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 ga lactose-related sugars specifically, Recombinant RBL (rRBL) with a his tidine tag was expressed in Escherichia coli, purified and characteriz ed. 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 th at residues Phe(130) and Asp(87) play key roles in the binding of carb ohydrate by RBL. When Thr(215), Leu(217) and Ser(218) in the carboxy-t erminal region were replaced by alanine, the respective replacements d ecreased the hemagglutinating activity, However, replacement by alanin e of Glu(219) did not decrease this activity. Three mutant rRBLs were generated by reference to the primary sequences of the proposed carboh ydrate- and metal-binding regions of mannose-specific lectins, Althoug h these rRBLs agglutinated rabbit erythrocytes, the hemagglutination w as not inhibited by mannose, Substitution or insertion that yielded a partial sequence similar to those of L-fucose-specific lectins and hem agglutinin from Maackia amurensis resulted in a complete loss of the h emagglutinating activity of rRBL. (C) 1997 Federation of European Bioc hemical Societies.