The interaction of Galectin-8 C -terminal domain with cell surface glycoconjugates modulates membrane elasticity to stimulate antigen uptake and presentation to CD4 T cells

Galectins constitute a family of soluble lectins with unique capacity to induce macroscale rearrangements upon interacting with cell membrane glycoconjugates. Galectin-8 is acknowledged for its role in facilitating antigen uptake and processing upon engaging with cell surface glycoconjugates on antigen-presenting cells. Galectin-8 consists of two covalently fused N- and C-terminal carbohydrate recognition domains, each exhibiting distinct glycan specificity. In this study, we utilized single N- and C-carbohydrate recognition domains recombinant proteins to dissect the nature of Galectin-8-glycan interactions during antigen internalization enhancement. Single C-carbohydrate recognition domain was able to replicate the effect of full-length Galectin-8 on antigen internalization in bone marrow-derived dendritic cells. Antigen uptake enhancement was diminished in the presence of lactose or when N-glycosylation-deficient macrophages served as antigen-presenting cells, underscoring the significance of glycan recognition. Measurement of the elastic modulus using Atomic Force Microscopy unveiled that full-length Galectin-8- and C-carbohydrate recognition domain-stimulated macrophages exhibited heightened membrane stiffness compared to untreated cells, providing a plausible mechanism for their involvement in endocytosis. C-carbohydrate recognition domain proved to be as efficient as full-length Galectin-8 in promoting antigen degradation, suggesting its implication in antigen-processing induction. Lastly, C-carbohydrate recognition domain was able to replicate full-length Galectin-8-induced antigen presentation in the major histocompatibility complex class II (MHC-II) context both in vitro and in vivo. Our findings support the notion that Galectin-8 binds through its C-carbohydrate recognition domain to cell surface N-glycans, thereby altering membrane mechanical forces conducive to soluble antigen endocytosis, processing, and presentation to cognate CD4 T cells. These findings contribute to a deeper comprehension of Galectin-8 and its mechanisms of action, paving the way for the development of more efficacious immunotherapies.