Galectin-8 C-terminal carbohydrate recognition domain is responsible for antigen uptake enhancement in dendritic cells

CECILIA ARAHÍ PRATO; JULIETA CARABELLI; CAMPETELLA, OSCAR; MARÍA VIRGINIA TRIBULATTI

Virtual

Congreso; Latin American Society of Glycobiology 6th Latin American Glycobiology Congress; 2021

Latin American Society of Glycobiology

Galectins comprise a family of mammalian lectins characterized by thepresence of conserved carbohydrate recognition domains (CRDs) with anaffinity for β-galactosides. Galectin-8 (Gal-8), from the?tandem-repeat? subgroup, possesses two CRDs, N-terminal (N-CRD) andC-terminal (C-CRD), covalently fused by a linker peptide. Each CRDdisplays distinct fine specificity: whereas N-CRD displays affinityfor sialylated and sulfated glycans, the C-CRD prefers blood antigensand poly-N-acetyl-lactosamine. Therefore, Gal-8 can exert manybiological functions with both domains acting in a concerted fashion,or separately, depending on the cellular context.Our group has demonstrated that Gal-8 enhances the elicitation ofadaptive immune responses by acting on both CD4+ T cells andantigen-presenting cells. Upon Gal-8 treatment, dendritic cells (DC)up-regulate the expression of co-stimulatory molecules, secreteincreased levels of inflammatory cytokines, and efficiently stimulateT cell response. Recently, we found that Gal-8-glycan interaction atDC surface results in antigen attachment and internalization, acrucial step during the initiation of a given immune response.Since soluble antigen association and subsequent internalization isdictated by the arrangement or ?lattice? formed by Gal-8 and theglycoconjugates present at the cell membrane, we aim to characterizethis interaction at the molecular level. To analyze the involvement ofeach isolated CRD and the requirement of the ?dimeric? structure, wegenerated single N- and C-CRD recombinant proteins as well as chimericproteins consisting of two covalently fused N-CRD (N-N) or C-CRD(C-C). Then, bone marrow-derived dendritic cells (BMDC) were culturedat 37°C in the presence of biotinylated-β-casein (antigen) togetherwith every single domain (C-CRD or N-CRD), the equimolar mixture ofboth (C-CRD + N-CRD) or each chimera (C-C or N-N). β-caseininternalization was determined after cell permeabilization andlabeling with fluorescent streptavidin, followed by flow cytometryanalysis.Both C-CRD (alone or in mixture with N-CRD) and the chimera C-C wereable to enhance antigen internalization, whereas the presence of N-CRDor the N-N chimera did not affect the internalization rate ofβ-casein. Remarkably, the only addition of single C-CRD was sufficientto recapitulate the Gal-8 effect on antigen internalization,indicating that the ?dimeric? structure is not required. Takentogether, these findings demonstrate that only the C-terminal domainparticipates in Gal-8-induced antigen internalization providing newinsights into the molecular characterization of this immunostimulatoryeffect.