Abstract
Endothelial lipase (EL) is a strong determinant of structural and functional properties of high-density lipoprotein (HDL). We examined whether the antioxidative capacity of HDL is affected by EL. EL-modified HDL (EL-HDL) and control EV-HDL were generated by incubation of HDL with EL- overexpressing or control HepG2 cells. As determined by native gradient gel electrophoresis, electron microscopy, and small-angle X-ray scattering EL-HDL is smaller than EV-HDL. Mass spectrometry revealed an enrichment of EL-HDL with lipolytic products and depletion of phospholipids and triacylglycerol. Kinetics of conjugated diene formation and HPLC-based malondialdehyde quantification revealed that EL-HDL exhibited a significantly higher resistance to copper ion-induced oxidation and a significantly higher capacity to protect low-density lipoprotein (LDL) from copper ion-induced oxidation when compared to EV-HDL. Depletion of the lipolytic products from EL-HDL abolished the capacity of EL-HDL to protect LDL from copper ion-induced oxidation, which could be partially restored by lysophosphatidylcholine enrichment. Proteomics of HDL incubated with oxidized LDL revealed significantly higher levels of methionine 136 sulfoxide in EL-HDL compared to EV-HDL. Chloramine T (oxidizes methionines and modifies free thiols), diminished the difference between EL-HDL and EV-HDL regarding the capacity to protect LDL from oxidation. In absence of LDL small EV-HDL and EL-HDL exhibited higher resistance to copper ion-induced oxidation when compared to respective large particles. In conclusion, the augmented antioxidative capacity of EL-HDL is primarily determined by the enrichment of HDL with EL-generated lipolytic products and to a lesser extent by the decreased HDL particle size and the increased activity of chloramine T-sensitive mechanisms.
Highlights
Numerous studies have demonstrated the relationship between structural and compositional features and antioxidative capacity of high-density lipoprotein (HDL) [1,2]
As revealed by non-denaturing gradient-gel electrophoresis and subsequent protein staining, Endothelial lipase (EL)-HDL particles were smaller compared to EV-HDL (Fig. 1A). This result was further confirmed by electron microscopy, showing greater abundance of small (0 nm–10 nm), and lower abundance of large (10 nm– 15 nm) particles in the population of EL-modified HDL (EL-HDL) compared to EV-HDL particles (Fig. 1B, C)
Impact of EL on the size and structure of HDL was assessed by Small-angle X-ray scattering (SAXS) analysis which showed a mean diameter of 133 Ǻ for EL-HDL, compared to 150 Ǻ for EV-HDL (Fig. 1D)
Summary
Numerous studies have demonstrated the relationship between structural and compositional features and antioxidative capacity of high-density lipoprotein (HDL) [1,2]. Besides HDL proteins, HDL lipids shape the antioxidative capacity of HDL [6] This is primarily due to the impact of lipids on the fluidity of the HDL surface and on the size of the HDL core. Enrichment of HDL with ceramide, sphingomyelin or cholesterol decreases the antioxidative capacity of HDL. This is thought to be due to a decreased HDL surface fluidity and in consequence reduced transfer of LOOH from LDL to HDL [7]. The antioxidative capacity of HDL is affected by alterations in triacylglycerol (TAG) and cholesteryl ester (CE) levels in the HDL core, whereby an increased TAG content diminishes apoA-I mediated inactivation of LOOH [8,9]
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