Journal 
Introduction
Calcitonin (CT) was first discovered in 1962 as a hormone that lowers blood calcium concentrations in mammals (Copp et al., 1962). CT is a peptide hormone consisting of 32 amino acids with an amidated COOHerminus and two Cys residues in the NH2-terminal region forming a disulfide bond (Neher et al., 1968;Potts et al., 1968). The hypocalcaemic response is mainly due to the potent inhibitory effect of CT on osteoclast-mediated bone resorption, leading to its widespread use in the treatment of bone diseases including Paget's disease, osteoporosis, and malignant hypercalcaemia (Sexton et al., 1999). In vertebrates, CT polypeptide in thyroid CT-producing cells was characterized as a 1050 nucleotide precursor, approximately 15,000 dalton mature peptide, intramolecular disulfide bonds, cyclic ring structure and recognition of dibasic amino acids (Jacobs et al., 1979;Amara et al., 1980). In non-mammalian vertebrates, CT orthologous peptides are synthesized in the ultimobranchial gland tissues of amphibians and light-bodied fish and are known to regulate calcium homeostasis, but detailed information on the role of CT is lacking (Niall et al., 1969;Mukherjee et al., 2004;Lafont et al., 2007).
Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide with a well conserved two Cys residues in the NH2-terminal region similar to CT and is distributed in a wide range of tissues and nerve fibers throughout the mammalian body where it plays an important role in nociception and afferent nervous system function (Russell et al., 2014). In addition to the nervous system, CGRP has recently been shown to affect a variety of tissues, including the cardiovascular, respiratory, gastrointestinal, immune and reproductive systems, and to regulate the metabolism of muscle and adipose tissue (Russo et al., 2023). In non-mammalian vertebrates, it is thought to be involved in osmotic regulation and the central and peripheral nervous systems, mainly in amphibians and light-bodied fish, but its detailed physiological functions are unclear (Martinez Garcia et al., 2002;Suzuki et al., 2002). In addition to CGRP, the CT-like peptides amylin, adrenomedullin, intermedin and CT receptor stimulating peptide are known, all of which share a well-conserved disulfide bond structure for linking Cys residues in the NH2-terminal region and a CT-specific signaling system (Cai et al., 2018;Sekiguchi, 2018;Russo et al., 2023).
The evolutionary origin of CT-type peptides in invertebrates is thought to be the common ancestor of bilaterians of both protostomes and deuterostomes, and a natriuretic peptide lacking the NH2-terminal Cys residue (DH31), consisting of 31 amino acid residues isolated from some insect species of the phylum Arthropoda, is classified as a CT-like peptide (Cai et al., 2018). Furthermore, the two types of peptides, CT-type and DH31-type, are thought to be homologous due to gene duplication in the ancestral animal (Conzelmann et al., 2013).
While CT and CGRP receptors have been relatively well studied in vertebrates, information on related receptors in invertebrates, especially mollusks, is very scarce (Hay et al., 2018;Sekiguchi, 2018). To date, studies have been carried out on the identification and activation of CT-type peptides and their receptors in bivalve river oysters (Crassostrea gigas) and Mediterranean mussels (Mytilus galloprovincialis), among the phylum of marine mollusks (Schwartz et al., 2019;Cardoso et al., 2020). On the other hand, some molecular biological information on the CT-type peptide signaling system of the giant clam (Lottia gigantea), which has the highest species diversity among the molluscan phylum, was revealed during genomic and transcriptomic analyses (Simakov et al., 2013). In order to understand the physiological and biochemical properties mediated by CT-type peptides in the gastropod abalone, an important marine fishery resource, this study aimed to isolate and identify CT-type peptide precursors and their receptors in Pacific abalone (Haliotis discus hannai, Hdh), and to obtain basic information on tissue-specific gene expression and receptor activation.
Materials and Methods
1. Sequence analysis
Transcripts encoding Hdh-CT precursor and Hdh-CT receptors (Hdh-CTRs) were identified in Pacific abalone Hdh transcriptome databases (Kim et al., 2017,2019) using the tBLASTn algorithm to search the databases of NCBI and UniProtKB/SwissProt. The amino acid sequences of Hdh-CT precursor and Hdh-CTRs were comparatively analyzed using the tBLASTn and MultAlin multiple sequence alignment (Corpet, 1988), then aligned using CLC Genomics Workbench. To generate a phylogenetic tree, the amino acid sequences of the receptors for CT and CGRP were aligned using MUSCLE and subsequently trimmed using the trimAl program with automated selection of parameters (https://ngphylogeny.fr). The trimmed sequences were used to construct a maximum likelihood tree using the W-IQ tree server 1.6.12.
