Phospholipase C‐δ3 binds with high specificity to phosphatidylinositol 4,5‐bisphosphate and phosphatidic acid in bilayer membranes

Open Access
Publisher Website
Google Scholar
Abstract
In order to acquire an understanding of phospholipase C–δ3 (PLC–δ3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC–δ3 to large, unilamellar phospholipid vesicles (LUVs). PLC‐δ3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5‐bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC‐δ3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 × 105m−1. High binding of PLC‐δ3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC‐δ3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC‐δ3 to PA and PtdSer vesicles was 1.6 × 104 m−1 and 9.4 × 102 m−1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC‐δ3 to the phospholipid membrane. Binding of PLC‐δ3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration‐dependent increase in activity of PLC‐δ3. The stimulatory effect of PA on PLC‐δ3 was calcium‐dependent. At Ca2+ concentrations lower than 1 µm, no effect of PA on the activity of PLC‐δ3 was observed. PA enhanced PLC‐δ3 activity by increasing the Vmax and lowering Km for PtdInsP2. As the mol fraction of PA increased from 0–40 mol% the enzyme Vmax increased 2.3‐fold and Km decreased threefold. Based on the results presented, we assume that PA supports binding of PLC‐δ3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium‐dependent interaction with the C2 domain of PLC‐δ3. We propose that binding of PLC‐δ3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC‐δ3 activity.