Limited proteolysis by a prostatic endopeptidase, the sperm-activating factor initiatorin, regulates the activation of pro-carboxypeptidase B in the seminal fluid of the silkworm, Bombyx mori

Abstract

A prostate trypsin-like serine endopeptidase called initiatorin (BmIni) is an essential factor in triggering the sperm maturation response of the silkworm, Bombyx mori. BmIni has been predicted to specifically cleave the carboxyl side of two consecutive arginine residues present in certain seminal plasma and sperm proteins, but the actual substrates are still unknown. In an attempt to elucidate the molecular mechanism underlying the sperm maturation signaling pathway, in this study, we examined whether BmIni activates the seminal carboxypeptidase B (BmCPB) protein through specific degradation. First, we confirmed in vitro that the inactive BmCPB present in unmated male vesicula (v.) seminalis is activated by treatment with BmIni or trypsin. Molecular cloning of the gene encoding the seminal BmCPB protein has shown that BmCPB is produced as a secreted proenzyme and may be activated after a trypsin-like protease cleaves the boundary between the prodomain and the enzyme site. In support of these findings, both trypsin and BmIni significantly activated recombinant Pro-BmCPB, which was successfully expressed and purified as a proenzyme in Escherichia coli; moreover, two specific cleavage forms appeared in the activation by BmIni that did not appear in that by trypsin. Therefore, a recombinant protein with a mutated diarginine motif (Arg¹⁰⁹-Arg¹¹⁰), which is presumed to be a pre-cleavage site of BmCPB based on its high homology with bovine CPB, was prepared and treated with BmIni. As a result, the two specific degraded peptides were no longer observed, and simultaneously the activation was suppressed. Taken together, these findings lead to the conclusion that zymogen BmCPB, which is synthesized and secreted in male reproductive organs, is activated by sequence-dependent proteolysis by BmIni during ejaculation and in the female reproductive organs, providing a clue to the mechanism underlying seminal plasma and/or sperm protein degradation by BmIni in the sperm maturation cascade of B. mori.

Keywords: Bombyx mori; Carboxypeptidase B; Initiatorin; Seminal fluid proteins; Sperm-activating factor; Trypsin-like protease.