Research Of Tartary Buckwheat Lectin With Activity Of RIPs Inhibit Proliferation Of HCT116 Cells

Lectin is a kind of protein or glycoprotein which can combine polysaccharides or monosaccharides reversibly,which usually contains one or more catalytic domain.Lectin are widely distributed in nature.The lectin with activity of type II ribosome inactivating protein(RIP)usually has two chains.A chain has RIP properties and B chain has lectin properties.The most of RIP can hydrolyze N-glycosidic bond between 4324 adenine base and ribose of ribosome 28S rRNA specifically.According to the previous research,Tartary Buckwheat Lectin(TBL)belongs to the type ? RIP,while the primary functions and enzyme activities are expecting for further research.The recombinant dual luciferase plasmid named psiCHECKTM-2-F28RNA was constructed in this study,which containing the sequence of A4324.TBL is a kind of ribosome inactivating protein with N-glycosidase activity.After the plasmid psiCHECKTM-2-F28RNA incubated with TBL,the agarose gel electrophoresis showed that the plasmid was changed to nicked type from supercoiled type.Meanwhile,the plasmid psiCHECKTM-2-F28RNA was transfected in HCT116 cells resulted in the fluorescence ratio(renilla/firefly)decreased obviously.These results indicated that the plasmid can be used to detectin toxicity assay of RIP to cell.Also,the GAGA sites were mutated using site-directed mutagenetis method,which suggested that the GAGA sequence was the site of RIP recognized.When the plasmid psiCHECKTM-2-F28RNA was incubated with several kinds of proteins from grain crops,the results showed that the plasmid can quickly screen RIP.Moreover,the plasmid psiCHECKTM-2-Wntl 3'UTR was constructed which contained the GAGA sequence and detected using agarose gel electrophoresis and dual luciferase detection.These findings suggested that the mRNA containing GAGA sequence could be a potential target.This study provides a simple method for identification of RIP,which can be used to detect and screen RIP quickly.To further understanding the biological functions and expanding the application range of TBL on antitumor.TBL was extracted and purified through affinity chromatography and molecular exclusion chromatography,and the ultimate density was about 2 mg/mL.The purificated protein was injected in rats three times.After 30 days,the polyclonal antibody of anti-TBL was obtained.The serum titer of antibody was detected by ELISA assay.The results showed the 1,2,3 rats serum titer reached 1:25 60000(P/N>2.1)and 4,5 rats serum titer reached 1:5120000(P/N>2.1),which provided materials for further research.The inhibition of cell proliferation by TBL was detected by MTT experiment in previous study,which showed that TBL could inhibit HCT116 cells obviously,while the effect on Hep G2 cells was relatively weakly and undetectable on normal FHC cell.By follow-up study,the phenomenon is associated with the expression of Galectin-3.Galectin-3 is one of the members of the Galectin family,which plays an important role on physiological and pathological process in tumor invasion and metastasis.In this study,Galectin-3 was distributed the cells membrane surface high quantity,and the expression of Galectin-3 in HCT116 cells was more than Hep G2 cells and normal cells FHC by immunefluorescence assay.After TBL treat three kinds of cells under the same condition,a lot of TBL distribut in the surface of HCT116 cells,while the distribution is not distinct in the other two cells.The result proved that TBL and Galectin-3 may have competive relationship in membrane surface by western blot assay.TBL competitively combine the receptors which Galectin-3 recognised in the cell membrane,which resulted the amount of Galectin-3 combined with the cell membrane surface greatly reduced,meanwhile reduced the cell invasion.At the same time,the membrane surface receptors recruited a large number of TBL,which increased the odds and concentration of the TBL into intracellular.Then,the TBL in cytoplasm can play the role of ribosome inactivating protein and the intracellular RNA was damaged,which inhibited translation process was and leaded to cell death.In recent years,lectin has become a research hotspot in the field of medicine and biology due to its possible cytotoxicity and unique biological function.For the first time,lectin was obtained from the Polygonaceae Fagopyrum tataricum,which has the RIP properities.Also,the possible anti-tumor activity was studied,while the exact mechanisms that how TBL recognised RNA and the effect on protein translation process and cell death need further research.