Background:
Ovastacin is an oocyte-specific protease involved in sperm adhesion and fertilization. Research into the ovastacin’s protease function suggest it acts on the post-fertilization cleavage of ZP2 via cortical granule exocytosis1. Cleaved ZP2 then causes hardening of the zona pellucida and is then unable to support further sperm binding2. Zona pellucida regulation is critical for correct fertilization3; potentially providing an avenue for non-hormonal contraception. Non-hormonal contraception has the potential to provide excellent contraception options without the problems and side-effects associated with current pharmaceutical options4.
Research into ovastacin activity shows fetuin B as a potent inhibitor of the protease5. Fetuin B knockout mice resulted in zona pellucida hardening before initial fertilization resulting in infertility. Fetuin B is part of the castatin super family of cysteine protease inhibitors which also include histidine-rich glycoprotein, kininogen6. Recombinant mouse fetuin B has been shown to inhibit ovastacin with an IC50 of 75 nM and it is assumed to relate closely with the activity of human fetuin B7.
Project Plan:
Ultimate goals of this project are to produce target enabling packages for both fetuin-B and ovastacin with a focus to identify possible avenues of non-hormonal-contraception drug development. Initial research will rely on the development of recombinant expression systems with yield high enough for various assays and experiments including (co-)crystallisation, ITC, SPR, activity assays, DSF. First cloning attempts have utilised Sf9 insect cells for expression. As expression of ovastacin has shown some stability issues, methods for co-expression of ovastacin and fetuin-B may be required.
Parallel investigations of mouse ovastacin (provided by Hagen Körschgen, Universitätsmedizin der Johannes Gutenberg-Universität, Mainz) will be used to relate the current scientific knowledge of mouse ovastacin to human. Obtaining usable quantities of ovastacin will allow for crystallization screening and optimisation experiments with the goal of structural determination of human ovastacin to below 2 Å; it is expected molecular replacement using the previously published homology models (AF_AFQ6HA08F1) should be successful.
Thermal shift assays will be used to identify possible inhibiting ligands which can then be used for crystal soaking experiments as well as kinetic determinations via ITC.
Further investigations will depend on the success of these initial goals.
For more details, please click the following Zenodo link: https://zenodo.org/record/7064370
1. Burkart, A. D., Xiong, B., Baibakov, B., Jiménez-Movilla, M. & Dean, J. Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. J. Cell Biol. 197, 37–44 (2012).
2. Sato, K. Polyspermy-preventing mechanisms in mouse eggs fertilized in vitro. J. Exp. Zool. 210, 353–359 (1979).
3. Gupta, S. K. et al. Mammalian zona pellucida glycoproteins: structure and function during fertilization. Cell Tissue Res. 349, 665–678 (2012).
4. Visser, J., Snel, M. & Vliet, H. A. V. Hormonal versus non‐hormonal contraceptives in women with diabetes mellitus type 1 and 2. Cochrane Database Syst. Rev. (2013) doi:10.1002/14651858.CD003990.pub4.
5. Stöcker, W. et al. Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B. Biol. Chem. 395, 1195–1199 (2014).
6. Lee, C., Bongcam-Rudloff, E., Sollner, C., Jahnen-Dechent, W. & Claesson-Welsh, L. Type 3 cystatins; fetuins, kininogen and histidine-rich glycoprotein. Front. Biosci. Landmark Ed. 14, 2911–2922 (2009).
7. Dietzel, E. et al. Fetuin-B, a liver-derived plasma protein is essential for fertilization. Dev. Cell 25, 106–112 (2013).