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     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and beta-casein with phospholipids.

The conformations adopted by beta-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being alpha-helical and the beta-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the beta-casein is in a compressed state whereas the apoprotein is extended as alpha-helices in the plane of the interface. The chain-length depedences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.     

Lipid transfers between reconstituted high density lipoprotein complexes and low density lipoproteins: effects of plasma protein factors

In this study we examined the transfer of lipids between reconstituted high density lipoprotein discs (r-HDL) and human low density lipoproteins (LDL) in the presence and absence of lecithin:cholesterol acyltransferase (LCAT) or of plasma phospholipid transfer protein (PLTP). We found that spontaneous transfer of phospholipids from r-HDL to LDL occurred by an apparent first order reaction with a half-time of 5.8 to 6.9 hr depending on the phospholipid. During the time of incubation of r-HDL with LDL (from 0 to 25 hr), the phospholipid content of r-HDL decreased more than 30%, the free cholesterol content increased 2.5-fold, and low levels of cholesteryl esters appeared in r-HDL. These compositional changes gave rise to small discoidal particles with a limiting diameter of 77 A and two molecules of apoA-I per particle. When LCAT was included in the reaction mixture, the r-HDL lost even more phospholipid, lost some free cholesterol, and gained cholesteryl esters relative to the apolipoprotein content, due to the enzymatic reaction. The products of the LCAT reaction had a diameter of 93 A and three, rather than two, apoA-I molecules per particle. Inclusion of PLTP into the reaction mixture accelerated the transfer of phospholipids (half-time of 1.7 hr) and the formation of the 77 A product. In addition to these compositional and morphological changes, which may be important in the interconversions of native HDL subspecies, the prolonged incubations revealed some slow reactions, such as the esterification of LDL cholesterol by LCAT, a background formation of cholesteryl esters in r-HDL, and an apparent hydrolysis of cholesteryl esters in LDL in the presence of r-HDL.     

Recycling of apoprotein E is associated with cholesterol efflux and high density lipoprotein internalization

After receptor-mediated endocytosis of triglyceride-rich lipoproteins (TRL) into the liver, TRL particles are immediately disintegrated in peripheral endosomal compartments. Whereas core lipids and apoprotein B are delivered for degradation into lysosomes, TRL-derived apoE is efficiently recycled back to the plasma membrane. This is followed by apoE re-secretion and association of apoE with high density lipoproteins (HDL). Because HDL and apoE can independently promote cholesterol efflux, we investigated whether recycling of TRL-derived apoE in human hepatoma cells and fibroblasts could be linked to intracellular cholesterol transport. In this study we demonstrate that HDL(3) does not only act as an extracellular acceptor for recycled apoE but also stimulates the recycling of internalized TRL-derived apoE. Furthermore, radioactive pulse-chase experiments indicate that apoE recycling is accompanied by cholesterol efflux. Confocal imaging reveals co-localization of apoE and cholesterol in early endosome antigen 1-positive endosomes. During apoE re-secretion, HDL(3)-derived apoA-I is found in these early endosome antigen 1, cholesterol-containing endosomes. As shown by time-lapse fluorescence microscopy, apoE recycling involves the intracellular trafficking of apoA-I to pre-existing and TRL-derived apoE/cholesterol-containing endosomes in the periphery. Thus, these studies provide evidence for a new intracellular link between TRL-derived apoE, cellular cholesterol transport, and HDL metabolism.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1

