Carbohydrate Binding Competition
Fucose-PAA-biotin binding to immobilized SPM was challenged by preincubating the SPM-coated wells (0.5 |xg SPM per well) with various concentrations (0-100 mM) of carbohydrates before addition of 0.25 |xg/ml fucose-PAA-biotin per well. The competition of fucose-PAA-biotin binding by fucose, glucose, maltose, mannose, galactose, inositol, 1- O-methyl-a-D-glycopyranoside, mannan, and fu-coidan (where mannan and fucoidan concentration was calculated from the values of mannose or fucose units, respectively) were tested. All tested carbohydrates inhibited fucose-PAA-biotin binding to immobilized SPM (P < 0.001, n = 3 for all inhibiting carbohydrates tested, Fig. 3).
The exception was inositol, which did not exert any binding competition even at the highest tested dose (P > 0.05, n = 3; Fig. 3). Galactose and 1-O-methyl-a-D-glycopyranoside only slightly inhibited this binding at concentrations higher than 10 mM, and 50% binding inhibition (EC50) was reached over 400 mM for both sugars. Fucoidan showed the highest competition for fucose-PAA-biotin binding to SPM (EC50 values of 41.7 ± 1.6 |xM), followed by mannan, which also effectively competed for this binding (EC50 values of 1.67 ± 0.02 mM) (Fig. 3).
FIG. 3. Carbohydrate competition of fucose-PAA-biotin binding to SPM from uncapacitated sperm cells. SPM (0.5 ^g/well) were incubated with 0.25 |xg/ml of fucose-PAA-biotin in the presence of increasing concentrations (0.0-100 mM) of fucose (Fuc), glucose (Glu), maltose (Mal), mannose (Man), galactose (Gal), mannan (Mann), inositol (Ino), 1-O-methyl-a-D-glycopyranoside (1-O-m), and fucoidan (Fdan). The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated.