Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides

Abstract

A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (Kd) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (ΔG) was compensated by a considerable gain in the entropic term. (1)H-(15)N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan-lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides.

Scheme 1. Glycosylated MUC1 Peptides Used in This Study

Scheme 2. Synthesis of Fmoc-Protected O-Glycosylated Thr Starting from Methyl 2-Azido-2-deoxy-β-d-galactopyranoside

Conditions: (a) PhSh, BF₃-OEt₂; (b) PhCH(OMe)₂, TsOH; (c) NIS, TfOH, −45 °C; (d) 80% AcOH, 80 °C; (e) Ac₂O-H₂SO₄, −20 °C; (f) TiBr₄; (g) AgClO₄, Fmoc-Thr-OPfp, −45 °C.

Scheme 3. Synthesis of Fmoc-Protected O-Glycosylated Thr/Ser Based on the One-Pot Azidochlorination Procedure Described by Plattner et al.

Conditions: (a) TBDPS-Cl, Et₃N, DMF, room temperature; (b) 9, CH₂Cl₂ at −30 °C then TMSOTf at room temperature; (c) TBAF, THF, AcOH, pH 7, room temperature; (d) Ac₂O, pyridine, CH₂Cl₂, room temperature; (e) NaN₃, FeCl₃, H₂O₂, CH₃CN, −30 °C; (f) AgClO₄, Fmoc-Thr/Ser-OPfp, −45 °C.

Figure 1

(A) Competitive binding assay. (B) Inhibition of binding of biotinylated ASF (5 nM) to galectin-3 (200 nM) by MUC1 glycopeptides (final concentrations of 0–1 mM). The final concentration of the beads was 25 μg/mL. The assay buffer consisted of 25 mM Hepes (pH 7.4) containing 100 mM NaCl and 0.05% Tween 20. Curves, AlphaScreen signal counts (counts per second) vs log [inhibitor, M], were plotted as means of five replicate measurements. The IC₅₀ values were obtained by nonlinear regression analysis using Graph Pad Prism 5.04.

Figure 2

ITC titration profile of (A) galectin-3 (140 μM) with LacNAc (2.6 mM), (B) galectin-3 (280 μM) with TF disaccharide (3.0 mM), (C) galectin-3 (140 μM) with Thr-TF (3.0 mM), and (D) galectin-3 (280 μM) with MUC1-Thr⁹ (2.0 mM) in buffer containing 20 mM phosphate, 0.15 M NaCl, and 10 mM BME (pH 7.2). Injections of ligand were performed every 240 s at 298 K. The top panels show the experimental ITC data and bottom panels a fit to a one-site model of the binding data using MicroCal analysis software (Origin 7.0). 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 the tables.

Figure 3

ITC titration profile of (A) the galectin-3 CRD (140 μM) with LacNAc (2.6 mM), (B) the galectin-3 CRD (140 μM) with TF disaccharide (2.6 mM), (C) the galectin-3 CRD (250 μM) with Thr-TF (3.0 mM), and (D) the galectin-3 CRD (280 μM) with MUC1-Thr⁹ (3.1 mM) in buffer containing 20 mM phosphate, 0.15 M NaCl, and 10 mM BME (pH 7.2). Injections of ligand were performed every 240 s at 298 K. The top panels show the experimental ITC data and bottom panels a fit to the one-site model of the binding data using MicroCal analysis software (Origin 7.0). 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 the tables.

Figure 4

(A) Thermodynamic signature of binding of full-length galectin-3 to LacNAc, TF disaccharide, Thr-TF, and MUC1-Thr⁹. (B) Thermodynamic signature of binding of the galectin-3 CRD to LacNAc, TF, Thr-TF, and MUC1-Thr⁹.

Figure 5

(A) Overlay of the ¹H–¹⁵N HSQC spectra of the galectin-3 CRD: black for the apo form and red upon addition of MUC1-Thr⁹ (10 equiv). (B) Overlay of the ¹H–¹⁵N HSQC spectra of the galectin-3 CRD: black for the apo form and green upon addition of MUC1 (10 equiv). (C) Average ¹H and ¹⁵N chemical shift perturbation {[ΔH² + (ΔN/5)²/2]^1/2}^, plotted for each amino acid of the galectin-3 CRD upon addition of MUC1-Thr⁹ (20 equiv). The horizontal line indicates a significant (>0.02) chemical shift perturbation. Secondary structural elements and corresponding names are shown at the top. (D) Ribbon diagram of the galectin-3 CRD complexed with the TF antigen (represented as balls and sticks) as deposited in the PDB (3AYA). The amino acids with a significant (>0.02) chemical shift perturbation for the interaction with MUC1-Thr⁹ are represented with a pink surface. (E) Comparison of the ¹H NMR spectra of the free and bound state of MUC1-Thr⁹ showing the protons affected by binding. Overlay of the ¹H NMR spectrum of MUC1-Thr⁹ in D₂O (black) and bound MUC1-Thr⁹ (red), obtained by the subtraction of the ¹H NMR spectrum of MUC1-Thr⁹–galectin-3CRD (2:1) and the ¹H NMR spectrum of the galectin-3 CRD, acquired under the same conditions. The red spectrum shows how certain proton resonances, corresponding to the Galβ1–3GalNAcα-Thr moiety, broaden significantly while the protons of the peptide part are hardly affected.

Figure 6

Semilogarithmic representation of fluorescent surface staining by galectin-3 of human SW480 colon adenocarcinoma cells. The control value of cell positivity by the second-step reagent in the absence of lectin is given as a gray-shaded area and the 100% value (lectin staining in the absence of inhibitor) as a thick black line. Quantitative characteristics of binding (percentage of positive cells/mean fluorescence intensity) in each panel are given in the order of listing (from bottom to top, in coding of gray scaling or using dashed lines): (A) dependence of binding on lectin concentration (10, 4, 2, and 1 μg/mL), (B and C) inhibition of the extent of binding at 5 μg/mL galectin-3 by increasing the concentration of lactose (B, 0.2, 0.5, 1, and 2 mM) or N-acetylgalactosamine (C, 0.5, 1, 2, and 5 mM), and (D) inhibition of galectin-3 binding (5 μg/mL) by the nonglycosylated peptide (MUC1) at 1 mM and glycopeptide MUC1-Thr⁴ at 0.5 and 1 mM.

Figure 7

Semilogarithmic representation of fluorescent surface staining by the N-terminal domain of galectin-9 (A and B) or galectin-4 (C and D) using CHO wild-type cells (A and B) or human Capan-1 pancreatic adenocarcinoma cells expressing tumor suppressor p16^INK4a (C and D). (A and B) Inhibition of binding of the N-terminal domain of galectin-9 (2 μg/mL) by test compounds used at a constant concentration of 1 mM (A, GalNAc, Lac; B, MUC1, MUC1-Thr⁹, MUC1-Thr⁴, MUC1-Thr¹⁶). (C and D) Inhibition of binding of galectin-4 (5 μg/mL) by test compounds at a constant concentration of 1 mM (C, GalNAc, Lac; D, MUC1, MUC1-Thr⁹, MUC1-Thr⁴, MUC1-Thr¹⁶).