The structure and properties of MFG-E8 and the In vitro assessment of its toxic effects on myoblast cells

Abstract

Four high-molecular-weight protein fractions of milk fat globule membrane (MFGM) were isolated from bovine milk. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), MALDI-TOF/TOF™ and Liquid chromatography electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) were used to measure the molecular sizes of the MFGM. Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) were performed to determine the conformations of the MFGM. The results showed that the main protein (98.33%) in MFGM protein fraction 2 was Milk fat globule epidermal growth factor-VIII (MFG-E8), with a molecular weight of 47.82 kDa. The secondary structural component measurements showed that the MFG-E8 consisted of 5% helix, 70% sheet and 25% random coil, and the results matched well with the prediction by SSPro 5.1 bioinformatic analysis. The thermograms analysis revealed that Td and△H of MFG-E8 were 60.50°Cand 132.29 kJ/mol. The in vitro digestibility of MFG-E8 showed that it can be enzymatically hydrolyzed in the stomach and relatively stable in the intestinal fluid. The in vitro C2C12 and Caco2 cell activity tests indicated that MFG-E8 promoted the proliferation of C2C12 myoblast cells without cytotoxicity. The biological functional properties of MFG-E8 may be related to the fact that MFG-E8 possesses a high level of β-sheet structure. Our results suggested that MFG-E8 possesses broad prospects not only for use in functional food products but also as a source of natural anti-sarcopenia drugs.