Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1.
To identify critical amino acid residues for carbohydrate binding of galectins (soluble beta-galactoside-binding lectins found in the animal kingdom). site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992) J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.
Hirabayashi
J
J
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Kasai
K
K
D000595:Amino Acid Sequence; D001212:Asialoglycoproteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D017124:Conserved Sequence; D017931:DNA Primers; D060748:Fetuins; D037483:Galectin 1; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D017931:DNA Primers; D060748:Fetuins; D037483:Galectin 1; D006388:Hemagglutinins; D037102:Lectins; D000509:alpha-Fetoproteins; C017029:asialofetuin