Lysin abalone

Figure 4. Scheme showing sperm-egg interaction in the abalone. 1. The sperm binds to the egg VE by the plasma membrane at the tip of the AV (AG), (F, flagellum M, mitochondrion N, nucleus). 2. The sperm acrosome reacts releasing lysin and the 18K protein from its anterior tip. 3. Lysin disrupts the fibers of the VE and the 18K coats the extending acrosome process as it extends. 4. The sperm passes through the hole in the VE and the membrane covering the tip of the acrosomal process fuses with the egg (from Vacquier and Lee, 1993). 

The abalone sperm AR can be artificially induced by raising the calcium ion concentration of seawater from the normal 10 mM to 50 mM in seawater buffered with 10 mM Tris at pH 8.2. Unlike other species used for fertilization studies, the abalone AR does not lead to the rapid death of the sperm. In abalone sperm, the acrosomal compartment is sealed off from the respiratory compartment acrosome-reacted, sperm will continue to swim for days if stored in the cold room at 4°C. The acrosomal exudate of these sperm is composed predominantly of soluble ly sin and 18K protein. Reducing and denaturing polyacrylamide gel electrophoresis of whole sperm, AV exudate, and purified lysin shows that abalone spermatocytes make a substantial investment in the synthesis of these two acrosomal proteins (Figure 6). 

Figure 6. Polyacrylamide gel electrophoresis of abalone sperm and AV contents. Lanes A and E are standard proteins of known molecular mass. Lane B, whole sperm dissolved in SDS. Lane C, the acrosome vesicle content released to seawater when exocytosis of the sperm is induced by high calcium ion concentration. Lane D, purified 16-kDa lysin. (from Lewis et al., 1982).

Figure 7. Species-specific dissolution of isolated VEs by purified lysins as determined by the light scattering assay. Vertical axis, percent VE dissolved horizontal axis, qg lysin added. R j, VEs from the red abalone, H. rufescens. B j, VEs from the black abalone, H. cracherodii. Pyj, VEs from the pink abalone, H. corrugata. ( ) red lysin (A) pink lysin and ( ) black lysin (from Vacquier and Lee, 1993).

Figure 8. The fusion of artificial phospholipid vesicles induced by 18K protein (a) and lysin (b) at the three concentrations indicated above (c, buffer alone). The upper panels are with sperm proteins from the red abalone (Hr H. refescens) the lower panels are with sperm proteins from the green abalone (Hf H. fulgens). The 18K proteins are more potent fusagens than lysin. Although the two 18K proteins are only 33.8% identical in primary structure, their five predicted amphipathic helices have similar hydrophobic moments (from Swanson and Vacquier, 1995a).

This first study encouraged us to continue to obtain additional lysin sequences. We found that ethanol fixation (50%-90%) of fragments of abalone testis preserved the lysin mRNA, making possible procurement of testis samples from approximately 30 species world wide. Following the synthesis of universal lysin primers in the 3 and 5 untranslated regions of lysin cDNA, it was a simple process to obtain lysin sequences from an additional 25 species using RT PCR (Lee et al., 1995 Lee and Vacquier, 1995). 

Figure 9. The alignments of lysin and the 18K proteins from five abalone species. Dots denote identity to the top sequence and dashes are inserted for alignment. Asterisks denote positions of perfect identity. In lysin,-18 to-1 is the signal sequence in18Kitis -17 to-1. The lengths of the mature proteins are given at the C-terminal ends. H. assimilis (threaded abalone) is closely related to H. kamtschatkana (pinto abalone). H. sorenseni is the white abalone (from Vacquier et al., 1997).

Primers were made to obtain the full length sequences of five species by PCR (Swanson and Vacquier, 1995b). Instead of presenting an alignment of all 27 lysin sequences and the five 18K sequences, we will present the sequences of both proteins from five species of California abalone (Figure 9 Vacquier et al., 1997). The sequences of both acrosomal proteins are known for four species. Haliotis kamtschatkana (known for lysin) and H. assimilis (known for 18K) are considered to be comparable, closely related species. 

Figure 11. The crystal structure of the red abalone lysin monomer. The a-carbon trace shows the five a-helices numbered a-1 to a-5 and the two basic tracks of Arg and Lys residues. The left basic track contains nine residues and the right track 14 residues (Arg and Lys are not visible in the crystal structure). The two termini are labeled N and C. The N-terminal segment of residues 1 to 12 extends away from the helical bundle and the hypervariable N- and C- termini are in proximity (from Shaw et al., 1993). In the Arg and Lys side chains, carbon atoms are white and nitrogen atoms dark gray.

The positions of the basic track residues are highly conserved in the sequences of lysins from 27 species of abalone (Figure 11 Lee and Vacquier, 1995). In the seven species of California abalones (Lee and Vacquier, 1992 Vacquier and Lee, 1993), among the common 18 positions comprising the basic tracks, four positions are exclusively held by Lys and eight by only Arg. The conservation and surface exposure of these basic track residues (Figures 11 and 13) suggests they could be involved in... 