2. Reverse transcription and cDNA cloning
The cerebral ganglion (CG) and pleuro-pedal ganglion (PPG) of Pacific abalone (7.8 cm shell length; 60.2 g body weight, BW) were dissected, immediately frozen in liquid nitrogen and stored at -80°C, until total RNA extraction. Total RNA was extracted using a Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA). Reverse transcription for cDNA synthesis was performed using the PrimeScrip RT reagent kit with gDNA eraser (Takara Bio, Shiga, Japan). Reverse transcription was performed as follows: total RNA (1 μg) was mixed with 5X gDNA eraser buffer (2 μl) and gDNA eraser (1 μl), and reacted at 42°C for 2 min. cDNA synthesis was done with 5X PrimeScrip buffer 2 (4 μl), PrimeScrip reverse transcriptase mix I (1 μl), RT Prime Mix (1 μl), and RNase free dH2O (4 μl) at 37°C for 15 min, followed by incubation at 85°C for 5 s to inactivate the reverse transcriptase.
Forward and reverse primers (Table 1) were designed to clone the sequences of two putative Hdh-CTRs (namely Hdh-CTR-L for long isoform and Hdh-CTR-S for short isoform). PCR was performed using cDNAs derived from the CG and PPG, Primestar HS DNA polymerase (TaKaRa Bio), dNTPs, and 5X Primestar buffer. The PCR cycling conditions were as follows: 98°C for 5 min; 35 cycles of 98°C for 30 s, 52°C for 30 s, and 72°C for 95 s; then 72°C for 5 min. PCR products were digested using MunI and XbaI, then cloned into EcoRI and XbaI restriction enzyme sites of the HA-tagged pcDNA3 expression vector (Invitrogen, Waltham, MA, USA). All constructs were verified using Sanger sequencing.
3. Real-time quantitative PCR (qPCR)
Mature abalone (73.9 ± 0.9 g BW; n = 5, each sex) were purchased from a local dealer (Gangneung, Gangwon-do, Korea). Neural tissues, CG and PPG, and ovary, testis, gills, intestine, hepatopancreas, adduct muscle, and mantle were dissected, immediately frozen in liquid nitrogen, then stored at -80°C. RNA extraction and reverse transcription for cDNA synthesis were performed as described in the earlier section of Reverse transcription and cDNA cloning. The cDNA (5 ng each) was mixed with TB Green Premix Ex Taq II (10 μl), 50× ROX Reference Dye II (0.4 μl), RNase Free dH2O (6 μl), and RT reaction solution (2 μl, Takara Bio). PCR was performed using a primer set (10 μM) designed by Express v3.0 software (Applied Biosystems, Boston, MA, USA). The ribosomal protein L5 gene (Hdh-RPL5) was used as a reference gene as previously described (Yoon et al., 2022). The gene-specific primers are listed in Table 1. SYBR-based qPCR was performed using the Applied Biosystems 7500 using the following reaction conditions: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
4. Luciferase reporter assay
HEK293 cells were grown in monolayer culture in Dulbecco’s modified Eagle medium (DMEM; Gibco, Loughborough, UK) with 10% fetal bovine serum (FBS; HyClone, GE Healthcare, Chicago, IL, USA) and seeded in 24-well plates (5 × 104 cells/well). After 24 h, pcDNA3-HA plasmid containing Hdh-CTR-S or Hdh-CTR-L cDNA, cAMP response element reporter (CRE-luc), and pRSV-β-galactosidase expression plasmid (100 ng each) were transfected into the cells as previously described (Yoon et al., 2022). After 4 h, the medium in the wells was removed and FBS-free DMEM was added. After 16 h, the cells were treated with Hdh-CT peptides or forskolin (Sigma-Aldrich, St. Louis, MO, USA); or the same volume of peptide-free DMEM for 6 h. The cells were harvested with a cell lysis buffer (Promega, Madison, WI, USA) and luciferase reporter assays were performed using a luminometer (Centro LB 960; Berthold, Bad Wildbad, Germany) and a microplate reader (Tecan Sunrise; Tecan, Mannedorf, Switzerland).
5. In silico docking model
Homology modeling of the Hdh-CTR-L structure was performed using SWISS-MODEL (https://swissmodel. xpasy.org). Peptide docking was performed using the HPEPDOCK server (http://huanglab.phys.hust.edu.cn/ pepdock/) to predict the binding mode of the Hdh-CT1 peptide in the Hdh-CTR-L model structure. The 3D structure of Hdh-CTR-L with Hdh-CT1 was visualized using PyMOL (http://www.pymol.org/).
