title: Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.
abstract: Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects.
authors.affiliation: Laboratorio de Inmunología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (UNSAM-CONICET), Av. 25 de Mayo y Francia, B1650HMP San Martín, Buenos Aires, Argentina.
authors.forename: Matías Nicolás
authors.initials: MN
authors.lastname: Schroeder
authors.affiliation: Laboratorio de Inmunología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (UNSAM-CONICET), Av. 25 de Mayo y Francia, B1650HMP San Martín, Buenos Aires, Argentina virginia@unsam.edu.ar.
authors.forename: María Virginia
authors.initials: MV
authors.lastname: Tribulatti
authors.affiliation: Laboratorio de Inmunología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (UNSAM-CONICET), Av. 25 de Mayo y Francia, B1650HMP San Martín, Buenos Aires, Argentina.
authors.forename: Julieta
authors.initials: J
authors.lastname: Carabelli
authors.affiliation: MaIAGE, INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
authors.forename: Gwenaëlle
authors.initials: G
authors.lastname: André-Leroux
authors.affiliation: Laboratorio de Biología Celular Estructural, Fundación Instituto Leloir, Av. Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
authors.forename: Julio Javier
authors.initials: JJ
authors.lastname: Caramelo
authors.affiliation: Laboratorio de Inmunología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (UNSAM-CONICET), Av. 25 de Mayo y Francia, B1650HMP San Martín, Buenos Aires, Argentina.
authors.forename: Valentina
authors.initials: V
authors.lastname: Cattaneo
authors.affiliation: Laboratorio de Inmunología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (UNSAM-CONICET), Av. 25 de Mayo y Francia, B1650HMP San Martín, Buenos Aires, Argentina.
authors.forename: Oscar
authors.initials: O
authors.lastname: Campetella
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D049109:Cell Proliferation; D037161:Galectins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D037161:Galectins; D011994:Recombinant Proteins
keywords: T-cell proliferation; T-cell receptor co-stimulation; galectin; glycan affinity; inflammation