Galectin–Glycan Interactions: Guidelines for Monitoring by ⁷⁷Se NMR Spectroscopy, and Solvent (H₂O/D₂O) Impact on Binding

Abstract

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance.T heir study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as Sand Se-glycosides.T he latter enable extensive analyses by specific 77 Se NMR spectroscopy,b ut stillr emain 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 as elenoglycoside spy ligand.B yf luorescence anisotropy,c ircular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDGb inding by galectins are similart ot hiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact.I TC data in D 2 Ov ersus H 2 Oa re similar for TDG and LacNAcb inding by both galectins, but as olvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAcd imers with extendedc hain length.

Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3

“…Furthermore, we also determined the strength of the interaction from the fluorescence anisotropy titrations and compared it to the values obtained by the NMR titrations and isothermal titration calorimetry (ITC) [ 40 ] as shown in Table 1 . The dissociation constants found by NMR were 123 ± 5 μM for SeDG and 8500 ± 2500 μM for DSeDG, which were similar to the values by fluorescence anisotropy of 35.2 ± 7.2 for SeDG with no value available for DSeDG.…”

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin

“…Of relevance in this context, extending the length of a canonical disaccharide ligand to two tetrasaccharides had recently revealed major solvent rearrangements to occur in the case of human galectins-1 and -3. 37 Finally, testing these glycopeptides as antigens will allow comparative thermodynamic studies with antibodies to be performed.…”

“…Solutions of both the ligand and the lectin (extracellular domain) were prepared in 10 mM HEPES sodium salt, 50 mM NaCl, and 2 mM CaCl 2 (pH 7.4) in both deionized H 2 O and D 2 O (heavy water, >99.8 atom % D), as shown previously to be practical for human galectins-1 and -3. 37 Thermodynamic analysis was performed using MicroCal PEAQ-ITC and Origin analysis software based on a one-set-of-sites model, and with the fitted offset parameter applied to each titration following the company’s guideline and previous applications. 38 − 40 Thermograms, integrated heat values, and signature plot analysis of the thermodynamic bindings are provided in Figures S28–S30 .…”

77Se-Enriched Selenoglycoside Enables Significant Enhancement in NMR Spectroscopic Monitoring of Glycan–Protein Interactions

“…77 Se is a spin-1 2 NMR isotope with broad chemical shift range (ca. 3000 ppm), which makes it promising as a selective probe [25,[27][28][29]. Direct 77 Se NMR detection [30,31] is, however, unfavorable owing to the low natural abundance (7.63%) and decreased sensitivity (0.7 of 1 H) of this nucleus.…”

“…To improve sensitivity, we proposed a 2D 1 H-77 Se HSQMBC (heteronuclear single quantum multiple-bond correlation) experiment via indirect 77 Se detection using CPMG-INEPT (Carr-Purcell-Meiboom-Gill insensitive nuclei enhanced by polarization transfer) out-and-back 1 H→ 77 Se→ 1 H polarization transfer. Theoretically, this approach can yield up to 60-fold ((γ H /γ Se ) 2.5 ) sensitivity enhancement, but, owing to competing relaxation and other transfer processes, a sensitivity gain about 20 can be realized in practice [25,28,32].…”