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.     

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.     

Metabolism of human apolipoproteins A-I and A-II: compartmental models

The metabolism of radioiodinated apolipoproteins (apo) A-I and A-II have been examined using the techniques of compartmental modeling. The model for apoA-I contains two plasma compartments decaying at different rates. One component of apoA-I has a residence time of 3.8 days and the second has a residence time of 6.1 days. In contrast, the apoA-II model has only one plasma component, with a residence time of 5.5 days, which decays through two distinct pathways. Twenty-seven percent of apoA-II decays through a pathway that takes 1.1 days longer to reach the urine than the remaining 73% which decays through the more direct path. These differences in the metabolism exist in both male and female populations. Comparison of fasting and nonfasting concentrations of apoA-I revealed that apoA-I concentration was elevated 0.5 standard deviations in the nonfasting samples while there was no significant difference in the apoA-II concentrations. The fasting apoA-I concentrations were found to be less stable over the study period when compared to fasting apoA-II concentrations. These findings are interpreted as indicating that apoA-I and apoA-II each have a separate metabolism which overlaps when they are present on the same lipoprotein particle. Furthermore, these findings are consistent with the concept that apoA-I metabolism is influenced more by perturbations such as dietary modulation.     

Expression of human apolipoprotein A-II and its effect on high density lipoproteins in transgenic mice.

Apolipoproteins A-I and A-II comprise approximately 70 and 20%, respectively, of the total protein content of HDL. Evidence suggests that apoA-I plays a central role in determining the structure and plasma concentration of HDL, while the role of apoA-II is uncertain. To help define the function of apoA-II and determine what effect increasing its plasma concentration has on HDL, transgenic mice expressing human apoA-II and both human apoA-I and human apoA-II were produced. Human apoA-II mRNA is expressed exclusively in the livers of transgenic animals, and the protein exists as a dimer as it does in humans. High level expression of human apoA-II did not increase HDL concentrations or decrease plasma concentrations of murine apoA-I and apoA-II in contrast to what was observed in mice overexpressing human apoA-I. The primary effect of overexpressing human apoA-II was the appearance of small HDL particles composed exclusively of human apoA-II. HDL from mice transgenic for both human apoA-I and human apoA-II displayed a unique size distribution when compared with either apoA-I or apoA-II transgenic mice and contain particles with both these human apolipoproteins. These results in mice, indicating that human apoA-II participates in determining HDL size, parallel results from human studies.     

The unique role of apolipoprotein A-I in HDL remodeling and metabolism

PURPOSE OF REVIEW: To rationalize the distinctive biological behavior of apolipoprotein (apo)A-I and apoA-II in light of differences in their respective structures, properties, and physico-chemical behavior. RECENT FINDINGS: The distinctive metabolic behavior of apoA-I compared with that of apoA-II, which are revealed as differences in their interactions with the HDL receptor, scavenger receptor class B type I, can be understood in terms of their physico-chemical properties. Detergent and chaotropic perturbation of HDL unmasks properties that distinguish apoA-I from apoA-II and emulate the secondary effects of lecithin: cholesterol acyltransferase, cholesteryl ester transfer protein, and phospholipid transfer protein - the key protein factors in HDL remodeling, that is, formation of lipid-free apoA-I but not apoA-II and particle fusion. Thus, of the two major HDL apolipoproteins, apoA-I is the more plastic and labile and this difference gives apoA-I a unique physiological role that has been verified in mouse models of HDL metabolism. SUMMARY: The compositions, structures, and properties of HDL particles are important determinants of the mechanisms by which these antiatherogenic lipoproteins are metabolized. Although the plasma lipid transfer proteins and lipid-modifying enzymes are important determinants of HDL processing, the distinctive structures and properties of apoA-I and apoA-II, the two major HDL proteins, determine in different ways the thermodynamic stability of HDL - the former through its greater plasticity and the latter by its higher lipophilicity. These distinctions have been revealed by physico-chemical studies of HDL stability in the context of numerous studies of enzyme and lipid transfer activities and of the interaction of HDL with its hepatic scavenger receptor.     

