Oxidation of high-density lipoprotein HDL3 leads to exposure of apo-AI and apo-AII epitopes and to formation of aldehyde protein adducts, and influences binding of oxidized low-density lipoprotein to type I and type III collagen in vitro

摘要

pThe changes in the immunological properties of apolipoprotein AI (apo-AI) and AII (apo-AII) during the oxidation of the high-density lipoprotein HDLsub3/sub and its influence on the binding of heavily oxidized low-density lipoprotein (LDL) to type I and III collagen were investigated. Oxidation of HDLsub3/sub or Eusup3+/sup-labelled HDLsub3/sub was performed with CuSOsub4/sub, varying the time of oxidation. Oxidation of HDLsub3/sub resulted in an increase in lipid hydroperoxides and enhanced the negative charge of this lipoprotein. Immunological studies with a solid-phase sandwich immunoassay revealed a strong increase in binding of Eusup3+/sup-labelled HDLsub3/sub to polyclonal antibodies against apo-AI and apo-AII within the first 4 h of oxidation. Neo-epitopes were also formed by interaction of the apolipoproteins with degradation products from the lipid peroxidation of polyunsaturated fatty acids, as evidenced by an immunoreaction of oxidized Eusup3+/sup-labelled HDLsub3/sub with antibodies to 4-hydroxynonenal (4-HNE)– and malondialdehyde (MDA)–protein adducts. Western blot analysis of oxidized HDLsub3/sub samples showed, as well as apo-AI and apo-AII bands, larger aggregated apolipoproteins, occurring after 0.5–2.5 h of oxidation. These aggregates were recognized by antibodies to apo-AI and apo-AII as well as by antibodies to 4-HNE- and MDA-protein adducts. Furthermore the original apo-AI monomers and apo-AII dimers decreased during the oxidation. The ability of native and oxidized HDLsub3/sub to prevent the binding of Eusup3+/sup-labelled 24 h-oxidized LDL to collagen on microtitration plates was estimated. Interestingly, 2 h-oxidized HDLsub3/sub competed most with the binding of 24 h-oxidized LDL on collagen type I and type III, followed by native HDLsub3/sub. However, 24 h-oxidized HDLsub3/sub was a weaker competitor. Thus oxidative modification of HDLsub3/sub strongly alters the immunological properties of this lipoprotein and its binding affinity for collagen./p