Overexpression And Purification Of Galectin-4 Truncates And Their Interactions With Ligands

Galectin is a family of animal lectin characterized by its specificity to decipher and respond to the information encoded by ?-galactoside sugars.To date,16 members have been identified and widely distributed in various bionts,which involve in almost all physiological processes.Galectin-4 is a member of tandem-repeat subtype,composed of two structurally conserved while sufficiently distinct carbohydrate recognition domains(CRDs,Galectin-4N at the N-terminus and Galectin-4C at the C-terminus)connected by a variable-length linker region that is susceptible to proteolytic degradation and another leading domain of 18 amino acids its N-termini.Sharing ca.35% of sequential identity,the two CRDs can bind to different ligands simultaneously,thus Galectin-4 can act as a crucial crosslinker and regulator in a large number of biological processes.Galectin-4 is able to cross-link combinations of sulfated glycosphingolipids and cholesterol,N-and O-glycosylated proteins and blood group antigens,which plays an important role in lipid raft stabilization,protein apical trafficking,cell adhesion,wound healing,intestinal inflammation,tumor progression,etc.In addition to CRDs,other domains of Galectin-4can also mediate the conformational stability,ligand binding and biological function.Human Galectin-4(hGal-4)expresses mainly in gastrointestinal epithelial cells and accompanies aberrant expression in tumor cells of alimentary canal which makes hGal-4attractive targets for the development of new strategies for diagnosis and for innovative drug design projects.We work on finding out the intracellular protein interacted with Galectin-4 and screening natural polysaccharide ligands,aiming at providing clues for development of natural drugs.Initially,pET28 a recombinant plasmids of full-length and seven truncated fragments of Galectin-4 were successfully constructed and overexpressed in E.coli BL21(DE3),then purified and obtained eight fragments by affinity chromatography,etc.There were significant differences in the yield of eight truncates,and the stability of the full-length was poor but the Galectin-4-19-323 lacking the first 18 amino acids could be steadily purified.Afterwards,through Hemagglutination and Differential Scanning Fluorimetry assay,we explored the activity and stability of truncated Galectin-4 as well as the interaction between natural polysaccharide ligands and truncates.Results comparing the minimum concentration of fragments mediating complete hemagglutination show that the agglutination of a single CRD is quite weak but the hemagglutinating activity of truncated fragments containing leading domain and linker observably increased,whichsuggested that traditional non-functional domains of Galectin-4 may play necessary roles in cross-linking.In the protein melting process of Differential Scanning Fluorimetry assays,the melting temperature(Tm)of Galectin-4C was much higher than Galectin-4N and Galectin-4-19-323,illustrating the better stability of Galectin-4C.In our research,MCP(pH-modified citrus pectin),PGA(Potato-galactan),RG-I(Rhamnogalacturonan I),AG(Arabinogalactan)and WGP(Water extraction of Ginseng penuche)were chosen as materials,according that the five natural polysaccharides have been reported interacted with Galectin-3.The polysaccharides interact with truncates of Galectin-4 could be semi-quantitative analysed by inhibiting the truncates-mediated agglutination,then comparing the minimum concentration(MIC)of complete inhibition of hemagglutination,thereby screening natural favorable polysaccharide-ligands of Galectin-4.The detection shows that the inhibition of WGP for Galectin-4 truncats was much stronger,while AG had no any inhibitory activity and the inhibitions of other three polysaccharides were quite week and incomplete Thus,WGP is a good natural polysaccharide ligand of Galectin-4.Performing lactose as control,comparing the melting shift temperature(?Tm)of the three fragments(Galectin-4N,-4C and-19-323)with polysaccharide additives,the result tested by Differential Scanning Fluorimetry assay indicated that none of the five polysaccharides had obvious interaction with these truncates.Finally,the intracellular protein ligands of were screened by Pull-down and co-immunoprecipitation(Co-IP)assaays.Pull-down assay of His6-Ni-NTA affinity system found that asialofetuin had varying degrees of interaction with Galectin-4truncates,and moreover completely inhibited by lactose.Our study screened many suspicious ligand proteins interacting with Galectin-4-19-323 in colon cancer HT29 cells by Co-IP.Furthermore,a target protein band of SDS-PAGE analysed by protein mass spectrometry,identified a complex fusion protein CAD of 245 kDa with abundance of up to 94.9%,namely Carbamoyl-phosphate II Synthetase-Aspartate TranscarbamoylaseDihydroorotase,which is a key enzyme catalyzing the synthesis of de novo pyrimidine.