Abstract
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.
Highlights
The results showed that neither cysteine nor tryptophan residues are directly involved in carbohydrate recognition, but some conservative hydrophilic residues such as
Construction and Expression of Mutant Lectin Genes-To assess the function of thiol groups, cysteine residues were substituted with serine residues
Rat and human 14-kDa lectins were not inactivated by chemical modification with monoiodoacetamide (22).* the stability of the modified lectin was significantly increased
Summary
Site-directed Mutagenesis-Site-directed mutagenesis was performed by the method of Nakamaye and Eckstein (23) by using an Amersham in vitro site-directed mutagenesis kit (version 2) and 5'phosphorylated mutagenic primers (Table I) synthesized by an Applied Biosystems 381A synthesizer. Bound lectin was eluted with 0.1 M lactose in EDTA/ME-PBS. Lectin-Asialofetuin Binding ASSQY-TO estimatethe affinity of purified mutant lectins for lactose in terms of ,1 (concentration required for 50% inhibition), the previously described lectin-asialofetuin binding assay (21) wasemployed with minor modifications. Purified mutant lectin (250 ng) was reacted with asialofetuin immobilized ona Nunc immunoplate in the presence of various concentrations of lactose (0.01-10mM), and the bound lectin was quantitated by a double antibody method using anti-human 14-kDa lectin antiserum and horseradish peroxidase-conjugated goat antirabbit IgG. Evaluation of Lectin Stability under Non-reducing ConditiomStability of mutant lectins was examined systematically in terms of the following criteria: (i) hemagglutinating activity in the absence of 8-mercaptoethanol; (ii)asialofetuin binding activity; and (iii) fluorescence intensity of the lectin peak in gel permeation chromatography. Thelectin peak was detected by fluorescence measurement (excitation, 280 nm; emission, 350 nm)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
