The Effects of Lignosulfonic Acid on Fertilization Events of the Sea Urchin, Strongylocentrotus purpuratus
Breakdown products of the wood structural polymer lignin are major components in the effluent from commercial pulp and paper manufacturing mills. Polar lignin-derived macromolecules (LDMs) isolated from vastly different pulping processes and from different source wood have surprisingly similar properties and biological activity. Our group has previously reported that a LDM from bleached Kraft mill effluent (BKME) inhibits sea urchin fertilization by binding to sperm cells and blocking the egg jelly induced acrosome reaction. Lignosulfonic acid (LSA), an LDM from sulfite mill effluent, is structurally distinct from BKME-LDM and data presented herein indicate that it was a more potent inhibitor of fertilization. LSA ranged in molecular size from 300K, with higher molecular size fractions being the more potent inhibitors of fertilization. The mode of action of LSA on sea urchin fertilization was characterized by examining its effects on two critical prerequisite sperm-egg interactions: acrosome reaction induction by egg jelly and sperm-to-egg binding. LSA inhibited both of these processes at low concentrations. Microscopically, a biotinylated LSA derivative (LSA-b) bound to localized domains of the sperm surface, particularly over the head and acrosomal regions. The interaction of LSA with binding sites on the sperm surface was further characterized using a quantitative solid phase whole-cell binding assay and the LSA-b probe. LSA-b behaved in a manner consistent with receptor-ligand binding models in that its interaction with sperm was specific, saturable, kinetically dependent, and reversible. LSA specifically inhibited the binding of antibodies to proteins that entirely co-localize within its binding domain, sea urchin receptor for egg jelly (suREJ3) and bindin. These results support the hypothesis that LSA exerts its biological action by binding to key molecules involved in gamete recognition.