Mechanism Of Antitumor Activity Of RAAL And Purification Of A New Active Protein From Edible/Medical Mushroom Agrocybe Aegerita

Our previous research has shown that protein components of edible/medicinal mushrooms possess high antitumor activity. An antitumor lectin AAL from fruiting bodies of Agrocybe aegerita was purified and its gene was cloned.The results of the three-dimensional structures of AAL and its complexes with carbohydrate ligand lactose revealed that the correlation between ligand binding and AAL bioactivity. In this thesis, we further investigated the relationship between antitumor activity of AAL and the glycosylation on cell surface. Moreover, we purified a new antitumor protein AAD.Part1:The mechanism of rAAL selective killing effectAlterations of protein and lipid glycosylation at the cell surface are consistent features of cancers, which allows tumor cells to invade and spread throughout the organism. The aberrant glycosylation is responsible for incomplete synthesis of the carbohydate chains, allowing higher expression of precursor carbohydrate. Sulfo-TF antigen ([3OSO3] Gal ?1-3GalNAc-Ser/Thr) is a member of tumor-associated antigens, which can be found on some cancer cells. Thus, sulfo-TF antigen can server as a target for tumor therapy.AAL is an antitumor lectin which is purified from Agrocybe aegerita. Our results showed that rAAL inhibited the proliferation of tumor cells by the induction of apoptosis with the activation of caspase-8,-9,-3, and it has a close relationship with the cell surface binding ability of rAAL. Our previous work showed that recombinant AAL (rAAL) had a higher affinity to sulfated glycans in the glycan array, in which it had the highest affinity to sulfo-TF antigen, than sialylated glycans. We investigated the mRNA expression levels of glycosyltransferases which synthesize TF-associated antigene. The results showed that the cells with higher ratio of GAL3ST2/ST3GAL1were more sensitive to rAAL. Down regulation the ratio of GAL3ST2/ST3GAL1by overexpressing ST3GAL1, reduced binding ability and sensitivity of jurkat cells to rAAL. Meanwhile, up regulation the ratio of GAL3ST2/ST3GAL1by overexpressing GAL3ST2rendered HL60and HeLa cells sensitive to rAAL. In addition, inhibiting the further elongation of TF antigen by benzyl-?-GalNAc decreased the antiproliferative activity of rAAL. These results confirmed that rAAL has the highest affinity to sulfo-TF antigen and the cells with higher ratio between GAL3ST2and ST3GALlare more sensitive to rAAL. These suggest that rAAL potentially serve for cancer diagnosis and targeted therapy.Part2:Purification and characterization of an antitumor protein with deoxyribonuclease activity from edible mushroom Agrocybe aegeritaOur previous research has shown that protein components of edible/medicinal mushrooms possess high antitumor activity. A lot of antitumor proteins have been identified, such as ubiquitin like proteases, ribosome inactivating proteins and lectins. Agrocybe aegerita is a popular and high nutritional edible mushroom all over the world and is used as a traditional herb in China. It has an abundant amount of proteins, which account for25%-30%of dry fruiting bodies. Two antitumor lectins, AAL and AAL-2, have been identified from the protein components of Agrocybe aegerita. In the current work, we have identified a novel antitumor protein (AAD) with deoxyribonuclease activity from Agrocybe aegerita. We also reveal that DNase is one of the active protein components in Agrocybe aegerita.Several Deoxyribonucleases (DNases) have also have been separated from fungus, but none were reported to have any antitumor activity. DNases are involved in DNA fragmentation during programmed cell death (PCD). They are proposed as attractive candidates for cancer therapy. In this report, to further identify the active ingredients of the protein components, we separated a novel antitumor DNase from it. These characterizations showed that the Agrocybe aegerita deoxyribonuclease (AAD) belonged to DNase I family. The amino acid sequence of AAD identified by tandem mass spectrometry against our Agrocybe aegerita transcriptome revealed97%homology with Aa—Pril from Agrocybe aegerita.