Study On Crystal Structure And Bioactivity Of Galectin-13

Galectins,widely expressed by cells within the animal kingdom,all have one or two evolutionarily conserved carbohydrate recognition domains(CRDs)that can bind?-galactosides.Galectin-13(Gal-13)is a member of prototype,which has one CRD in its monomeric form and two in its homodimeric form linked by disulfide bonds.Gal-13 is predominantly expressed in placenta,specifically in the syncytiotrophoblast,it can also induce apoptosis of activated T cells and regulate immune tolerance between maternal and fetal tissues.Gal-13,including intrauterine growth restriction(IUGR),early IUGR,pre-eclampsia,and I plays an important role in pregnancy,and could be valuable at discriminating obstetrical complications UGR associated with pre-eclampsia.Despite extensive biochemical studies on Gal-13,its three-dimensional structure has remained unknown.The present study is focused on gaining a better understanding of Gal-13structure-function relationships.Here,we study the crystal structure of Gal-13,as well as biochemical and cellular studies on ligand binding specificity and distribution of the lectin in cells.The results were shown as follows:Initially,p ET28 was combinant plasmid of wild type and Site-directed mutagenesis of Gal-13 were successfully constructed and overexpressed in Escherichia coli BL21(DE3)cells.The wild type,mutant R53 H and mutant C136A/C138 A were extracted and purified using a Ni-NTA agarose column.Afterwards,we crystallized wild type Gal-13 and its R53 H mutant using two different conditions.As with all other galectins,the crystal structure of wild type adopts the typical“jelly-roll” fold formed by sandwiching of two anti-parallel ?-sheets that consist of six(S1-S6)and five(F1-F5)?-strands and a short ?-helix connecting strands S2 and F5.The crystal structure discovered that its dimer is stabilized by two disulfide bridges between Cys136 and Cys138 and six hydrogen bonds involving Val135,Val137 and Asn139.Native PAGE and gel filtration demonstrate that artifact Gal-13 dimers also form in solution.Finally,Gal-13 can induce erythrocyte agglutination by the hemagglutination assay.However,our research shows that galactose,maltose,xylose,arabinose,glucose,mannose,lactose,fructose,sucrose,and cellulose do not inhibit Gal-13-mediated agglutination.In addition,affinity chromatography studies show that Gal-13 could not bind to sepharose 6b covalently-linked with lactose,arabinose,maltose,or sucrose seems inconsistent with reports that Gal-13 can bind to sugar-modified agarose gels.Biolayer interferometry(BLI)results indicate that Gal-13 also can not bind to RG-I-4,WGPA,and mushroom polysaccharide.Ourbiochemical studies indicate that the ligand binding specificity of Gal-13 is different from that of other galectins in that it does not bind ?-galactosides.Mutating Arg53 to histidine does not re-establish normal ?-galactoside binding,but rather traps molecules of the cryo-protectant glycerol within the ligand binding site in crystals of the R53 H mutant.Moreover,we also found that GFP-tagged Gal-13 is predominantly localized within the nucleus of He La and293 T cells by transfection and fluorescence microscopy.