Study On The Structures And Activity Of Galectin-10 Mutants

Galectins are a class of classical galactose-binding proteins in the lectin family,the structurally comprises 1 to 2 glycosyl recognition domains with a specific affinity for β-galactoside,which domain consists of approximately 140 amino acids with a high degree of recognition and a highly conserved amino acid sequence.Galectins play important physiological functions in many aspects,such as growth regulation,cell adhesion,apoptosis and immune response,and play a key role in the treatment of diseases.Among them,Galectin-10(Gal-10)is a prototype galectin in the galectin family,which contains only a single glycosyl recognition domain,mainly in the form of homodimers,mostly distributed in eosinophils and basophils.And Galectin-10(Gal-10)can characteristically form a double pyramidal hexagonal crystal in the body,also known as Charcot-Leyden crystal protein(CLC).Eosinophils are found in various tissues,such as the respiratory tract,lymph nodes,and spleen,and also are associated with various diseases,including asthma,allergic rhinitis,and acute myeloid leukemia(AML).Since the discovery of Galectin-10,it has been more than 160 years,it was originally identified as that Galectin-10 can spontaneously formprotein crystals in human body.Although the three-dimensional structure of the crystal has been resolved,the current results show that Galectin-10 can not bind lactose for unknown reasons.The research ideas in this paper focus on the relationship between the structure and activity of mutants by mutating the key sites of Galectin-10 wild type.The research on the level of dimerization of the mutant,the structural analysis of the crystal,the related activity and the distribution in the cell can bring new ideas and directions for the treatment of related diseases.The specific research contents,methods and research results of this paper are introduced as follows.First,by reading the literature and analyzing the structure,five key amino acid positions of Galectin-10 wild type are determined,and then the mutations are separately made to synthesize five recombinant plasmids,which are independently expressed in E.coli.Extraction and purification are carried out by Ni-NTA affinity chromatography to obtain a protein with higher purity.The obtained Galectin-10 wild type and 5 mutant proteins are separately crystallized,screening conditions suitable for crystal growth by sitting drop method,and then the conditions such as solution concentration and pH are optimized by a hanging drop method to obtain crystals having a regular shape.By comparing the structure of the Galectin-10 mutants with the wild-type protein structure,the structure of the co-crystal of the mutant and lactose is analyzed,further,the binding ability of these mutants to lactose and the effect on protein dimerization are analyzed.The results show that the mutant E33 A is able to bind lactose weakly,while other proteins can not.The multimeric forms of these Galectin-10 mutants are studied by protein molecular sieve chromatography experiments.The results show that the mutant W127 A is a monomeric structure,indicating that the 127-tryptophan(Trp127)is a key amino acid for the dimerization of Galectin-10.In addition,the red blood cell agglutination experiments,sugar repression experiments,and agarose gel affinity chromatography experiments are used to supplement the related activities of Galectin-10 wild type and mutants.In the red blood cell agglutination experiment,the mutant W127 A shows higher hemagglutination activity than the wild type Galectin-10 and other mutants.In the erythrocyte agglutination inhibition experiment,the mutant E33 A is sensitive to lactose while the other mutants are not sensitive.Moreover,the mutant W127 A can be combined with a lactose-modified agarose gel,Which indicates that the open carbohydrate binding site of the W127 A monomer can bind to lactose.In addition,the red blood cell agglutination experiments,sugar repression experiments,and agarose gel affinity chromatography experiments are used to supplement the related activities of Galectin-10 wild type and mutants.In the red blood cell agglutination experiment,the mutant W127 A shows higher hemagglutination activity than the wild type Galectin-10 and other mutants.In the erythrocyte agglutination inhibition experiment,the mutant E33 A is sensitive to lactose while the other mutants are not sensitive.Moreover,the mutant W127 A can be combined with a lactose-modified agarose gel.In addition,the red blood cell agglutination experiments,sugar repression experiments,and agarose gel affinity chromatography experiments are used to supplement the related activities of Galectin-10 wild type and mutants.In the red blood cell agglutination experiment,the mutant W127 A shows higher hemagglutination activity than the wild type Galectin-10 and other mutants.In the erythrocyte agglutination inhibition experiment,the mutant E33 A is sensitive to lactose while the other mutants are not sensitive.Moreover,the mutant W127 A can be combined with a lactose-modified agarose gel.Immunofluorescence microscopy of eosinophils reveale a significant amount of Galectin-10 concentrated particles in the nucleus.In this paper,pEGFP-N1 with green fluorescent tag is selected as the expression vector,and the recombinant plasmid is successfully constructed by molecular cloning technology.At the same time,the recombinant plasmid is transferred into cervical cancer cells(HeLa cells)by means of cell biology techniques including cell subculture,cell transfection,and nuclear fluorescent staining,and its distribution and expression are observed.The results show that the EGFP-tagged Galectin-10 wild type and each mutant are evenly distributed in the nucleus of HeLa cells,indicating that Galectin-10 may be dynamically exchanged in the nucleus,while the mutant W72 A is distributed in the cytoplasm and cannot enter the nucleus,infer 72-position tryptophan is the decisive key amino acid.