Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin.
The roles of selected amino acid residues of human 14-kDa beta-galactoside-binding lectin were studied by site-directed mutagenesis. Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate beta-galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted. All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin. In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions. It retained asialofetuin binding activity for over a week in the absence of beta-mercaptoethanol, while the wild-type lectin lost it within a day. This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin. Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose. Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation. Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential.
Hirabayashi
J
J
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Kasai
K
K
D000595:Amino Acid Sequence; D001212:Asialoglycoproteins; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D060748:Fetuins; D005697:Galactosides / Q000378:metabolism*; D037161:Galectins; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D005697:Galactosides; D037161:Galectins; D006388:Hemagglutinins; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin