Preparation Of Nine Galectins And Their Interaction With Fungal Polysaccharides

Galectins are a class of animal lectins that can specifically bind to ?-galactoside,and 16 species have been found so far,which are divided into three types: prototype,chimeric and tandem repeat.Galectins are widely involved in various physiological activities of organisms,have functions of regulating intracellular glycoprotein transport,cell adhesion,migration,growth and apoptosis,and are related to various diseases such as immunity,inflammation and cancer.In terms of cancer detection,galectins may be important immunohistochemical markers for diagnosing cancer,and are one of the current drug targets for cancer treatment.However,the preparation methods and storage conditions of various galectins are still imperfect,which limits the research on the structure and function of galectins.Therefore,in this paper,by constructing a prokaryotic expression system,we studied the stable and large-scale preparation of nine galectins,and optimized their storage conditions,laying a material foundation for the study of galectins.Fungal polysaccharides have multiple activities,can be used as an important resource for the development of galectin inhibitors,and have the advantages of weak toxic and side effects and wide sources.At present,there are few reports on the interaction between galectin and fungal polysaccharides.In order to promote the development of galectins inhibitors,the interaction between five kinds of galectin and seven kinds of fungal polysaccharides was studied to find the fungal polysaccharides ligands of galectins.It lays a theoretical foundation for the research of galectins inhibitors and the drug development of fungal polysaccharides and tumor treatment.The specific research content and results are as follows:Nine kinds of galectins were prepared.By constructing prokaryotic recombinant plasmid of galectin and inducing expression in E.coli BL21(DE3),proteins including Gal-1,Gal-2,Gal-3,Gal-4,Gal-7,Gal-8,Gal-9M,Gal-10,Gal-13 were purified by affinity chromatography using Ni-NTA gel and CL-6B lactose,and the purity of above protein was more that 90%.The yield of each galectin was different,with the highest yield of gal-7(200 mg/L),followed by Gal-3(35 mg/L),Gal-1,Gal-2 and Gal-8.These four yields were similar(30 mg/L),and the lower rates were Gal-4(25 mg/L),Gal-10(22 mg/L)and Gal-13(15 mg/L).Gal-9M was an inclusion body,the yield was only 20 mg/L after refolding.The activity of the recombinant protein was detected by the hemagglutination experiment.The results showed that the galectin-induced erythrocyte agglutination ability was Gal-7(0.49 ?g/mL),Gal-10(0.59 ?g/mL),Gal-13(0.60 ?g/mL),Gal-4(1.46 ?g/mL),Gal-1(1.50 ?g/mL),Gal-8(2.10 ?g/mL),Gal-2(2.19 ?g/mL),Gal-9M(3.90 ?g/mL)and Gal-3(5.80 ?g/mL).The storage conditions of different galectins were optimized.The activity of the recombinant protein was detected by SDS-PAGE method and the hemagglutination experiment.The results showed that the galectin-induced erythrocyte agglutination ability was Gal-7(0.49 ?g/mL),Gal-10(0.59 ?g/mL),Gal-13(0.60 ?g/mL),Gal-4(1.46 ?g/mL),Gal-1(1.50 ?g/mL),Gal-8(2.10 ?g/mL),Gal-2(2.19 ?g/mL),Gal-9M(3.90 ?g/mL)and Gal-3(5.80 ?g/mL).The storage conditions of different galectins were also optimized.The results showed that the addition of glycerin,DTT and other protective agents to the storage solution can stabilize the protein,which had little side effect for the activity of the protein.After optimization,Gal-1,-2,-3,and-8 are stored in the state of lyophilized powder,and Gal-4,-7,-9M,-10,and-13 are stored in the liquid state.The interaction between galectin and fungal polysaccharides was studied.Five kinds of proteins including prototype Gal-1 and Gal-7,chimeric Gal-3,tandem repeat Gal-4 and Gal-8 were choosed to explore and clarify the regularity of the interaction between galectins and fungal polysaccharides included seven fungal polysaccharides,such as Penicillium polysaccharides(DPS6OR-2-a,WPDP-b and WPOP-b),Pleurotus eryngii polysaccharides(APEP-A-b,APEP-N-b),Pleurotus ostreatus polysaccharides(APOP-A-b,APOP-N-b)using erythrocyte agglutination inhibition methods.The results of erythrocyte agglutination inhibition experiments showed that Penicillium polysaccharide DPS6OR-2-a can be used as the dominant ligand of Gal-1,penicillium polysaccharides WPDP-b and WPOP-b can be used as the dominant ligands of Gal-3,and Penicillium polysaccharides WPOP-b,Pleurotus ostreatus polysaccharides APOP-N-b and APOP-A-b can be used as Gal-4 dominant ligands to inhibit Gal-4-induced erythrocyte agglutination.Pleurotus eryngii polysaccharide APEP-A-b as the dominant ligand of Gal-7 and Gal-8 can effectively inhibit the erythrocyte agglutination.There was no significant correlation between the function and the molecular weight of polysaccharide.These polysaccharides are mainly glucan,glucogalactan and mannan galactosan,and the content of galactose has no obvious correlation with their function.Similarly,there was no obvious correlation between this inhibitory effect and the molecular weight,and these polysaccharides were mainly dextran,galactosan and mannogalactan,and the content of galactose had no obvious correlation with its function.The fungal polysaccharides mentioned above can effectively inhibit the erythrocyte agglutination induced by tandem repeat Gal-4 and Gal-8.Galactose-based WPDP-b can also bind Gal-3.Both mannose-based DPS6OR-2-a and glucose-based APEP-A-b,APOP-A-b can bind Gal-1 and Gal-7,and APEP-A-b can also bind Gal-3.The glucose-based APOP-N-b can bind Gal-7.The results provide a theoretical basis for the development of galectin inhibitors and drug development based on fungal polysaccharides.