Galectins are small human proteins participating in inflammation processes, immune response, and cancerogenesis. Tandem-repeat galectins comprising Gal-4, Gal-8, and Gal-9 are a vital yet less studied part of the galectin fingerprint in cancer-related processes. The present work studies a library of prepared multivalent neo-glycoproteins decorated with poly-N-acetyllactosamine and human-milk-type oligosaccharides as ligands of this underexplored family of tandem-repeat galectins. A thorough binding evaluation by ELISA and biolayer interferometry was complemented with a detailed epitope mapping both from the galectin and the glycoconjugate viewpoints by nuclear magnetic resonance. The found interactions in the galectin binding site were correlated to in silico data from molecular modeling. The present work reveals pioneer information on the binding of tandem-repeat galectins to multivalent glycoconjugates carrying complex carbohydrate ligands and represents an invaluable starting point for the development of new high-affinity tailored ligands of tandem-repeat galectins, needed both for diagnosis and therapy.

Centro de Investigación Biomedica En Red de Enfermedades Respiratorias C Monforte de Lemos 3 5 Pabellón 11 Planta 0 Fuencarral El Pardo E 28029 Madrid Spain

CICbioGUNE Basque Research and Technology Alliance Parque Científico Tecnológico de Bizkaia Building 801A E 48160 Derio Spain

Department of Analytical Chemistry Faculty of Science Charles University Hlavova 8 CZ 128 43 Prague 2 Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 3 CZ 166 28 Prague 6 Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 CZ 128 43 Prague 2 Czech Republic

Department of Inorganic and Organic Chemistry Faculty of Science and Technology University of the Basque Country UPV EHU E 48940 Leioa Spain

Ikerbasque Basque Foundation for Science Plaza Euskadi 5 E 48009 Bilbao Spain

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 CZ 142 00 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Náměstí 2 CZ 166 10 Prague 6 Czech Republic

Laboratory for Biomaterials Institute of Biotechnology and Helmholtz Institute for Biomedical Engineering RWTH Aachen Pauwelstr 20 D 52079 Aachen Germany

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