Mass spectral measurements of the apoHDL in horse (Equus caballus) cerebrospinal fluid

As a continuation of our proteogenomic studies of equine apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with the HDL in horse cerebrospinal fluid (CSF). Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I and A-II, as well as acylated apoA-I. In comparison with our previously published data on equine plasma apolipoproteins, there appears to be a higher percentage of acylated apoA-I in the CSF than in plasma. As was the case in plasma, apoA-II circulates as a homodimer. These studies also revealed a protein with a mass of 34,468 Da that we are speculating is the value for horse apoE.     

The secondary structure of apolipoproteins in human HDL3 particles after chemical modification of their tyrosine, lysine, cysteine or arginine residues. A Fourier transform infrared spectroscopy study

Fourier transform infrared spectra of apolipoprotein E-depleted human HDL3 have been obtained in H2O and 2H2O buffers. The absorption bands in the protein amide I and amide II regions (1700-1500 cm-1) were assigned to alpha-helical, disordered and beta-strand/beta-turn structures of apolipoproteins A-I and A-II (apoA-I and apoA-II), the apolipoprotein constituents of HDL3. Modification of HDL3 by tetranitromethane (TNM) treatment, acetylation, reduction plus alkylation and 1,2-cyclohexanedione treatment derivatised tyrosine, lysine, cysteine and arginine residues, respectively, and caused alteration of the secondary structure of the HDL3 apolipoproteins to different extents. Each of the chemical modifications caused changes in the frequency of bands associated with beta-strands/beta-turns, but only TNM treatment of HDL3, as judged by the second- and fourth-derivative spectra, resulted in a shift of the band assigned to the alpha-helical structure of the proteins. In agreement with other workers, only TNM treatment of HDL3 particles was found to inhibit their binding by high-affinity cell membrane receptors. It is proposed, therefore, that receptor recognition of HDL3 particles is dependent on conservation of the alpha-helix structures within apoA-I and apoA-II, and that beta-strand/beta-turn structures are not involved. This conclusion is consistent with the predominance of amphipathic alpha-helical structures in both apolipoproteins and with the relaxed specificity of the receptors which are thought to recognise both apoA-I and apoA-II.     

Synthesis, transport, and processing of apolipoproteins of high density lipoproteins

Cell biology methods have greatly influenced the elucidation of the biosynthetic pathways of apolipoproteins. In vitro and tissue culture systems allow the study, to a large extent, of the process of synthesis, intracellular processing, secretion, and extracellular processing of the major high density lipoprotein apoproteins apoA-I and A-II and also of a minor component, apoA-IV. Whereas the latter apoprotein is equipped only with a signal sequence, the primary translation products of apoA-I and apoA-II carry N-terminal extensions of preprosequence of 24 amino acids for apoA-I and 23 amino acid residues for apoA-II. The pro-form of apoA-I characterized by a hexapeptide extension is completely stable intracellularly and is secreted as such. The pro-form is further processed by a serum protease specific for an unusual -Gln-Gln-Asp-Glu-sequence site. Pro-apoA-II, a pentapeptide sequence, is partially processed intracellularly to its mature form and secreted together with the residual pro-form. The cleavage site of pro-apoA-II is characterized by two basic amino acid residues Arg-Arg, present also in other known pro-proteins. The biological function of the N-terminal pro-sequences and details of their final processing by the serum protease(s) have yet to be established.     

