Location and motion of free cholesterol molecules in high density lipoprotein

Human high density lipoprotein (HDL₃) was reconstituted with the free cholesterol molecules replaced with 4-[¹³C]-cholesterol. 90 MHz [¹³C]-NMR spectra were obtained and two cholesterol resonances at chemical shifts of 41.73 and 42.20 ppm could be resolved. The former signal arises from the C-4 atom of cholesterol molecules associated with phospholipids and located in the surface of the HDL₃ particle while the latter resonance is due to cholesterol molecules associated with cholesterol ester and triglyceride molecules in the core. HDL₃ reconstituted without any cholesterol ester or triglyceride gave a single resonance at 41.73 ppm indicating that all the free cholesterol molecules are in the surface. 60% of the free cholesterol molecules present in normal HDL₃ are in the phospholipid monolayer around the surface where they undergo relatively restricted motion compared to the remaining 40% situated in the liquid core. The free cholesterol molecules can equilibrate between the two pools in the timescale 10ms–700s.     

Location and interactions of phospholipid and cholesterol in human low density lipoprotein from 31P nuclear magnetic resonance.

The major phospholipids, phosphatidylcholine and spingomyelin, of low density lipoprotein (LDL) are accessible to small amounts of Pr3+, suggesting that the head groups of all mobile phospholipids are on the surface of the particle in contact with the aqueous medium. The major source of the nuclear Overhauser effect enhancement of 31P resonances is the N-methyl proton of the choline moiety, indicating close N-methyl phosphate group interactions, probably similar to those found previously in phospholipid vesicles. This behavior of the phospholipid head groups in LDL is similar to that in small vesicles without cholesterol, suggesting that in LDL most of the cholesterol is not associated with mobile, surface phospholipids. In contrast to LDL, where the presence of a large protein immobilizes some phospholipid head groups, immobilization does not occur in high density lipoprotein, consistent with occurrence of smaller peptides in the latter.     

Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Lateral diffusion of phospholipids in the lipid surface of human low-density lipoprotein measured with a pyrenyl phospholipid probe

Human low-density lipoprotein (LDL) was labelled with the excimeric fluorescent phospholipid analogue 1-palmitoyl-2-(1'-pyreneoctanoyl)-sn-glycero-3-phosphocholine by using phosphatidylcholine-specific transfer protein for the probe insertion. The lateral diffusivity of the probe in the phospholipid/cholesterol surface monolayer of LDL was determined from the measured dependence of the pyrene monomer fluorescence yield on probe concentration. The data were analyzed by the milling-crowd model (J. Eisinger et al. (1986) Biophys. J. 49, 987-1001] to obtain the short-range lateral diffusivity of the probe. The lateral mobility of the probe in LDL was compared to that in model lipid systems, i.e. in protein-free LDL-like lipid particles and in small unilamellar vesicles, with a phospholipid/cholesterol composition characteristic of LDL. This analysis with the probability PE = 1 for excimer production between nearest-neighbour probes gives the lower limits for f, the frequency of translational lipid--lipid exchanges of the probe of 0.62 x 10(8), 0.19 x 10(8) and 0.19 x 10(8)s-1 in LDL, LDL-like lipid particles, and small unilamellar vesicles, respectively. The lower limits for the corresponding lateral diffusion constants are 16, 5 and 5 microns 2 s-1. The results suggest that the translational mobility of phospholipid molecules in the lipid--protein surface of LDL is not constrained by the apolipoprotein B-100 moiety or the neutral lipid core of the lipoprotein. Instead, the protein moiety may perturb the lipid order with the lipid--associating peptide domains and thus fluidize the amphiphilic surface monolayer of LDL relative to the protein-free model systems. In general, lateral diffusivity of the pyrenyl phospholipid probe in LDL and the model lipid systems is comparable to the lateral mobility of lipid analogue probes in a variety of model and biological membranes.     

Fusion of low density lipoproteins with cholesterol ester-phospholipid microemulsions. Prevention of particle fusion by apolipoprotein A-I

