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 (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 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 PLCζ/PLCδ1 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 PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker 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 Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. 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.

The sperm phospholipase C-ζ and Ca2+ signalling at fertilization in mammals

“…The luciferase tag allows quantitative assessment of relative PLC protein expression at the same time as measuring intracellular Ca 2 + oscillations [30]. Using the PLC-luciferase fusion proteins it is possible to quantify the greater potency of PLCζ over PLCδ1 in causing Ca 2 + oscillations [28]. One distinctive feature of PLCζ that distinguishes it from PLCδ1 is that it is very sensitive to Ca 2 + , as previously noted for sperm extracts.…”

“…PLCζ is unusual in many respects in its ability to cause Ca 2 + oscillations in mammalian eggs since other PLCs of the β, γ and δ class are unable to cause Ca 2 + release at the same concentration range as PLCζ [15,28]. PLCδ is the isoform most similar to PLCζ , however approximately 40 times more PLCδ1 than PLCζ is required to initiate Ca 2 + oscillations in mouse eggs and the frequency of PLCδ1 oscillations is very low [28].…”

“…PLCδ is the isoform most similar to PLCζ , however approximately 40 times more PLCδ1 than PLCζ is required to initiate Ca 2 + oscillations in mouse eggs and the frequency of PLCδ1 oscillations is very low [28]. The domain structure of PLCζ is relatively simple compared with other mammalian PLCs, it has both the X and the Y catalytic domains found in all PI-specific PLCs, flanked by a C-terminal C2 domain and 4 EF hand domains at the N-terminus [16,17].…”

“…Replacement of the EF hand domains of PLCζ with the EF hands of PLCδ1 produces a chimeric protein that still can cause Ca 2 + oscillations. In contrast, replacement of the C2 domain or a region between the X and Y domains (the X-Y linker) leads to a loss of ability to cause Ca 2 + oscillations [28,31]. The X-Y linker is particularly significant because it appears to play a key role in binding to PIP 2 [34,35].…”

Novel signalling mechanism and clinical applications of sperm-specific PLCζ

“…We previously proposed that the EF-hand domains are vital for PLCζ Ca 2 + sensitivity, because deletion of both EF-hands dramatically increased the EC 50 of PLCζ from 80 nM to 30 mM [26]. Furthermore, replacement of PLCζ EF-hand domains with that from PLCδ1 caused a ∼10-fold decrease in PLCζ Ca 2 + sensitivity, without affecting Ca 2 + oscillation-inducing activity or PIP 2 affinity [27]. The reduced Ca 2 + sensitivity of a PLC chimera was used in a simple model to explain the different frequencies of Ca 2 + oscillations in eggs.…”