peroxide, 0.05% diaminobenzidine (VWR Chemicals, Leicestershire, UK) and 0.05% nickel chloride (Sigma-Aldrich, Gillingham, UK) in PBS. When strong staining was observed, sections were washed in distilled water, dehydrated through a graded ethanol series (50%, 70%, 90% and 100%) and washed in xylene before being mounted with coverslips on DPX mounting medium (Thermo Fisher Scientific, Waltham, MA).
Having investigated the specificity of immunostaining with antisera, a more extensive immunohistochemical analysis was performed using the same methods as described above, but employing the use of affinity-purified antibodies to ArASTP, ArLQP and ArCRZP (TEA fraction diluted 1:20, 1:15 and 1:15, respectively, in 5% NGS/PBST). For experiments with ArASTP antibodies, adjacent sections were incubated with affinity-purified rabbit antibodies to asterotocin (TEA fraction diluted 1:15 in 5% NGS/PBST), which have been reported previously (Odekunle et al. 2019 (link)).