Little is known about the mechanism and control of lipoprotein particle fusion, although apoproteins are presumed to be important in maintenance of particle structure. This study characterizes the interaction of apo-B-containing low density lipoproteins (LDL) with cholesterol ester microemulsions (CEME) in the presence and absence of apo-A-I to determine if a role for these apoproteins in particle integrity could be ascertained. CEME are an apoprotein-free analog of LDL formed by sonication of radiolabeled phospholipid (surface) and cholesterol ester (core). Incubation of CEME with LDL followed by precipitation of LDL with MnCl2 resulted in coprecipitation of CEME with LDL that was time-, temperature-, and concentration-, but not pH (pH 6-9)-, dependent and occurred over a wide range of CEME and LDL particle compositions. Particles from the incubation were larger than the unincubated particles and intermediate in density and electrophoretic mobility between the starting LDL and CEME. Differential scanning calorimetry experiments suggested that CEME surface and core lipids had mixed with those of LDL. When particles from incubations were exposed to an anti-apo-B column, radiolabeled surface and core molecules originating from the CEME particles bound to the column. Particles eluted at low pH from the anti-apo-B column were irregularly shaped and had excess surface material as judged by electron microscopy. Incubation of CEME with LDL in the presence of 3 M KBr or 4% bovine serum albumin did not alter the interaction of the particles. However, incubation of CEME with LDL in the presence of apo-A-I (2:1 CEME cholesterol-to-apo-A-I mass ratio) greatly reduced the interaction of the LDL and CEME particles. We conclude that the incubation of CEME with isolated LDL resulted in particle fusion that was prevented by apo-A-I.     

Enhancing the contrast of ApoB to locate the surface components in the 3D density map of human LDL

A 26 Å resolution map of the structure of human low-density lipoprotein (LDL) was obtained from electron cryomicroscopy and single-particle image reconstruction. The structure showed a discoidal-shaped LDL particle with high-density regions mainly distributed at the edge of the particle and low-density regions at the flat surface that covers the core region. To determine the chemical components that correspond to these density regions and to delineate the distribution of protein and phospholipid located at the particle surface at the resolution of the map, we used Mono-Sulfo-NHS-Undecagold labeling to increase preferentially the contrast of the apolipoprotein B component on the LDL particle. In the three-dimensional map from the image reconstruction of the undecagold-labeled LDL particles, the high-density region from the undecagold label was distributed mainly at the edge of the particle, and lower density regions were found at the flat surfaces that cover the neutral lipid core. This suggests that apolipoprotein B mainly encircles LDL at the edge of the particle and the phospholipid monolayers are located at the flat surfaces, which are parallel to the cholesterol ester layers in the core and may interact with the core lipid layers through the acyl chains.     

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.     

Molecular dynamics of the local anesthetic tetracaine in phospholipid vesicles

Upon introduction into phosphatidylcholine vesicles, the 13C magnetic resonance peaks of the aromatic resonances of tetracaine are broadened while the T1 relaxation times show little change. Addition of tetracaine to vesicles containing 30% cholesterol produces a similar broadening in the 13C NMR spectrum of tetracaine. Nuclear magnetic resonance parameters of phosphatidylcholine in vesicles which are unchanged by the addition of equimolar tetracaine include 13C T1 relaxation time and 31P linewidth, T1 relaxation time, and nuclear Overhauser effect enhancement. These results are interpreted as indicating a hydrophobic interaction between hydrocarbon portions of the anesthetic and phospholipid bilayer. The rotational correlation time of tetracaine about its long axis in the vesicles has been calculated from the 13C NMR spin lattice relaxation times to be about 10(-10.3) s and is unchanged by incorporation into the phospholipid bilayer. The positively charged ammonium group of tetracaine interacts with the negatively charged phosphate group of the vesicle lipids. Using shift reagents and 31P NMR, tetracaine has been shown to displace cations from the bilayer surface, and does not undergo fast flip-flop across the vesicle bilayer.     

Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.     

Association of apolipoprotein E with the low density lipoprotein receptor: demonstration of its co-operativity on lipid microemulsion particles

When human apolipoprotein E (apoE), which forms a self-associated tetramer in an aqueous solution, bound to the surface of triolein/phosphatidylcholine microemulsion with a particle diameter of 26 nm, it became monomeric on the lipid particle surface without strong evidence for its accumulation on a particular particle that might be expected from its tetramer formation in the aqueous phase. ApoE in the form of the self-associated tetramer did not inhibit binding of human low density lipoprotein (LDL) to its receptor on cultured human skin fibroblast. LDL binding was inhibited only when apoE was bound to the lipid particle surface. The affinity of the apoE-containing lipid particle to the LDL receptor was of the same order as that of LDL on the basis of particle molarity when the surface of the particle was covered with apoE up to 40 to 50% of the saturation level. When the particle was covered more with apoE, the affinity increased by some 20 times. Since the surface of the lipid particle was saturated with 7 apoE molecules, the particle seemed to require to have at least 4 apoE molecules on its surface in order to obtain high binding affinity to LDL receptor.     

Temperature-dependent 13C nuclear magnetic resonance studies of human serum low density lipoproteins.

