Male Bombyx mori has a trypsin-type protease, called initiatorin, in the secretion from the posterior
segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility,
although this inference remains to be demonstrated. Here, we revised the experimental procedures
including that for purification and definitely identified the purified initiatorin protein as an activation
factor of B. mori sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme.
Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine
trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera
but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also
showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that
initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is
synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic
peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate,
BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate
the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation
cascade in that it releases free arginines, which in turn become an energy resource for sperm motility.
Male Bombyx mori has a trypsin-type protease, called initiatorin, in the secretion from the posterior
segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility,
although this inference remains to be demonstrated. Here, we revised the experimental procedures
including that for purification and definitely identified the purified initiatorin protein as an activation
factor of B. mori sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme.
Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine
trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera
but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also
showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that
initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is
synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic
peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate,
BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate
the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation
cascade in that it releases free arginines, which in turn become an energy resource for sperm motility.
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