Oxidized Phosphatidylcholines Regulate Secretory Phospholipase A₂ Through Membrane Nanodomain Remodeling

Oxidative stress generates oxidized phospholipids (OxPLs) that alter membrane structure and inflammatory lipid signaling, yet the underlying biophysical mechanisms remain poorly understood. Here, we examine how two structurally distinct truncated oxidized phosphatidylcholines (OxPCs), 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), remodel membrane lateral organization and regulate secretory phospholipase A₂ (sPLA₂) activity. Large unilamellar vesicles composed of sphingomyelin, cholesterol, and either monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or polyunsaturated 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) were used to reconstitute the liquid-ordered/liquid-disordered (L_o/L_d) phase coexistence characteristic of eukaryotic plasma membranes. Fluorescence spectroscopy revealed that OxPLs modulate lipid packing and nanodomain organization in a structure- and composition-dependent manner. POVPC promoted pronounced membrane ordering and L_o domain stabilization compared with PGPC, particularly in monounsaturated membranes with low cholesterol content. In contrast, PDPC-containing membranes, especially at elevated cholesterol, exhibited enhanced structural resilience to OxPL-induced perturbations. These biophysical changes were associated with distinct functional outcomes. Notably, the relationship between membrane structural parameters and sPLA₂ activity was not linear, indicating a decoupling between bulk membrane properties and enzymatic response. sPLA₂ activity was linked to membrane lateral organization: the size of L_o domains modulate hydrolysis by influencing the physicochemical properties of L_o/L_d interfaces, which may represent preferential sites for enzyme activation. Consistent with this, POVPC reduced sPLA₂ activity through stabilization of ordered domains at both low and high cholesterol, while PGPC enhanced hydrolysis at high cholesterol. Importantly, PDPC-containing membranes attenuated sPLA₂ activity and exhibited a protective effect against OxPC-induced enzymatic activation. Together, these findings identify membrane lateral organization as a key regulator of sPLA₂ function and provide mechanistic insight into how oxidative stress can differentially modulate inflammatory lipid signaling depending on membrane composition. This work highlights membrane organization as an active determinant of enzyme activity and a potential target in pathologies associated with oxidative stress, including atherosclerosis, neuroinflammation, and metabolic disease.