Novel peptides with calcium-binding capacity from antler bone hydrolysate, its bioactivity on MC3T3-E1 cells, and the possible chelating mode

In this study, peptide-calcium chelate was screened from antler bone hydrolysate, and its bioactivity on MC3T3-E1 cells and its chelating mechanism were investigated. In vitro experiments showed that peptide-calcium chelate promoted the differentiation and mineralization of MC3T3-E1 cells. Subsequently, three novel calcium-chelating peptides were obtained from antler bone hydrolysate using hydroxyapatite chromatography (HAC), Sephadex G-25 gel filtration chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Meanwhile, this work determined peptides' amino acid sequences as TKLGTQLQL, LETVILGLLKT, and KMVFLMDLLK based on LC-MS/MS. Then the present work prepared the three peptides, with the corresponding calcium-chelating rates being verified as 87.68 +/- 2.86%, 80.72 +/- 0.93%, and 67.96 +/- 0.98%, respectively. Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible absorption (UV-vis) spectroscopy, X-ray diffraction (XRD), circular dichroism (CD), zeta potential, and molecular dynamics (MD) simulations were adopted to investigate the chelating mode of peptides with calcium ions. As a result, oxygen in the carboxyl group and the nitrogen in the amino group were related to calcium binding. In addition, the chelation site preferred the negatively charged carboxylate groups of Leu or Thr. The present work revealed that antler bone might be the new calcium-chelating peptide source and elucidated their positive role in osteogenesis. This study identified that antler bone peptide-calcium chelate promotes MC3T3-E1 cell differentiation and mineralization. Two novel calcium-chelating peptides were identified from antler bone hydrolysate with high chelation rates. Oxygen and nitrogen in the peptides' carboxyl and amino groups are involved in calcium binding, with a preference for negatively charged carboxylate groups of Leu or Thr.image