The natural abundance 13C nuclear magnetic resonance (NMR) spectrum of human serum low density lipoproteins (LDL) shows significant temperature-dependent changes. These temperature-dependent spectra have been used to monitor changes in the organization of cholesterol esters within the LDL particle. Comparison with 13C NMR spectra of both cholesterol linoleate and an aqueous codispersion of cholesterol linoleate and egg phosphatidylcholine suggests that at low temperatures (10 degrees C), the cholesterol esters in LDL are organized in a smectic-like, liquid-crystalline arrangement. At temperatures above the order-disorder transition exhibited by the cholesterol esters of LDL, the cholesterol esters appear to be partially melted but still are motionally restricted compared with liquid cholesterol esters.     

Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins: role of lipoprotein and hepatic lipase.

To explore the interactions of triacylglycerol and phospholipid hydrolysis in lipoprotein conversions and remodeling, we compared the activities of lipoprotein and hepatic lipases on human VLDL, IDL, LDL, and HDL2. Triacylglycerol and phospholipid hydrolysis by each enzyme were measured concomitantly in each lipoprotein class by measuring hydrolysis of [14C]triolein and [3H]dipalmitoylphosphatidylcholine incorporated into each lipoprotein by lipid transfer processes. Hepatic lipase was 2-3 times more efficient than lipoprotein lipase at hydrolyzing phospholipid both in absolute terms and in relation to triacylglycerol hydrolysis in all lipoproteins. The relationship between phospholipid hydrolysis and triacylglycerol hydrolysis was generally linear until half of particle triacylglycerol was hydrolyzed. For either enzyme acting on a single lipoprotein fraction, the degree of phosphohydrolysis closely correlated with triacylglycerol hydrolysis and was largely independent of the kinetics of hydrolysis, suggesting that triacylglycerol removed from a lipoprotein core is an important determinant of phospholipid removal via hydrolysis by the lipase. Phospholipid hydrolysis relative to triacylglycerol hydrolysis was most efficient in VLDL followed in descending order by IDL, HDL, and LDL. Even with hepatic lipase, phospholipid hydrolysis could not deplete VLDL and IDL of sufficient phospholipid molecules to account for the loss of surface phospholipid that accompanies triacylglycerol hydrolysis and decreasing core volume as LDL is formed (or for conversion of HDL2 to HDL3). Thus, shedding of whole phospholipid molecules, presumably in liposomal-like particles, must be a major mechanism for losing excess surface lipid as large lipoprotein particles are converted to smaller particles. Also, this shedding phenomenon, like phospholipid hydrolysis, is closely related to the hydrolysis of lipoprotein triacylglycerol.     

Cholesterol rotation in phospholipid vesicles as observed by 13C-NMR

¹³C-NMR spectra of cholesterol 90% enriched at C-4 with ¹³C have been obtained in CHCl₃ and in sonicated egg phosphatidylcholine vesicles. ¹³C spin-lattice relaxation times, nuclear Overhauser effects and spin-spin relaxation times have been measured for the C-4 carbon of cholesterol in phosphatidylcholine bilayers as a function of cholesterol content and temperature. All the data are consistent with a correlation time for axial rotation of about 10^?10 s. This rotation is one or two orders of magnitude faster than axial rotation of the phospholipid molecule.     

Lipid transfer between human plasma low-density lipoprotein and a triolein/phospholipid microemulsion catalyzed by insect hemolymph lipid transfer particle

Lipid transfer between human plasma low-density lipoprotein (LDL) and an LDL-size microemulsion of triolein and phosphatidylcholine stabilized with human apolipoprotein A-I was catalyzed by the lipid transfer particle from hemolymph of the tobacco hornworm (Manduca sexta). Net transfer of phospholipid and triacylglycerol from the emulsion to LDL was observed and the apparent initial rates of transfer were dependent on the amount of catalyst. Net transfer of phospholipid mass was twice as much as that of triacylglycerol with respect to both the initial rate and the final equilibrium state. The final amount of net transfer of both lipids was dependent upon the initial ratio of LDL: microemulsion present in the incubation mixture up to 1:1 on the basis of phospholipid. The microemulsion lipid composition was maximally altered from an initial weight ratio of 1.09 +/- 0.08 (phospholipid/triolein) to 0.90 +/- 0.03 by this reaction. Further increase of LDL in the incubation caused neither further net transfer nor further change in the lipid composition of the microemulsion. The catalyst neither affected spontaneous transfer of free cholesterol between the emulsion and LDL nor enhanced cholesteryl ester transfer in this reaction system. As a result of the facilitated reaction, LDL gained a significant amount of phospholipid and triacylglycerol causing up to an 8% increase in core lipids and 14% in phospholipid. Some free cholesterol is recovered in the emulsions via spontaneous exchange. Transfer or exchange of apolipoproteins during the course of facilitated lipid transfer did not occur.     

