Reconstituted High Density Lipoprotein Particles Research Articles

The carboxyl-terminal hydrophobic residues of apolipoprotein A-I affect its rate of phospholipid binding and its association with high density lipoprotein.

We performed a series of mutations in the human apolipoprotein A-I (apoA-I) gene designed to alter specific amino acid residues and domains implicated in lecithin:cholesterol acyltransferase (LCAT) activation or lipid binding. We used the mutant apoA-I forms to establish nine stable cell lines, and developed strategies for the large scale production and purification of the mutated apoA-I proteins from conditioned media. HDL and dimyristoyl phosphatidylcholine binding assays using the variant apoA-I forms have shown that replacement of specific carboxyl-terminal hydrophobic residues Leu222, Phe225, and Phe229 with lysines, as well as replacement of Leu211, Leu214, Leu218, and Leu219 with valines, diminished the ability of apoA-I to bind to HDL and to lyse dimyristoyl phosphatidylcholine liposomes. The findings indicate that Leu222, and Phe225, Phe229 located in the putative random coil region, and Leu211, Leu214, Leu218, and Leu219 located in the putative helix 8, are important for lipid binding. In contrast, substitutions of alanines for specific charged residues in putative helices 7, 8, or 9 as well as various point mutations in other regions of apoA-I, did not affect the ability of the variant apoA-I forms to bind to HDL or to lyse dimyristoyl phosphatidylcholine liposomes. Cross-linking experiments confirmed that the carboxyl-terminal domain of apoA-I participates in the self-association of the protein, as demonstrated by the inability of the carboxyl-terminal deletion mutants delta185-243 and delta209-243 to form higher order aggregates in solution. Lecithin:cholesterol acyltransferase analysis, using reconstituted HDL particles prepared by the sodium cholate dialysis method, has shown that mutants (Pro165-->Ala,Gln172-->Glu) (Leu211-->Val,Leu214-->Val, Leu218-->Val,Leu219-->Val), Leu222-->Lys,Phe225-->Lys, Phe229-->Lys) and delta209-243 reduced LCAT activation (38-68%). Mutant (Glu191-->Ala,His193-->Ala,Lys195-->Ala) enhanced LCAT activation (131%), and mutant (Ala152-->Leu, Leu159-->Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms [corrected]. The apparent catalytic efficiency (Vmax(app)/Km(app)) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the Km and the Vmax in the different mutants. These findings indicate that putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltransferase. In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for the identification of the domains and residues of apoA-I involved in binding the scavenger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.

Efflux of Cellular Cholesterol and Phospholipid to Apolipoprotein A-I Mutants

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(-6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(-6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(-6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(-6-222)) and 210 (apoA-I-(-6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(-6-150)) and 135 (apoA-I-(-6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(-6-243), apoA-I-(-6-222), and apoA-I-(-6-210), but slightly higher for apoA-I-(-6-150) and apoA-I-(-6-135). When complexed with phospholipid, all forms except apoA-I-(-6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent.

The role of apolipoprotein AI domains in lipid binding.

Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apo AI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI delta 44-126, apoAI delta 139-170, and apoAI delta 190-243). Circular dichroism spectroscopy showed that the alpha-helical content of lipid-free apoAI delta 44-126 was 27% while the other mutants and native apoAI averaged 55 +/- 2%, suggesting that the missing N-terminal portion contains most of the alpha-helical structure of lipid-free apoAI. ApoAI delta 44-126 exhibited the largest increase in alpha-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for alpha-helix stability in the lipid-free state, whereas the C terminus is required for alpha-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the alpha-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in alpha-helical content observed when native apoAI binds lipid results from the formation of alpha-helix primarily in the C-terminal half of the molecule.

