Purification, Identification and Neuroprotective Effects of Proteins from Bombyx batryticatus in Glu-Stimulated PC12 Cells

Abstract

Bombyx batryticatus (BB) is one of the most commonly used Traditional Chinese Medicines (TCMs) in the treatment of convulsions and epilepsy. The antiepileptic effects of total proteins from BB (BBPs) have been proven in our previous research. In this study, BBPs were further purified, the neuroprotective effects were evaluated in Glu-stimulated PC12 cells, and the structure was identified by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six subfractions (PF-1 to PF-6) were obtained by DEAE-52 Sepharose FF ion-exchange chromatography. It was found that PF-1, PF-2, and PF-3, with similar protein compositions, possessed neuroprotective effects in Glu-stimulated PC12 cells by significantly increasing the GABA level, and decreasing the levels of IL-1β and TNF-α. The most active fraction (PF-2) was further separated by Sephadex G-75 gel filtration chromatography, and an effective protein component named PF-2-2 was obtained. Fluorescein isothiocyanate-labeled PF-2-2 (FITC-PF-2-2) was prepared, and the binding of FITC-F-2-2 to the PC12 cells was directly observed with a confocal microscope. PF-2-2 was found to first bind to the surface of PC12 cells and then internalize into the cells. The main band of PF-2-2 was then analyzed by MALDI-TOF/TOF-MS and searched in the MASCOT database; finally a protein named Low molecular mass 30 kDa lipoprotein 21G1 was identified. In conclusion, PF-2-2 and purified proteins isolated from BBPs have potential application prospects in the treatment of epilepsy.