Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.
A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.
Department of Surgical Oncology, Roswell Park Memorial Institute, Buffalo, New York 14263.
H
H
Ahmed
H J
HJ
Allen
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A
Sharma
K L
KL
Matta
D001665:Binding Sites; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D037161:Galectins; D006388:Hemagglutinins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D013154:Spleen; D000509:alpha-Fetoproteins
D037161:Galectins; D006388:Hemagglutinins; D000509:alpha-Fetoproteins