Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus.
Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp32-->Glu32), rPL-N34S (Asn34-->Ser34) and rPL-V36A (Val36-->Ala36) whereas binding site 2; rPL-D61E (Asp61-->Glu61), rPL-N63S (Asn63-->Ser63) and rPL-V65A (Val65-->Ala65). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.
Tsutsui
Shigeyuki
S
Fisheries Laboratory, The University of Tokyo, Shizuoka 431-0211, Japan. tsutsui@kitasato-u.ac.jp
Tasumi
Satoshi
S
Suetake
Hiroaki
H
Kikuchi
Kiyoshi
K
Suzuki
Yuzuru
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009093:Mucus / Q000378:metabolism; D037121:Plant Lectins / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000378:metabolism; D023741:Takifugu / Q000235:genetics* / Q000378:metabolism
D029941:Fish Proteins; D037601:Mannose-Binding Lectin; D037121:Plant Lectins; D008358:Mannose