Galectin-4 N-Terminal Domain: Binding Preferences Toward A and B Antigens With Different Peripheral Core Presentations

Abstract

The tandem-repeat Galectin-4 (Gal-4) contains two different domains covalently linked through a short flexible peptide. Both domains have been shown to bind preferentially to A and B histo blood group antigens with different affinities, although the binding details are not yet available. The biological relevance of these associations is unknown, although it could be related to its attributed role in pathogen recognition. The presentation of A and B histo blood group antigens in terms of peripheral core structures differs among tissues and from that of the antigen-mimicking structures produced by pathogens. Herein, the binding of the N-terminal domain of Gal-4 toward a group of differently presented A and B oligosaccharide antigens in solution has been studied through a combination of NMR, isothermal titration calorimetry (ITC), and molecular modeling. The data presented in this paper allow the identification of the specific effects that subtle chemical modifications within this antigenic family have in the binding to the N-terminal domain of Gal-4 in terms of affinity and intermolecular interactions, providing a structural-based rationale for the observed trend in the binding preferences.

Keywords: NMR; blood type antigen; galectin-4; lectin—carbohydrate interaction; molecular recognition.

Scheme 1

(A) The A and B histo blood group antigens and their possible peripheral disaccharide core structures (the core types studied herein are in bold). X stands for the linked atom at the reducing-end residue (X = 3 for types-1 and -4; X = 4 for types-2 and -6). (B) Tetra- and pentasaccharide representative structures of the A and B blood group antigens of type-1, -2, -4, and -6 studied herein. The difference between A and B antigens is the substituent at C2 of the terminal residue (highlighted in blue): -OH (Gal) for the B antigen, and NHAc (GalNAc) for the A antigen. The type depends on the peripheral disaccharide core structure whose structural differences are highlighted in orange.

Figure 1

(A) ¹H STD NMR experiment for the complex formed by the A type-6 tetrasaccharide and Gal-4N (50:1 molar ratio). Top: the reference spectrum (black, off-resonance). Bottom: the STD NMR spectrum (green, on-resonance at the aromatic region). The ¹H-NMR signals showing STD effect are annotated. The epitope mapping (relative STD) is shown in the ligand structure. (B) Details of the off-resonance and the STD NMR spectra showing the NAc region obtained for the HBGAs in the presence of Gal-4N: from left to right: A type-6, B-type-1, B-type-2, and B-type-4. Terminal refers to the terminal α-GalNAc residue. Red-end refers to the reducing-end GlcNAc or GalNAc residues. The intensities of the STD spectra are 75-fold incremented with respect to the reference off-resonance spectra.

Figure 2

¹H STD NMR experiment for the complex formed by the B type-4 pentasaccharide and Gal-4N (50:1 molar ratio). Top: the reference spectrum (black, off-resonance). Bottom: the STD NMR spectrum (green, on-resonance at the aromatic region). The ¹H-NMR signals showing STD effect are annotated. The epitope mapping (relative STD) is shown in the ligand structure. *overlapping proton signals.

Figure 3

¹H-NMR-based ligand chemical exchange (EXSY) analysis from the trROESY experiments. (A) trROESY spectrum of the B type-6 tetrasaccharide antigen in the presence of Gal-4N (molar ratio 20:1). The cross-peaks in black correspond to those arising from regular nuclear Overhauser effects in the rotating frame, while those in red arise from chemical exchange. Their chemical shifts at the different ¹H dimensions in the 2D spectrum correspond to those in the free (down-field) and bound (up-field) states. (B) Plot for the measured differences in chemical shifts between the free and bound states (Δδ[¹H] free-bound) for those selected protons of the antigens that experience slow exchange in the ROESY spectrum in the presence of the lectin.

Figure 4

Protein backbone CSP analysis for the interaction of Gal-4N with A and B type-6 tetrasaccharide antigens. (A) Molecular model (MD simulation) for the complex of Gal-4N with A type-6 antigen. The residues that significantly differ in their chemical shift perturbation upon binding to group A or B antigens are highlighted in orange, as well as F47 (unknown resonance assignment). Left: zoom at the S2–S3 region showing residues F46, V138, D139, and G140. (B) Superimposition of the ¹H-¹⁵N HSQC spectra of Gal-4N in the presence of A type-6 (blue) and B type-6 antigens (red). (C) Expansion of the ¹H-¹⁵N HSQC spectra at residues V138, D139, and G140: in green, Gal-4N apo; in blue, Gal-4N/A type-6 (12 eq.); and in red Gal-4N/B type-6 (10 eq.).

Figure 5

¹H-¹⁵N HSQC: chemical shift perturbation of residues V75 and F76 of Gal-4N in the presence of the B antigens types-1, -2, -4, and -6.

Figure 6

Schematic representation of the affinity trends for the binding of the A and B blood group antigen tetrasaccharides (types-1, -2, and -6) to Gal-4N as deduced by ITC. The NMR-based data follow the same trend although the estimated binding affinities are ca. 1.5-fold weaker. The type-4 oligosaccharides are not displayed since their affinities are even weaker than those of the type-1 analogs.

Figure 7

Different perspectives of the superimposition of the 3D models for the complexes Gal-4N/A type-6 (orange) and Gal-4N/B type-6 (green) according to the MD simulations. Reducing-end and Fuc residues are faded. Key residues of the protein are highlighted.

Figure 8

Molecular models of the complexes between Gal-4N and group B types-1, -2, -4, and -6 antigens according to MD simulations. Key residues are highlighted.