Core modification of human low-density lipoprotein by artificial triacylglycerol emulsion

To determine whether an apolipoprotein-free artificial triacylglycerol emulsion can substitute for VLDL in studying cholesterol ester-triacylglycerol exchange processes between triacylglycerol-rich lipoproteins and cholesterol ester-rich lipoproteins, we used Intralipid to modify human plasma LDL. Intralipid was incubated with LDL in the presence of lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. Intralipid served as an acceptor for cholesterol ester and as a donor of triacylglycerol, modifying the low-density lipoproteins so that triacylglycerol became the major core lipid in the particle - the contribution of cholesterol ester to LDL mass decreased from 38% to 18%, while that of triacylglycerol increased from 4.9% to 26%. On lipolysis most added LDL triacylglycerol (59-72%) was hydrolyzed, resulting in a smaller particle than the "native' LDL particle with net loss of cholesterol ester. Incubation of LDL with the original Intralipid emulsion resulted in modified LDL with a high relative weight of phospholipid (27.7%). On removal of excess phospholipid from Intralipid and incubation of the resultant "washed' Intralipid with LDL, the relative weight of phospholipid in modified LDL decreased to 20%, which was similar to that observed after incubation of LDL with VLDL. We demonstrate that artificial triacylglycerol emulsion can indeed substitute for VLDL in neutral lipid exchange processes, and further confirm that transfer of core cholesterol ester and triacylglycerol occurs independently of the apolipoproteins present in triacylglycerol-rich lipoproteins and LDL.     

Reconstitution of lipoproteins. II. Lipid-protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5'-, 12'-, and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid-protein interactions. The temperature-induced phospholipid phase transition, as measured by 5'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-iii2, is very broad and has a small cooperative unit indicative of extensive lipid-protein interactions occurring at the head group region of the phospholipid bilayer. When 12"- and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.     

Mechanisms and consequences of cellular cholesterol exchange and transfer

It is apparent from consideration of the reactions involved in cellular cholesterol homeostasis that passive transfer of unesterified cholesterol molecules plays a role in cholesterol transport in vivo. Studies in model systems have established that free cholesterol molecules can transfer between membranes by diffusion through the intervening aqueous layer. Desorption of free cholesterol molecules from the donor lipid-water interface is rate-limiting for the overall transfer process and the rate of this step is influenced by interactions of free cholesterol molecules with neighboring phospholipid molecules. The influence of phospholipid unsaturation and sphingomyelin content on the rate of free cholesterol exchange are known in pure phospholipid bilayers and similar effects probably occur in cell membranes. The rate of free cholesterol clearance from cells is determined by the structure of the plasma membrane. It follows that the physical state of free cholesterol in the plasma membrane is important for the kinetics of cholesterol clearance and cell cholesterol homeostasis, as well as the structure of the plasma membrane. Bidirectional flux of free cholesterol between cells and lipoproteins occurs and rate constants characteristic of influx and efflux can be measured. The direction of any net transfer of free cholesterol is determined by the relative free cholesterol/phospholipid molar ratios of the donor and acceptor particles. Cholesterol diffuses down its gradient of chemical potential generally partitioning to the phospholipid-rich particle. Such a surface transfer process can lead to delivery of cholesterol to cells. This mechanism operates independently of any lipoprotein internalization by receptor-mediated endocytosis. The influence of enzymes such as lecithin-cholesterol acyltransferase and hepatic lipase on the direction of net transfer of free cholesterol between lipoproteins and cells can be understood in terms of their effects on the pool sizes and the rate constants for influx and efflux. Excess accumulation of free cholesterol in cells stimulates the rate of cholesteryl ester formation and induces deposition of cholesteryl ester inclusions in the cytoplasm similar to the situation in the 'foam' cells of atherosclerotic plaque. Clearance of cellular cholesteryl ester requires initial hydrolysis to free cholesterol followed by efflux of this free cholesterol. The rate of clearance of cholesteryl ester from cytoplasmic droplets is influenced by the physical state of the cholesteryl ester; liquid-crystalline cholesteryl ester is removed more slowly than cholesteryl ester in a liquid state.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

Lipoprotein nanoplatform for targeted delivery of diagnostic and therapeutic agents

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI) contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs). Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL]) to over a micron (chylomicrons) is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains), surface loading (intercalation into the phospholipid monolayer), and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core). Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (相似文献