Study On Crystal Structure And Bioactivity Of Galectin-13 Mutant Protein

The galectin family are widely expressed and defined by two properties:conservative amino acid sequences(one or two highly conserved carbohydrate recognition domains(CRDs)made up of about 135 amino acid residues)and affinity for ?-galactoside sugars.Galectin-13 is highly expressed in placenta,specifically in the syncytiotrophoblast,where its lower expression is related to pre-eclampsia.During pregnancy,the Gal-13 concentration in maternal serum may be useful for predicting pre-eclampsia and HELLP syndrome.Recently,crystal structures of wild type Gal-13 and its variant R53 H have solved at high resolution.Crystallographic and biochemical results showed that Gal-13 and R53 H could not bind lactose.So we identified several residues that were likely responsible for the inability of Gal-13 and then used site-directed mutagenesis to re-engineer the sugar binding site of wild type Gal-13 so that it could bind lactose.This paper is devoted to studying the crystal structure,activity,affinity of glycoligand and distribution in the HeLa cells of Gal-13 six mutant proteins.The following are specific research contents and results:Firstly,the recombinant plasmids of Gal-13 six mutant proteins were constructed and transformed into competent cell Ecoli BL21(DE3)respectively,and the high purity mutant proteins were obtained via IPTG-induced expression and Ni-NTA affinity chromatography.Of six newly engineered mutants,we were able to solve the crystal structures of three(R53HH57R,R53HH57RD33 G,R53HR55NH57RD33G).Three variants had the same two mutations(R53 to H and H57 to R)and were able to bind lactose in the crystal,indicating these two amino acid residues are essential for their binding to lactose.Furthermore,the structures of R53 H and R53HR55 N showed that these variants could co-crystallize with a molecule of Tris.Afterwards,bioactivities of Gal-13 variants were functionally assessed using the hemagglutination assay.The results showed that variant R53HH57 R demonstrated greater bioactivity than wild-type Gal-13,and all other variants had similar activities as wild-type protein.Overall,the mutations within the ligand binding site of Gal-13 did not profoundly change the activities of the variants in terms of promoting hemagglutination.However,high concentrations of lactose and sucrose(200 mM)did not inhibit aggregation and also did not bind to lactose-modified Sepharose CL-6B.This is in contrast to the results of crystallography,suggesting that Gal-13 induced erythrocyte agglutination not via the canonical ligand binding site and Gal-13 could strongly bind unknown ligands on the erythrocyte membrane that lactose cannot effectively inhibit and the actual cause needs further investigation.Finally,to study the distribution of Gal-13 variants within HeLa cells,EGFP N-terminal tagged recombinant plasmids of variants of Gal-13 were successfully constructed.The results showed that they are concentrated in the nucleus and could be co-localized within filamentary materials,possibly actin.In conclusion,we demonstrated that Gal-13 cannot naturally bind lactose.Although the primary and tertiary structure of Gal-13 is similar to all other galectins,Gal-13 appears to be a different galectin.Up to now,the molecular function of Gal-13 remains unknown,aside from it being related to pre-eclampsia.Future work should focus on identifying more physiological ligands of Gal-13 and to determine whether Gal-13 is a signaling transduction protein involved in regulating gene replication or transcription.Answers to these questions might help to treat pre-eclampsia.