HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is un- known, and was determined in the present study. In hu- mans, we found serum HDL PON activity and HDL sus- ceptibility to oxidation to be inversely correlated ( r 2 5 0.77, n 5 15). Supplementing human HDL with purified PON in- hibited copper-induced HDL oxidation in a concentration- dependent manner. Adding PON to HDL prolonged the ox- idation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initia- tion. When purified PON was added to whole serum, essen- tially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in per- oxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage choles- terol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H 2 O 2 ), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we an- alyzed serum PON activity in the atherosclerotic apolipo- protein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity de- creased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the anti- atherogenicity of this lipoprotein. ( J. Clin. Invest. 1998. 101: 1581-1590.) Key words: paraoxonaseHDLLDLlipid peroxidationapolipoprotein E deficient mice

/pdf/paraoxonase-inhibits-high-density-lipoprotein-oxidation-and-3zbj3ex19e.pdf

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II.

In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1's association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic N-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable N-terminus, we demonstrate that PON1 associates with lipoproteins through its N-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1's retained N-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic N-terminus to phospholipids optimally presented in association with apoA-I.

Human Serum Paraoxonase/Arylesterase’s Retained Hydrophobic N-Terminal Leader Sequence Associates With HDLs by Binding Phospholipids Apolipoprotein A-I Stabilizes Activity

https://pure.uva.nl/ws/files/3851100/2853_27272y.pdf

The molecular pathology of lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes.

The Farnesoid X-receptor Is an Essential Regulator of Cholesterol Homeostasis

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity.

Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

Distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma lipoprotein fractions. Evidence for the association of active LCAT with low density lipoproteins

Further characterization of purified lecithin: cholesterol acyltransferase from human plasma.

Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Canadian kindred of Italian and Swedish descent with familial LCAT deficiency. Four subjects had LCAT mass of 5.21±0.87 μg/ml (mean±SD) and LCAT activity of 98.8±12.0 nmol/h/ml, well within their respective normal ranges. Five family members, including the parents, the maternal grandmother, and two of four siblings of the LCAT deficient subjects, had enzyme mass (2.85±0.32 μg/ml) and activity (50.8±6.3 nmol/h/ml) approximately one-half that of normal levels. These presumed heterozygotes had normal levels of apolipoproteins A-I, A-II, B and D. The two subjects with LCAT deficiency had no detectable LCAT mass (below 0.1 μg/ml) or LCAT activity (below 0.76 nmol/h/ml), apolipoprotein A-I and D levels approximately 50% of normal, and apolipoproteins B and A-II levels only 30–35% of normal. LCAT deficiency in this family is determined by an autosomal recessive mode. Furthermore, LCAT levels and activity are determined by two autosomal codominant alleles, LCATn, the normal LCAT gene, and LCATd, the LCAT deficiency gene.

Ching Hong Chen

Papers

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity.

Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

Distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma lipoprotein fractions. Evidence for the association of active LCAT with low density lipoproteins

Further characterization of purified lecithin: cholesterol acyltransferase from human plasma.

Familial lecithin-cholesterol acyltransferase: identification of heterozygotes with half-normal enzyme activity and mass.