Interaction of apoA-II from human high density lipoprotein with lysolecithin.

The effects of lysolecithin and hexadecyltrimethylammonium bromide on the structure and stability of apoA-II from human high density lipoprotein have been evalued by circular dichroism and fluorescence measurements. There is a profound enhancement in the stability of apoA-II to guanidinium hydrochloride denaturation when it forms a phospholipid complex with lysolecithin micelles. This complex is not only resistant to guanidinium hydrochloride denaturation, but it can be formed in a 6 M solution of this denaturant. The behavior of apoA-II in the native human high density protein is much closer to that of the lysolecithin apoA-II complex than to that of the free apoA-II.     

The molecular behavior of apoA-I in human high density lipoproteins.

The properties of apoA-I in human high density lipoprotein (1.063 smaller than d smaller than 1.210 gm per ml) have been examined by fluorescence and difference absorption spectroscopy, while the behavior of the total complement of apoproteins has been evaluated by far ultraviolet circular dichroism. Marked increases in stability to temperature, pH, and guanidine hydrochloride were observed for apoA-I in the native particle as compared to the isolated state.     

Self-association of apo-C-I from the human high density lipoprotein complex.

The molecular properties of apo-C-I, isolated from the human high density lipoprotein complex, have been evaluated as a function of pH, solvent composition, and protein concentration by sedimentation equilibrium and circular dichroic measurements. This protein self-associates in aqueous solution at neutral pH with concomitant changes in secondary structure. In contrast, in the acid pH range, apo-C-I is monomeric and its ellipticity is independent of protein concentration. The results are discussed in terms of the interpretation of experiments where changes in the physical properties of apolipoproteins have been used to monitor ligand binding and lipid-apolipoprotein recombination.     

Chromatofocusing of apolipoproteins from human serum high density lipoprotein

Human HDL was delipidated and the apolipoproteins were fractionated by chromatofocusing. Chromatofocusing, which separates proteins due to their differing isoelectric points, resulted in 8 peaks with corresponding pI values of 7.40, 6.92, 6.64, 5.48, 5.30, 5.18, 4.92 and 4.63. By one single chromatofocusing run four apolipoproteins were obtained in pure form. Two additional polypeptides could be purified during the desalting step using phenyl-Sepharose.     

Thermal behavior of human plasma high density lipoprotein.

Human plasma low density lipoprotein displays a reversible thermal transition between 20 and 40 degrees C, due to a phase transition of its core cholesterol ester from a smectic to a more liquid-like state. To determine if the cholesterol of high density lipoprotein (HDL) displays similar thermal behavior, the human lipoprotein and its extracted lipid have been examined by differential scanning calorimetry, low angle X-ray scattering and polarizing microscopy. Neither HDL2**(d 1.063--1.125--1.21 g/ml) nor HDL3(d1.125--1.21g/ml) show thermal transitions between O and 60 degrees C. By contrast cholesterol ester isolated from HDL and mixtures of cholesterol oleate and linoleate show reversible liquid crystalline transitions between 20 and 40 degreesC. X-ray scattering studies of HDL2 and HDL3 performed at 10 degreesC show no scattering fringes attributable to a smectic phase of cholesterol ester. When HDL is heated to temperatures above 60 degreesC a broad, double-peaked endotherm is observed. The first component (peak temperature=71 degreesC) corresponds to a selective release of apoprotein A-1 from the lipoprotein, and the second component (peak temperature=90 degreesC) to a more generalized disruption of lipoprotein structure with release of cholesterol ester and apoprotein A-2. Following the thermal disruption of HDL, reversible liquid crystalline transitions of cholesterol ester can be seen by differential scanning calorimetry and polarizing microscopy, showing the presence of large domains of cholesterol ester. The absence of cholesterol ester transitions in intact HDL may indicate an interaction of cholesterol ester molecules with the protein-phospholipid surface of HDL that prevents the formation of an organized lipid phase. The high temperature behavior of HDL indicates that apoprotein A-1 is less important than apoprotein A-2 in maintaining the HDL apolar lipids in the form of a stable miroemulsion.     

Dynamic properties of human high density lipoprotein apoproteins.

