Proteomic analysis of human low‐density lipoprotein reveals the presence of prenylcysteine lyase, a hydrogen peroxide‐generating enzyme

Abstract

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid-phase IEF has been used to resolve LDL proteins into well-defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL-associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol-binding protein, and paraoxonase-1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time-dependent generation of H 2 O 2 which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H 2 O 2 generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.Abbreviations: FAD, flavin adenine dinucleotide; FC, farnesylcysteine; H 2 O 2 , hydrogen peroxide; HDL, high density lipoprotein; LDL, low density lipoprotein; PCL1, prenylcysteine lyase; RBP, retinol-binding protein; SAA, serum amyloid A; VLDL, very low density lipoprotein * These authors contribute equally to this work.

Aggregated Electronegative Low Density Lipoprotein in Human Plasma Shows a High Tendency toward Phospholipolysis and Particle Fusion

“…The origin of such phospholipolytic activity is unknown. Studies using proteomic approaches have reported the presence of more than 20 minor proteins in total LDL (37,38), but none of these proteins have a known PLC activity. The only proteins in LDL with a well established phospholipolytic activity are PAF-AH and lecithin:cholesterol acyl transferase.…”

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring

“…The input compartment is the plasma amino acid precursor pool (D3-Leu tracer enrichment in plasma) expressed as a forcing function that drives the appearance of plasma D3-Leu tracer in the model. Each participant's D3-Leu tracer by guest, on www.jlr.org that contain low levels of apoA-I (36)(37)(38). Group II represents the proteins that predominate in ␣ 1 and ␣ 2 size HDL and contains 11 proteins including apoE, apoM, apoL-I, apoC-IV, cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP).…”

Overview of Current Proteomic Approaches for Discovery of Vascular Biomarkers of Atherosclerosis

“…However, the high ionic strength and the high centrifugal field forces might cause either the dissociation of proteins or their exchange between different lipoprotein classes, altering the pattern of associated exchangeable apolipoproteins. Indeed, some of these studies reported a loss of proteins after a second step of ultracentrifugation (Banfi et al, 2009;Davidson et al, 2009;Mancone et al, 2007). Some others employed two ultracentrifuge procedures, using both salts and other compounds, such as sucrose and iodixanol (Bondarenko et al, 1999;Sun et al, 2010), reporting comparable results.…”

“…Traditional methods, established in the 1950s (Havel et al, 1955), imply ultracentrifugation in high-salt media containing KBr or NaBr. Several lipoproteomic studies have been published using these procedures of lipoprotein isolation (Banfi et al, 2009;Davidson et al, 2009;Green et al, 2008;Heller et al, 2005Heller et al, , 2007Hortin et al, 2006;Karlsson et al, 2005aKarlsson et al, , 2005bKhovidhunkit et al, 2004;Mancone et al, 2007;Mazur et al, 2010;Rezaee et al, 2006;Vaisar et al, 2007Vaisar et al, , 2010Alwaili et al, 2011). However, the high ionic strength and the high centrifugal field forces might cause either the dissociation of proteins or their exchange between different lipoprotein classes, altering the pattern of associated exchangeable apolipoproteins.…”

“…The authors analysed PCL1 distribution in all the lipoprotein classes isolated by ultracentrifugation from 6 healthy subjects showing a decline from VLDL to LDL to HDL and its absence in lipoprotein-depleted plasma. Due to the oxidizing role of PCL1, they hypothesized that lipoproteins can themselves generate pro-oxidant species, thus suggesting a new role for lipoprotein in the development of atherosclerosis (Banfi et al, 2009). Bancells et al analysed the proteome of LDL subfractions isolated by anion exchange chromatography after sequential ultracentrifugation of pooled healthy subjects plasma.…”