18K protein

XIII. Lysin and the 18K Protein Arose by Gene Duplication.70... 

Figure 3. (Upper Panel). Scanning electron micrograph of an H. rufescens spermatozoon. The sperm head, from mitochondrion (M) to tip of the acrosome vesicle (granule AV) is 7 pm. The width of the nucleus (N) is 1 pm. (Lower Panel). Transmission electron micrograph of the acrosomal vesicle showing it attached to the nucleus (NF) by the rod of actin filaments (AF). The darker material labelled 1 shows the location of the 18K protein and 2 shows the location of lysin (from Lewis et al., 1980).

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). 

Purified 18K protein will aggregate liposomes composed of PS PC PE (1 1 1), but liposomes made of PC are not aggregated. The 18K protein also induces the release of dye from liposomes. Fluorescence-based assays showed that both lysin and 18K are capable of fusing negatively charged liposomes however, the 18K is a much more potent fusagen than is lysin (Figure 8). Analysis for secondary structure... 

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).

We purified the 18K protein by CM cellulose ion exchange chromatography and obtained the sequence of the first 41 residues by gas phase protein sequencing. 

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).

Figure 10. Neighbor-joining trees of lysin and 18K proteins from the five species. The scale bar shows amino acid p-distances. The topology of both trees is the same, however 18K is two to three times more divergent than lysin (from Vacquier et al., 1997).

XIII. LYSIN AND THE 18K PROTEIN AROSE BY GENE DUPLICATION... 

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.

---