Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Apolipoproteins regulate the kinetics of endothelial lipase-mediated hydrolysis of phospholipids in reconstituted high-density lipoproteins

Endothelial lipase (EL) is a newly identified member of the triglyceride lipase gene family that hydrolyzes high-density lipoprotein (HDL) phospholipids. This study investigates the ability of the major apolipoproteins of rHDL to regulate the kinetics of EL-mediated phospholipid hydrolysis in well-characterized, homogeneous preparations of spherical rHDL. The rHDL contained either apoA-I as the only apolipoprotein, (A-I)rHDL, apoA-II as the only apolipoprotein, (A-II)rHDL, or apoA-I as well as apoA-II, (A-I/A-II)rHDL. The rHDL were comparable in terms of size and lipid composition and contained cholesteryl esters (CE) as their sole core lipid. Phospholipid hydrolysis was quantitated as the mass of nonesterified fatty acids (NEFA) released from the rHDL during incubation with EL. The V(max) of phospholipid hydrolysis for (A-I/A-II)rHDL [391.9 +/- 12.9 nmol of NEFA formed (mL of EL)(-1) h(-1)] was greater than (A-I)rHDL [152.8 +/- 4.7 nmol of NEFA formed (mL of EL)(-1) h(-1)]. The energy of activation (E(a)) for the hydrolysis reactions was calculated to be 52.1 and 34.8 kJ mol(-1) for (A-I)rHDL and (A-I/A-II)rHDL, respectively. Minimal phospholipid hydrolysis was observed for the (A-II)rHDL. Kinetic analysis showed that EL has a higher affinity for the phospholipids in (A-I)rHDL [K(m)(app) = 0.10 +/- 0.01 mM] than in (A-I/A-II)rHDL [K(m)(app) = 0.27 +/- 0.03 mM]. Furthermore, (A-I)rHDL is a competitive inhibitor of the EL-mediated phospholipid hydrolysis of (A-I/A-II)rHDL. These results establish that apolipoproteins are major determinants of the kinetics of EL-mediated phospholipid hydrolysis in rHDL.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.     

Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.     

Role of apolipoprotein A-II in the structure and remodeling of human high-density lipoprotein (HDL): protein conformational ensemble on HDL

High-density lipoproteins (HDL, or "good cholesterol") are heterogeneous nanoparticles that remove excess cell cholesterol and protect against atherosclerosis. The cardioprotective action of HDL and its major protein, apolipoprotein A-I (apoA-I), is well-established, yet the function of the second major protein, apolipoprotein A-II (apoA-II), is less clear. In this review, we postulate an ensemble of apolipoprotein conformations on various HDL. This ensemble is based on the crystal structure of Δ(185-243)apoA-I determined by Mei and Atkinson combined with the "double-hairpin" conformation of apoA-II(dimer) proposed in the cross-linking studies by Silva's team, and is supported by the wide array of low-resolution structural, biophysical, and biochemical data obtained by many teams over decades. The proposed conformational ensemble helps integrate and improve several existing HDL models, including the "buckle-belt" conformation of apoA-I on the midsize disks and the "trefoil/tetrafoil" arrangement on spherical HDL. This ensemble prompts us to hypothesize that endogenous apoA-II (i) helps confer lipid surface curvature during conversion of nascent discoidal HDL(A-I) and HDL(A-II) containing either apoA-I or apoA-II to mature spherical HDL(A-I/A-II) containing both proteins, and (ii) hinders remodeling of HDL(A-I/A-II) by hindering the expansion of the apoA-I conformation. Also, we report that, although endogenous apoA-II circulates mainly on the midsize spherical HDL(A-I/A-II), exogenous apoA-II can bind to HDL of any size, thereby slightly increasing this size and stabilizing the HDL assembly. This suggests distinctly different effects of the endogenous and exogenous apoA-II on HDL. Taken together, the existing results and models prompt us to postulate a new structural and functional role of apoA-II on human HDL.     

