Different roles of the heterodimer architecture of galectin-4 in selective recognition of oligosaccharides and lipopolysaccharides having ABH antigens.

The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) towards the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by employing a combined experimental approach including NMR, solid-phase and hemagglutination assays and molecular modeling. The data show that Gal-4C prefers A- over B-antigens (twofold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length tandem-repeat form (Gal-4FL) has been additionally scrutinized. ITC and NMR data demonstrate that both domains within Gal-4FL bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B-antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide (LPS) containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross link multivalent ligands, beyond the glycan binding capacity of the dimer.