Sugar-Binding Properties of the Two Lectin Domains of LEC-1 with Respect to the Galβ1-4Fuc Disaccharide Unit Present in Protostomia Glycoconjugates
Biological and Pharmaceutical Bulletin
Galectins are a group of animal lectins characterized by their specificity for b-galactosides and by an evolutionarily conserved sequence motif in their carbohydrate-binding site. Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune function. 1-6) A Galb1-4GlcNAc (N-acetyllactosamine) disaccharide unit is thought to be the endogenous glycoepitope recognized by vertebrate
... galectins. This recognition has also been observed in galectins from invertebrate species such as Caenorhabditis elegans (C. elegans), including the galectins LEC-1 and LEC-6, which we have isolated previously. 7-9) However, the existence of a glycan containing Nacetyllactosamine units has not yet been confirmed in C. elegans.       Recently, we isolated N-type glycoproteins captured by the C. elegans galectin LEC-6 and analyzed their sugar structure by matrix-assisted laser desorption-time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We found that LEC-6 interacts strongly with N-glycans containing a Gal-Fuc disaccharide unit attached to position 6 of the innermost GlcNAc residue. 16) In order to identify the linkage between the galactose and fucose residues, and examine the structure of the endogenous glycoepitope recognized by LEC-6, we compared the binding strengths of the chemically synthesized Galb1-4Fuc and Galb1-3Fuc, 17) and found that LEC-6 interacts more strongly with Galb1-4Fuc than with Galb1-3Fuc. 18) LEC-1, another major galectin from C. elegans, also showed preferential binding to Galb1-4Fuc. 18) These results suggest that Galb1-4Fuc is the endogenous unit recognized by the C. elegans galectins. LEC-1 is a tandem-repeat type galectin, wherein two homologous carbohydrate recognition domains exist within a single polypeptide chain. 7, 8) We previously reported that although the two domains of LEC-1 showed affinity for sugars containing Galb1-4GlcNAc units, detailed analysis using fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars) in frontal affinity chromatography (FAC) 19) revealed these domains had different sugar binding properties. 20) However, the oligosaccharides used in the previous study 20) differed in that they were mainly of vertebrate origin and commercially available in pyridylaminated forms. For this report, we used recombinant proteins of the whole molecule (LEC-1), the N-terminal lectin domain (LEC-1Nh), and the C-terminal lectin domain (LEC-1Ch), and compared their binding affinities for the Galb1-4Fuc and Galb1-3Fuc disaccharide units. Although the two lectin domains showed preferential binding to Galb1-4Fuc compared to Galb1-3Fuc and N-acetyllactosamine-containing structure LNnT (Galb1-4GlcNAcb1-3Galb1-4Glc), the binding profiles of the two domains were different. While LEC-1Nh demonstrated a very high affinity to Galb1-4Fuc with a very low affinity for Galb1-3Fuc and LNnT, LEC-1Ch displayed a moderate affinity for both Galb1-3Fuc and LNnT. Furthermore, both LEC-1Nh and LEC-1Ch had significantly lower binding affinities for the LEC-6-binding glycans. These results suggest that the two domains of LEC-1 bind to different N-glycans containing the Gal-Fuc disaccharide unit, and that the ligands for the two domains of LEC-1 are different from the LEC-6-binding glycans. MATERIALS AND METHODS Materials Galb1-4Fuc labeled with pyridylamine (PA) via a spacer (PA-Galb1-4Fuc) and Galb1-3Fuc labeled with pyridylamine via a spacer (PA-Galb1-3Fuc) (Fig. 1A) were chemically synthesized, as reported previously. 17) Chemical Galb b1-4Fuc disaccharide unit was recently reported to be the endogenous structure recognized by the galectin LEC-6 isolated from the nematode Caenorhabditis elegans. LEC-1, which is another major galectin from this organism, is a tandem repeat-type galectin that contains two carbohydrate recognition domains, the N-terminal lectin domain (LEC-1Nh) and the C-terminal lectin domain (LEC-1Ch), and was also found to have an affinity for the Galb b1-4Fuc disaccharide unit. In the present study, we compared the binding strengths of LEC-1, LEC-1Nh, and LEC-1Ch to Galb b1-4Fuc, Galb b1-3Fuc, and Galb b1-4GlcNAc units as well as to LEC-6-ligand Nglycans by using frontal affinity chromatography (FAC) analysis. The two lectin domains of LEC-1 exhibited the highest affinity for Galb b1-4Fuc, though sugar-binding properties differed somewhat between LEC-1Nh and LEC-1Ch. Furthermore, these two domains had significantly lower affinities for the LEC-6-binding glycans. These results suggest that the endogenous recognition unit of LEC-1 is likely to be Galb b1-4Fuc, and that the endogenous ligands for LEC-1 are different from those for LEC-6.