Regulation of reconstituted high density lipoprotein structure and remodeling by apolipoprotein E

Apolipoprotein E (apoE) enters the plasma as a component of discoidal HDL and is subsequently incorporated into spherical HDL, most of which contain apoE as the sole apolipoprotein. This study investigates the regulation, origins, and structure of spherical, apoE-containing HDLs and their remodeling by cholesteryl ester transfer protein (CETP). When the ability of discoidal reconstituted high density lipoprotein (rHDL) containing apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein to act as substrates for LCAT were compared with that of discoidal rHDL containing apoA-I [(A-I)rHDL], the rate of cholesterol esterification was (A-I)rHDL > (E2)rHDL approximately (E3)rHDL > (E4)rHDL. LCAT also had a higher affinity for discoidal (A-I)rHDL than for the apoE-containing rHDL. When the discoidal rHDLs were incubated with LCAT and LDL, the resulting spherical (E2)rHDL, (E3)rHDL, and (E4)rHDL were larger than, and structurally distinct from, spherical (A-I)rHDL. Incubation of the apoE-containing spherical rHDL with CETP and Intralipid(R) generated large fusion products without the dissociation of apoE, whereas the spherical (A-I)rHDLs were remodeled into small particles with the formation of lipid-poor apoA-I. In conclusion, i) apoE activates LCAT less efficiently than apoA-I; ii) apoE-containing spherical rHDLs are structurally distinct from spherical (A-I)rHDL; and iii) the CETP-mediated remodeling of apoE-containing spherical rHDL differs from that of spherical (A-I)rHDL.     

The structure of apolipoprotein A-II in discoidal high density lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.     

Protein-lipid interactions in reconstituted high density lipoproteins: apolipoprotein and cholesterol influence.

Two fluorescent probes-cis- and trans-parinaric acids were used to study the dimensions, lipid dynamics and apolipoprotein location in the reconstituted discoidal high density lipoproteins (rHDL). The rHDL particles made from apolipoprotein A-I (apoA-I), dipalmitoylphosphatidylcholine (DPPC), with or without cholesterol (Chol) were compared with the analogous particles with two other apolipoproteins-apoE and apoA-II. The data obtained for apoA-I-containing rHDL were as follows: (1) the inclusion of 8 mol.% of cholesterol did not significantly change the particle dimensions (13+/-1 nm) or the mean distance between apoA-I and the disc axis; (2) the phospholipid domains-boundary lipid region in the close vicinity to apoA-I molecule and the remaining part of the bilayer-existed at temperatures both lower and above DPPC transition temperature T(t); (3) at T相似文献   

In vivo metabolism of apolipoprotein A-I on high density lipoprotein particles LpA-I and LpA-I,A-II.

Apolipoprotein (apo) A-I is the major protein in high density lipoproteins (HDL) and is found in two major subclasses of lipoproteins, those containing apolipoprotein A-II (termed LpA-I,A-II) and those without apoA-II (termed LpA-I). The in vivo kinetics of apoA-I on LpA-I and LpA-I,A-II were investigated in normolipidemic human subjects. In the first series of studies, radiolabeled apoA-I and apoA-II were reassociated with autologous plasma lipoproteins and injected into normal subjects. LpA-I and LpA-I,A-II were isolated from plasma at selected time points by immunoaffinity chromatography. By 24 h after injection, only 52.8 +/- 1.0% of the apoA-I in LpA-I remained, whereas 66.9 +/- 2.7% of apoA-I in LpA-I,A-II remained (P less than 0.01). In the second series of studies, purified apoA-I was labeled with either 131I or 125I and reassociated with autologous plasma. Isolated LpA-I and LpA-I,A-II particles differentially labeled with 131I-labeled apoA-I and 125I-labeled apoA-I, respectively, were simultaneously injected into study subjects. The plasma residence time of apoA-I injected on LpA-I (mean 4.39 days) was substantially shorter than that of apoA-I injected on LpA-I,A-II (mean 5.17 days), with a mean difference in residence times of 0.79 +/- 0.08 days (P less than 0.001). These data demonstrate that apoA-I injected on LpA-I is catabolized more rapidly than apoA-I injected on LpA-I,A-II. The results are consistent with the concept that LpA-I and LpA-I,A-II have divergent metabolic pathways.     