Spur cells in patients with alcoholic liver cirrhosis are associated with reduced plasma levels of apoA-II, HDL3, and LDL

The precise nature and origin(s) of the abnormalities in lipoprotein and apolipoprotein profile associated with severe hepatic dysfunction and the presence of spur cells remain poorly defined. To shed light on this question, we have analyzed the plasma lipoprotein and apolipoprotein profiles in five patients with alcoholic cirrhosis and spur cells, and compared them with those of a group with similar hepatocellular dysfunction, but lacking spur cells, and with that of a control group. Lipoproteins were subfractionated by density gradient ultracentrifugation and their physicochemical properties were determined; apolipoprotein A-I, A-II, and B contents in plasma and the respective subfractions were quantitated by radial immunodiffusion, while the complement of low molecular weight apolipoproteins in each subfraction was analyzed by isoelectric focusing and electrophoresis in alkaline-urea polyacrylamide gels. Spur cell plasma was distinguished by reduced levels of apoA-II and elevated ratios of apoA-I/apoA-II (approximately 13:1 as compared to 3.3-3.9:1 in the other two groups), and by reduced concentrations of HDL3. Gradient fractionation showed the apoA-II content of HDL3 to be dramatically and significantly diminished in spur cell plasma; in addition, apoA-II content was reduced relative to apoA-I in this subclass (4.7:1 as compared to 1:1 in cirrhotics lacking spur cells and 1.9:1 in controls). Spur cell HDL2 was similarly deficient in apoA-II, with elevated ratios of apoA-I:apoA-II (9.8:1 in comparison with 1.9-2.5:1 in the two other groups). Nonetheless, high HDL2 concentrations were seen in both series of cirrhotic patients, irrespective of red cell morphology. Spur cell HDL2 thus appears to consist primarily of particles possessing only apoA-I, with a minor population containing both apoA-I and apoA-II. The free cholesterol content of all lipoprotein subfractions from spur cell plasma was increased, as indeed was the molar ratio of free cholesterol to phospholipid, in comparison with that of corresponding fractions from alcoholic cirrhotics lacking spur cells and of control subjects. LDL levels were reduced in spur cell plasma, thereby distinguishing this group from the cirrhotics without spur cells who displayed elevated LDL levels. Markedly reduced plasma levels of apoA-II, HDL3, and LDL appear characteristic of alcoholic cirrhotics presenting with spur cells. Our findings suggest that apoA-II may be essential to the normal function and metabolism of HDL, one aspect of which may be the transport of free cholesterol and thereby the direct or indirect maintenance of red cell morphology.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins

The two major apolipoproteins associated with human and chimpanzee (Pan troglodytes) high density lipoproteins (HDL) are apoA-I and dimeric apoA-II. Although humans are closely related to great apes, apolipoprotein data do not exist for bonobos (Pan paniscus), western lowland gorillas (Gorilla gorilla gorilla) and the Sumatran orangutans (Pongo abelii). In the absence of any data, other great apes simply have been assumed to have dimeric apoA-II while other primates and most other mammals have been shown to have monomeric apoA-II. Using mass spectrometry, we have measured the molecular masses of apoA-I and apoA-II associated with the HDL of these great apes. Each was observed to have dimeric apoA-II. Being phylogenetically related, one would anticipate these apolipoproteins having a high percentage of invariant sequences when compared with human apolipoproteins. However, the orangutan, which diverged from the human lineage between 16 and 21 million years ago, had an apoA-II with the lowest monomeric mass, 8031.3 Da and the highest apoA-I value, 28,311.7 Da, currently reported for various mammals. Interestingly, the gorilla that diverged from the lineage leading to the human–chimpanzee branch after the orangutan had almost identical mass values to those reported for human apoA-I and apoA-II. But chimpanzee and the bonobo that diverged more recently had identical apoA-II mass values that were slightly larger than reported for the human apolipoprotein. The chimpanzee A-I mass values were very close to those of humans; however, the bonobo had values intermediate to the molecular masses of orangutan and the other great apes. With the already existing genomic data for chimpanzee and the recent entries for the orangutan and gorilla, we were able to demonstrate a close agreement between our mass spectral data and the calculated molecular weights determined from the predicted primary sequences of the respective apolipoproteins. Post-translational modification of these apolipoproteins, involving truncation and oxidation of methionine, are also reported.     

