The N-terminal cleavage peptide of sardine procalcitonin has different effects from calcitonin and promotes the activity of osteoblasts in goldfish scales (2023)

Bond 211,

September 2017

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heNucleotide sequenceIn this study, the content of procalcitonin precursors in sardines has been determined from their terminal branchial glands. From our analysis of this sequence, we found that the sardineprocalcitoninConsists of amino-terminal procalcitonincleavage peptide(N-proCT) (53 amino acids),Connecticut(32 amino acids) and procalcitonin carboxy-terminal cleavage peptide (C-proCT) (18 amino acids). Compared with C-proCT, N-proCT is highly conserved among teleosts, reptiles and birds, indicating that N-proCT has certain biological activity. Therefore, sardine N-proCT and sardine CT were synthesized, and their effects on osteoblasts andBone massStudies were performed using our goldfish scale assay system composed of osteoblasts and osteoclasts. As a result, sardine N-proCT (10−7M) Activated osteoblast markersEnzyme activity, while the sardine CT has no change. On the other hand, sardines CT (10−9at 10−7M) Inhibition of enzymatic activity of osteoclast markers, although sardine N-proCT did not affect enzymatic activity. In addition, the mRNA expression of osteoblast markers, such asType 1 Collagenyesbone calciumSardine N-proCT (10−7m) Processing; however, CT sardines did not affect their expression. The osteoblastic effect of N-proCT was inconsistent. In this study, we could accurately assess the role of osteoblasts because our scale assay system has high sensitivity and is a co-culture system of osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT appear to affect osteoblasts and osteoclasts and promote bone formation through different actions in teleosts.


Calcitonin (CT) consists of 32 amino acid residues and acts as a calcium-lowering hormone that inhibits osteoclast activity in mammals (Azria, 1989). In addition, CT in teleosts and mammals has been shown to inhibit osteoclast activity in freshwater and marine teleosts (Suzuki et al., 2000; Sekiguchi et al., 2009). CT is mainly synthesized in C cells of the mammalian thyroid and in the terminal branchial gland (UBG) of non-mammalian vertebrates (Sasayama, 1999). During CT production, the hormone is first produced as a procalcitonin precursor, which is subsequently cleaved into 3 peptides: procalcitonin amino-terminal cleavage peptide (N-proCT), CT, and carboxy-cleavage peptide. Terminal procalcitonin (C-proCT) in salmon, chicken, rat, and humans (Burns et al., 1989a, Burns et al., 1989b). In cultures of neoplastic C cells, nearly equimolar concentrations of N-proCT and CT in cell extracts and basal media have been reported (Burns et al., 1989a). These facts strongly suggest that N-proCT has some biological activity.

To date, there is no consensus in the literature on whether N-proCT has an osteogenic effect. That is, human N-proCT has no mitogenic activity on rat osteoblasts or preosteoblasts (Guenther and Fleisch, 1991), and has no accelerating effect on human osteoblast mitosis and cell differentiation (Hassager et al., 1991) . In contrast, Burns et al. (1989b) found that N-proCT at nanomolar concentrations (duration of treatment 20-22 hours) was mitogenic in human osteosarcoma cell lines.

On the other hand, we have previously determined the CT peptide sequence of UBGs from Japanese sardines (sardine) using reversed-phase high-performance liquid chromatography (RP-HPLC) (Suzuki et al., 1994). The calcium-lowering activity of synthetic sardine CT was investigated by comparing it with synthetic salmon CT in a rat bioassay. We found that the calcium-lowering activity of sardine CT was more than twice that of salmon CT (Suzuki et al., 1994). Therefore, sardine N-proCT may be highly active in osteoblasts because sardine CT is highly active in calcium metabolism. In general, however, sardines are difficult to keep long-term in aquariums, although the fish is easily collected in Japan. Therefore, this species is not suitable for physiological experiments. To analyze the bioactivity of CT and N-proCT, we developed an originalin vitrogoldfish detection system, it has been previously shown that fish CT inhibits osteoclast activity in goldfish scales (Caracio Dorado) (Suzuki et al., 2000, Sekiguchi et al., 2009). In addition, the osteoclast-activating hormone parathyroid hormone acts on osteoblasts and osteoclasts and then induces active osteoclasts (multinucleated osteoclasts) in goldfish (Suzuki et al., 2011). Then we scalein vitroGoldfish bioassays are suitable for elucidating hormone function.

In this study, we determined the full-length sequence of the sardine procalcitonin precursor from its UBG using the rapid amplification of 5' and 3' cDNA ends (RACE) method. Furthermore, sardine N-proCT and sardine CT were synthesized and their bioactivity on osteoblasts and osteoclasts was investigated using our original goldfish scale assay system.

partial excerpt


Japanese sardines (sardine) were purchased from Choshi Fisheries Association. Fish were used to determine procalcitonin precursors.

goldfish(Caracio Dorado), purchased from a commercial source (Higashikawa Fish Farm, Yamato Koriyama, Japan), and artificially inseminated from one female and one male goldfish (20–30 g) at the Graduate School of Marine Science and Technology, University of Science and Technology. Tokyo Marine Technology. The fish were fed commercial pellets.

Sequencing of the sardine UBG procalcitonin precursor

The sequence of the sardine procalcitonin precursor was obtained from the RNA of its UBG using the RACE method. The sardine procalcitonin precursor consists of 134 amino acid residues, including the signal peptide (25 amino acids) (Figure 1). We predicted the signal peptide sequence using the SignalP 4 program (Petersen et al., 2011). Sardine procalcitonin consists of N-proCT (53 amino acids), CT (32 amino acids) and C-proCT (18 amino acids) (Figure 1).

These vertebrate N-proCT


Using the RACE method, we determined the total length of the sardine procalcitonin precursor (Figure 1). Our results showed that sardine procalcitonin consists of N-proCT (53 amino acids), CT (32 amino acids) and C-proCT (18 amino acids) (Figure 1). The amino acid sequences of 20 N-proCTs including sardine N-proCT were calculated by the neighbor-joining method using MEGA 6.06 software (Tamura et al., 2013). Molecular dendrograms of CT and N-proCT showed that these peptides were

Express thankfulness

This research was supported in part by an N.S. grant. (Research Scholarship[C]No.16K07871JSPS), a Y.T. (Research Scholarship[B]No.24310046af JSPS), en A.H. (Grant-in-Aid for Scientific Research [C] No.24570068JSPS), a T.S. (scientific research[C]No.15K07126JSPS) and K.K. (Scientific Research [C] No.15K01705via JSPS). This work was partially supported byNorwegian Institute for Nature and Environment Research Collaborative Research Project


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