The surface properties of apolipoproteins A-I and A-II at the lipid/water interface

The monolayer system was employed to investigate the relative affinities of apolipoproteins A-I and A-II for the lipid/water interface. The adsorption of reductively 14C-methylated apolipoproteins to phospholipid monolayers spread at the air/water interface was determined by monitoring the surface pressure of the mixed monolayer and the surface concentration of the apoprotein. ApoA-II has a higher affinity than apoA-I for lipid monolayers; for a given initial surface pressure, apoA-II adsorbs more than apoA-I to monolayers of egg phosphatidylcholine (PC), distearoyl-PC and human high-density lipoprotein (HDL3) surface lipids. Comparison of the molecular packing of apolipoproteins A-I and A-II suggests that apoA-II adopts a more condensed conformation at the lipid/water interface compared to apoA-I. The ability of apoA-II to displace apoA-I from egg PC and HDL3 surface lipid monolayers was studied by following the adsorption and desorption of the reductively 14C-methylated apolipoproteins. At saturating subphase concentrations of the apoproteins (3.10(-5) g/100 ml), two molecules of apoA-II absorbed for each molecule of apoA-I displaced. This displacement was accompanied by an increase in surface pressure. An identical stoichiometry for the displacement of apoA-I from HDL particles by apoA-II has been reported by others. At low subphase concentrations of apoproteins (5.10(-6) g/100 ml), the apoA-I/lipid monolayer was not fully compressed and could accommodate the adsorbing apoA-II molecules without displacement of apoA-I molecules. ApoA-I molecules were unable to displace apoA-II from the lipid/water interface. The average residue hydrophobicity of apoA-II is higher than that of apoA-I; this may contribute to the higher affinity of apoA-II for lipids compared to apoA-I. The probable helical regions in apolipoproteins A-I and A-II were located using a secondary structure prediction algorithm. The analysis suggests that the amphiphilic properties of the alpha-helical regions of apoA-I and apoA-II are probably not significantly different. Further understanding of the differences in surface activity of these apolipoproteins will require more knowledge of their secondary and tertiary structures.     

Influence of phospholipid depletion on the size, structure, and remodeling of reconstituted high density lipoproteins

This study shows that phospholipid depletion has a major impact on the size and structure of spherical, reconstituted high density lipoproteins (rHDL) and their remodeling by cholesteryl ester transfer protein (CETP). Spherical rHDL, 9.2 nm in diameter with a phospholipid/cholesteryl ester/unesterified cholesterol/apolipoprotein A-I (apoA-I) (PL/CE/UC/A-I) molar ratio of 37.3/24.5/4.1/1.0, were depleted progressively of phospholipids by incubation with phospholipase A(2). After 30 min of incubation the PL/CE/UC/A-I molar ratio of the rHDL was 8.0/31.2/4.4/1.0 and their diameter had decreased to 8.0 nm. Comparable changes in rHDL size and composition were also apparent when the incubations were carried out in the presence of other lipoprotein classes and lipoprotein-deficient plasma. The changes in size and composition were not accompanied by the dissociation of apoA-I from the rHDL. Phospholipid depletion did not affect rHDL surface charge or the structure and stability of apoA-I. The remodeling of unmodified and phospholipid-depleted rHDL by CETP was also investigated. When the rHDL were incubated for 3 h with CETP and Intralipid, transfers of core lipids between the phospholipid-depleted rHDL and Intralipid were decreased relative to unmodified rHDL. This difference was no longer apparent when the incubations were extended beyond 3 h. In these incubations apoA-I dissociated from the phospholipid-depleted and unmodified rHDL at 3 and 12 h, respectively. At 24 h the respective diameters of the unmodified rHDL and phospholipid-depleted rHDL were 8.0 and 7.8 nm. In conclusion, phospholipid depletion has a major impact on rHDL size and their remodeling by CETP.     

