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

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.

Development of an AlphaScreen assay for discovery of inhibitors of low-affinity glycan-lectin interactions.

The development of high-throughput screening (HTS) assays with increased sensitivity for the identification of potent and selective inhibitors of galectins has been hampered by the weak binding affinities between galectins and their carbohydrate ligands. To circumvent this obstacle, we have developed an AlphaScreen assay for a 384-well plate format in a competitive binding configuration for discovery of new inhibitors of galectin-3. His-tagged galectin-3 was bound to nickel chelate acceptor beads, whereas biotinylated asialofetuin (biotin-ASF), a galectin-3 nanomolar binding partner, was bound to streptavidin-coated donor beads. Inhibitors of the carbohydrate-galectin interaction lead to a reduction of the AlphaScreen signal by competing with the biotin-ASF. The obtained IC50 values for known carbohydrate ligands of galectin-3 are in good agreement with the Kd values reported and measured for galectin-3 by isothermal titration calorimetry (ITC). Thus, the developed AlphaScreen assay in a competitive binding configuration offers several advantages over the existing screening assays for inhibitors of glycan-lectin interactions. In addition, the assay format for the galectin-3/ASF pair could be easily applied in screening for glycan- and/or small molecule-based inhibitors of other members of the galectin family.