Studies by site-directed mutagenesis of the carbohydrate-binding properties of a bark lectin from Robinia pseudoacacia.
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 Thu215, 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.
Nishiguchi
M
M
Forestry and Forest Products Research Institute, Ibaraki, Japan.
Yoshida
K
K
Sumizono
T
T
Tazaki
K
K
D000818:Animals; D050260:Carbohydrate Metabolism; D004912:Erythrocytes; D004926:Escherichia coli; D006386:Hemagglutination Tests; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008358:Mannose / Q000494:pharmacology; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014197:Trees / Q000737:chemistry*
D037102:Lectins; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; D008358:Mannose