Identification Of The AAL Gene From Agrocybe Aegerita And The Study Of The Mechanism For Its Inducing Cell Apoptosis

According to the N-terminal sequence of the AAL, the degenerate primers were designed and the cDNA of AAL was cloned through RT-PCR using the mycelia of Agrocybe aegetira as the template. The length of the cDNA is 635bp which encode a protein which is consisted of 158 amino acids, the M.W. of the protein is 16993.75D. in which we can find the sequenced amino acids fragments QGVNIYNI and QPDGPWLVEQR,therefore,the cDNA is correct.Analysis of the cDNA sequence indicated that AAL belongs to the Galectin family, it is the first Galectin found from Agrocybe aegerita and the fourth Galectin found from Fungi. After cloning, the fuse protein rAAL was expressed and purified from prokaryotic cell, and has the same Hemagglutination activity as the natural AAL.In order to know how AAL can induce the death of tumor cells and the mechanism to induce apoptosis, we try to find its interactive protein. Through the 15 random peptides biopanning using AAL as the bait protein, 6 clones were identified and sequenced and the 15 peptides QADGPNSVVRPFTLT was shared by them.. When searching the GeneBank with the polypeptides, a protein subunit which was shared by Farnesyltransferase (FTase) and geranylgeranyltransferase I(GGTase I) was found called FTase/ α . This two enzymes are two key transferases for prenylation of Ras the gene FTase/ α was cloned by RT-PCR from the HeLa cDNA.The vector pGAD-FTase/ α ,pGBK-α al were constructed for the yeast two-hybrid system assay.pHM-αal and pCMV-FTase/ α were constructed for the co-immnoprecipitation assay.The interaction between AAL and the subunit was confirmed using the yeast two-hybrid system and co-immnoprecipitation assay. It is the first time found that Galectin can interact with the subunit of FTase/GGTase.The next thing is know what will happened after AAL interact with FTase/ α . Firstly, the HeLa cells were treated with AAL in different concentration. After 24hours, some cells were extracted the membrane proteins for the detection of Ras on membrane, some cells were dealt in order to find the change of Fas on membrane using the Flow cytometric analysis , others were extracted total RNA for the real-time PCR to detect Fas in mRNA level. Finally, it is demonstrated that Ras was downregulated on memebrane, while Fas were upregulated on membrane, mRNA of Fas was regulated for 100 times.Therefore, we can demonstrate the mechanism of inducing apoptosis of tumor cells by AAL and its antitumor activity: AAL interacts FTase and inhibit it, which induce the upregulation of Ras and downregulation of Fas ,the downregulation of Ras lead to the loss of its GTPase ,so tumor cells cannot grow unlimitedly; Fas was the member of death receptor family, so its upregulation will lead tumor cells to apoptosis.