Ascidian Sperm Acrosin and Spermosin: Structures and Roles in Fertilization

Summary

We have previously reported that two sperm trypsin-like proteases, acrosin and spermosin, participate in the fertilization of the ascidian Halocynthia roretzi. In the present study, we isolated the cDNA clones encoding these enzymes. The isolated cDNAs of precursors for ascidian acrosin and spermosin have single open reading frames, which encode 505 and 388 amino acid residues, respectively. The mRNAs of both enzymes are expressed in the gonads but not in other tissues. Ascidian acrosin has paired basic residues (Lys⁵⁶-His⁵⁷), which were found to be responsible for the binding of (pro)acrosin to the vitelline coat. It was also found that ascidian proacrosin contains two CUB domains in the C-terminal portion and that at least CUB domain 1 is involved in its binding to the vitelline coat. SDS-PAGE of the purified spermosin gave two bands with molecular masses of 33-kDa and 40-kDa under nonreducing conditions. N-terminal sequence analyses of both bands revealed that the 33-kDa spermosin is made up by a heavy chain (residues of 130–388) and an L1 light chain (97–129), while the 40-kDa spermosin consists of a heavy chain and an L2 light chain (23–129). L1, unlike L2, contains a Pro-rich region (L1(ΔL2)). This Pro-rich region appears to be responsible for the binding of spermosin to the vitelline coat, since GST-L1 and GST-L1(ΔL2) fusion proteins, but not GST-L2 fusion protein, are capable of associating with the 28-kDa vitelline coat component.