Membrane Deformation and Mechanism of Stimulation of Phospholipase A2 by Oxidized Lipids

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The focus of this project is the biophysical characterization of mixed assemblies of oxidized phospholipids (OxPL) and heart and brain phosphatidylcholine (PC) lipids in Aim 1 and elucidation of the mechanism of OxPL induced human group V (hgV) and group IIa (hgIIa) secretory phospholipase A2 (sPLA2) activity in Aim 2. Published work and preliminary Laurdan emission spectroscopy on mixtures of OxPL and bilayer PL show lipid compositional ordering in the form of OxPL rich domains or micelles and bilayer PL rich domains or vesicles. A physicochemical basis for demixing is hypothesized to be (i) the hydrogen bond mediated bonding between one OxPL polar terminal group and another OxPL ester group which brings the OxPL together to form OxPL rich domains; (ii) the molecular shape difference between the inverse cone shaped OxPL and the conical bilayer lipid which induces positive curvature in the OxPL domains and negative curvature in the bilayer PL domains respectively. Curvature stresses eventually lead to separation of the OxPL rich domains as micelles. Temperature and lipid unsaturation increase the bilayer cone angle because of increased chain mobility and further accentuate the shape difference and promote demixing. Mixtures of the OxPL 1-palmitoyl-2-glutaryl-sn- glycero-3-phosphocholine (carboxyl terminal group) and 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (aldehyde terminal group) and heart and brain PC lipids will be investigated by Dynamic Light Scattering (DLS) for aggregate sizes and by Laurdan fluorescence to detect segregation. States of mixing predicted are: homogenous mixing, bilayer with OxPL and bilayer PL rich domains, coexisting bilayer lipid vesicles and OxPL micelles. Micelle / vesicle coexistence is readily detected by DLS, but mixed bilayers with segregated domains are reported simply as vesicles. Using the sensitivity of Laurdan fluorescence excitation and emission to inter-lipid bonding in domains and deconvolution using lognormal distributions will be novel approaches to better detect domains. The hypothesis predicts that demixing will be more pronounced when the terminal group is the more polar carboxyl rather than aldehyde. Membrane oxidation is known to be a leading cause in triggering proinflammatory sPLA2 activity. Segregation stimulates enzymatic activity because the high curvature OxPL domains or micelles are highly accessible substrates. The end group in the truncated tail of OxPL is hydrophilic and points to the interface making the lipid protrude, further increasing its accessibility. HgV sPLA2 hydrolyzes PC membranes and increases in its activity for these reasons. The hgIIA sPLA2 does not bind to and does not hydrolyze PC membranes. However interfacial presence of charged truncated tail end groups of OxPL creates a charged interface, to which this enzyme can bind, and stimulate hydrolysis. The present research using pure PC bilayers will resolve the question of whether OxPL stimulates sPLA2 irrespective of phosphatidylserine or other charged bilayer lipid exposure. Enzymatic activity will be measured by pH-Stat methods to determine correlation between increased activity and formation of domains.