Influence of development, estrogens, and food intake on apolipoprotein A-I, A-II, and E mRNA in rat liver and intestine

The influence of development and ethinylestradiol (EE) on apolipoprotein (apo) A-I, A-II, and E mRNA in rat liver and intestine was studied by dot blot hybridization and Northern blot analysis. ApoA-I mRNA levels were maximal in the perinatal period and declined after day 15. An opposite trend was noted for the apoA-II mRNA levels, whereas apoE mRNA remained fairly constant. Liver apoA-I mRNA levels increased after ovariectomy (OVX). A further rise was observed when EE was given at 2000 micrograms/day. When the influence of OVX and EE was controlled for food intake by pair-feeding, OVX still increased hepatic apoA-I mRNA. The rise in liver apoA-I mRNA after EE, however, was no longer significant. Under the same conditions OVX slightly increased intestinal apoA-I mRNA. EE (2000 micrograms/day) decreased intestinal apoA-I mRNA to 80% of the pair-fed controls. Liver apoA-II mRNA levels did not change after OVX when the animals were fed ad libitum, but decreased slightly when the rats were pair-fed. EE caused a dose-dependent decrease in liver apoA-II mRNA, irrespective of food intake. None of these treatments caused any change in liver apoE mRNA levels. Serum apoA-I levels increased upon OVX, while serum apoE did not change. EE provoked a dose-dependent decrease of both apolipoproteins in serum. In conclusion: 1) Changes in food intake play an important role in the in vivo effects of estrogens on apolipoprotein mRNA levels. 2) The stimulatory effect of OVX on hepatic apoA-I mRNA as well as the inhibitory effect of EE on hepatic apoA-II mRNA are independent of food intake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides.     

Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency

Our purpose was to compare HDL subpopulations, as determined by nondenaturing two-dimensional gel electrophoresis followed by immunoblotting for apolipoprotein A-I (apoA-I), apoA-II, apoA-IV, apoCs, and apoE in heterozygous, compound heterozygous, and homozygous subjects for cholesteryl ester transfer protein (CETP) deficiency and controls. Heterozygotes, compound heterozygotes, and homozygotes had CETP masses that were 30, 63, and more than 90% lower and HDL-cholesterol values that were 64, 168, and 203% higher than those in controls, respectively. Heterozygotes had approximately 50% lower pre-beta-1 and more than 2-fold higher levels of alpha-1 and pre-alpha-1 particles than controls. Three of the five heterozygotes' alpha-1 particles also contained apoA-II, which was not seen in controls. Compound heterozygotes and homozygotes had very large particles not observed in controls and heterozygotes. These particles contained apoA-I, apoA-II, apoCs, and apoE. However, these subjects did not have decreased pre-beta-1 levels. Our data indicate that CETP deficiency results in the formation of very large HDL particles containing all of the major HDL apolipoproteins except for apoA-IV. We hypothesize that the HDL subpopulation profile of heterozygous CETP-deficient patients, especially those with high levels of alpha-1 containing apoA-I but no apoA-II, represent an improved anti-atherogenic state, although this might not be the case for compound heterozygotes and homozygotes with very large, undifferentiated HDL particles.     

Apolipoprotein specificity for lipid efflux by the human ABCAI transporter

ABCAI, a member of the ATP binding cassette family, mediates the efflux of excess cellular lipid to HDL and is defective in Tangier disease. The apolipoprotein acceptor specificity for lipid efflux by ABCAI was examined in stably transfected Hela cells, expressing a human ABCAI-GFP fusion protein. ApoA-I and all of the other exchangeable apolipoproteins tested (apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, apoE) showed greater than a threefold increase in cholesterol and phospholipid efflux from ABCAI-GFP transfected cells compared to control cells. Expression of ABCAI in Hela cells also resulted in a marked increase in specific binding of both apoA-I (Kd = 0.60 microg/mL) and apoA-II (Kd = 0.58 microg/mL) to a common binding site. In summary, ABCAI-mediated cellular binding of apolipoproteins and lipid efflux is not specific for only apoA-I but can also occur with other apolipoproteins that contain multiple amphipathic helical domains.     

Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein

Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at -20 degrees C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues. Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Local/bulk determinants of conformational stability of exchangeable apolipoproteins

GuHCl-induced denaturation of human plasma apoA-I, apoA-II, apoA-IV, apoE3 and three recombinant apoE isoforms in solution and discoidal complexes with phosphatidylcholine (only plasma proteins) was studied. The protein conformational stability (ΔG(H(2)O)) and a slope of linear dependence of free energy of unfolding on GuHCl concentration (m-value) were estimated with the three equilibrium schemes. The data for all proteins, except apoA-II, fit with the three-state model, thus evidencing two-domain structure. The predicted folding rate of the four apoE in solution correlated with conformational stability. The dependence disappeared at the inclusion of apoA-I and apoA-IV into analysis and the m-values, adjusted for residue number in helices (m(rh)), differed between those for apoE and apoA-I/apoA-IV. However, the m(rh)-values for six proteins correlated positively with the fractional change in accessible surface area at unfolding for Phe, Lys and Asn, while negatively for Arg, Ala and Gly residues. The difference between the adjusted ΔG(rh)(H(2)O) values for apolipoproteins in complexes and in solution decreased at the increase of reduced temperature (T(obs)-T(t))/T(t). The induction of intrinsic disorder by arginine residues may be of primary importance in metabolism and function of exchangeable apolipoproteins, while their stability in nascent discoidal HDL is controlled by the physical state of phosphatidylcholine.     

A quantitative analysis of apolipoprotein binding to SR-BI: multiple binding sites for lipid-free and lipid-associated apolipoproteins

Competitive binding experiments were performed using Y1-BS1 adrenal cells to provide information about the interaction of HDL apolipoproteins with scavenger receptor class B, type I (SR-BI). Exchangeable apolipoproteins apolipoprotein A-I (apoA-I), apoA-II, apoE-2, apoE-3, and apoE-4 as phospholipid complexes bind like HDL3 to SR-BI via their multiple amphipathic alpha-helices; the concentrations required to reduce the binding of HDL3 to SR-BI by 50% (IC50) were similar and in the range of 35-50 microgram protein/ml. In the case of apoA-I, peptides corresponding to segments 1-85, 44-65, 44-87, 149-243, and 209-241 all had the same IC50 as each other (P = 0.86), showing that a specific amino acid sequence in apoA-I is not responsible for the interaction with SR-BI. The distribution of charged residues in the amphipathic alpha-helix affects the interaction, with class A and Y helices binding better than class G* helices. Synthetic alpha-helical peptides composed of either l or d amino acids can bind equally to the receptor. Association with phospholipid increases the amount of apolipoprotein binding to SR-BI without altering the affinity of binding. Lipid-free apolipoproteins compete only partially with the binding of HDL to SR-BI, whereas lipidated apolipoproteins compete fully. These results are consistent with the existence of more than one type of apolipoprotein binding site on SR-BI.     

Interaction of the apoproteins of very low density and high density lipoproteins with synthetic phospholipids.

The interaction of synthetic dimyristoyl phosphatidylcholine (lecithin) liposomes with isolated apoC-I and apoC-III proteins from very low density lipoproteins has been studied by microcalorimetry. Complex formation is a highly exothermal process characterized by a maximal enthalpy of -130 kcal/mol (-544 kJ) apoC-III-1 and -65 kcal/mol apoC-I proteins (-272 kJ). The complex composition determined after its isolation by ultracentrifugal flotation agrees with the value derived from the enthalpy binding curves. The binding of a constant amount of dimyristoyl lecithin to apoprotein mixtures containing various proportions of apoA-I and apoC-III failed to demonstrate the existence of any preferential association between the two apoproteins, in contrast with results obtained previously with apoA-I/apoA-II protein mixtures. Finally the various contributions to the enthalpy of binding such as that arising from an increase in apoprotein helicity have been evaluated. A classification of the apolipoproteins according to their lipid-binding affinity is proposed as: apoA-II congruent to apoC-III greater than apoC-I greater than apoA-I proteins.