Correlation of Antiphospholipid Antibody Recognition with the Structure of Synthetic Oxidized Phospholipids: IMPORTANCE OF SCHIFF BASE FORMATION AND ALDOL CONDENSATION

Peter Friedman,Sohvi Hörkkö,Daniel Steinberg,Joseph L Witztum,Edward A Dennis

doi:10.1074/jbc.m108860200

Abstract

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.

Highlights

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways
The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines to yield Schiff base products
Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation

Summary

Introduction

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. Polyunsaturated fatty acids are converted to fatty acid hydroperoxides, which decompose to form highly reactive breakdown products such as malondialdehyde and 4-hydroxynonenal [14] Such reactive aldehydes can form covalent Schiff base and Michael-type adducts with lysine residues of apoB, the protein moiety of LDL [15]. In the case of the common phospholipid 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphorylcholine (PAPC), a near terminal oxidation product would yield an aldehyde at the ⑀-carbon of the sn-2 oxidized arachidonic acid, yielding the reactive compound POVPC (1-palmitoyl-2-(5Ј-oxo)valeroyl-sn-glycero-3-phosphorylcholine) This reactive phospholipid, termed a “phospholipid core aldehyde” [21,22,23], could form Schiff base adducts with lysine residues of apoB and presumably other proteins and with amine-containing phospholipids such as phosphatidylethanolamine and phosphatidylserine. These data strongly suggested that EO6 recognized the oxidized phospholipid either as the nonconjugated lipid or as a covalent adduct with lysine or lysine residues of apoB

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