To summarize, variable structural features of lysins suggest ways to attack the elucidation of the molecular mechanism of species-specific sperm-egg recognition in abalones. The invariant structural features of lysins suggest ways to explore the molecular mechanism lysin uses to destroy nonenzymatically the integrity of the VE to allow the sperm to pass through this protective envelope and contact the egg cell membrane. 

Figure 75. Alignment of lysin and 18K proteins from red abalone. Both proteins have five exons (numbered) and four introns (positions shown by arrows). The position of intron three in lysin could not be determined and is shown by a question mark corresponding to the third 18K intron. For both proteins, the known introns are in the exact same positions and have the same phase in the interrupted codon (phase numbered above arrow head). These data indicate the two proteins arose by gene duplication.

When the lysin sequences of the first seven species were obtained, we were impressed at how much divergence had occurred between their primary structures (Figure 9 Table 1). We also discovered that amino acid replacement was mainly nonconservative regarding the class of residue replaced. Next, we made pairwise comparisons of the aligned cDNA sequences and scored the numbers of amino acid altering (nonsynonymous) and silent (synonymous) nucleotide changes in the 21 pairwise comparisons of the seven sequences. The data (Table 2) showed that the vast majority of codon differences between any two lysins are amino acid altering. For example, in the comparison of mature red and pinto abalone lysins of 136 codons, 25 of the codon differences are nonsynonymous and only one is silent (Lee and Vacquier, 1992). 

Figure 17. The vitelline envelope receptor for lysin (VERL) is a giant glycoprotein. (Left panel), electrophoresis of VERL on 2.5% acrylamide gels (silver staining) shows it resolves as two sharp bands between titin (2,800K) and nebulin (770K), Lane 1, rabbit muscle extract myosin (205) lanes 2-6, different loads of pink abalone VERL resolved into two components. (Right panel), electron micrography of VERL molecules negatively stained with uranyl acetate. The VERL fibers are 13 nm in diameter (from Swanson and Vacquier, 1997).

Baginsky, M.L., Stout, C D., and Vacquier, V.D. (1990). Diffraction quality crystals of lysin from spermatozoa of the red abalone (Haliotis rufescens). J. Biol. Chem. 265 4958-4962. 

Diller, T.C., Shaw, A., Stura, E.A., Vacquier, V.D., and Stout, C.D. (1994). Acid pH crystallization of the basic protein lysin from the spermatozoa of red abalone Haliotis rufescens). Acta Crystallog., Sect. D 50 620-627. 

Fridberger, A., Sundelin, J., Vacquier, V. D., and Peterson, P.A. (1985). Amino acid sequence of an egg-lysin protein from abalone spermatozoa that solubilizes the vitelline layer. J. Biol. Chem. 266 9092-9099. 

Haino-Fukushima, K. and Usui, N. (1986). Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa. Dev. Biol. 115 27-34. 

Lee, Y.-H. and Vacquier, V.D. (1992). The divergence of species-specific abalone sperm lysins is promoted by positive Darwinian selection. Biol. Bull. 7S2 97-105. 

Y.-H. and Vacquier, V.D. (1995). Evolution and systematics in Haliotidae (Mollusca, Gastropoda) Inferences from DNA sequences of sperm lysin. Marine Biology 724 267-279. Y.-H., Ota,T., and Vacquier, V.D. (1995). Positive selection is a general phenomenon in the evolution of abalone sperm lysin. Molecular Biology and Evolution 72 231-239. 

Swanson, W.J. and Vacquier, V.D. (1997). The abalone egg vitelline envelope receptor for sperm lysin is a giant, multivalent molecule. Proc. Natl. Acad. Sci. USA. 94 6724-6730. 

Vacquier, V.D. and Lee, Y.-H. (1993). Abalone sperm lysin Unusual mode of evolution of a gamete recognition protein. Zygote 7 181-196. 

Vacquier, V. D., Gamer, K.R., and Stout, C D. (1990). Species-specific sequences of abalone lysin, the sperm protein that creates a hole in the egg envelope. Proc. Natl. Acad. Sci. USA 87 5792-5796. 

Sperm penetrate the zona pellucida only after completion of the acrosome reaction. A similar process occurs in nonmammalian species, where sperm must penetrate the vitelline coat. In abalone this is accomplished by release of lysin, an acrosomal protein that disperses the vitelline coat by a noncatalytic mechanism (Lewis et al., 1982 Shaw et al., 1993). In contrast, the generally accepted model for mammalian sperm penetration of the zona pellucida is the acrosin hypothesis in which proteolysis of zona pellucida matrix glycoproteins by acrosin, the acrosomal serine esterase, plays a trailblazing role in the sperm penetration process (Yanag-... 

---