Binding Characteristics of Galectin-3 Fusion Proteins

Abstract

Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.

Glycopolymers by RAFT Polymerization as Functional Surfaces for Galectin‐3

“…Gal‐3 binding to the immobilized glycopolymer yielded typical sensorgrams (Figure A). This is in contrast to SPR measurements using an immobilized standard glycoprotein, asialofetuin, that gave plateau‐like curves without distinct association and dissociation phases …”

“…Protein concentrations were measured by the Bradford assay using bovine serum albumin (BSA, Carl Roth, Karlsruhe, Germany) for calibration. For testing the full binding properties, an ELISA‐type binding assay was performed using immobilized asialofetuin …”

“…For testing the full binding properties, an ELISA-type binding assay was performed using immobilized asialofetuin. [22] Surface Plasmon Resonance Spectroscopy: SPR spectroscopy was performed with Reichert SR7000DC System using gold sensor chips (XanTec bioanalytics GmbH, Düsseldorf, Germany). The (glyco) polymer at a concentration of 2 mg mL −1 was immobilized on a chip via trithiocarbonate residues of the polymer.…”

“…This is in contrast to SPR measurements using an immobilized standard glycoprotein, asialofetuin, that gave plateau-like curves without distinct association and dissociation phases. [21,22] Overall, very low unspecific binding to the reference poly mer pHEMA was observed. The reference values were subtracted from the binding data for both lectins, ECL and Gal-3.…”

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

“…We chose APEX2 over other peroxidase enzymes, due to its monomeric nature, small size (28 kDa), and improved activities in various cellular compartments [16], especially for proteomic applications [20]. We fused APEX2 to the N-terminus of galectin-3, as previous work has shown that such fusion proteins remain active towards binding glycans [21]. Both fusion proteins were generated efficiently (Figs.…”