This study was designed to identify a method for the measurement of human high density lipoprotein subfraction (HDL2 and HDL3) metabolism. Apolipoproteins A-I, A-II, and C, the major HDL apoproteins, were radioiodinated and incorporated individually into HDL2 and HDL3 in vitro. Using a double label technique, the turnover of apoA-I in HDL2 and HDL3 was measured simultaneously in a normal male. The apoprotein exchanged rapidly between the two subfractions, evidenced by equilibration of their apoA-I specific activity. Radiolabeled apoA-II, incorporated into the subfractions, showed a similar exchange in vitro. Incubation of 131I-labeled very low density lipoproteins (VLDL) with HDL or its subfractions resulted in transfer of C proteins from VLDL to the HDL moiety. The extent of transfer was dependent on the HDL subfraction present; 50% of the VLDL apoC was transferred to HDL3, while the transfer to total HDL and HDL2 was 69% and 78%, respectively. ApoC also exchanged between HDL2 and HDL3, again showing a preference for the former and suggesting a primary metabolic relationship between VLDL and HDL2. Overall, the study indicates that apoA-I, apoA-II, and the C proteins exist in equilibrium between HDL2 and HDL3. This phenomenon precludes their use as probes for HDL subfraction metabolism in humans.     

The self-association of the reduced ApoA-II apoprotein from the human high density lipoprotein complex.

The molecular properties of the single linear chain form of human apoA-II, i.e., Cm apoA-II, have been evaluated by circular dichroism, polarization of fluorescence, difference absorption, and sedimentation equilibrium. The self-association of Cm apoA-II to a dimer resembles closely that of apoA-II though the free energy change is somewhat smaller. The dimerization of Cm apoA-II is accompanied by major changes in secondary and tertiary structure. The apoA-II molecule, therefore, represents a molecular association where the intramolecular structure is strongly dependent on the quaternary structure.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Reconstitution of apolipoprotein A-I from human high density lipoprotein with bovine brain sphingomyelin

Reconstitution of apolipoprotein A-I was found to occur readily with bovine brain sphingomyelin (BBSM), with a maximum rate occurring at a temperature of 28 degrees C, a temperature approximating the phase transition temperature for this naturally occurring phospholipid. At BBSM:A-I weight ratios of 7.5:1 or less, a single recombinant product was observed which contained three A-I molecules per particle, which had a BBSM:A-I molar ratio of 360 to 1 and which appeared in the electron microscope as a discoidal complex with a thickness of 68 A and a diameter of 217 A. By these criteria, as well as by gel filtration, this product appears very similar to that obtained by recombination of A-I with phosphatidylcholine at elevated ratios of phospholipid/protein. No evidence was found for the existence of any BBSM:A-I complexes comparable to the smaller lecithin:A-I complex containing 200-250 mol of phospholipid and two A-I molecules per complex which has been previously reported. At BBSM:A-I ratios of 15:1 (w/w), a new type of complex was observed which was discoidal by electron microscopy but possessed a larger diameter (390 A) and higher phospholipid:protein molar ratio (535:1) than has been observed previously for recombinant complexes. The BBSM:A-I complexes were found to be significantly more resistant to denaturation by guanidine hydrochloride than the dimyristoyl phosphatidylcholine:A-I recombinant complexes. It is concluded that the mechanisms of interaction between apolipoprotein A-I and either bovine brain sphingomyelin or phosphatidylcholines are similar, but that the nature of the protein-lipid interactions with BBSM are such as to produce larger and more stable complexes than are observed with the phosphatidylcholines.     

Interaction of human serum high density lipoprotein apoprotein with phospholipids

The interaction of the apoprotein of human serum high density lipoprotein-3 (apo HDL₃)¹¹ with aqueous dispersion of natural and synthetic phospholipids (PL) was investigated at a temperature above the transitions of the PL hydrocarbon chains and also above their critical micellar concentration. This protein is known to contain two major polypeptides: apo A-I and apo A-II. The protein:PL mixtures (weight ratio, 2, 1 or 0.5) were subjected to sonic irradiation and then fractionated by either CsCl or D₂O-sucrose density gradient ultracentrifugation. Usually three bands were obtained, the relative mass distribution of which depended upon the nature of the PL and the ratio of the interacting components: one band contained the PL-poor protein (d 1.28 g/ml), another, the uncombined PL (d ? 1.08 g/ml), and the third band, both protein and PL. This band, which was considered to represent the actual complex, had a hydrated density intermediate between those of either apo HDL₃ or PL alone, a particle weight of 80,000 to 170,000 (i.e., less than that of intact HDL₃), a morphology by electron microscopy which depended on the materials and experimental conditions employed and circular dichroic spectra     

