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

ABSTRACT: Phospholipase C-zeta (PLC?) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLC? is very similar to the somatic PLC?1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLC?/PLC?1 chimeras. We examined their ability to cause Ca(2+) 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 Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLC? EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLC? completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLC? XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLC? C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLC? and that membrane PIP2 binding involves the C2 domain and XY-linker of PLC?. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

SUBMITTER: Theodoridou M 

PROVIDER: S-EPMC3843027 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature