High-affinity binding of recombinant human galectin-4 to SO₃^-→3Galβ1-→3GalNAc

Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of β-galactose in these oligosaccharides with α-fucose, α-GaINAc, α-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K_d ≅ 8 × 10^-4 M). Galectin-4 showed weak binding ability to core 1 and C-2′ or -3′-substituted lactose, type 1, and type 2 with α-fucose, α-GaINAc, or sulfate (K_d : 5 × 10^-5 ∼ 3 × 10^-4 M). Interestingly, the K_d value, 3.4 × 10^-6 M, of SO₃^-→3Galβ1→3GalNAc-O-Bn to galectin-4 at 25°C was two orders of magnitude lower than that of core 1-O-Bn. 3′-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3′-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.