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Functional Structure/Activity Relationships
Isolation and characterization of peptides with osteogenic activity in mouse MC3T3-E1 pre-osteoblast cells from Mytilus edulis Zhe Xu, Hui Chen, Zhenyu Wang, Fengjiao Fan, Pujie Shi, Maolin Tu, and Ming Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06530 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019
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Journal of Agricultural and Food Chemistry
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Isolation and characterization of peptides with osteogenic
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activity in mouse MC3T3-E1 pre-osteoblast cells from
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Mytilus edulis
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Zhe Xua, Hui Chena, Zhenyu Wanga, Fengjiao Fanb, Pujie Shib, Maolin
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Tub, Ming Dua, *
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a
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Seafood, Dalian Polytechnic University, Dalian 116034, China;
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b
School of Food Science and Technology, National Engineering Research Center of
Department of Food Science and Engineering, School of Chemistry and Chemical
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Engineering, Harbin Institute of Technology, Harbin 150001, China
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Corresponding author:
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Dr. Ming Du; E-mail:
[email protected];
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Postal address: School of Food Science and Technology, National Engineering
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Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China;
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Tel: +86-411-86332275;
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Fax: +86-411-86323262
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List of Abbreviations: α-MEM, α-Minimum Essential Medium; FBS, Fetal Bovine Serum; MTT, 3-
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(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
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phosphatase; DMSO, dimethyl sulfoxide; HPLC, High-Performance Liquid
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Chromatography; CESI-Q-TOF, Capillary Electrophoresis Electrospray Ionization-
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Quadrupole-Time of Flight; MS/MS, tandem mass spectrometry; RGD, Arginine-
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Glycine-Aspartic acid; MAPK, mitogen activated protein kinase; ERK, extracellular
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regulated protein kinase; FAK, focal adhesion kinase; FA, formic acid; ACN,
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acetonitrile; WPM, water-soluble protein from Mytilus edulis; TH, trypsin enzymatic
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hydrolysate; PH, pancreatin enzymatic hydrolysate; NH, neutral proteinase enzymatic
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hydrolysate; NH-1, neutral proteinase enzymatic hydrolysate more than 3 kDa; NH-2,
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neutral proteinase enzymatic hydrolysate less than 3 kDa; NHA, neutral proteinase
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enzymatic hydrolysate after alcohol precipitation; NHA-1, neutral proteinase
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enzymatic hydrolysate more than 3 kDa after alcohol precipitation ; NHA-2, neutral
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proteinase enzymatic hydrolysate less than 3 kDa after alcohol precipitation.
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bromide;
ALP,
alkaline
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ABSTRACT
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Seafood provides a range of health benefits due to its high-protein level. In this
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study, a novel peptide, YPRKDETGAERT, was identified from NHA-2 of Mytilus
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edulis by Capillary Electrophoresis Electrospray Ionization-Quadrupole-Time of
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Flight (CESI-Q-TOF). Peptide YPRKDETGAERT showed the highest affinity among
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all the peptides with “-CDOCKER Energy” values of 204.482 kcal/mol on integrin
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(PDB: 3VI4) and 210.16 kcal/mol on another integrin (PDB: 1L5G). The secondary
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mass spectrogram at the m/z of peptide YPRKDETGAERT was 1422.53 Da, which
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was determined by CESI-Q-TOF. Peptide YPRKDETGAERT induced an increase of
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28.27±3.66% in mouse MC3T3-E1 pre-osteoblast cells growth. The alkaline
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phosphatase activity of peptide YPRKDETGAERT was 2.79±0.07 mU, which had an
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increase by 21.25% compared to control. These results provided theoretical and
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practical insights for the preparation and application of osteogenic peptides in the
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functional foods industry.
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KEYWORDS: Mytilus edulis; peptides; proliferation; osteoblast; integrin
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GRAPHICAL ABSTRACT
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INTRODUCTION
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Bone is a tissue of dynamic biological structure that is highly regulated by the
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dynamic processes of bone resorption and bone formation. Bone formation is a
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complex process that involves a combination of many biological factors 1. Some
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bioactive peptides or proteins were reported to be beneficial to bone regeneration,
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which may affect the cell adhesion, proliferation and differentiation of bone,
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connective tissue and epithelial cells 2.
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In the course of bone growth and development, osteoblasts (OBs) can regulate
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bone mineralization by secreting bone matrix and noncollagen proteins, and
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osteoclasts (OCs) are directly involved in the bone resorption process 3. Osteoblasts
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are important for bone formation and resorption both directly and indirectly and are
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closely connected with osteoporosis. Therefore, those peptides and proteins with
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activities promoting bone formation have been attracting more and more attention 4-5.
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Concurrently, the integrins are comprised of noncovalently linked α and β
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heterodimeric subunits, which are transmembrane adhesion receptors, and mediate the
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adhesion dependence and adhesion of cells to extracellular matrix for cell
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proliferation
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migration, adhesion, proliferation , differentiation and mediating cells to interact with
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some bone matrix proteins 8. Particularly, integrins such as αvβ3 and αvβ5 are
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important for bone resorption, tumor angiogenesis and inflammation 9. The Arg-Gly-
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Asp (RGD)-binding integrins αvβ3 and α5β1 mediated pre-osteoblast adhesion to
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partially-denatured protein, but not to native. Moreover, pre-osteoblasts cultured on
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partially-denatured had a significant mineralization activity. Some denatured
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collagens expose RGD sequences, triggering integrin α5β1 and αv- binding, which in
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turn promotes a series of processes such as adhesion of osteoblasts proliferation and
6-7.
The β1- and αvβ- containing integrins are important for osteoblast
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differentiation
Taken together, this strong evidence reveals one approach for the
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bone formation: α5β1 and αvβ3 play important roles in growth factor-induced cell
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cycle progression 11. At the same time, peptides and proteins regulate integrins for cell
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signaling, showing the increase of bone formation in vivo 12.
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The shellfish shelling process is similar to the mechanism of the bone formation
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process 13. Mytilus edulis is a kind of nutrient-rich seafood. The water-soluble protein
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of Mytilus edulis accounts for 65% of its mass on a dry basis, some of which can be
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converted to bioactive peptides by enzymatic hydrolysis. Some studies suggest that
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there are many health benefits of this seafood due to its high protein content. The
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cleavage of proteins to small bioactive peptides from marine mussels
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protein hydrolysate of Mytilus edulis illustrate some of the benefits for promoting
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bone formation
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inhibition, antihypertensive effect and blood pressure reduction 16-18. In addition, some
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reports using molecular docking to screen bioactive peptides for intermolecular
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interactions
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by molecular docking with integrin as a receptor.
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19-20.
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and the
Peptides (
