Impact of mutations in carbohydrate binding sites of tandem-repeat type galectin from Takifugu obscurus on its antimicrobial activity

Galectins belong to a family of galactoside-binding proteins and exhibit diverse biological functions. In the present research, a tandem-repeat type galectin (named ToGalectin) was identified from obscure puffer Takifugu obscurus. The 296 amino acids ToGalectin contained two carbohydrate recognition domains (CRDs), one of which possessed two conserved carbohydrate binding motifs. Phylogenetic analysis showed that ToGalectin clustered tightly with other galectin-8 proteins from teleost fish. ToGalectin transcripts were ubiquitously expressed in all tissues examined and its expression was significantly upregulated in the liver, kidney, and intestine after Vibrio harveyi or Staphylococcus aureus infection. To investigate the effect of carbohydrate binding sites on biological activity, ToGalectin and its mutant (MUT-ToGalectin) were expressed and purified. The recombinant ToGalectin and MUT-ToGalectin proteins showed strong agglutinating activity against both V. harveyi and S. aureus. rToGalectin could bind to all tested carbohydrates and bacteria, whereas rMUT-ToGalectin bound to some carbohydrates and bacteria with specific and relatively strong affinity. rToGalectin significantly suppressed the growth of all six bacteria detected and promoted bacterial clearance in vivo, whereas MUT-ToGalectin inhibited the growth of only two bacterial species, which could be attributed to the differences in conserved motifs within the CRDs. Our results suggested that ToGalectin is involved in the immune response against bacterial infection and the clearance of pathogens in T. obscurus.