Effects of chum salmon stanniocalcin,a calcium regulatiug hormone in teleosts and its synthetic N-terminal peptide fragment on bone metabolism

• Main Author: Yoshiko, Yuji
• Format: Thesis
• Language: English
• Published: 2014
• Subjects: 490; Keta; atlantic cod; Gadus morhua
• Online Access: https://ir.lib.hiroshima-u.ac.jp/00031885; https://ir.lib.hiroshima-u.ac.jp/files/public/3/31885/20141016184016628881/diss_otsu2836.pdf; https://doi.org/10.11501/3120004

Stanniocalcin (STC) from chum salmon (Oncorhynclucs keta) was isolated by extracting the corpuscles of Stannius (CS) with ethanol-ammonium, followed by ion-exchange chromatography, gel filtration and reversed phase high performance liquid chromatography. STC migrated as a 46-kDa product under nonreducing conditions and as a 23-kDa product under reducing conditions on sodium dodecylsulphate-polyacrylamide gel electrophoresis. The protein is likely to be a homodimer composed of two subunits of 23 kDa each. Purified STC decreased the intestinal calcium uptake in a dose related manner in the Atlantic cod (Gadus morhua). A cDNA was cloned from cDNAs of the CS by means of PCR using two primers corresponding to the N- and C-terminal amino acid sequence of STC. Sequence analysis of the protein and the eDNA revealed that chum salmon STC is a homodimer, and that the monomer consists of 179 amino acids including 11 half-Cys residues and one N-linked glycosylation site, which is 44, 52 and 35 residues smaller at the C-terminal region than the sequences deduced from coho salmon, Australian eel and human STC cDNA, respectively. A synthetic peptide (peptide N) corresponding to the N-terminal amino acid residues which had a high amino acid sequence identity with coho salmon and Australian eel STC, was prepared from chum salmon. Its effects were compared with those of intact STC on mammalian bone metabolism in vitro. Peptide N (10-10-10-11 M) slightly decreased the rate of loss of radioactivity from fetal rat calvariae labeled with 45Ca, both with and without stimulation by N-terminal peptide fragment of human parathyroid hormone (hPTH1-34). Intact STC had no effect on the release of 45Ca from the calvariae. Peptide N (10-10-10-12 M) also inhibited increases in the number of tartrate-resistant acid phosphatase-positive multinucleated cells promoted by hPTH1-34 in cultures of murine hemopoietic cells, although intact STC had no effect on the number of the cells. The accumulation of cyclic AMP induced by hPTH 1-34 in ROS 17/2.8-5 cells was suppressed by peptide N (10-10-10-12 M). Peptide N (10- 11-10-13 M) increased the rate of incorporation of [3H]proline into the collagenase-digestible protein of calvariae in newborn mice. These results indicated that the highly conserved amino-terminal region of STC from teleosts has diverse effects on the metabolism of mammalian bone, causing a biphasic response. Such effects have not been observed with materials from the CS and intact STC and they also differ from the effects of hPTH1-34. CONTENTS 1. INTRODUCTION / p1 2. MATERIALS AND METHODS / p4 2.1. Preparation of chum salmon stanniocalcin (STC) / p4 2.2. Intestinal Ca²⁺influx of Atlantic cod / p4 2.3. Preparation of peptide fragments of STC / p5 2.4. Amino acid sequence analysis / p5 2.5. Cloning of cDNA encoding STC / p6 2.6. Synthesis of N-terminal peptide fragment of STC (peptide N) / p7 2.7. Calvarial bone resorption assay / p7 2.8. Generation of osteoclast-like cells / p8 2.9. Production of cyclic AMP in osteoblast-like cells / p8 2.10. Synthesis of collagen in calvarial bone / p9 2.11. Statistical analysis / p9 3. RESULTS / p10 3.1. Isolation and characterization of STC / p10 3.2. Amino acid and cDNA sequence analysis of STC / p11 3.3. Synthesis of peptide N / p12 3.4. Effects of STC and peptide N on the calvarial bone resorption / p12 3.5. Effects of STC and peptide N on generation of osteoclast-like cells / p13 3.6. Effects of peptide N on the production of cyclic AMP in osteoblast-like cells / p14 3.7. Effects of peptide N on the synthesis of calvarial bone collagen / p14 4. DISCUSSION / p16 4.1. Chemical properties of STC / p16 4.2. Effects of STC and peptide N on the metabolism of mammalian bone / p18 5. SUMMARY / p23 6. REFERENCES / p25 広島大学(Hiroshima University) 博士(歯学) Dentistry doctoral