Long-chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine decrease the stability of recombinant high density lipoprotein

The lecithin: cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I (apoA-I) were investigated using recombinant HDL (rHDL) containing phosphatidylcholine (PC), ['H]cholesterol, and apo A-I. The PC component of the rHDL contained sn-1 16:O and sn-2 18:l (POPC), 20:4 (PAPC), 20:s n-3 (PEPC), or 22:6 n-3 (PDPC) or 10% of the respective PC: species and 90% sn-1 18: 1, sn-2 16:O PC ether (OPPC ether). The a p ~ b ' , > ~ , ~ of the rHDL containing 100% PC varied 10-fold and was ordered POPC>PEPC>PAPC>PDPC, whereas the appK,,< values varied 19-43 &M PC. The ether-containing rHDL had appV,,,,values 17-40% of their respective 100% PC rHDL, but maintained the same rank order. The activation energy of LCAT was lower for rHDI, containing long chain polyunsaturated fatty acids (PUFA) compared to rHDL containing 100% POPC or 10% PC/90% OPPC ether. The concentration of guanidine HCI (D1,2) required to denature onehalf' of the apoA-I on rHDL containing long chain PUFA was reduced ( 1 2-1.6 M ) compared to those containing 100% POPC or 10% PC/90% OPPC (2.2-2.4 M ) and there was a strong correlation ( P = 0.71) between LCAT activation energy and the stability of apoA-I (Le., D, ,? ) .U We conclude that long chain PUFA in the .sn-2 position of PC decreases the catalytic efficiency of LCAT, the activation energy of the LCAT reaction and the stability of apoA-I on the rHDL particles. The strong association between rHDL apoA-1 stability and LCAT activation energy suggests that the temperaturedependent step of the LClAT reaction may be sensitive to the strength of the interaction of apoA-I with rHDL PC:.-Parks, J. S., and A. K. Gebre. Long-chain polyunsaturated fatty acids in the sa2 position of phosphatidylcholine decrease the stability of recombinant high density lipoprotein apolipoprotein AI and the activation energy of the 1ecithin:cholesterol acyltransferase reaction. ,]. Lipid &.s. 1997. 38: 266-275. Supplementary key words PDPC OPPC ether cholesterol POPC PAPC PEPC Lecithin: cholesterol acyltransferase (LCAT) is a 6365 kD plasma glycoprotein that is responsible for the synthesis of most cholesterol esters (CE) found in plasma (1 ) . The enzyme catalyzes the hydrolysis of a sn-2 fatty acid from phospholipid and the transesterification of the fatty acid to the 3-hydroxyl group of cholesterol to form CE and lysophospholipid ( 1). Apolipoprotein A-I (apoA-I) serves as a cofactor for the reaction and is the m?jor structural apolipoprotein of high density lipoproteins (HDL), which are the preferred macromolecular substrate particle for the LCAT reaction (2-4). LCAT is essential for the maturation of HDL that are secreted from the liver and intestine or formed during lipolysis of triglyceride-rich lipoproteins (5). The generation of CE by LCAT converts nascent discoidal HDL to spherical particles, which are the predominant form of HDL in plasma. The importance of LCAT in this HDL maturation process is best illustrated by genetic (6) or induced (7) LCAT deficiency states in Abbreviations: PUFA, po~yunsaturated fatty acids; PC, phosphatidylcholine: CX, cholesteryl ester; K A T , lecithin : cholesterol acyltransferase; apoA-I, apolipoprotein A-I; rHDL, rerombinant HDL; (D, 2 ) , concentration of guanidine HCI required to denature one half of the apoA-I on rHDL; HDL, high density lipoprotein; LDL, low density lipoprotein; LysoPC, I-palmitoyl-slf-glycero-3-phosphorholine; OPPC ether-, loleyl-2-palniityl phosphatidylcholine ether; POPC, l-palmitoyl-2-oleoyl-.m-glycero-3-phosphorholine; PAF'C, I -palmitoyl-2-arachadonoyl-,~~-glycertr9-phosph~1choline: PEPC l-palmitoyl-2-eicosapentaenoyl-sn;glycero-3-phosphocholine; PDPC, l-palmitoyl-2-docosahexaenoyl-~~glycero-3-phosphocholine: (AG'Id), frce energy of denaturation at 7ero guanidine HCI concentration; Ea. activation energy. 'To whoin correspondence should be addressed. 266 Journal of Lipid Research Volume 38, 1997 by gest, on S etem er 0, 2017 w w w .j.org D ow nladed fom which nascent discoidal HDL accumulate in plasma and CE concentrations are very low. LCAT is also thought to play an important role in reverse cholesterol transport, the process by which excess free cholesterol is removed from peripheral tissues by HDL and transported to the liver for excretion (1). The role of LCAT in this process appears to be the generation of a chemical gradient for the flux of free cholesterol from cells into HDL by decreasing the concentration of free cholesterol in HDL through its conversion to CE. Studies of the enzymatic mechanism of LCAT have been significantly advanced by the ability to form discoidal substrates of known composition that mimic nascent HDL in size and composition (8). These are referred to as recombinant HDL (rHDL) and are made by a cholate dialysis procedure (9). Using rHDL, investigators showed that LCAT activity in vitro can vary 100fold depending on the fatty acyl composition of rHDL (10). This variation in LCAT activity could be mediated through changes in enzyme binding to the interface of the rHDL particle and/or through changes in the binding and catalysis of monomeric substrate lipids (i.e., cholesterol and phospholipid). Studies by Jonas et al. (10) and Pownall, Pao, and Massey (11) used ether phosphatidylcholine (PC) analogues, which cannot be hydrolyzed by LCAT, to form rHDL that contained a uniform, inert interface to distinguish between interfacial versus active site effects of various PC species on LCAT activity. These studies demonstrated that systematically changing the fatty acyl composition of PC modulated LCAT activity through changes in the interfacial binding of LCAT to rHDL as well as changes in the binding and catalysis of substrate molecules at the active site of the enzyme. These studies have increased our mechanistic understanding of the control of LCAT enzymatic activity. The purpose of this study is to better understand the mechanism by which long-chain polyunsaturated fatty acids affect LCAT activity. The impetus for the present study originated with the observation that feeding nonhuman primates a diet in which fish oil was isocalorically substituted for lard resulted in a decreased concentration of CE in plasma LDL and HDL (12). Plasma LCAT mass and activity, measured with rHDL-containing egg yolk PC, were the same for both diet groups (1 3). However, when phospholipids from the plasma of both diet groups were extracted, purified, and used to make rHDL, the LCAT activity of rHDL made from phospholipids derived from the plasma of the fish oil group was less than half that of the lard group (13). Later studies using defined, synthetic PC species demonstrated that n-3 fatty acids in the sn-2 position of PC were responsible for the decreased reactivity of plasma LCAT (14). However, mechanistic details for the decreased reactivity of LCAT with n-3 fatty acids are lacking and are the subject of this study. EXPERIMENTAL PROCEDURES