pGalectins, a family of β-galactoside-specific endogenous lectins, are involved in regulating diverse activities such as proliferation/apoptosis, cell—cell (matrix) interaction and cell migration. It is presently unclear to what extent the carbohydrate fine specificities of the combining sites of mammalian galectins overlap. To address this issue, we performed an analysis of the carbohydrate-recognition domain (CRD-I) near the N-terminus of recombinant rat galectin-4 (G4-N) by the biotin/avidin-mediated microtitre plate lectin-binding assay with natural glycoproteins (gps)/polysaccharide and by the inhibition of galectin—glycan interactions with a panel of glycosubstances. Among the 35 glycans tested for lectin binding, G4-N reacted best with human blood group ABH precursor gps, and asialo porcine salivary gps, which contain high densities of the blood group Ii determinants Galβ1-3GalNAc (the mucin-type sugar sequence on the human erythrocyte membrane) and/or GalNAcα1-Ser/Thr (bTn/b), whereas this lectin domain reacted weakly or not at all with most sialylated gps. Among the oligosaccharides tested by the inhibition assay, Galβ1-3GlcNAcβ1-3Galβ1-4Glc was the best. It was 666.7 and 33.3 times more potent than Gal and Galβ1-3GlcNAc, respectively. G4-N has a preference for the β-anomer of Gal at the non-reducing ends of oligosaccharides with a Galβ1-3 linkage, over Galβ1-4 and Galβ1-6. The fraction of bTn/b glycopeptide from asialo ovine submandibular glycoprotein was 8.3 times more active than Galβ1-3GlcNAc. The overall carbohydrate specificity of G4-N can be defined as Galβ1-3GlcNAcβ1-3Galβ1-4Glc (lacto-iN/i-tetraose)&Galβ1-4GlcNAcβ1-3Galβ1-4Glc (lacto-iN/i-neo-tetraose) and bTn/b clusters&Galβ1-4Glc and GalNAcβ1-3Gal&Galβ1-3GalNAc&Galβ1-3GlcNAc&Galβ1-4GlcNAc&GalNAc&Gal. The definition of this binding profile provides the basis to detect differential binding properties relative to the other galectins with ensuing implications for functional analysis./p
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