Chemical modification and NMR studies on a mushroom lectin Ischnoderma resinosum agglutinin (IRA)

Abstract

Chemical modification and NMR studies on a β-galactosyl-specific lectin which was isolated from the fruiting bodies of a mushroom, Ischnoderma resinosum, has been carried out in order to investigate the amino acid residues involved in its sugar-binding sites. Modification of amino groups with succinic anhydride greatly affected the hemagglutinating activity. Inhibitory sugar lactulose could prevent the loss of the activity. Modification of car☐yl groups with glycine ethyl ester led to a 75% loss of the activity, the presence of inhibitory sugar being protective against the modification. Treatment with cyclohexane-1,2-dione for modification of arginine residues was accompanied by a complete loss of the activity. The arginine residues modification could also be protected by the inhibitory sugar. N-Bromosuccinimide treatment for modification of tryptophan residues caused a loss of the activity, although the inhibitory sugar exhibited no protective effect against this treatment. Modification of thiol groups with 5,5′-dithiobis(2-nitrobenzoic acid) resulted in a 50% loss of the activity. Modification of histidine residues with ethoxyformic anhydride led to a complete loss of the activity. The loss of the activity could be protected by the inhibitory sugar. Treatment with N-acetylimidazole for modification of tyrosine residues was accompanied by a loss of the activity. This modification was completely prevented in the presence of the inhibitory sugar. The activity of the tyrosine-modified lectin was recovered by the treatment with hydroxylamine. Furthermore, in the NOESY spectrum of the mixture of IRA and its inhibitory sugar, methyl β-galactoside, an NOE cross peak between H-3 and/or 5 of the p-hydroxyphenyl group of a tyrosine in the lectin, and H-5 of the galactoside could be observed. These results indicate that a tyrosine residue is involved in the carbohydrate-binding site of the lectin. In addition, line broadening and down-field shifts of the galactoside-protons were observed in the presence of the lectin.