Premium
Studies by site‐directed mutagenesis of the carbohydrate‐binding properties of a bark lectin from Robinia pseudoacacia
Author(s) -
Nishiguchi Mitsuru,
Yoshida Kazumasa,
Sumizono Toshiro,
Tazaki Kiyoshi
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00068-9
Subject(s) - biochemistry , lectin , site directed mutagenesis , mannose , hemagglutination , mutant , chemistry , fucose , agglutinin , galactose , mutagenesis , alanine , histidine , biology , microbiology and biotechnology , amino acid , antibody , gene , immunology
A bark lectin, RBL, from Robinia pseudoacacia (black locust), binds galactose‐related sugars specifically. Recombinant RBL (rRBL) with a histidine tag was expressed in Escherichia coli , purified and characterized. rRBL agglutinated rabbit erythrocytes and the hemagglutination was inhibited by galactose and related sugars. To elucidate the mechanism of the binding of carbohydrate by RBL, 16 mutant rRBLs were produced by site‐directed mutagenesis. The analysis of the mutants indicated that residues Phe 130 and Asp 87 play key roles in the binding of carbohydrate by RBL. When Thr 215 , Leu 217 and Ser 218 in the carboxy‐terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity. However, replacement by alanine of Glu 219 did not decrease this activity. Three mutant rRBLs were generated by reference to the primary sequences of the proposed carbohydrate‐ and metal‐binding regions of mannose‐specific lectins. Although these rRBLs agglutinated rabbit erythrocytes, the hemagglutination was not inhibited by mannose. Substitution or insertion that yielded a partial sequence similar to those of l ‐fucose‐specific lectins and hemagglutinin from Maackia amurensis resulted in a complete loss of the hemagglutinating activity of rRBL.© 1997 Federation of European Biochemical Societies.