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. 1H–15N 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.

Molecular Recognition of a Thomsen–Friedenreich Antigen Mimetic Targeting Human Galectin‐3

“…The average of the K D yielded an apparent K D ( K D app ) of 210 μ m . This result is in the same range as those reported by ITC for the TF disaccharide 7 (248 μ m ) and a TF bearing only the Thr residue, TF‐Thr 8 (272 μ m ) . The obtained data strongly suggest that the chemical modification at the reducing unit of the TF mimetic 2 does not affect the global affinity for Gal‐3.…”

“…[20] The resultsy ielded similar K D valueso f208, 188, and 233 mm for Asn160, Glu184,and the lateral chain of Trp181, respectively.The average of the K D yielded an apparent K D (K Dapp )o f2 10 mm.T his result is in the same range as those reportedb yI TC for the TF disaccharide 7 (248 mm)a nd aT Fb earing only the Thr residue,T F-Thr 8 (272 mm). [26] Theobtained data strongly suggest that the chemical modification at the reducing unit of the TF mimetic 2 does not affect the globalaffinity for Gal-3.…”

“…In detail, the amide cross-peaks of residues His158,A sn160, Arg162, Asn174, Thr175, Glu184, Glu185, Arg186, as wella st he indole NH group of Trp181 were found to be the major residues perturbed upon addition of the TF mimetic 2.T hese perturbed residues constitute the NMR-deduced binding site, which is mostly coincident to thatr eported for the naturalT Fa ntigen 1 and which has previously been fully characterized by X-ray crystallography and NMR studies. [25,26,20] Thus, the two ligands likely display as imilar binding mode to Gal-3 CRD. The observed effects on the chemical shifts were then used to determine the dissociation constants, K D .…”

Protein–Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy

“…As well, the addition of sialic acid (Siaα2‐3) is also tolerated and also slightly improves the affinity with respect to the primary β‐galactoside. Furthermore, the molecular features that govern Gal‐3/β‐galactosides recognition events have been well characterized by using in vitro studies . In this work, we have monitored the Gal‐3/crowder interactions and the Gal‐3/lactose binding event in crowding environment.…”

“…Furthermore, the molecular features that govern Gal-3/b-galactosides recognition eventsh ave been well characterizedb yu sing in vitro studies. [28][29][30][31] In this work, we have monitored the Gal-3/crowder interactions and the Gal-3/lactose binding event in crowding environment. Distinct protein and polymer crowders were employed such as bovine and human serum albumins (BSA and HSA), Ficoll 70 and PEG3350.S erum albumins (BSA-I and HSA-I) were selected due to their glycosylationc ontent.…”