The alpha-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

Abstract

Galectins are a sub-family of lectins, defined by their highly conserved beta-sandwich structures and ability to bind to beta-galactosides, like Gal beta1-4 Glc (lactose). Here, we used (15)N-(1)H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of beta1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via alpha1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (K(d)) of 10 x 10(-6) M, compared to 260 x 10(-6) M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the beta-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1-carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences.

Fig. 1

Chemical structure of the repeat unit in Davanat. Davanat is a galactomannan, whose backbone is composed of (1→4)-linked β-d-mannopyranosyl units to which single α-d-galactopyranosyl residues are periodically attached via a (1→6)-linkage, with an average repeating unit of 17 β-d-Man residues and 10 α-d-Gal residues, and an average polymeric molecule containing approximately 12 such repeating units.

Fig. 2

¹H-¹⁵N HSQC spectra for gal-1 with and without Davanat. HSQC spectra are shown for ¹⁵N-enriched gal-1 (1 mg/mL) alone (A) and at gal-1:Davanat molar ratios of 30:1 (B) and 15:1 (C). Spectral expansions are provided below each plot to better visualize the observed resonance broadening as Davanat is added to the gal-1 solution. Resonances in the expansion plots are labeled with assignments reported by Nesmelova, Pang, et al. (2008).

Fig. 3

Gal-1/Davanat binding from HSQC resonance broadening map. Fractional changes in gal-1 resonance intensities observed at a gal-1:Davanat molar ratio (20:1) where most gal-1 resonances are still apparent versus the amino acid sequence of gal-1. 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. The 11 β-strands in gal-1 are identified below the residue number. The inset shows the average fractional change (±SE) in gal-1 HSQC resonance intensities versus the concentration (mg/mL) of Davanat. The solid line represents a sigmoidal fit to the average of these values at each point.

Fig. 4

Davanat binding domain on gal-1. (A) Residues on the folded structure of gal-1 that have been most affected by binding to Davanat are highlighted in red and orange as discussed in the text. The x-ray structure of lactose-bound human galectin-1 has been used in this figure (pdb access code: 1gzw, Lopez-Lucendo et al.) . The orientation at the left shows the face of the dimer where Davanat binds. The gal-1 dimer interface is also indicated. The orientation at the right shows the opposite side of the dimer where lactose binds. (B) Illustration of gal-1 residues in the Davanat binding domain. Polar, positively charged, and hydrophobic residues are colored in orange, blue, and green, respectively. For reference, the lactose molecule in its binding site is shown in purple.

Fig. 5

HSQC resonance broadening map for binding of mannan to gal-1. Fractional changes in gal-1 (4 mg/mL) HSQC resonance intensities in the presence of mannan (16 mg/mL) versus the amino acid sequence of gal-1. 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. The 11 β-strands in gal-1 are identified below the residue number. The inset shows the average fractional intensity change (±SE) of cross-peaks versus the mannan concentration (mg/mL). The solid line represents a polynomial fit to these data.

Fig. 6

Lactose binds gal-1 in presence of Davanat. (A) Overlay of expansions from two HSQC spectra of ¹⁵N-enriched gal-1 (1 mg/mL) in the presence of Davanat (molar ratio of 10:1, gal-1:Davanat) (black cross-peaks) and with the addition of lactose at 1 mM (magenta), 3 mM (red), and 10 mM (blue). (B) Overlay of expansions from two HSQC spectra of ¹⁵N-enriched gal-1 (1 mg/mL) alone (black cross-peaks) and with the addition of lactose at 1 mM (magenta), 3 mM (red), and 10 mM (blue). Resonances in expansion plots in A and B are labeled as assigned previously by Nesmelova, Pang, et al. (2008)_. (C) ¹⁵N-¹H-weighted chemical shift differences (Δδ) of gal-1 resonances observed upon the addition of lactose to a solution of gal-1 containing Davanat as in A versus the amino acid sequence of gal-1. (D) ¹⁵N-¹H-weighted chemical shift differences (Δδ) of gal-1 resonances observed upon the addition of lactose to a solution of gal-1 alone as in B versus the amino acid sequence of gal-1. (E) The fractional chemical shift changes of the 20 most shifted resonances of gal-1 in the presence of Davanat (from data as exemplified in A) as a function of lactose concentration. (F) The fractional chemical shift changes of the 20 most shifted resonances of gal-1 alone (from data as exemplified in B) as a function of lactose concentration. The solid lines in both E and F represent sigmoidal fits to the averages of these values at each point, as discussed in the text.

Fig. 7

Lactose reduces Davanat-induced broadening. (A) The top plot shows fractional changes in resonance intensities of gal-1 in the presence of Davanat (molar ratio of 10:1 gal-1:Davanat) versus the amino acid sequence of gal-1. 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. The bottom plot shows fractional changes in resonance intensities of gal-1 in the presence of Davanat (molar ratio of 10:1 gal-1:Davanat) and 1 mM lactose versus the amino acid sequence of gal-1. (B) Fractional broadening for 28 resonances of gal-1 in the presence of Davanat is plotted versus the lactose concentration. Lines shown simply connect data points as visual aids.

Fig. 8

¹H NMR of Davanat, along with diffusion decay curves for titration with gal-1. (A) A ¹H NMR spectral trace for Davanat is shown. The inset illustrates the diffusion-mediated gradient-induced decay of a few resonance envelopes as discussed in the text. Lines below the spectrum provide ranges for chemical shifts of protein resonances for Gal and Man rings, as discussed in the text. Diffusion decay curves for Davanat resonances at 3.93 ppm (B) and 3.694 ppm (C) are shown as a function of the gal-1 concentration in mg/mL. The glycan concentration was held constant at 4.6 mg/mL, and gal-1 was titrated into solution. Dashed lines show extrapolation to the Y-intercept of the slow component of the decay curve, as discussed in the text.

Fig. 9

Diffusion coefficients for Davanat. (A) D-values for the slow component of the deconvoluted diffusion decay curves are plotted versus gal-1 concentration. (B) Lines indicating diffusion coefficients for standard glycans and proteins versus their molecular weights are shown as discussed in the text. The dashed line indicates a hypothetical situation for protein bound to glycan, where the complex is neither protein nor glycan, but some combination of both.