A novel peptide purified from Low molecular weight Ark shell (Scapharca subcrenata) protein hydrolysates enhances osteogenic differentiation through bone morphogenetic protein signaling

Abstract

Aging of human bone is characterized by decreased bone formation and bone mass. These changes demolish a balance between bone formation by osteoblast and bone resorption by osteoclast in bone tissues. The imbalance causes bone-related diseases such as osteoporosis, hypercalcemia, and Paget’s disease. Recently, various therapeutic approaches to the diseases are investigated, but the conventional drugs have side effects because most of the remedies are principally based on anti-resorptive agents (e.g. bisphosphonates, raloxifen). A number of natural bioactive peptides have diverse biological activity and certain products have shown beneficial effects on osteoblast differentiation unlike previously other studies. Thus, these peptides can be useful as alternative agents to treat bone-related diseases. In this study, it was suggested that ark shell protein hydrolysates (ASPHs) with low molecular weight contributed to osteoblast differentiation and the effects of ASPHs through modulating bone morphogenetic protein (BMP) signaling were investigated by measuring osteoblastic biomarkers such as BMP-2, p-Smad1/5, runt-related transcription factor-2 (Runx-2), Dlx5, osterix, and MAPKs in mouse mesenchymal stem cells (MSCs, D1 cells). ASPHs were generated by pepsin at E/S ratio of 1:500 with 2 h hydrolysis indicated the highest ALP activity. Further ASPHs were separated according to molecular weight using molecular weight cut-off (MWCO) membrane with 10, 3, and 1 kDa. Low molecular weight peptide fraction (ASPH<1 kDa) exhibited the highest stimulation effects among the others in a dose-dependent manner. Also ASPH<1 kDa significantly increased activity of alkaline phosphatase (ALP) and amount of hydroxyapatite related bone mineralization in MSCs as indicated by ALP staining and Alizarin red S staining. The most bioactive peptide fraction (ASPH<1 kDa) was fractionated by ion exchange chromatography using Sephadex C-25 and purified by RP-HPLC on Hypersil GOLD column. At each stage of isolation, one of potential peaks was selected by measuring ALP activity and finally the single most active peak was identified. The purified peptide enhanced osteoblast diff-erentiation by regulating BMP signaling. Thus, Ark shell peptides offer the possibility to be highly valuable application as therapeutic substances.