AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2
Rajib Majumder, Samudra Prosad Banik, Suman Khowala
International Journal of Biological Macromolecules
Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of 34 industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not 35 been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. 36 clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal 37 hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage 38 chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal 39 sequencing, MALDI -TOF/MS-MS analysis and enzyme kinetics studies. AkP could cleave the growth 40 promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 41 75±1.18 nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of 42 apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein 43 expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl 44 fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings 45 demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration 46 and surface proteoglycans cleavage.
Circular dichroism, Secondary structure, Biochemistry