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL₂ and HDL₃ were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K_d) measured for WT human apoA-I interactions with HDL₂ and HDL₃ are about 10 µM, indicating that the binding is low affinity. This K_d value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.Understanding the structure and function of HDL is significant because of the beneficial cardioprotective properties of this lipoprotein (1). The anti-atherogenic effects of HDL arise, in part, from its participation in the reverse cholesterol transport pathway where the principal HDL protein, apolipoprotein A-I (apoA-I), plays a central role (2). As a result, the structure-function relationships of apoA-I have been studied extensively (for reviews, see Refs. 3–5). Perhaps the most important characteristic of the apoA-I molecule is its ability to bind lipids; this interaction is mediated by the amphipathic α-helices present in the protein molecule (6). ApoA-I binds well to phospholipid (PL)-water interfaces and, under appropriate conditions, can solubilize the PL to create discoidal HDL particles (7, 8). The binding of apoA-I to a PL surface involves a two-step mechanism. First, α-helices in the C-terminal domain of the protein interact with the surface, and, second, the N-terminal helix bundle domain opens to allow more helix-lipid interactions to occur (5, 9). Although the binding of apoA-I to model PL particles has been studied extensively, the binding of apoA-I to HDL particles has not been investigated much because of the difficulty of separating free and bound apoA-I in this system. This lack of information about apoA-I/HDL interactions is significant because the cycling of apoA-I molecules on and off HDL particles occurs during the metabolism of HDL particles (10, 11), in particular to release apoA-I molecules into the preβ-HDL pool (10, 12). This recycling is consistent with the well-established ability of apolipoproteins, such as apoA-I, to exchange spontaneously between different populations of lipoprotein particles (13–16) and PL vesicles (17, 18). As a rule, any remodeling event that depletes HDL particles of PL induces particle fusion and dissociation of that fraction of the apoA-I molecules that is in a labile pool (19). At this stage, quantitative understanding of the kinetics of apoA-I interactions with HDL particles is unavailable.Here, we exploit surface plasmon resonance (SPR) to monitor in real time the association and dissociation reactions in the apoA-I/HDL system. SPR has been used to derive quantitative information about the binding of both lipoproteins (20) and apoE (21–23) to proteoglycans. As far as the application of SPR to the HDL system is concerned, the binding of several plasma remodeling factors to HDL immobilized on a sensor chip has been investigated successfully (24–26). Also, the conformation of apoA-I in HDL was explored by comparing the binding of HDL particles to anti-apoA-I monoclonal antibodies immobilized on an SPR chip (27). We have extended these approaches to study the binding of apoA-I to HDL particles. The results show that apoA-I can bind reversibly and with low affinity to HDL particles by a two-step mechanism.     

Forms of human serum high density lipoprotein protein

Delipidation by ethanol-diethyl ether at -10 degrees C of human serum high-density lipoprotein (HDL, d 1.063-1.21) or of its subclasses HDL(2) (d 1.063-1.120) and HDL(3) (d 1.120-1.21), yielded proteins-alphaP, alphaP(2), and alphaP(3)-containing 3% phospholipid (largely lecithin) and 3.3% carbohydrate (glucosamine:L-fucose:D-galactose, D-mannose:sialic acid, 1.00:41 : 0.56:0.31). Solubility data and analytical ultracentrifugal analyses indicated that, upon lipid removal, HDL protein aggregates readily; the aggregation is dependent upon pH and ionic strength of the solvent medium. Subunits of 21,000 mol wt were obtained by acetylation or addition of sodium dodecyl sulfate (SDS). HDL and alphaP elicited in the rabbit a similar immunological response. By agar gel immunoelectrophoresis both anti-HDL and anti-alphaP sera detected a major and two minor antigenic determinants in HDL, HDL(3), alphaP, alphaP(2), and alphaP(3). HDL(2), antigenically homogeneous, gave an immunoelectrophoretic pattern of HDL(3) upon mixing with alphaP. alphaP, alphaP(2), and alphaP(3) exhibited a single antigenic determinant after treatment with SDS (0.5 M) or upon acetylation. Native or delipidated forms of HDL, HDL(2), and HDL(3) were separated by vertical starch gel electrophoresis into several components, which showed identical reactions against anti-HDL or anti-alphaP sera. The data suggest that (a) the proteins of HDL, HDL(2), and HDL(3) are made of subunits, probably identical, of an average molecular weight of 21,000; (b) the difference in antigenic behavior between HDL(2) and HDL(3) is due to the presence in the latter of a lipid-poor protein; (c) antigenic polymorphism of alphaP is probably related to the presence in solution of monomeric and polymeric forms having different reactivity against anti-HDL and anti-alphaP sera.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL.(ABSTRACT TRUNCATED AT 250 WORDS)