Functional and biochemical characterization of a modified Galectin-8 protein

SCHROEDER, MATÍAS NICOLÁS; CARABELLI, JULIETA; CATTANEO, VALENTINA; CARAMELO, JULIO; CAMPETELLA, OSCAR; MARÍA VIRGINIA TRIBULATTI

Honolulu

Congreso; Joint Meeting of The Society For Glycobiology and the Japanese Society of Carbohydrate Research; 2014

Galectins (Gals) constitute a family of mammalian lectins with affinity for beta-galactosides, characterized by the presence of conserved carbohydrate-recognition domains (CRDs). Gal-8, from tandem-repeat group, contains two CRDs joined by a linker peptide. We found that Gal-8 exerts two different actions on CD4+T cells: antigen-independent proliferation and, at lower concentration, antigen-specific T cell costimulation. To study the relevance of glycan-lectin interaction on these activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, as to abolish sugar-binding capacity. Mutant protein was properly folded as determined by comparative circular dicroism assays. As expected, the absence of lactose competition observed in pull down and immunoprecipitation tests confirmed that recognition of this sugar was precluded. However, preservation of lectin activity was suspected since Gal-8mut still displayed binding capacity to T cell surface and hemoagglutination effect. To test this hypothesis, glycan affinity analysis using glycochips from the Consortium For Functional Glycomics was conducted. Interestingly, the screening revealed that Gal-8mut lost low and intermediate, but maintained higher affinity interactions. Regarding biological activity, Gal-8mut was unable to induce T cell proliferation, but it retained costimulation of antigen-specific response efficiently. Therefore, Gal-8mut dissects the Gal-8 activities on T cells and represents a useful tool to identify the specificity of lectin-glycan interactions underlying the lectin activities on T cell biology.