engleski

CHARACTERIZATION OF EPITOPES FOR OB/04 ANTIBODY ON HUMAN PLASMA LOW DESNITY LIPOPROTEINS

Human plasma low density lipoproteins (LDLs) are supramolecular lipid-protein assemblies involved in the cholesterol transport in the bloodstream. They exhibit interesting structural complexity with the surface monolayer organization of phospholipids surrounding the hydrophobic core. Their structure is stabilized by apolipoprotein B100 (apoB), one of the largest monomeric proteins known with its 4536 aminoacid residues. Tertiary structure of the complex at atomic resolution has not yet been elucidated. LDL is prone to various modifications, oxidative one being the most important and physiologically relevant. Oxidized LDL (oxLDL) is believed to be involved in the development of atherosclerosis. To investigate lesions for the existence of oxidized LDL, polyclonal antisera or monoclonal antibodies have been raised against Cu2+ - oxidized LDL or LDL modified by lipid peroxidation– derived breakdown products such as malondialdehyde (MDA) or 4– hydroxinonenal (4-HNE). However, the antibodies characterized so far also showed cross– reactivity with other proteins modified with MDA or 4-HNE. By the strategy of mice immunization with entire oxLDL particles an immune response directed against certain surface structures on LDL or apoB not formed on other proteins has been obtained. As a result, the antibody OB/04 has been purified. The aim of the present study is to obtain an insight into the formation of epitopes for this antibody on LDL particles. The kinetics of the epitope formation during copper– induced LDL oxidation has been monitored by immunoassays and intrinsic fluorescence spectroscopy. The titration of LDL with the antibody has been performed in several time points of LDL oxidation. The affinities of different oxLDLs for the antibody will be discussed. Modification of free thiol groups at the LDL surface has been performed in order to investigate their involvement in the antibody binding. Intrinsic fluorescence quenching with potassium iodide (KI) has been measured to follow the conformational changes of different oxLDLs upon antibody binding. The results are discussed in the framework of the proposed model of the LDL copper– induced oxidation as well as proposed pentapartite model of apoB domain structure. Characterization of the epitopes recognized by this antibody could contribute new detailes to the knowledge of the structural changes caused by oxidative modification of LDLs.

ldl; antibody; fluorescence spectroscopy

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