6. Statistical analysis
Analyses were performed with SPSS software (version 25.0; SPSS Inc., Chicago, IL, USA). Significance was determined using one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. Differences were considered statistically significant at P values < 0.05.
Results
1. Sequence analysis of CT peptides and CTRs
A BLAST search of the Hdh transcriptome database identified of two transcripts coding for the precursors of Hdh-CTs. Alignment of Hdh-CTs with CT and CGRP orthologs from other species exhibited the typical two Cys residues forming a disulfide bond in their N-terminal regions common to CT-type peptides (Fig. 1). In protostome CTs, there were highly conserved features, two Cys spacing patterns, CX7C or CX8C, and hydrophobic residues in the 2nd and 4th residues after the first Cys. These CT-type peptides are similar in length at 31 or 32 amino acids, except for oyster CT1a and human CGRP.
Two CTR isoforms, Hdh-CTR-S and Hdh-CTR-L, were identified in the Hdh transcriptome databases. Comparison of Hdh-CTR isoforms with other molluscan CTR orthologs revealed high homology of transmembrane domains with variable extracellular N-terminal residues and intracellular C-terminal residues (Fig. 2). A phylogenetic analysis revealed that gastropod CTRs including Hdh-CTRs are positioned in a large molluscan CTR subfamily with a bootstrap support of 96% (Fig. 3). In particular, Hdh-CTRs showed a sister relationship with the CTR subfamily members of bivalves.
2. mRNA expression of Hdh-CT precursors
Precursor mRNAs of CT peptide was analyzed in the neural tissues, CG and PPG, and the peripheral tissues, ovary, testis, gills, intestine, hepatopancreas, adduct muscle, and mantle in mature abalone. In general, qPCR analyses of the Hdh-CT1 and -CT2 precursors revealed significantly higher levels of the transcripts in the CG and PPG than in the other examined tissues in mature female and male abalone (Fig. 4A, B). Relatively higher expression levels were measured in the hepatopancreas of males, although there were no significant differences between hepatopancreas and other peripheral tissues.
3. Reporter assay
The CRE-luc reporter activities were measured in Hdh-CTR-L and Hdh-CTR-S-transfected HEK293 cells. Treatments of Hdh-CT1 significantly activated the reporter in Hdh-CTR-L-transfected cells (Fig. 5A). However, Hdh-CT2 could not significantly alter basic reporter activities both Hdh-CTRs (Fig. 5A, B). The response of Hdh-CT1 was specific for Hdh-CTR-L, because HEK293 cells transfected with maternal plasmid pcDNA3-HA did not respond to Hdh-CT1 treatment (Fig. 5C).
4. In silico docking model
A docking simulation was performed to predict the binding mode of the Hdh-CT1 peptide, and critical interactions were estimated in the Hdh-CTR-L binding pocket. The Hdh-CT1 docking model showed that the N-terminal residues in Hdh-CT1 were deeply inserted inside the binding pocket of Hdh-CTR-L and two hydrogen bonds between Thr14/Glu14 of Hdh-CT1 and Thr163/Tyr419 of Hdh-CTR-L (Fig. 6). The docking score of Hdh-CT1 and Hdh-CTR-L was −244.3 Rosetta energy unit (REU).
Discussion
In this study, we compared the amino acid sequences of Pacific abalone CT-1 and CT-2 with multiple CT and CT-like peptides in protostomes and deuterostomes. We also compared the mRNA expression of CT precursors in different tissues of Pacific abalone. Using luciferase reporter assays, we determined the reactivity of CT peptides for CT receptors in heterologous HEK293 cells. In addition, we showed in silico docking model between CT and CT receptor.
