Role of basic amino acids in the interaction of bindin with sulfated fucans.
Bindin, the acrosomal sperm adhesion protein of the sea urchin Strongylocentrotus purpuratus, binds specifically and with high affinity (Kd = 10(-8) M) to egg sulfated fucans in the high ionic strength milieu of seawater (0.55 M salt). Previous studies indicated that the negatively charged sulfate groups of the polysaccharide are critical for binding which suggested a binding mechanism involving basic residues of bindin. We found that the binding of fucan to bindin or polyarginine is stable at the ionic strength of seawater, whereas the binding of fucan to polylysine or polyhistidine is inhibited by 50% or more at this ionic strength. Group-specific modification of either arginine, lysine, or histidine residues of bindin results in a substantial inactivation of fucan binding activity. Preincubation of bindin with fucan can almost completely protect bindin from inactivation by arginine-specific reagents, butanedione and phenylglyoxal, but only moderately slowed the inactivation by the histidine reagent diethyl pyrocarbonate. In contrast, prior fucan binding could not prevent loss of activity by the reaction of citraconic anhydride with lysine residues. Other sulfated polysaccharides which do not interact strongly with bindin did not protect binding from phenylglyoxal-mediated inactivation when 800-3000-fold more polysaccharide than fucan was used during the preincubation before modification. We found that the larger and more hydrophobic arginine-modifying reagents, camphorquinone-10-sulfonic acid and cyclohexanedione, fail to inactivate fucan binding, suggesting that essential arginine residues may reside in an environment with restricted accessibility to these reagents. Parallel kinetic studies monitoring [14C]phenylglyoxal incorporation and fucan binding inactivation indicate that several of the four total arginine residues may be critical for fucan binding.(ABSTRACT TRUNCATED AT 250 WORDS)
DeAngelis
P L
PL
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Glabe
C G
CG
D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010658:Phenylglyoxal / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions
D006023:Glycoproteins; D011134:Polysaccharides; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor; C007789:fucoidan; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal