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Proteolytic processing of human zona pellucida proteins
Susan McLeskey Kiefer1, Patricia Saling
1Department of Obstetrics and Gynecology, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biology of Reproduction
|January 24, 2002
Summary
Zona pellucida (ZP) glycoproteins are essential for fertilization. This study reveals that regulated proteolysis by furin convertase family members is crucial for forming the zona pellucida matrix.
Area of Science:
- Reproductive biology
- Molecular and cell biology
- Protein biochemistry
Background:
- The zona pellucida (ZP) is a critical extracellular matrix surrounding mammalian oocytes, essential for fertilization and embryonic development.
- ZP glycoproteins (ZP1, ZP2, ZP3) are secreted and assemble into this matrix.
- Mammalian ZP sequences contain a furin consensus cleavage site, suggesting processing by furin-like endoproteases.
Purpose of the Study:
- To investigate the role of furin-like proteases in the processing and assembly of ZP glycoproteins.
- To determine if the furin cleavage site is utilized during the formation of the zona pellucida matrix.
- To elucidate the post-translational modifications and proteolytic events involved in ZP formation.
Main Methods:
- Recombinant expression of human ZP1, ZP2, and ZP3 (hZP1, hZP2, hZP3).
- Site-directed mutagenesis of the furin cleavage site in hZP3.
- Treatment with a furin inhibitor and Brefeldin A to disrupt Golgi processing.
- SDS-PAGE analysis and immunoblotting.
- "hZP3 rescue" mouse model to study ZP assembly in vivo.
Main Results:
- Recombinant hZP proteins were secreted and processed, with secreted forms lacking C-terminal regions present in cell-associated forms.
- Mutagenesis of the furin cleavage site and furin inhibition altered hZP3 processing and secretion.
- Cleavage of cell-associated hZP3 by exogenous furin mimicked the processing of secreted hZP3.
- In vivo, assembled hZP3 in the zona pellucida matrix lacked the furin cleavage site, indicating processing occurred prior to or during matrix assembly.
Conclusions:
- The formation of the mammalian zona pellucida matrix involves regulated proteolysis of ZP glycoproteins by a member of the furin convertase family.
- This proteolytic processing is essential for proper ZP assembly and function.
- Understanding ZP processing provides insights into reproductive biology and potential targets for contraception or infertility treatments.