Apolipoprotein E genotype as a most significant predictor of lipid response at lipid-lowering therapy: mechanistic and clinical studies.

APOE alleles and apolipoprotein E isoforms control plasma cholesterol level on population level. Among three ɛ2, ɛ3, ɛ4 alleles, ɛ4 allele is associated with the increase in cholesterol level, risk of atherosclerosis and Alzheimer disease, while ɛ2 allele is associated with the decrease in cholesterol level and risk of atherosclerosis. The increase in plasma triglyceride is an independent risk factor of atherosclerosis and triglyceride-high density lipoprotein coupling determines the efficiency of reverse cholesterol transport. The impairment of this coupling specifically at hypertriglyceridemia may be followed by specific lipoprotein markers. The influence of major lipid-lowering drugs on lipoprotein metabolism and association of apoE isoforms with the efficiency of therapy by statins and fibrates are summarized both at isolated and combined increase in plasma triglyceride and cholesterol. APOE polymorphism seems to be a single genetic variant with a confirmed stratification both at candidate gene and at wide genome analyses.

Role of ApoE in conformation-prone diseases and atherosclerosis.

Three isoforms of human plasma apolipoprotein E (apoE) are ligands to lipoprotein receptors and influence in different manner the synthesis and catabolism of pro-atherogenic triglyceride-rich lipoproteins. Among three isoforms, the apoE4 isoform is associated with increased frequency of atherosclerosis and Alzheimer's disease (AD). The conformational transitions of beta-amyloid (Abeta) influenced by apoE and serum amyloid P (SAP) component are key events in AD development, the accumulation of intermediate diffusible and soluble oligomers of Abeta being of particular significance. SAP and apoE, in a different manner for the three isoforms, serve as "pathological" chaperones during the aggregation of Abeta considered as a conformation-prone process. In turn, apoE consisting of two domains self-associates in solution and intermediate structures differently populated for the three isoforms exist. The different structures of the three isoforms determine their different distribution among various plasma lipoproteins. The structural and metabolic consideration of the common apoE pathway(s) in two pathologies assumes four molecular targets for AD correction: (i) inhibition of the accumulation of diffusible soluble Abeta oligomers; (ii) inhibition of apoE synthesis and secretion by astrocytes, in particular, under lipid-lowering therapy; (iii) inhibition of the binding of apoE and/or SAP to Abeta; (iv) stimulation of the expression of cholesterol transporter ABCA1.

Apolipoprotein E structure and substrate and receptor-binding activities of triglyceride-rich human plasma lipoproteins in normo- and hypertriglyceridemia.

Cysteine-arginine interchanges along the primary sequence of human plasma apolipoprotein E (apoE) play an important role in determining its biological functions due to a high mutation frequency of cytosine in CGX triplet that codes 33 of 34 apolipoprotein arginine residues. The contribution of apoE secondary structure to apolipoprotein-lipid interaction is described. The significance of apolipoprotein in triglyceride synthesis, lipoprotein lipolysis, and receptor-mediated clearance of lipolytic remnants of triglyceride-rich lipoproteins is discussed as well. The metabolic flow of lipoproteins in normo- and hypertriglyceridemia can be described by separate compartments that contribute to lipoprotein interaction with at least six different receptors: 1) low density lipoprotein (LDL) receptor; 2) LDL receptor-related protein (LRP); 3) apoB(48) macrophage receptor for hypertriglyceridemic very low density lipoproteins (VLDL); 4) scavenger receptors; 5) VLDL receptor; 6) lipolysis-stimulated receptor. The contribution of the exposure of apoE molecules on the surface of triglyceride-rich particles sensitive both to lipolysis and plasma triglyceride content to the interaction with LDL receptor and LRP is emphasized.

Protein-lipid interactions in reconstituted high density lipoproteins: apolipoprotein and cholesterol influence.

Two fluorescent probes-cis- and trans-parinaric acids were used to study the dimensions, lipid dynamics and apolipoprotein location in the reconstituted discoidal high density lipoproteins (rHDL). The rHDL particles made from apolipoprotein A-I (apoA-I), dipalmitoylphosphatidylcholine (DPPC), with or without cholesterol (Chol) were compared with the analogous particles with two other apolipoproteins-apoE and apoA-II. The data obtained for apoA-I-containing rHDL were as follows: (1) the inclusion of 8 mol.% of cholesterol did not significantly change the particle dimensions (13+/-1 nm) or the mean distance between apoA-I and the disc axis; (2) the phospholipid domains-boundary lipid region in the close vicinity to apoA-I molecule and the remaining part of the bilayer-existed at temperatures both lower and above DPPC transition temperature T(t); (3) at T

Apolipoprotein A-I localization and dipalmitoylphosphatidylcholine dynamics in reconstituted high density lipoproteins.