To study the assembly of newly synthesized lipids with apoprotein A1, we administered [2-3H]glycerol to young chickens and determined the hepatic intracellular sites of lipid synthesis and association of nascent lipids with apoprotein A1. [2-3H]glycerol was rapidly incorporated into hepatic lipids, reaching maximal levels at 5 min, and this preceded the appearance of lipid radioactivity in the plasma. The liver was fractionated into rough and smooth endoplasmic reticulum and Golgi cell fractions. The isolated cell fractions were further subfractionated into membrane and soluble (content) fractions by treatment with 0.1 M Na2CO3, pH 11.3. At various times, the lipid radioactivity was measured in each of the intracellular organelles, in immunoprecipitable apoprotein A1, and in materials that floated at buoyant densities similar to those of plasma lipoproteins. Maximal incorporation occurred at 1 min in the rough endoplasmic reticulum, at 3-5 min in the smooth endoplasmic reticulum, and at 5 min in the Golgi cell fractions. The majority (66-93%) of radioactive glycerol was incorporated into triglycerides with smaller (4-27%) amounts into phospholipids. About 80% of the lipid radioactivity in the endoplasmic reticulum and 70% of that in the Golgi cell fractions was in the membranes. The radioactive lipids in the content subfraction were distributed in various density classes with most nascent lipids floating at a density less than or equal to 1.063 g/ml. Apoprotein A1 from the Golgi apparatus, obtained by immunoprecipitation, contained sixfold more nascent lipids than did that from the endoplasmic reticulum. These data indicate that [2-3H]glycerol is quickly incorporated into lipids of the endoplasmic reticulum and the Golgi cell fractions, that most of the nascent lipids are conjugated with apoproteins A1 in the Golgi apparatus, and that very little association of nascent lipid to apoprotein A1 occurs in the endoplasmic reticulum.     

Interchange of apoprotein components between the human plasma high density lipoprotein subclasses HDL2 and HDL3 in vitro.

The major apoproteins of human high density lipoproteins (HDL) labeled with 125I have been shown to exchange between the two major HDL subclasses HDL2 and HDL3 in vitro. This bidirectional exchange process is inhibited by cross-linking with bifunctional reagents and is apparently dependent upon the formation of collision complexes. This exchange has been demonstrated both when the subclasses of HDL are free in solution and also when one of them is covalently bound to Sepharose. Using system involving Sepharose-bound HDL, it could be shown that not only free apoprotein molecules but subunits consisting of lipid-apoprotein combinations were exchanged between HDL2 and HDL3. The rate of exchange in these processes is significant in the lifetime of the protein particles in vivo equalling approximately 2.5% per h for apoprotein exchange. These experiments suggest that there is a dynamic relationship between HDL2 and HDL3 even though each of them exists alone in vitro as stable separate entities; when they are placed together in solution significant interaction occurs between the particles. Apoprotein exchange occurs between HDL2:HDL2 and HDL3:HDL3 as well as between HDL2 and HDL3 molecules. These data also suggest that the interconversion of HDL2 and HDL3 may be affected by the availability of lipids.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

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.     

Apoproteins of avian very low density lipoprotein: demonstration of a single high molecular weight apoprotein.

The high molecular weight apoproteins of very low density lipoprotein (VLDL) were compared after preparation of VLDL from plasma and sera of diethylstilbestrol-treated roosters. When prepared from plasma with adequate control of endogenous proteolytic activity, VLDL contained a single high molecular weight apoprotein (apo-VLDL-B) as judged by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Serum VLDL contained multiple apoprotein species, the largest of which corresponded to apo-VLDL-B. Immunological analyses showed that the multiple apoproteins of serum VLDL were quantitatively and qualitatively indistinguishable from plasma apo-VLDL-B. These data indicate that apo-VLDL-B can be cleaved during VLDL isolation to produce an apparent heterogeneity of high molecular weight apoproteins. The molecular weight of plasma apo-VLDL-B was estimated to be 350 000. This protein was stable to reduction and S-carboxymethylation and showed no association with apo-VLDL-II [Chan, L., Jackson, R.L., O'Malley, B. W., & Means, A.R. (1976) J. Clin. Invest. 58, 368] through disulfide linkage. Apo-VLDL-B and apo-VLDL-II represented 54% and 46%, respectively, of the total VLDL protein recovered following gel filtration chromatography in sodium dodecyl sulfate. Protein recovery in the chromatographic analyses (92%) was sufficient to conclude that apo-VLDL-B and apo-VLDL-II are the major and possibly the only apoproteins of chicken VLDL. The molar ratio of the apo-VLDL-II monomer to apo-VLDL-B was estimated to be 32.     

