Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites.
Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed. Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity. These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites. Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity. Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.
Amano
Koh
K
Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Japan.
Fujihashi
Masahiro
M
Ando
Akikazu
A
Miki
Kunio
K
Nagata
Yoshiho
Y
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018076:DNA, Complementary / Q000235:genetics; D005643:Fucose / Q000378:metabolism*; D006384:Hemagglutination / Q000187:drug effects / Q000235:genetics; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D037102:Lectins; C064549:lectin, Aleuria aurantia; D005643:Fucose; D014443:Tyrosine; D013774:Tetranitromethane