Sequence comparisons suggested that Hdh-CT-1 and Hdh-CT-2 have conserved N-terminal characteristic cysteines (C2-C10) in the CT/CGRP peptide family members (Cai et al., 2018;Sekiguchi, 2018). In accordance to the Hdh-CT peptides, two CT precursors of oyster containing two CT-type peptides and encoded by two distinct genes with a similar organization exhibited the two N-terminal paired cysteine residues and, except a CT2-derived peptide, the C-terminal proline-amide motif typical of deuterostome CT-type peptides (Schwartz et al., 2019). In addition, sequence alignment of Hdh-CTRs demonstrated that the CTRs have conserved 6 Cys residues at the N-terminal regions. This N-terminal conserved intradomain and putative disulfide-bonded cysteines within the receptor are similar to the general structure of the extracellular juxtamembranous regions of the class B GPCRs (Dong et al., 2014). In mollusks, CTR number was variable and arose through lineage and species-specific duplications (Cardoso et al., 2020). In this study, we have found two CTR isoforms, Hdh-CTR-L and Hdh-CTR-S, although the short isoform Hdh-CTR-S could not show significant activation in a reporter assay. Similar to this result, a previous study demonstrated that three endogenous CTRs have shown specific responses for three CT peptides in the Pacific oysters (Schwartz et al., 2019). Molecular phylogenetic analysis showed that Hdh-CTRs are members of a gastropod CTR clade in a large molluscan CTR family with a bivalve CTR clade. The phylogenetic analysis clearly shows that vertebrate CTRs cluster separately from protostome CTR sequences. Within the CTR cluster, the arrangement of the lophotrochozoan members suggested that two main receptor sub-clusters (two types) exist: CTR-type I and CTR-type II and that they have emerged from a specific gene duplication event early in this lineage (Cardoso et al., 2020). Together, it is suggested that additional types of Hdh-CTR are present in Pacific abalone.
Tissue-specific expression of CT-1 and CT-2 precursor mRNAs in abalone was analyzed using RT-qPCR and found to be significantly higher in the neural ganglia than in other examined tissues. In the Pacific oyster, CT precursor mRNA expression was also higher levels in the neural ganglia (Schwartz et al., 2019). In bivalves, CT precursors are most likely coupled to the regulation of calcium transport and osmotic metabolism in common with vertebrates, probably to build their shell (Schwartz et al., 2019;Cardoso et al., 2020). Considering the similar shell formation between gastropods and bivalves (McDougall and Degnan, 2018), mechanism by which Hdh-CT peptides regulate calcium movements requires investigations to understand the coupling with ion transporters and shell matrix protein secretion in abalone. Both Hdh-CT precursors tended to higher levels in hepatopancreas of male abalone, although there are no significant differences between hepatopancreas and other peripheral tissues. Since basal CT levels of males are higher than females in mammals and the CT family members are expressed in digestive glands including hepatopancreas in oysters (Kiriakopoulos et al., 2022;Schwartz et al., 2019), It would be interesting to further investigate the relationship between CT expression and gender.
The Gs protein linked to GPCR activated by ligand binding activates adenylate cyclase, which converts ATP to cAMP, increasing the intracellular concentration of cAMP and activating CRE-luc (Cheng et al., 2010). In this study using CRE-luc, we demonstrated that the Hdh-CT1 peptide activates Hdh-CTR-L in HEK293 cells, suggesting that the Hdh-CT1 system is coupled to cAMP/Gs/PKA signaling pathway. The short isoform Hdh-CTR-S had a truncated intracellular domain compared to the Hdh-CTR-L, suggesting the importance of Gs protein-interacting domain in the Hdh-CTRs. The oyster CT peptides, Cragi-CT1b and Cragi-CT2, specifically and exclusively activated two distinct CTRs through the cAMP and phosphoinositide transduction pathways (Schwartz et al., 2019), although cAMP/Gs/PKA signaling represents the most common pathway to mediate the CT/CGRP/DH31 peptides (Iga and Kataoka, 2015). The Mediterranean mussel (M. galloprovincialis) CTRs were also stimulated by the endogenous CT peptides via the cAMP and phosphoinositide transduction pathways (Cardoso et al., 2020). Considering that Hdh-CT2 could not activate CTRs, it will be interesting to elucidate a Hdh-CT2-specific CTR in Pacific abalone.
In silico docking simulation of Hdh-CTR-L with Hdh-CT1 suggested that the main interactions are likely to be with the N-terminal amino acids of Hdh-CT1 and the extracellular loops of Hdh-CTR-L. Although it is difficult to predict the contact points on the receptor, the Hdh-CT1 docking model showed two polar interactions between the N-terminal residues of Hdh-CT1 and the Hdh-CTR-L’s binding pocket. In human and rodents, the N-terminal residues 8–18 of CGRP are likely to contact the CGRP receptor in a pocket formed by extracellular loops (Barwell et al., 2012). Highly conserved Thr12 residue of Hdh-CT1 contacts with Thr163 of N-terminal extracellular loop via a hydrogen bond. Interestingly, mutation of Thr9 in human CGRP (corresponding to Thr12 in Hdh-CT1) resulted in decrease in affinity (Barwell et al., 2012). These observations suggest that the tertiary structure with a disulfide bond and conserved residues in CT-type peptides are important for activation of CTRs in bilaterians.
In summary, we identified CT signaling system in Pacific abalone and this study will be used as information to reveal the physiological function of the CT signals in gastropods.