Specific targeting of a lipophilic prodrug of iododeoxyuridine to parenchymal liver cells using lactosylated reconstituted high density lipoprotein particles

We recently reported the conversion of the water-soluble antiviral drug iododeoxyuridine (IDU) into the lipophilic prodrug dioleoyl-iododeoxyuridine (IDU-Ol 2). The prodrug was incorporated into reconstituted high-density lipoprotein (NeoHDL) particles with physical and biological properties similar to those of native HDL. We also found, in initial experiments, that lactosylation of the prodrug-loaded NeoHDL increases its liver uptake. Because this offers the attractive perspective of using these particles for the delivery of drugs to the liver, we now analyze the characteristics and biological fate of lactosylated IDU-Ol 2-loaded NeoHDL. The particles (containing approximately 25 prodrug molecules) have the same size and charge as native HDL, indicating that lactosylation does not cause aggregation or oxidative modification. At 10 min after intravenous injection of lactosylated [ 3H]IDU-Ol 2-loaded NeoHDL into rats, only 13.5 ± 2.8% of the dose was left in plasma and 75.9 ± 2.4% of the dose was recovered in the liver. The relative specific uptake by the liver was 1–2 orders of magnitude higher than that of any other tissue. The hepatic uptake of lactosylated [ 3H]IDU-Ol 2-loaded NeoHDL was much higher than that of free [ 3H]IDU (< 20% of the dose). Both parenchymal liver cells and Kupffer cells express galactose-specific receptors. By isolating liver cells after injection of the prodrug-loaded particles, it was established that hepatic uptake occurred mainly (for 84.4 ± 3.8%) in parenchymal liver cells. Preinjection with asialofetuin substantially reduced the liver uptake of lactosylated [ 3H]IDU-Ol 2-loaded Neo-HDL, which points to uptake by the asialoglycoprotein receptor. Subcellular fractionation of the liver indicated that lactosylated [ 3H]IDU-Ol 2-loaded NeoHDL does not merely associate to cells, but is internalized and delivered to the lysosomes. In conclusion, we show that IDU can be specifically targeted to the parenchymal liver cell. Conversion of the water-soluble parent drug into a lipophilic prodrug that is incorporated into a lactosylated reconstituted HDL particle, is an approach that may also be used to deliver other water-soluble drugs to the parenchymal liver cells. This may lead to more effective therapy for liver diseases such as hepatitis B.

Neutralization and Transfer of Lipopolysaccharide by Phospholipid Transfer Protein

Phospholipid transfer protein (PLTP) and lipopolysaccharide-binding protein (LPB) are lipid transfer proteins found in human plasma. PLTP shares 24% sequence similarity with LBP. PLTP mediates the transfer and exchange of phospholipids between lipoprotein particles, whereas LBP transfers bacterial lipopolysaccharide (LPS) either to lipoprotein particles or to CD14, a soluble and cell-surface receptor for LPS. We asked whether PLTP could interact with LPS and mediate the transfer of LPS to lipoproteins or to CD14. PLTP was able to bind and neutralize LPS: incubation of LPS with purified recombinant PLTP (rPLTP) resulted in the inhibition of the ability of LPS to stimulate adhesive responses of neutrophils, and addition of rPLTP to blood inhibited cytokine production in response to LPS. Transfer of LPS by rPLTP was examined using fluorescence dequenching experiments and native gel electrophoresis. The results suggested that rPLTP was able to mediate the exchange of LPS between micelles and the transfer of LPS to reconstituted HDL particles, but it did not transfer LPS to CD14. Consonant with these findings, rPLTP did not mediate CD14-dependent adhesive responses of neutrophils to LPS. These results suggest that while PLTP and LBP both bind and transfer LPS, PLTP is unable to transfer LPS to CD14 and thus does not mediate responses of cells to LPS.

High yield overexpression and characterization of human recombinant proapolipoprotein A-I.

