Fusion of liposomes induced by a cationic protein from the acrosome granule of abalone spermatozoa

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Abstract
Lysin, a protein of Mr 16,000 from the acrosome granule of the abalone, is responsible for the dissolution of the egg vitelline layer. The primary structure of this cationic protein projects some hydrophobic domains in the secondary structure. Lysin was found to associate nonselectively with phospholipid bilayers and cause a spontaneous release of encapsulated carboxyfluorescein in liposomes. The association of lysin with phosphatidylcholine liposomes suggests that there is a hydrophobic interaction between lysin and lipid bilayers. Binding of lysin to phospholipid resulted in the aggregation of phosphatidylserine-containing liposomes, but aggregation was not observed in neutral phosphatidylcholine liposomes. Resonance energy transfer and dequenching of fluorescent 1-palmitoyl-2-cis-parinaroylphosphatidylcholine were both used to determine the fusogenic activity of lysin in aggregated liposomes. Results from both assays are consistent. Lysin-induced fusion was observed in all the phosphatidylserine-containing liposomes, and the general trend of fusion susceptibility was phosphatidylserine/phosphatidylcholine (1:2) .simeq. phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine (1:1:1) > phosphatidylserine/phosphatidylethanolamine (1:2). Cholesterol up to 30% did not affect the intrinsic fusion susceptibility. A hydrophobic penetration by protein molecules and the packing of phospholipid bilayers are used to interpret the fusion susceptibility. Lysin-induced liposome aggregation was highly independent of the state of self-association of lysin in ionic medium. However, the fusogenic activity of self-associated lysin was found to be much less than the monodispersed one. Liposomes preincubated with Ca2+ did not fuse initially as readily as those without Ca2+ treatment. This early inhibition was subsequently reduced while lysin-induced fusion was taking place, and the same level of lipid mixing was reached. Our observations suggest that positively charged lysin may utilize its hydrophobic domain to interact with the hydrophobic region of lipid bilayers and facilitate the fusion of negatively charged membranes, which are in close apposition to each other, because of the polycationic nature of lysin. We propose that lysin may play two roles in fertilization: dissolution of the egg vitelline layer and promotion of fusion between sperm and egg plasma membrane.

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