Differential Stability of High-density Lipoprotein Subclasses: Effects of Particle Size and Protein Composition

High-density lipoproteins (HDLs) are complexes of proteins (mainly apoA-I and apoA-II) and lipids that remove cholesterol and prevent atherosclerosis. Understanding the distinct properties of the heterogeneous HDL population may aid the development of new diagnostic tools and therapies for atherosclerosis. Mature human HDLs form two major subclasses differing in particle diameter and metabolic properties, HDL₂ (large) and HDL₃ (small). These subclasses are comprised of HDL(A-I) containing only apoA-I, and HDL(A-I/A-II) containing apoA-I and apoA-II. ApoA-I is strongly cardioprotective, but the function of the smaller, more hydrophobic apoA-II is unclear. ApoA-II is thought to counteract the cardioprotective action of apoA-I by stabilizing HDL particles and inhibiting their remodeling. To test this notion, we performed the first kinetic stability study of human HDL subclasses. The results revealed that the stability of plasma spherical HDL decreases with increasing particle diameter; which may facilitate preferential cholesterol ester uptake from large lipid-loaded HDL₂. Surprisingly, size-matched plasma HDL(A-I/A-II) showed comparable or slightly lower stability than HDL(A-I); this is consistent with the destabilization of model discoidal HDL observed upon increasing the A-II to A-I ratio. These results clarify the roles of the particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of apoA-II in this remodeling.     

Apolipoprotein composition and particle size affect HDL degradation by chymase: effect on cellular cholesterol efflux

Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal pre beta-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase. We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.     

Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 degrees C from 0.5 to 24 h. The products were analyzed by non-denaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 A A-II-POPCrHDL was a rearrangement into 78 A particles containing apoA-I and/or apoA-II, and 90 A particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 A A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 A to 105 A containing apoA-I and/or apoA-II, with main particles of 78 A, 88 A, and 98 A. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent V(m)ax and a 6-fold decrease in apparent K(m), while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent K(m); there was no significant change in apparent V(m)ax. Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism.     

Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high-density lipoprotein phospholipids

Hepatic lipase (HL) and endothelial lipase (EL) are both members of the triglyceride lipase gene family. HL hydrolyzes phospholipids and triglycerides in triglyceride-rich lipoproteins and high-density lipoproteins (HDL). EL hydrolyzes HDL phospholipids and has low triglyceride lipase activity. The aim of this study was to determine if HL and EL hydrolyze different HDL phospholipids and whether HDL phospholipid composition regulates the interaction of EL and HL with the particle surface. Spherical, reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoylphosphatidylcholine (PDPC) as the only phospholipid, apolipoprotein A-I as the only apolipoprotein, and either cholesteryl esters (CE) only or mixtures of CE and triolein (TO) in their core were prepared. The rHDL were similar in size and had comparable core lipid/apoA-I molar ratios. The CE-containing rHDL were used to determine the kinetics of HL- and EL-mediated phospholipid hydrolysis. For HL the V(max) of phospholipid hydrolysis for (POPC)rHDL > (PLPC)rHDL approximately (PDPC)rHDL > (PAPC)rHDL, while the K(m)(app) for (POPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (PAPC)rHDL. For EL the V(max) for (PDPC)rHDL > (PAPC)rHDL > (PLPC)rHDL approximately (POPC)rHDL, while the K(m)(app) for (PAPC)rHDL approximately (PLPC)rHDL > (POPC)rHDL > (PDPC)rHDL. The kinetics of EL- and HL-mediated TO hydrolysis was determined using rHDL that contained TO in their core. For HL the V(max) of TO hydrolysis for (PLPC)rHDL > (POPC)rHDL > (PAPC)rHDL > (PDPC)rHDL, while the K(m)(app) for (PLPC)rHDL > (POPC)rHDL approximately (PAPC)rHDL > (PDPC)rHDL. For EL the V(max) and K(m)(app) for (PAPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (POPC)rHDL. These results establish that EL and HL have different substrate specificities for rHDL phospholipids and that their interactions with the rHDL surface are regulated by phospholipids.     

