Abstract
The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.
Highlights
We found a significant elevation in ONE-ketoamide adducts in the HDLs of patients with familial hypercholesterolemia, somewhat greater than the levels of IsoLG adducts in these same samples
Crosslinking of HDL proteins may hinder the ability of HDLbound apoA-I to exchange with exogenously added lipid-free apoA-I associated with the particles, resulting in decreased HDL–apoA-I exchange
HDL–apoA-I exchange correlates with cholesterol efflux capacity, we find that ONE-modification of HDL or apoA-I did not alter the ability of HDL to induce cholesterol efflux from macrophages at any of the tested concentrations
Summary
Crosslinking of HDL proteins may hinder the ability of HDLbound apoA-I to exchange with exogenously added lipid-free apoA-I associated with the particles, resulting in decreased HDL–apoA-I exchange. HDL–apoA-I exchange correlates with cholesterol efflux capacity, we find that ONE-modification of HDL or apoA-I did not alter the ability of HDL to induce cholesterol efflux from macrophages at any of the tested concentrations. Crosslinking of HDL proteins by IsoLGs correlated with the significant reduction of its cholesterol efflux capacity [20].
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