Glycosaminoglycan—lipoprotein interactions: 2. Structure of heparin-releted variants with high affinity for low density lipoprotein

A particular heparan sulphate fraction which possessed the largest proportion of high affinity variants for human low density lipoprotein contained almost equal proportions of the repeating units l-iduronosyl(O-sulphate)N-sulphamidoglucosamine and d-glucoronosyl-N-acetylglucosamine. The heparan sulphate was fractionated on lipoprotein-agarose into three populations. Results of periodate oxidation—alkaline elimination indicated that the size of the completely N-sulphated block regions increased with increasing affinity. In contrast, the number of consecutive l-iduronosyl(O-sulphate)-containing repeats decreased with increasing affinity towards lipoprotein. After selective periodate oxidation—alkaline scission of d-glucoronic acid residues only a portion of the heparan sulphate fragments retained high affinity for lipoprotein. This portion consisted of fragments larger than dodecasaccharide which contained both l-iduronic acid-O-sulphate and non-sulphated uronic acid residues (−) 2:1). No affinity or little affinity was displayed by fragments (of comparable size) that contained only sulphated l-iduronic acid residues.     

Hepatic CTP:phosphocholine cytidylyltransferase-alpha is a critical predictor of plasma high density lipoprotein and very low density lipoprotein

CTP:phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC). We previously generated a mouse in which the hepatic CTalpha gene was specifically inactivated by the cre/loxP procedure. In CTalpha knock-out mice, plasma high density lipoprotein (HDL) and very low density lipoprotein (VLDL) levels were markedly lower than in wild type mice (Jacobs, R. L., Devlin, C., Tabas, I., and Vance, D. E. (2004) J. Biol. Chem. 279, 47402-47410.) To investigate the mechanism(s) responsible for the decrease in plasma lipoprotein levels, we isolated primary hepatocytes from knock-out and wild type mice. ABCA1 expression was reduced in knock-out hepatocytes and apoAI-dependent cholesterol, and PC efflux was impaired. When knock-out hepatocytes were infected with an adenovirus expressing CTalpha, apoAI-dependent PC efflux returned partially, whereas cholesterol efflux and ABCA1 levels were not restored to normal levels. Adenoviral expression of CTalpha did not increase VLDL secretion in knock-out hepatocytes, even though cellular PC levels returned to normal. However, in vivo adenoviral delivery of CTalpha normalized plasma HDL and VLDL levels in knock-out mice. The observations demonstrate that hepatic PC biosynthesis is a key player in maintaining plasma VLDL and HDL, and further underscores the importance of the liver in HDL formation.     

Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer

The interactions of high density lipoprotein (HDL) and acetylated high density lipoprotein (acetyl-HDL) with isolated rat sinusoidal liver cells have been investigated. Cellular binding of 125I-acetyl-HDL at 0 degrees C demonstrated the presence of a specific, saturable membrane-associated receptor. This receptor was affected neither by formaldehyde-treated albumin nor by low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by the cells, indicating that the receptor for acetyl-HDL constitutes a distinct class among the scavenger receptors for chemically modified proteins. Parallel binding experiments using 125I-HDL also revealed the presence on these cells of a receptor for unmodified HDL. The ligand specificities of these two receptors were similar to each other except that the acetyl-HDL receptor was sensitive to polyanions such as dextran sulfate and fucoidin. Interaction of HDL with the cells at 37 degrees C was totally different from that of acetyl-HDL. Cellular binding of HDL was not accompanied by subsequent intracellular degradation of its apoprotein moiety, whereas its cholesterol moiety was significantly transferred to the cells. In contrast, acetyl-HDL was endocytosed and underwent lysosomal degradation as a holoparticle. This shift in receptor-recognition from the HDL receptor to the acetyl-HDL receptor was accomplished by acetylation of approximately 8% of the total lysine residues of HDL apoprotein. This unique difference in endocytic behavior between HDL and acetyl-HDL suggests a potential link of the HDL receptor to HDL-mediated cholesterol transfer in sinusoidal liver cells.     

Lipid transfer particle-induced transformation of human high density lipoprotein into apolipoprotein A-I-deficient low density particles

