Dissecting molecular determinants of the Galectin-4 glycan interactions and their potential relevance in intestinal inflammation

CAGNONI, ALEJANDRO J.; MASSARO, MORA; MANSELLE COCCO, MONTANA; CUTINE, ANABELA; PÉREZ SÁEZ, JUAN M.; RABINOVICH, GABRIEL A.; MARIÑO, KARINA V.

Congreso; 2020 Virtual Society for Glycobiology Annual Meeting; 2020

Inflammatory Bowel Diseases (IBD), including Crohn?s Disease and Ulcerative Colitis, constitute a group of chronic inflammatory disorders that affect the gastrointestinal tract, in which the perpetual state of inflammation constitutes a risk factor for the development of colitis-associated colorectal cancer (CACRC). In this context, and even though galectin-glycan interactions play a central role as a mediator of pathophysiological processes, their role in IBD and CACRC has been scarcely explored. Galectins, a family of evolutionary-conserved soluble lectins, are involved in immunomodulation, inflammation, pathogen recognition, tumor escape, resistance and metastasis. In particular, Galectin-4 (Gal-4), a tandem-repeat type galectin with two distinct carbohydrate recognition (CRD) domains is expressed in the gastrointestinal tract. This lectin exerts a predominantly pro-inflammatory role, stimulating T-cells and exacerbating inflammation via recognition of a colitis-associated aberrant glycome. In this work, we optimized the production and purification of human Gal-4, as well as each of its isolated CRDs. From the different approaches assayed, the expression vector pET-28a-SUMO was finally selected based on the reproducible protocol for tag cleavage using ULP1 protease. Thus, corresponding DNA constructs containing inserts for hGal-4, hGal-N-terminal-CRD or hGal-4-C-terminal-CRD were expressed in E. coli BL21 (DE3), and the purification of recombinant proteins was achieved in two chromatography steps, by Ni-NTA affinity and size-exclusion. Then, the activity and glycosidic preferences of recombinant full-length Gal-4 and its CRDs were evaluated by hemagglutination, solid-phase assays and isothermal titration calorimetry. In doing so, we identified selective recognition of Gal-4 for particular glycan moieties, which were recognized by full length Gal-4 and/or specific carbohydrate recognition domains with high affinity and specificity when compared to other galectins. Furthermore, by molecular docking and molecular dynamics simulations, we characterized the biochemical and biophysical determinants of these interactions in silico, identifying key amino acid residues responsible for glycan recognition. Finally, we evaluated the inhibitory role of these specific glycans in vitro, demonstrating that these oligosaccharides inhibited Gal-4-dependent IL-6 and Granzyme B secretion by splenocytes, and in consequence, hampering Gal-4 proinflammatory activity. In summary, our results provide new insights on the molecular determinants of Gal-4-glycan interactions, and postulate a potential regulation of intestinal inflammation via Gal-4 inhibitors.