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. 2021 Jan 4;27(1):316-325.
doi: 10.1002/chem.202003143. Epub 2020 Dec 2.

Galectin-Glycan Interactions: Guidelines for Monitoring by 77 Se NMR Spectroscopy, and Solvent (H2 O/D2 O) Impact on Binding

Tammo Diercks  1 , Francisco J Medrano  2 , Forrest G FitzGerald  3 , Donella Beckwith  3 , Martin Jaeger Pedersen  4 , Mark Reihill  4 , Anna-Kristin Ludwig  5 , Antonio Romero  2 , Stefan Oscarson  4 , Maré Cudic  3 , Hans-Joachim Gabius  5
Affiliations

Galectin-Glycan Interactions: Guidelines for Monitoring by 77 Se NMR Spectroscopy, and Solvent (H2 O/D2 O) Impact on Binding

Tammo Diercks et al. Chemistry. .

Abstract

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance. Their study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as S- and Se-glycosides. The latter enable extensive analyses by specific 77 Se NMR spectroscopy, but still remain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and -3, we have evaluated diverse 77 Se NMR detection methods and propose selective 1 H,77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against a selenoglycoside spy ligand. By fluorescence anisotropy, circular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDG binding by galectins are similar to thiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact. ITC data in D2 O versus H2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAc dimers with extended chain length.

Keywords: 77Se NMR spectroscopy; calorimetry; circular dichroism; galectins; selenoglycosides.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
1H NMR spectra of SeDG (4 mm, assignments in black) and SeDGlc (4 mm, assignments in grey) in the absence (black) and presence (red) of hGal‐3 (0.125 mm). A) 1H Spectrum. Strong signal broadening and attenuation indicates binding only for SeDG, where the maximal attenuation (−76 %) is observed for H1. B) [1H→]1H STD spectrum in the presence of hGal‐3. The difference 1H spectrum (blue, 32‐fold upscaled) reveals maximal STD effects of ≥3 % for SeDG protons H2, H4, H5, and H6 while STD effects on H3 and especially on H1 are much weaker. No STD effects are observed for SeDGlc. Residual hGal‐3 1H signals were suppressed by a 100 ms T1ρ filter.
Figure 2
Figure 2
77Se NMR spectrum (1H decoupled) of SeDG and SeDGlc (4 mm) in the absence (black) and presence (red, acquired with double number of scans) of hGal‐3 (0.125 mm). A) Direct detection with 1024 scans, 3 s interscan delay, 52 min experiment time. B) Indirect detection by 2D 1H,77Se CPMG‐HSQMBC (positive F1 projection) with 70 ms 1H→77Se transfer delay, 8 scans, 1.5 s interscan delay, 26 min experiment time. Spectra were processed with 3 Hz line broadening. Signal‐to‐noise ratios for same experiment time: 5 (A) vs. 97 (B).
Figure 3
Figure 3
1D 1H traces from 2D 1H,77Se correlation spectra taken at the 77Se chemical shift (393.7 ppm) of SeDG (4 mm) in the absence (black) or presence (red) of hGal‐3 (0.125 mm). A) CPMG‐HSQMBC (Δ opt(H1)=70 ms, B CPMG=1143 Hz). The indirect H3 correlation signal (4 J H3,Se=0) derives from HoHaHa (TOCSY) transfer. B) HeHaHa (B DIPSI2=1450 Hz, Δ opt(H1)=80 ms). The average signal intensity is −60 % lower than in the HSQMBC spectrum. C) H1 selective HeHaHa (B DIPSI2=285 Hz, Δ opt(H1)=100 ms). The H1 signal now shows pure in‐phase splitting (3 J H1,H2=10 Hz) and 10 % (175 %) sensitivity gain relative to the corresponding CPMG‐HSQMBC (HeHaHa) spectrum. Corresponding 1H traces for the nonbinding SeDGlc are shown in Figure S4.
Figure 4
Figure 4
Interaction of hGal‐1 with TDG and SeDG monitored by intrinsic Trp FA. Binding isotherms for increasing concentrations of A) TDG and B) SeDG at 25 °C. Equilibrium K D values were derived by data fitting to a nonlinear regression model for single site‐specific binding using GraphPad Prism 8 and assuming one binding site per monomer. Error bars represent standard deviations from five repeated FA measurements. K D errors are estimates from the fitting algorithm.
Figure 5
Figure 5
ITC titration profile of A) hGal‐3 (110 μm) with SeDG (6.0 mm), B) hGal‐3 (70 μm) with DSeDG (10.0 mm), C) hGal‐3 (135 μm) with DiLacNAc (6.0 mm), and D) hGal‐3 (40 μm) with DiLacNAc (1.0 mm) in phosphate buffer (pH 7.2) containing 20 mm phosphate, 10 mm NaCl, and 10 mm BME in water (A–C) or D2O (D). Ligand was injected every 150 s at 25 °C. The top panels show the experimental ITC data and bottom panels a fit to a one‐site model of the binding data using MicroCal PEAQ‐ITC analysis software. Resulting values for the stoichiometry (n), binding affinity (K A), dissociation constant (K D), enthalpy (ΔH), and change in entropy with respect to temperature (TΔS) are shown in Table 3.
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