Kinetic measurements of binding of galectin 3 to a laminin substratum.
Galectin 3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal segment including multiple repeats of a proline/tyrosine/glycine-rich motif. Previous work has shown that galectin 3 but not the isolated CRD binds to laminin, a multivalent ligand, with positive cooperativety indicating the formation of multiple interactions although the lectin in solution is monomeric. Using surface plasmon resonance, we find that hamster galectin 3 at sub-micromolar concentrations or its isolated CRD at all concentrations binds to a laminin substratum with similar association (k(ass); 10-30,000 M(-1) S(-1)) and dissociation (k(diss); 0.2-0.3 S1(-1)) rates and weak affinity (Ka; 1-3 x 10(5) M(-1)). At higher concentrations of galectin 3 the off rate decreases ten fold leading to increased affinity. Ligation of an N-terminal epitope of galectin 3 with a monoclonal Fab fragment increases association and dissociation rates ten fold. A recombinant protein obtained by deletion of the first 93 N-terminal residues binds to laminin with positive cooperativity and a slowly dissociating fraction (K(diss); 0.002 S(-1)) accumulates on the substratum. The data suggest that homophilic interactions between CRD as well as N terminal domains are implicated in galectin 3 aggregation on the substratum leading to positive binding cooperativity.
Barboni
E A
EA
National Institute for Medical Research, Mill Hill, London, UK.
Bawumia
S
S
Hughes
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D006224:Cricetinae; D037502:Galectin 3; D006801:Humans; D007700:Kinetics; D007797:Laminin / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D000943:Antigens, Differentiation; D037502:Galectin 3; D007797:Laminin; D008562:Membrane Glycoproteins