Abstract
Galectins are involved in the regulation of divergent physiological and pathological processes and are increasingly recognized to play important roles in a number of diseases. However, a simple and effective way in assessing galectin-ligand interactions is lacking. Our examination of the sequence of all 12 human galectin members reveals the presence of one or more tryptophan residues in the carbohydrate-recognition domains of each galectin. This led us to investigate the possibility that alteration of the galectin intrinsic tryptophan fluorescence could be used in determining the strength of galectin-ligand interactions. One representative member from each of the three subtype galectins, galectin-2 (proto-), galectin-3 (chimera-) and galectin-4 (tandem repeat-type), was selected and analysed for galectin interaction with three ligands of different affinities: galactose, lactose and N-acetyl-lactosamine using tryptophan fluorescence spectroscopy (TFS) and, as a comparison, isothermal titration calorimetry (ITC). Good agreement between TFS and ITC measurements were revealed in ligand bindings of all galectin members. Moreover, TFS detected very weak galectin binding where ITC could not reliably do so. The reliability of TFS in determining galectin-ligand interactions was further validated by analysis of galectin-3 interaction with a semisynthetic ligand, F3. Thus, TFS can be used as a simple, sensitive and reliable way to determine galectin-ligand interactions and also as a drug-discovery platform in developing galectin-targeted therapeutic drugs.
Highlights
Galectins are a family of 15 galactoside-binding mammalian proteins (12 in human)
It was found that the binding of three galectin binding ligands with different binding affinities to each example of the three subtype galectins galectin-2, galectin-3 and galectin-4 can be quickly determined by intrinsic tryptophan fluorescence spectroscopy
The binding affinities obtained from tryptophan fluorescence spectroscopy (TFS) were in good agreement with that obtained by isothermal titration calorimetry (ITC), the “gold-standard” measurement in assessing protein-ligand interactions
Summary
Galectin-3-ligand interactions determined by TFS and ITC. Galectin-3, the only chimera type galectin, contains three tryptophan residues; one in the CRD (Trp181), which is well conserved among all human galectins and is directly involved in galectin carbohydrate binding (Figs 1 and 2), and two in the N-terminal domain (Trp[22], Trp26) (Fig. 2). The presence of lactose showed no effect at all on the maximum tryptophan fluorescence of BSA (bovine serum albumin) (345 nm), a non-glycan binding and three tryptophan residue-containing protein, and, as expected, caused only a small reduction of fluorescence intensity due to buffer dilution (Fig. S1) This provides further support to the effectiveness of TFS analysis in determining galectin-3-ligand interactions. The consistent agreement of bindings obtained by TFS and ITC for all the three subtypes of galectins indicates that measurements of intrinsic tryptophan fluorescence changes can serve as a reliable way to determine galectin-ligand interactions. To demonstrate this further, we assessed the interaction of galectin-3. As F3 is a heparin derivative of complex glycan, these results indicate that TFS analysis can be used in assessing galectin interactions with simple ligand as well as complex glycans
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