Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

Theodoridou, Maria, Nomikos, Michail, Parthimos, Dimitris ORCID: https://orcid.org/0000-0003-3852-323X, Gonzalez-Garcia, J., Elgmati, K., Calver, Brian Lewis, Sideratou, Z., Nounesis, G., Swann, Karl ORCID: https://orcid.org/0000-0002-4355-1449 and Lai, Francis Anthony ORCID: https://orcid.org/0000-0003-2852-8547 2013. Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization. Molecular Human Reproduction 19 (12) , pp. 852-864. 10.1093/molehr/gat070

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Abstract

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca2+ oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca2+ in eggs. To investigate how discrete protein domains contribute to Ca2+ release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca2+ oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca2+ sensitivity and enzyme kinetics. Insertion of a PHdomain or replacement of thePLCζ EF hands domain had no deleterious effect on Ca2+oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCz completely abolished Ca2+ releasing activity. Notably, chimeras containing the PLCζ XYlinker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca2+ sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XYlinker ofPLCζ.Toinvestigate the relationship betweenPLCenzymatic properties andCa2+oscillations in eggs,wehave developed a mathematical model that incorporatesCa2+-dependent InsP3 generation by thePLCchimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency ofCa2+oscillatory activity associated with specificPLCvariants.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Subjects:	R Medicine > R Medicine (General) R Medicine > RZ Other systems of medicine
Uncontrolled Keywords:	Animals; Calcium; Calcium Signaling; Female; Fertilization; Kinetics; Male; Mice; Models, Theoretical; Oocytes; Phosphatidylinositol 4,5-Diphosphate; Protein Isoforms; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sperm-Ovum Interactions; Spermatozoa; Type C Phospholipases
Publisher:	Oxford University Press
ISSN:	1360-9947
Funders:	Wellcome Trust
Date of Acceptance:	16 October 2013
Last Modified:	24 Feb 2024 02:08
URI:	https://orca.cardiff.ac.uk/id/eprint/76341

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