Human apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) where it defines the particle structure and stability and functions as the main activator of the enzyme lecithin:cholesterol acyltransferase (LCAT). ApoA-I is expressed in the liver as a preproprotein that is targeted to the endoplasmic reticulum for secretion; in plasma, an unknown protease removes the six amino acid long propeptide. In this study, the cDNA coding the human proapoA-I was cloned into an Escherichia coli vector; the overexpressed protein was purified to 99% homogeneity and was extensively characterized together with mature apoA-I purified from plasma. SDS-PAGE, mass spectrometry, and Edman sequence analysis showed that the initial Met residue needed for translation in E. coli is posttranslationally removed from the N-terminal sequence of the proapoA-I. The structural and functional analyses were carried out on the lipid-free and the lipid-bound proteins. ProapoA-I self associated, interacted with dimyristoyl phosphatidylcholine vesicles, and formed secondary structures very similar to the lipid-free apoA-I. Reconstituted HDL particles made with two initial molar ratios of palmitoyloleoyl phosphatidylcholine/cholesterol/apolipoprotein/Na-cholate had identical particle sizes and distributions when apoA-I or proapoA-I were used. Particles having diameters of 79 A and 98 A, containing two apoA-I or proapoA-I molecules per particle, were isolated and characterized. The particles contained the same amounts of alpha-helical structure, had very similar fluorescence properties, and activated LCAT equally well. We conclude that proapoA-I expressed and purified from E. coli is functionally and structurally indistinguishable from mature apoA-I purified from plasma when analyzed in vitro. Therefore, this recombinant proapoA-I and mutants derived from it will be important sources of protein for analyzing apoA-I structure and function, as well as for studies of proapoA-I processing.

Cholesterol Transfer from Low Density Lipoproteins to Reconstituted High Density Lipoproteins Is Determined by the Properties and Concentrations of Both Particles

Cholesterol spontaneously transfers from low density lipoproteins (LDL) to high density lipoproteins (HDL). This transfer is important physiologically as it supplies the major portion of cholesterol for the lecithin:cholesterol acyltransferase reaction and is one mechanism for the reduction of atherogenic LDL cholesterol. The objective of this work was to examine the properties of both HDL and LDL which modulate cholesterol transfer, as well as to obtain the relevant kinetic constants for the transfer at concentrations of lipoproteins approaching those existing in vivo. To examine the effects of HDL structural parameters on cholesterol transfer, we prepared reconstituted HDL particles with saturated or unsaturated phospholipid, with apolipoprotein AI or apolipoprotein AII, with increasing size and phospholipid content, and with increasing initial contents of cholesterol. We also prepared five LDL subfractions of variable density and size. The kinetics of cholesterol mass transfer were measured by incubating LDL with rHDL at 37 degrees C, separating the lipoproteins by dextran sulfate/Mg2+ precipitation of LDL at timed intervals, and analyzing rHDL cholesterol content. The cholesterol content of rHDL at equilibrium, Ceq, and the half-time for transfer, t1/2, as well as the ratio of the lipid surface areas of LDL to rHDL were used in the analysis of the kinetic data by the aqueous diffusion model for lipid transfer developed by Nichols and Pagano [(1982) Biochemistry 21, 1720-1726]. The only variables that significantly affect the C(eq) and/or t1/2 are the phospholipid content and composition of the rHDL and the size or density of the LDL particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein.

We have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS-sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R-HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two-step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL. We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30-fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP.

Synthesis of the dioleoyl derivative of iododeoxyuridine and its incorporation into reconstituted high density lipoprotein particles.

We investigated the potential use of reconstituted HDL particles (NeoHDL) as a carrier for lipophilic (pro)drugs. The antiviral drug iododeoxyuridine (IDU) was used as model compound. [3H]-IDU was derivatized with two oleoyl residues to dioleoyl[3H]iododeoxyuridine ([3H]IDU-Ol2), and the lipophilic prodrug was incorporated into NeoHDL by cosonication of [3H]IDU-Ol2 with lipids and HDL apoproteins. NeoHDL particles with the same density, size, and electrophoretic mobility as native HDL were obtained, which contained 7.3 +/- 0.8% (w/w) [3H]IDU-Ol2 (about 30 molecules of prodrug per particle). NeoHDL-associated [3H]IDU-Ol2 was stable during 2 h of incubation with human plasma; the prodrug was not appreciably hydrolyzed, nor exchanged with LDL. After intravenous injection of [3H]-IDU-Ol2-loaded 125I-NeoHDL into rats, [3H]IDU-Ol2 disappeared more rapidly from the circulation than the 125I-apoproteins (78.0 +/- 8.0% vs 30.1 +/- 4.5% of the dose cleared from plasma in 60 min, respectively). The hepatic association of the prodrug was higher than that of the apoproteins (21.6 +/- 0.5 vs 5.2 +/- 1.0% of the dose at 10 min after injection, respectively). As selective clearance and uptake of lipid esters is also observed with native HDL, this suggests that, in vivo, prodrug-loaded NeoHDL may be subject to physiological HDL-specific processing. Lactosylated [3H]IDU-Ol2-loaded 125I-NeoHDL, which contains galactose residues that can be recognized by galactose receptors on parenchymal liver cells, was rapidly cleared from plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS.

Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.

Hepatic lipase promotes the uptake of HDL esterified cholesterol by the perfused rat liver: a study using reconstituted HDL particles of defined phospholipid composition.

The role of hepatic triacylglycerol lipase (H-TGL) in promoting the liver uptake of high density lipoprotein (HDL) free and esterified cholesterol was studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and is active at the vascular bed. For this purpose, reconstituted HDL of defined phospholipid composition were prepared, containing either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, a substrate for H-TGL, or 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine, a non-hydrolyzable analog. Reconstituted HDL were then used in the perfused rat liver system. The main results are the following. 1) Reconstituted HDL were obtained by sonication of lipids and apolipoproteins and isolated by ultracentrifugation in the 1.07-1.21 g/ml density interval. Reconstituted HDL containing either diacylphosphatidylcholine or alkyl-acyl-phosphatidylcholine were similar in terms of chemical composition, apparent size, and apolipoprotein A-I immunoreactivity, and were comparable to native HDL3. 2) Reconstituted HDL were labeled with free [14C]cholesterol and [3H]cholesteryl ether, a non-hydrolyzable tracer of esterified cholesterol, and were perfused through the rat liver. Liver uptake of [3H]cholesteryl ether was 2.5-fold higher from reconstituted HDL containing diacylphospholipid than from HDL reconstituted with alkyl-acyl-phospholipids. Liver uptake of free [14C]cholesterol was identical in both cases. 3) H-TGL-depleted rat livers were obtained by a 12-min preperfusion in the presence of heparin, displacing 90% of the enzymatic activity. The residual activity in the perfusate was inhibited by a specific antibody directed against rat H-TGL. Liver uptake of [3H]cholesteryl ether from reconstituted HDL containing diacylphospholipid was reduced by 35% in hepatic lipase-depleted livers compared to controls. On the other hand, hepatic lipase depletion had no effect on the liver uptake of esterified cholesterol from HDL reconstituted with alkyl-acyl-phospholipids. The above findings support a role for the phospholipase A1 activity of H-TGL in stimulating the delivery of HDL esterified cholesterol to liver cells.

Structure of the apolipoprotein A-IV / lipid discoidal complexes: an attenuated total reflection polarized Fourier transform infrared spectroscopy study

Discoidal lipid particles were prepared from a reaction mixture containing apo A-IV and dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) in the molar ratio of 185:1 (lipid/protein). The complexes were isolated by gel filtration and characterized in terms of composition and size. Infrared attenuated total reflection spectroscopy was used to estimate the secondary structure of apolipoprotein A-IV and the orientation of its amphipatic α-helices with respect to the lipid hydrocarbon chains. In addition, infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the particles. This approach has been applied successfully to reconstituted HDL particles prepared from a reaction mixture containing DPPC and apo A-I in the molar ratio of 150:1 (Wald, J.H., Goormaghtigh, E., De Meutter, J., Ruysschaert, J.M. and Jonas, A. (1990) J. Biol. Chem. 265, 20044–20050). Apo A-IV helecity increased for the protein bound to DMPC or DPPC but the increase was more pronounced for the apo A-IV/DMPC particles. In both complexes, the α helical amphipathic segments of the protein were parallel to the lipid acyl chains and no significant modification of the overall organization of the lipid molecules in the lipid bilayer was observed. The presence of apo A-IV seems only to affect the conformation of the lipid hydrocarbon chains in close contact with the protein in the discoidal particles.

