Substrate specificity of ascidian sperm trypsin‐like proteases, spermosin and acrosin

Abstract

In order to investigate systematically the substrate or subsite specificity of two sperm proteases, acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization.

Spermosin, a trypsin‐like protease from ascidian sperm

“…Here we show that there are two molecular forms of the molecule made up of a common heavy chain and either a short or a long light chain depending on the processing sites. We previously reported that ascidian spermosin is a novel sperm trypsin‐like protease, distinct from acrosin, a well‐known sperm trypsin‐like protease that is widely distributed in mammalian sperm, in terms of substrate specificity and inhibitor susceptibility [9,12]. The present study clearly showed that ascidian spermosin is a novel protease and is distinct from ascidian acrosin on the basis of amino‐acid sequence [16].…”

“…We have reported previously that spermosin inhibitors, Z‐Val‐Pro‐Arg‐H [10] and Dns‐Val‐Pro‐Arg‐H [12], and anti‐spermosin antibody [11] are capable of inhibiting fertilization in a concentration‐dependent manner indicating that spermosin plays an important extracellular role in ascidian fertilization and that the proteolytic activity of spermosin is required for ascidian fertilization. As a proline‐rich region (residues 28–88) of spermosin L1 light chain is able to associate with the vitelline coat of the egg, it is inferred that spermosin is involved not only in the sperm penetration of the vitelline coat but also in the sperm binding to the vitelline coat.…”

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane, the other involving the follicle cells

“…We conclude that (1) suramin acts mainly at the VC level, since we never obtained any electrical response on intact eggs which had not been penetrated by sperm while we frequently detected a fertilization current and sperm penetration in naked eggs; and (2) suramin also affects the process of gamete fusion, since while 35% of the naked eggs responded electrically in contact with this drug, 100% of naked eggs inseminated normally in SW displayed a fertilization current. Possibly suramin inhibits one of the sperm proteases involved in ascidian fertilization (De Santis et al, 1992;Koch et al, 1994;Takizawa et al, 1993;Sawada and Someno, 1996); additional experiments are planned to check this possibility.…”

Sperm penetration through cumulus mass and zona pellucida

“…Two trypsin-like serine proteases, acrosin and spermosin, and ubiquitin-proteasome system in sea squirts and sea urchins are demonstrated to play key roles in sperm binding to and penetration through the egg vitelline envelope (Sawada et al, 1984 andSawada and Someno, 1996;Yokota and Sawada, 2007). Ascidian sperm contain 26S or 20S proteasome, ubiquitin, and ubiquitin-conjugating enzyme that are released from the acrosome to ubiquitinate and degrade a sperm receptor, HrVC70, as an analogue of mammalian ZP3 on the surface of the vitelline coat during fertilization (Sawada et al, 2002).…”