Proteomic Identification of the bovine sperm protease BSp66

KATUNAR MR, CESARI A, RONCHI PV , FORNÉS MW

Misiones. Argentina

Congreso; XL Annual Meeting. SAIB 2004; 2004

BSp66 is a glycoprotein with trypsin-like activity located in the acrosome of mammalian sperm. The aim of this work was to determine BSp66 sub-cellular localization and to characterize it by proteomic studies. After sub-cellular fractionation, BSp66 proteolytic activity was detected in the cytosolic fraction of bovine sperm. BSp66 was developed by silver staining and by immunodetection using a polyclonal antibody against bovine BSp66 on 2D-IEF gel from cytosolic extracts. The spot corresponding to BSp66 revealed a Mr of 66 kDa and pI 5.27 which had homology to Testase 1 (ADAM 24). A 2D-functional analysis of bovine cytosolic proteases showed 10 proteolytic activities, with Mr 40-66 kDa and pI<6.1. The gelanolytic spot corresponding to BSp66 was not observed suggesting the lost of activity under the assayed conditions. Then, we tested the presence of BSp66 on human (Ho) and hamster (Ha) total sperm extract using proteomic tools. A conserved major protein with Mr 66 kDa and pI 5.7 was observed in both Ho and Ha although it was not recognized by the heterologous anti-BSp66. However, the immunoblot assays revealed a spot of Mr 50 kDa and pI 4,9 in Ha sperm. In Ho sperm, the antibody revealed a set of spots with Mr around 36 kDa and pI near to 6,13 homologue to a testis-specific mammalian serine kinase-3. The results suggest that BSp66 in both Ho and Ha slightly differ from bovine BSp66, and that there are several acidic proteases within bovine cytosol. In order to confirm that BSp66 is conserved among species we must carry out the sequencing of this protease. (Funded by UNMdP, CONICET)