title: Characterization of monomeric forms of galectin-1 generated by site-directed mutagenesis.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma Center for Molecular Medicine, Oklahoma City 73190, USA.
authors.forename: M
authors.initials: M
authors.lastname: Cho
authors.forename: R D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000378:metabolism; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003956:Dialysis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D037483:Galectin 1; D006384:Hemagglutination; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D037102:Lectins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013388:Succinimides
chemical_list: D000606:Amino Sugars; D003432:Cross-Linking Reagents; D037483:Galectin 1; D006388:Hemagglutinins; D037102:Lectins; D011994:Recombinant Proteins; D013388:Succinimides; C000458:N-acetyllactosamine; C019358:disuccinimidyl suberate