Influence of Lipid Oxidization on Structures and Functions of Biological Membranes

Abstract

The primary aim of this thesis is to clarify how the structures and functions of biological membranes are influenced by the oxidative damage mediated by free radicals. As a precisely defined model systems, artificially reconstituted lipid membranes (Langmuir monolayers, vesicles, supported membranes, multilamellar membranes) incorporating two oxidized phospholipids bearing aldehyde or carboxyl groups at the end of truncated sn-2 acyl chains were fabricated. By the combination of various experimental methods, the generic impact of chain oxidization on physical characteristics of membranes (e.g. lateral cooperativity, fine-structures perpendicular to membrane planes, electrostatics) and the specific interactions of oxidized phospholipids with EO6 peptides and acute immune response proteins was investigated. In the first step, the influence of oxidized phospholipids (OxPL) on the thermodynamics and electrostatics were investigated using Langmuir film balance at the air-water interface. The pressure-area (π-A) isotherms and surface potential (Δψ-A) measurements implied that both OxPLs lead to a decrease in the isothermal compression modulus. In fact, surface potential measurements suggest changes in the orientation of oxidized moieties that decrease the lateral cooperativity. Further increase in the fraction of oxidized lipids resulted in the loss of molecules into bulk water, which seems consistent with the destabilization of cell membranes under oxidative stresses. In the second step, the impact of lipid oxidization on the electrostatics of membranes was examined by the combination of high-energy specular X-ray reflectivity (XRR) and grazing-incidence X-ray fluorescence (GIXF). The scattering length density profiles reconstructed from XRR results suggested that both OxPL leads to membrane thinning, which seems plausible from the decrease in the lateral cooperativity suggested by Langmuir isotherms. GIXF offers an unique possibility to localize specific target elements within A accuracy, suggesting that the binding affinity (Ca2+ > Cs+ > K+) could be interpreted in terms of the solvation entropy (Hofmeister series). Further, the impact of oxidization on the vertical structural ordering of vertically stacked membrane models was investigated by off-specular neutron scattering. A decreased lamellar periodicity d indicated that incorporation of OxPL into the membrane displace water molecules from the inter-membrane region due to the reorientation of oxidized moieties. In the third step, the combination of experimental techniques was utilized to shed light on specific interactions of OxPLs with peptides and proteins; C-reactive protein that is characteristic for the acute immune responses and monoclonal antibody EO6 to oxidized lipids. Following the fundamental characterization of membrane-protein interactions using isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) of vesicle suspensions, in addition to XRR, GIXF, off-specular neutron scattering, dual waveguide polarization interferometry (DPI) was used to monitor the changes in thickness, refractive index, and the optical anisotropy (birefringence) of lipid membranes simultaneously. Furthermore, the specific binding of EO6 was verified from the fluorescence imaging of glioblastoma multiforme cells undergoing apoptosis, where a clear accumulation of OxPLs could be identified in apoptotic blebs. The obtained results demonstrated that the combination of well defined membrane models and unique physical techniques is a powerful tool to shed a new quantitative light on the generic and specific impacts of lipid oxidization on the lateral cooperativity, vertical fine-structures, electrostatics, and specific interactions in inflammation and apoptosis.