Sugar binding properties of the two lectin domains of the tandem repeat-type galectin LEC-1 (N32) of Caenorhabditis elegans -: Detailed analysis by an improved frontal affinity chromatography method

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14kDa-type galectins, To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Oh preferred Gal beta1-3GlcNAc to Gal beta1-4GlcNAc. Because the Fuc alphal-2Gal beta1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K-d value of pentasaccharide Fuc alpha1-2Gal beta1-3GlcNAc beta1-3Gal beta1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 x 10(-5), 1.3 x 10(-4), and 6.5 x 10(-5) M, respectively. The most significant difference between Nh and Oh was in their affinity for GalNAc alpha1-3 (Fuc alpha1-2) Gal beta1-3GlcNAc beta1-3Gal beta1-4Glc-PA, which contains the blood group A antigen; the K-d value for immobilized Nh was 4.8 x 10(-5) M, and that for Ch was 8.1 x 10(-4) M, The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.