Structural features for α-galactomannan binding to galectin-1

Abstract

Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-D-galacto-β(1 → 4)-D-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with (15)N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-D-galactopyranosyl doublets surrounded by regions of about four or more "naked" mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [6(1)-α(1 → 6)-D-galactosyl-β-D-mannotriaose and 6(3),6(4)-di-α(1 → 6)-D-galactosyl-β-D-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.

Fig. 1.

¹H–¹⁵N HSQC spectral expansions are shown for ¹⁵N-gal-1 (1 mg/mL) alone (cross peaks in black in A–C) and in the presence of 3 mg/mL of GM 2 (A), GM 4 (B) and β(1 → 4)-mannan (C). Resonances are labeled in (A) with assignments reported previously by Nesmelova, Pang, et al. (2008).

Fig. 2.

HSQC resonance broadening maps are shown for GM binding to gal-1. Fractional changes in gal-1 resonance intensities observed for gal-1 in the presence of GMs are exemplified with resonance broadening maps for GM 1.8 (3 mg/mL, A) and GM 2 (1.8 mg/mL, B) vs the amino acid sequence of gal-1. Data are shown under conditions where most gal-1 resonances are still apparent. A value of 1 indicates that the resonance associated with that particular residue is no longer apparent, and a value of zero indicates no change in resonance intensity.

Fig. 3.

HSQC chemical shift mapping is shown for GM binding to gal-1. ¹⁵N- and ¹H-weighted chemical shift changes, Δδ, between ¹⁵N-gal-1 (the absence of ligands) and ¹⁵N-gal-1 in the presence of near saturating concentrations of GM 2 (A), GM 2.4 (B) and GM 3 (C) are plotted vs the amino acid sequence of gal-1.

Fig. 4.

The proposed GM-binding domain on gal-1 is shown. Segments containing residues that are most affected by binding to these GMs in general are indicated and are highlighted in blue on the structure of the gal-1 dimer, as discussed in the text. The X-ray structure of lactose-bound human gal-1 was used in this figure (pdb access code: 1gzw, Lopez-Lucendo et al. 2004), and bound lactose molecules are shown in purple.

Fig. 5.

The average fraction of resonance broadening from ¹⁵N-gal-1 HSQC resonance intensity changes vs the concentration (mg/mL) of GMs and mannans are shown. Solid lines are used as visual aids and simply connect data points in each curve.

Fig. 6.

(A) Probabilities (relative distributions) P(n) of isolated sequences (Gal/Man)_n for n between 1 and 6 along the galactomannan backbone. “Isolated” in this context means surrounded by “naked” (unsubstituted) single or multiple (Man) residues. Only one Gal residue can be attached to one Man residue, forming a (Gal/Man) pair. A random distribution of Gal residues along the galactomannan backbone, hence, a random distribution of (Gal/Man)_n combinations (n = 1–6) is assumed for these calculations. This random distribution is described by formula P = q² × (1 − q)ⁿ, where q is a fraction of “naked” Man residues in the galactomannan, and (1 − q) is a fraction of Gal/Man pairs in the galactomannan. (B) The effect of 1,4-β-d-galactomannans (GMs) with different degrees of attachment of (1 → 6)-α-d-Gal residues to (1 → 4)-β-d-Man residues in the GM backbone, described as the (Man/Gal) ratio, on the binding avidity of these GMs to gal-1. Binding avidity here is defined as the average broadening of ¹⁵N-gal-1 HSQC spectral peaks upon the interaction with these GMs.

Fig. 7.

¹⁵N-gal-1 HSQC spectral expansions are shown, with eight overlays each, one for gal-1 (0.3 mM) alone (black cross-peaks) and the others for ¹⁵N-gal-1 in the presence of 1.1–72 mM (red) of GM mannopentaose 6³,6⁴-di-α(1 → 6)-d-galactosyl-β-d-mannopentaose (mannopentaose, A); 0.4–67 mM (red) 6¹-α(1 → 6)-d-galactosyl-β-d-mannotriaose (mannotriaose, B) and 0.04–10 mM (red) lactose (C). Inserts to each panel plots the average chemical shift changes over the top 30 shifting residues vs the saccharide concentration, respectively. The mid-points on these curves allow estimation of equilibrium dissociation constants, K_d, for the binding of each saccharide to gal-1, as discussed in the text.

Fig. 8.

HSQC chemical shift maps are shown for GM mannotriaose and GM mannopentaose binding to gal-1. ¹⁵N- and ¹H-weighted chemical shift changes (ppm), Δδ, between gal-1 (absence of ligands) and gal-1 (0.3 mM) in the presence of 35 mM mannopentaose (A), 10 mM lactose (C) and 70 mM mannotriaose (E) are plotted vs the amino acid sequence of gal-1. The other two panels show difference maps for Δδ mannopentaose minus Δδ lactose (B) and Δδ mannotriaose minus Δδ lactose (D), as discussed in the text.