A mutation in the human apolipoprotein A-I gene. Dominant effect on the level and characteristics of plasma high density lipoproteins

Epidemiologic and genetic data suggest an inverse relationship between plasma high density lipoprotein (HDL) cholesterol and the incidence of premature coronary artery disease. Some of the defects leading to low levels of HDL may be a consequence of mutations in the genes coding for HDL apolipoproteins A-I and A-II or for enzymes that modify these particles. A proband with plasma apoA-I and HDL cholesterol that are below 15% of normal levels and with marked bilateral arcus senilis was shown to be heterozygous for a 45-base pair deletion in exon four of the apoA-I gene. This most likely represents a de novo mutation since neither parent carries the mutant allele. The protein product of this allele is predicted to be missing 15 (Glu146-Arg160) of the 22 amino acids comprising the third amphipathic helical domain. The HDL of the proband and his family were studied. Using anti-A-I and anti-A-II immunosorbents we found three populations of HDL particles in the proband. One contained both apoA-I and A-II, Lp(A-I w A-II); one contained apoA-I but no A-II, Lp(A-I w/o A-II); and the third (an unusual one) contained apoA-II but no A-I. Only Lp(A-I w A-II) and (A-I w/o A-II) were present in the plasma of the proband's parents and brother. Analysis of the HDL particles of the proband by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two protein bands with a molecular mass differing by 6% in the vicinity of 28 kDa whereas the HDL particles of the family members exhibited only a single apoA-I band. The largely dominant effect of this mutant allele (designated apoA-ISeattle) on HDL levels suggests that HDL particles containing any number of mutant apoA-I polypeptides are catabolized rapidly.     

ApoA-I structure on discs and spheres. Variable helix registry and conformational states

Apolipoprotein A-I (apoA-I) readily forms discoidal high density lipoprotein (HDL) particles with phospholipids serving as an ideal transporter of plasma cholesterol. In the lipid-bound conformation, apoA-I activates the enzyme lecithin:cholesterol acyltransferase stimulating the formation of cholesterol esters from free cholesterol. As esterification proceeds cholesterol esters accumulate within the hydrophobic core of the discoidal phospholipid bilayer transforming it into a spherical HDL particle. To investigate the change in apoA-I conformation as it adapts to a spherical surface, fluorescence resonance energy transfer studies were performed. Discoidal rHDL particles containing two lipid-bound apoA-I molecules were prepared with acceptor and donor fluorescent probes attached to cysteine residues located at specific positions. Fluorescence quenching was measured for probe combinations located within repeats 5 and 5 (residue 132), repeats 5 and 6 (residues 132 and 154), and repeats 6 and 6 (residue 154). Results from these experiments indicated that each of the 2 molecules of discoidal bound apoA-I exists in multiple conformations and support the concept of a "variable registry" rather than a "fixed helix-helix registry." Additionally, discoidal rHDL were transformed in vitro to core-containing particles by incubation with lecithin:cholesterol acyltransferase. Compositional analysis showed that core-containing particles contained 11% less phospholipid and 633% more cholesterol ester and a total of 3 apoA-I molecules per particle. Spherical particles showed a lowering of acceptor to donor probe quenching when compared with starting rHDL. Therefore, we conclude that as lipid-bound apoA-I adjusts from a discoidal to a spherical surface its intermolecular interactions are significantly reduced presumably to cover the increased surface area of the particle.