Characterization of monomeric forms of galectin-1 generated by site-directed mutagenesis.

Galectin-1 is a beta-galactoside-binding protein secreted by animal cells, and it exists in a monomer-dimer equilibrium (Kd approximately 7 microM). The function(s) of galectin-1 is(are) not yet defined, but dimerization and divalency are presumably important. Crystal structures of the mammalian galectin-1 dimer predict N- and C-terminal interactions at the subunit interface. To examine the mechanism of dimer formation and possibly generate active monomeric galectin-I, mutations were made in the N- and C-termini of recombinant hamster galectin-1. N-Gal-1 contains disruptions of three hydrophobic amino acids at the N-terminus; V5D-Gal-1 contains a single mutation of Val5 to Asp; N/C-Gal-1 contains multiple changes in hydrophobic amino acids at both the N- and C-termini. All mutants behave as monomers in size-exclusion HPLC and native gel electrophoresis. N-Gal-1 and V5D-Gal-1 bind weakly to lactosyl-Sepharose, but N/C-Gal-1 is nonfunctional. In equilibrium dialysis, N-Gal-1 and V5D-Gal-1 bind N-acetyllactosamine with a Kd approximately 90 microM, which is similar to that of native lectin. At high concentrations, V5D-Gal-1 and N-Gal-1 dimerize and can be covalently cross-linked with disuccinimidyl suberate. The Kd values of the monomer-dimer equilibrium for V5D-Gal-1 and N-Gal-1 are estimated to be approximately 60 microM and approximately 250 microM, respectively. The cross-linked dimers of V5D and N-Gal-1 were isolated and were similar to native lectin in both hemagglutinating activity and high-affinity binding to lactosyl-Sepharose. Thus, specific mutations in galectin-1 can alter monomer-dimer equilibrium without affecting carbohydrate-binding activity. The availability of active monomers and functional covalent dimers of galectin-1 should aid in future studies aimed at understanding the biological function(s) of the lectin and the role of divalency.