Fertilization and Early Embryogenesis
(M340) CHARACTERIZATION OF TRYPSIN-LIKE PROTEOLYTIC ACTIVITY FROM THE THELYCUM OF THE Penaeus monodon FEMALE.
Kruevaisayawan, Hathairat1, 2, Vanichviriyakit, Rapeepun1, 2, Murphy, Melinda 3, Hennebold, Jon3, Basak, Ajoy1, Weerachartyanukul, Wattana2, Sobhon, Prasert2, Tanphaichitr, Nongnuj1, 4, 1 Ottawa Health Research Institute, Ottawa, ON, Canada2 Mahidol University, Bangkok, Thailand3 Oregon Health & Science University, Beaverton, OR4 University of Ottawa, Ottawa, ON, Canada
ABSTRACT- In penaeoid shrimp, sperm packaged in spermatophores in the male are inserted into and stored in the thelycum of the female prior to their release at the spawning time. Our published work indicates that the thelycum plays an active role in endowing fertilizing competence to sperm during their residence in the thelycum. After a prolonged period in the thelycum, the spermatophore surface is disrupted and sperm are released into the thelycum lumen for further molecular and physiological modifications. We hypothesize that the thelycum may secrete proteases that digest the spermatophore capsule, thus allowing the release of sperm into the lumen. The objective of this study was to investigate the presence of serine proteases in the thelycum lumen. Serine protease activities of the thelycal fluid (TF) were determined using four fluorogenic peptidyl–4–methylcoumaryl–7–amides (MCA) substrates; Boc–QAR–MCA, pERTKR–MCA, Boc–RVRR–MCA, and Suc–AAPF–MCA. Our results revealed the highest specific activity of trypsin–like protease(s) based on their specificity to Boc–QAR–MCA, a trypsin substrate (∼170 U/g protein). This protease activity was also reduced by trypsin inhibitors; soy bean trypsin inhibitor (SBTI, ∼80%), 4–amidino phenyl methane sulfonyl fluoride (APMSF, ∼75%), and N––tosyl–L–lysine chloromethyl ketone (TLCK, ∼45%). These TF trypsin–like proteases were partially purified by SBTI–affinity column chromatography. Peak trypsin–like activity was eluted by 0.1 M sodium acetate, pH 3.0, and corresponded to two major bands with molecular weights of ∼30 and 31.5 kDa following SDS–PAGE and silver staining. The doublet bands were cut and subjected to tryptic digestion and liquid chromatography/mass spectrometry (LC/MS). The MS results showed 41 spectra with no homology to any proteins in publicly accessible databases. In order to verify whether these proteins are novel, their peptide sequences are being obtained via MS/MS. This work is funded by National Science and Technology Development Agency, Higher Education Commission, Thailand (HK, WW, NT, PS), Thailand Research Fund (RV, PS) and the National Institutes of Health, U.S.A. (JH: NCRR RR00163 and NICHD HD42000).
KEY WORDS: Shrimp fertilization, Thelycum, Serine proteases