Studies by site‐directed mutagenesis of the carbohydrate‐binding properties of a bark lectin from Robinia pseudoacacia

FEBS Letters

Yoshida, Kazumasa¹  Sumizono, Toshiro¹  Nishiguchi, Mitsuru¹  Tazaki, Kiyoshi¹ 

[1] Forestry and Forest Products Research Institute, P.O. Box 16, Tsukuba Norin Kenkyu Danchi-nai, Ibaraki 305, Japan

关键词: Robinia pseudoacacia;    Bark lectin;    Carbohydrate binding;    Site-directed mutagenesis;    BSA;    bovine serum albumin;    IPTG;    isopropyl-β-d-thiogalactopyranoside;    PBS;    phosphate-buffered saline;    PMSF;    phenylmethylsulphonyl fluoride;    RBL;    bark lectin from Robinia pseudoacacia;    rRBL;    recombinant RBL;   

DOI  :  10.1016/S0014-5793(97)00068-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 Phe¹³⁰ and Asp⁸⁷ play key roles in the binding of carbohydrate by RBL. When Thr²¹⁵, Leu²¹⁷ and Ser²¹⁸ in the carboxy-terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity. However, replacement by alanine of Glu²¹⁹ 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.

【 授权许可】

Unknown