Crystal Structure Of Galectin-10 And Interaction With Ligands

Galectins are a family of animal lectin with binding specificity for ?-galactosides.Up to date,16 members have been identified.These galectins have structural similarity and are widely involved in various physiological processes.Galectin-10 is a member of prototype galectins,the gene of which are mapped to human chromosome 19.It is composed of 142 amino acids.Galectin-10 is mainly present in eosinophils and it can spontaneously form crystals in vivo.Some new researches show that gelctin-10 are considered hallmarks of eosinophil involvement in several diseases,such as asthma,eosinophilic cystitis,mastocytoma and atopic dermatitis,and galectin-10 may play a key role in regulating the function of lymph cell.Therefore,galectin-10 can be used as a target for developing new diagnostic strategies and innovative drugs.In this paper,we work on studing the structure of galectin-10,attempt to identify its polysaccharide ligands and research the binding mechanism,aiming at providing clues for development of drugs which can cure disease associate with galectin-10.Initially,pET28 a recombinant plasmids of wide-type and seven variants of galecint-10 were successfully constructed and overexpressed in E.coli BL21(DE3),then purified and obtained eight proteins by affinity chromatography.The purity and content between wide-type galectin-10 and variants are basically similar.The hemagglutination assay was used to evaluate the biological activities of wide-type galectin-10 and variants.The results show that these proteins we purified can induce agglutination,and it indicates that these eight proteins all have biological activities.Afterwards,the crystallization of eight proteins were processed.Through the initial crystallization screen,optimization of crystals,X-ray power diffraction,data collection and analysis,we have solved the crystal structures of galectin-10 and seven variants at resolutions of 1.55-2.00 ?.The crystal structures of galectin-10 and seven variants show that all proteins cannot co-crystallize with lactose,mannose,fructose,glucose,sucrose,N-acetyl-D-glucosamine and N-acetyl-D-lactosamine,whereas some of these proteins were found to be complexed with one molecule of glycerol.Further research indicates that there is a Glu33 occupied the carbohydrate binding site of galectin-10,which inhibiting disaccharide binding.Careful inspection of variants,we found that His53?Asn65?Trp72?Lys73 and Gln75 can effect on binding glycerol,but Cys57 and Gln74 cannot.Structural analysis and size exclusion chromatography demonstrated that galectin-10 dimerizes with a novel global shape that is different from that of galectin-1,-2 and-7.Finally,the results of hemagglutination assay,affinity chromatography and thermal shift assay indicate that galectin-10 cannot bind lactose,mannose,glucose,sucrose,N-acetyl-D-glucosamine and N-acetyl-D-lactosamine.