Apoprotein A-1 is a cofactor independent substrate of protein kinase C

Apoprotein A-1 (apo A-1), the predominant protein constituent of high density lipoproteins (HDL), was phosphorylated by protein kinase C (PKC). Optimal phosphorylation of lipid-free apo A-1 occurs in the absence of calcium, phosphatidyl serine (PS), and diolein (DO). However, HDL-bound apo A-1 was not phosphorylated by PKC. Furthermore, addition of either native or reconstituted HDL particles to lipid-free apo A-1 resulted in a concentration-dependent inhibition of phosphorylation. It appears that the phosphorylatable sites on apo A-1 are involved in hydrophobic interaction with the lipids of HDL. Apo A-1 is a novel substrate of PKC because it does not require calcium and lipid cofactors for optimal phosphorylation.

Transformations of reconstituted high-density lipoprotein subclasses as a function of temperature or LDL concentration

The objectives of this study were to determine the structural changes in defined, reconstituted high density lipoproteins (rHDL) resulting from spontaneous phospholipid depletion in the presence or absence of low-density lipoproteins (LDL), to establish the precursor-product relationships among the rHDL particles and to assess the differences in behavior of rHDL particles containing apo A-I or apo A-II. The rHDL particles were prepared by the sodium cholate dialysis method, and were incubated in buffer at 50 degrees C, or in buffer containing different concentrations of LDL at 37 degrees C, for up to 24 h. The changes in the rHDL particle distributions with time were followed by non-denaturing gradient gel electrophoresis, and the rHDL were isolated at various time points for chemical analysis. We found that rHDL particles containing apo A-I or apo A-II lose phospholipid and gain cholesterol when incubated with LDL. Increasing LDL concentrations remove increasingly larger amounts of phospholipid. With phospholipid loss the apo A-I containing particles undergo major structural rearrangements that give rise to 78 A and 106 A particles from 86 A and 94 A precursors. The 78 A products appear to be the most stable, lipid-poor species. Reconstituted HDL particles prepared with apo A-II (94 and 101 A in diameter) are more resistant to structural rearrangements than the apo A-I counterparts under similar reaction conditions.

Structure of apolipoprotein A-I in three homogeneous, reconstituted high density lipoprotein particles.

To elucidate further the conformation of human apolipoprotein A-I (apoA-I) in lipid-bound states and its effect on the reaction with lecithin cholesterol acyltransferase (LCAT), we prepared reconstituted HDL (rHDL) particles from a reaction mixture containing dipalmitoylphosphatidylcholine/cholesterol/apoA-I in the molar ratios of 150:7.5:1. The particles were separated by gel filtration into three classes of highly homogeneous and reproducible discs with diameters of 97, 136, and 186 A, containing 2, 3, and 4 molecules of apoA-I/disc, respectively, and increasing proportions of phospholipid and cholesterol. These three classes of particles were then investigated by a variety of fluorescence techniques, to probe the average environment and mobility of the tryptophan (Trp) residues in the structure of apoA-I. We found small, gradual changes in the fluorescence parameters with changes in the size of the rHDL, consistent with a shift of Trp residues to a more hydrophobic and more rigid environment, as well as an increased resistance of apoA-I to denaturation by guanidine hydrochloride in the larger particles. In contrast, circular dichroism measurements and binding studies with seven monoclonal antibodies indicated a similar alpha-helical structure (73%) for apoA-I in all the particles, and similar exposure of apoA-I epitopes in the COOH-terminal two-thirds of the apolipoprotein. Thus the structure of apoA-I is comparable for the three classes of particles and is consistent with the presence of eight alpha-helical segments per apoA-I in contact with the lipid. In addition, we obtained the apparent kinetic parameters for the reaction of the rHDL particles with lecithin cholesterol acyltransferase. The apparent Km values were similar but the apparent Vmax decreased almost 8-fold, going from the 97- to the 186-A particles; therefore, the decreasing reactivity for the larger particles can be attributed mainly to differences in the catalytic rate constant. The rate limiting step is probably affected by local structural differences in the apoA-I, or by the interfacial properties of the lipid.