Study On Crystal Dissolution And Interaction Mechanism With Ligand Of Galectin-10

Galectin-10（Gal-10）,alias Charcot-Leyden crystal protein,is a member of the galectin family.It is recognized as a marker for many eosinophilic diseases such as asthma,rhinitis,sinusitis,and atopic dermatitis.Furthermore,it induces pathogenesis in the organism mainly by crystallizing to form Charcot-Leyden crystals（CLC）,although a number of researchers have developed antibodies that can specifically inhibit CLC formation within minutes or hours,no specific drug has yet been emerged.In this thesis,the effects of glutathione（GSH）on the formation of CLCs,the binding activity of Gal-10s to lactose,and the interaction with Tubulinα-1B（TUBA1B）,were investigated as follows:Firstly,Gal-10s with high purity was obtained by affinity chromatography,and CLCs with good growth was optimized.subsequently,it was found that GSH can lyse human CLC（h CLC）and inhibit the crystallization of human Gal-10（h Gal-10）in a short period of time by chemical compound lysis CLCs experiments.However,GSH had no effect on CLCs of two monkeys,Macaca fascicularis（M.fascicularis）and Macaca mulatta（M.mulatta）.According to a previous study,Gal-10s in the two monkey CLCs contain one cysteine residue,Cys29 and Cys32,respectively,while h Gal-10 contains another cysteine residue,Cys57.Therefore,in this paper,we investigated the effect of GSH on CLCs formed by h Gal-10 mutant C57A（G10-C-57a）and found that GSH could not solubilize CLC formed by G10-C-57a or inhibit its crystallization.This suggests that the effect of GSH on h Gal-10 or h CLC is produced by chemical modification of Cys57.Next,using hemagglutination assays,M.fascicularis Gal-10 and M.mulatta Gal-10,unlike h Gal-10,were found to bind lactose and inhibit erythrocyte agglutination,in combination with the previous studies in this experiment,this was shown to be due to evolutionary mutations in two key lactose-binding residues at the 57 and 75 loci.Secondly,protein blotting experiments showed that TUBA1B binds specifically to Gal-10 and that GSH,GSSG,GTP and Mg²⁺can change the overall conformation of Gal-10or stabilize this interaction by stabilizing the structure of TUBA1B.Whereas,colchicine binding to TUBA1B or colchicine-induced microtubule breakage inhibits this effect.It is noteworthy mentioning,the effect of these compounds on binding is also concentration dependent.In addition to,Fluorescence co-localization experiments with Gal-10 and TUBA1B disclosed that their action sites were distributed near the cell membrane.Overall,this paper revealed that GSH can be used as a drug to improve Gal-10/CLC-induced diseases,while TUBA1B,as a novel protein chaperone of Gal-10,is stabilized in its binding by the presence of GSH.This will provide new clues to resolve the molecular mechanism of Gal-10/CLC in cells.