Evidence for involvement of tryptophan residue in the low-affinity saccharide binding site of ricin D.
The nature of the saccharide-binding site of ricin D, which is a galactose- and N-acetylgalactosamine-specific lectin, was studied by chemical modification and spectroscopy. With excitation at 290 nm, ricin D displayed a fluorescence spectrum with a maximum at 335 nm. Upon binding of the specific saccharides, the spectrum shifted to shorter wavelength by 3 nm. However, binding of galactosamine and N-acetylgalactosamine failed to induce such a change in the fluorescence spectrum. The interaction of ricin D with its specific saccharides was analyzed in terms of the variation of the intensity at 320 nm as a function of saccharide concentration. The results indicate that the change in the fluorescence spectrum induced by saccharide binding is attributable to the binding of saccharide to the low-affinity (LA-) binding site of ricin D. The cytoagglutinating activity of ricin D decreased to 2% upon modification of two tryptophan residues/mol with N-bromosuccinimide at pH 4.0, but in the presence of galactose or lactose one tryptophan residue/mol remained unmodified, and a fairly high cytoagglutinating activity was retained. Galactosamine and N-acetylgalactosamine did not show such a protective effect. Spectroscopic analyses indicate that the decrease in the cytoagglutinating activity of ricin D upon tryptophan modification is principally due to the loss of the saccharide binding activity of the LA-binding site. The results suggest that one tryptophan residue is essential for saccharide binding at the LA-binding site, which can bind galactose and lactose but lacks the ability to bind N-acetylgalactosamine and galactosamine.
T
T
Hatakeyama
N
N
Yamasaki
G
G
Funatsu
D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D005688:Galactosamine / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D009005:Monosaccharides / Q000378:metabolism*; D011887:Raffinose / Q000378:metabolism; D012276:Ricin / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014364:Tryptophan / Q000494:pharmacology*
D009005:Monosaccharides; D005688:Galactosamine; D014364:Tryptophan; D012276:Ricin; D008553:Melibiose; D007785:Lactose; D001974:Bromosuccinimide; D011887:Raffinose; D005690:Galactose