Incubation of human high density lipoprotein (HDL) particles (density = 1.063-1.21 g/ml) with catalytic amounts of Manduca sexta lipid transfer particle (LTP) resulted in alteration of the density distribution of HDL protein such that the original HDL particles were transformed into new particles with an equilibrium density = 1.05 g/ml. Concomitantly, substantial amounts of protein were recovered in the bottom fraction of the density gradient. The LTP-induced alteration in HDL protein density distribution was dependent on the LTP concentration and incubation time. Electrophoretic analysis revealed that the lower density fraction contained apolipoprotein A-II (apoA-II) as the major apoprotein component while nearly all of the apoA-I was recovered in the bottom fraction. Lipid analysis of the HDL substrate and product fractions revealed that the apoA-I-rich fraction was nearly devoid of lipid (less than 1%, w/w). The lipid originally associated with HDL was recovered in the low density, apoA-II-rich, lipoprotein fraction, and the ratios of individual lipid classes were the same as in control HDL. Electron microscopy and gel permeation chromatography experiments revealed that the LTP-induced product lipoprotein population comprised particles of larger size (19.7 +/- 1.4-nm diameter) than control HDL (10.6 +/- 1.4-nm diameter). The results suggest that facilitated net lipid transfer between HDL particles altered the distribution of lipid such that apoprotein migration occurred and donor particles disintegrated. Similar results were obtained when human HDL3 or HDL2 density subclasses were employed as substrates for LTP. The lower surface area to core volume ratio of the larger, product lipoprotein particles compared with the substrate HDL requires that there be a decrease in the total exposed lipid/water interface which requires stabilization by apolipoprotein. Selective displacement of apoA-I by apoA-II or apoC, due to their greater surface binding affinity, dictates that apoA-I is preferentially lost from the lipoprotein surface and is therefore recovered as lipid-free apoprotein. Thus, it is conceivable that the structural arrangement of HDL particle lipid and apoprotein components isolated from human plasma may not represent the most thermodynamically stable arrangement of lipid and protein.     

Binding of high density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues

Cultured extrahepatic cells possess a specific high affinity binding site (receptor) for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. To characterize the binding recognition site(s) on HDL, the ability of HDL to interact with cultured human fibroblasts was assayed after chemical alteration of specific apoprotein amino acid residues. Reduction and alkylation, acetylation, and cyclohexanedione treatment of HDL3 had little or no effect on its cellular binding. Treatment of HDL3 with tetranitromethane (TNM), however, caused a large dose-dependent decrease in binding, with maximum inhibition at 3 mM. Amino acid analysis of the TNM-treated particles showed specific alteration of tyrosine residues, but sodium dodecyl sulfate-gel electrophoresis demonstrated apoprotein cross-linking coincident with decreased binding. These results suggest that modification of HDL tyrosine residues and/or cross-linking of HDL apoproteins alters the ligand site recognized by the HDL receptor. Gradient gel electrophoresis, molecular sieve chromatography, and electron microscopy showed only minor changes in size distribution and shape of HDL3 particles after treatment with 3 mM TNM, but at higher TNM concentrations, coalescence and aggregation of particles was evident. Treatment of HDL3 with 3 mM TNM affected neither its promotion of the low affinity (receptor-independent) cholesterol efflux from cells nor its ability to accept cholesterol from an albumin suspension, yet promotion of high affinity (receptor-dependent) cholesterol efflux from cells was abolished. The finding that TNM treatment of HDL3 decreases both its receptor binding and its promotion of cholesterol efflux from cells without substantial alteration of its physical properties supports the hypothesis that the HDL receptor functions to facilitate cholesterol transport from cells.     

Lipid and apolipoprotein distribution as a function of density in equine plasma lipoprotein

1. Equine lipoproteins were isolated from plasma by density gradient ultracentrifugation and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. VLDL and IDL were present at low concentration (0.2 mg/ml). Two apoB components of Mr corresponding to human apoB-100 and one apoB-48-like component were represented in VLDL fraction. 3. LDL-1 and LDL-2 subfractions have displayed an almost equal concentration (0.4 mg/ml). Two apoB-100-like components were the major apolipoproteins in each fraction. Small amounts of apoB-48-like component were detectable in LDL-1 and LDL-2. 4. HDL-2 represented a major class of equine lipoproteins (1.8 mg/ml). ApoA-1-like component was the dominant protein in HDL-1, HDL-2 and HDL-3. Dimeric apoA-II-like components were slightly represented in HDL subfractions. 5. HDL-3 displayed the same apolipoprotein pattern as HDL-1 and HDL-2, but two further minor proteins of Mr 20,000 and 14,000 were detected. 6. VHDL represented a minor class of lipoprotein (0.2 mg/ml). ApoA-I-like component was the major apolipoprotein of VHDL. Small amounts of apoA-IV-like, apoE-like, and Mr 55,000 protein were detectable. 7. ApoC-like of Mr lower than 10,000 was represented in all equine lipoprotein classes.