Lysin, a protein from abalone sperm, creates a hole in the envelope of the egg, permitting the sperm to pass through the envelope and fuse with the egg. The structure of lysin, refined at 1.9 angstroms resolution, reveals an alpha-helical, amphipathic molecule. The surface of the protein exhibits three features: two tracks of basic residues that span the length of the molecule, a solvent-exposed cluster of aromatic and aliphatic amino acids, and an extended amino-terminal hypervariable domain that is species-specific. The structure suggests possible mechanisms of action.

Abstract, Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A. resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 ~M and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.URING fertilization, spermatozoa must penetrate the extracellular investments surrounding the egg before the plasma membranes of the two gametes can make contact and fuse. To accomplish this, most animal species have evolved sperm with acrosomal vesicles that undergo exocytosis to release lyric proteins used in penetration of the egg coat. Abalone spermatozoa possess an enormous acrosomal vesicle (Lewis et al., 1980). These sperm bind to the elevated egg vitelline envelope (VE) 1 and release concentrated 16-kD lysin from the exocytosing acrosome. Lysin creates a hole in the VE by a nonenzymatic mechanism to allow the sperm cell to pass through the VE and fuse with the egg (Lewis et al., 1982).Purified lysin exhibits species-selectivity in the dissolution of isolated VE. For example, 12 p~g of red abalone (Haliotis rufescens) lysin will dissolve 95 % of red abalone egg VE, whereas 12 ~g of lysin from the black abalone (1t.

Abalone eggs are contained within a rigid, elevated vitelline envelope through which the sperm must pass before reaching the egg cell membrane. Abalone spermatozoa possess an acrosomal protein called iysin that creates a hole in the egg vitelline envelope by a nonenzymatic mechanism. Lysins from two species ofabalone, termed pink and red, which share the same habitat, exhibit species specificity in the dissolution of isolated egg envelopes. Cloning and sequencing the cDNAs for pink and red abalone lysins reveal transcript lengths of -660 nucleotides. The open reading frames of465 (pink) and 462 (red) nucleotides show a 13% difference. The 3' untranslated regions before the poly(A) tails are 170 (pink) and 165 (red) nucleotides long and differ from each other by about 7%. The protein sequences show nearly identical signal sequences of 18 amino acids for both lysins. The mature protein is 137 amino acids in the pink abalone and 136 in the red abalone; the two mature lysins differ in 29 of 137 amino acids (21%). The most variable region, which may account for lysin's species specificity, is at the NH2 terminus, where 11 of the 15 amino acids differ between the two species. Predictions of secondary structure indicate that both lysins contain four homologous amphiphilic a-helices.In animals the fusion of sperm and egg during fertilization is preceded by several events mediated by gamete recognition proteins. These events include the adhesion of sperm to the egg envelope, the induction of the acrosome reaction by components ofthe egg's extracellular matrix, and penetration of the sperm through the egg envelope. When cross-species fertilization is attempted, blocks to these events can be demonstrated depending on the particular species combinations being studied. Such blocks to cross-species fertilization can be readily demonstrated in congeneric species of marine invertebrates that share the same habitat, have overlapping breeding seasons, and release their gametes to the seawater where fertilization occurs. Knowledge of the proteins (and their genes) that mediate the species-specific events of sperm-egg interaction is important for understanding the molecular mechanisms of fertilization. It will also be important in understanding the evolution of the mechanisms that prevent successful sperm-egg interaction and ultimately create barriei to reproduction between species.Abalone Lre large archeogastropod molluscs of the genus Haliotis, eight species of which occur on the Pacific Coast of North America. The abalone egg is enclosed in an elevated glycoproteinaceous vitelline envelope (VE; -1 u.m in thickness) through which the spermatozoon must pass before it can fuse with the egg. During fertilization the abalone spermatozoon attaches to the VE, its acrosome granule opens, and two proteins of Mr -16,000 and =18,000 are released onto the VE. A hole 3 gm in diameter appears in seconds in the VE to allow the 1-Am (diameter) sperm to pass through this extracellular barrier (1). The Mr 16,000 protein (hereafter called ...

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