The structure and molecular dynamics of recombinant high density lipoproteins (rHDL) were studied by non-radiative energy transfer (NRET), fluorescence anisotropy and intensity measurements. The rHDL particles contained human plasma apolipoprotein (apo) A-I and dipalmitoylphosphatidylcholine (DPPC). Fluorescent cis- and trans-parinaric acids were used both as probes of molecular motion in the particle lipid phase and as acceptors in the Forster's energy transfer from apo A-I tryptophan residues to determine particle dimensions, apolipoprotein localization and lipid dynamics. The probes are sensitive to thermal wobbling (macromobility) and conformational deformations (micromobility) of phospholipid acyl chains. The experimental data fitted to various models of the particle structure are compatible with the following: (a) at T < Tt the particles appeared as lens-like discs with a radius of the lipid phase of 5 nm and a mean thickness of 4 nm, the value being more by 20% in the particle centre, the alpha-helices of about 1 nm thickness were located around the edge of the lipid core. Compared to liposomes, both macro- and micromobility of DPPC molecules in rHDL were more rapid due to a significant disorder of the boundary lipid molecules close to the apo A-I molecule. This disorder led to the increase of the specific surface area per one lipid molecule, S(o). The lipid phase can be divided into three regions: (i) zone I of the most tightly packed lipid (0-1.7 nm from the disc axis) with a S(o) value small as 0.5 nm2; (ii) intermediate zone II (from 1.7 to 4.0 nm); and (iii) boundary lipid zone III (4-5 nm) of significantly disordered lipid with a S(o) value large as 0.65 nm2. (b) at T> Tt the S(o) heterogeneity disappeared, the radius of the lipid phase did not increase significantly, not exceeding 5.2-5.4 nm, but protein-induced immobilization of lipid molecules which affected about half or more of the total lipid, became remarkable. The overall effect was the suppression of the transition amplitude in rHDL compared to liposomes. The structural inhomogeneity might underlie the function of the native plasma HDL as the key component of the transport and metabolism of plasma lipids.

Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study.

Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followed by dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hours at 24 degrees C) interconversion of a self-associated 'open' conformer into a more dense 'closed' conformer. The hydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of 'active' lipid-bound apoE molecules in the reaction of binding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant K(a) averaged for both PCs and plotted against this ratio abruptly increased from 3.8 x 10(7) to 3.8 x 10(8) M(-1) with a transition midpoint of 150-180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density lipoproteins (VLDL) or low density lipoproteins (LDL) isolated from patients with E3/3 phenotype, for binding to the LDLr. Again, the competition efficiency abruptly increased at the increase in PC content with a transition midpoint of 130 PC/apoE, mole ratio. The transitions observed both in direct and competitive binding assay probably correspond to the abrupt increase in the number of 'active' apoE molecules on the complex surface accompanying the change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDL bind to LDLr following a simple encounter complex model, while LDL binding was characterized by a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of the continuum from several (2-3) apoE molecules on the surface of TG-rich particles that resulted in the increased binding affinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous and different lipid-bound forms of apoE is proposed, in particular, the formation of a transient disc-lipoprotein particle structure during the interaction with LDLr in vivo as well as in LPL-stimulated lipolysis of the lipid phase of the particle.

Structural peculiarities of the binding of very low density lipoproteins and low density lipoproteins to the LDL receptor in hypertriglyceridemia: role of apolipoprotein E.

Very low (VLDL) and low density lipoproteins (LDL) were isolated from plasma of patients with the E3/3 phenotype which were divided into three groups based on their plasma triglyceride content: low (TG<200 mg/dl, TG(l)), intermediate (200<300 mg/dl, TG(i)300 mg/dl, TG(h)). The protein density (PD) on the VLDL and LDL surface was calculated from lipoprotein composition and protein location was studied by tryptophan fluorescence quenching by I(-) anions at 25 degrees C and 40 degrees C. A comparison of the TG(h) with the TG(l) group revealed a significant (<0.05) increase of the PD parameter as much as 21% for VLDL, but not for LDL where this parameter did not change for any group; generally, PD(LDL) values were 3.2-3.8-fold lower than PD(VLDL). In accordance with this difference, the tryptophan accessibility f in VLDL vs. LDL was lower at both temperatures. There were temperature-induced changes of the f parameter in opposite directions for these lipoproteins. The difference in f value gradually decreased for VLDL in the direction TG(l)TG(i)TG(h) while for LDL there was a U-shaped dependence for these groups. The Stern-Volmer quenching constant K(S-V) which is sensitive to both temperature and viscosity, did not change for VLDL, but K(S-V)(LDL) was 2-3-fold higher for the TG(i) group compared to the other two. The efficiencies of VLDL and LDL binding to the LDL receptor (LDLr) in vitro were compared by solid-phase assay free of steric hindrance observed in cell binding. The maximal number of binding sites did not change for either type of particles and between groups. The association constant K(a) and apolipoprotein (apo) E/apoB mole ratio values all increased significantly for VLDL, but not for LDL, in comparison of the TG(i+h) with the TG(l) group. Based on VLDL and LDL concentrations in serum and on the affinity constant values obtained in an in vitro assay, VLDL concentrations corresponding to 50% inhibition of LDL binding (IC(50)) were calculated in an assumption of the competition of both ligands for LDLr in vivo; the mean values of IC(50) decreased 2-fold when plasma TG exceeded 200 mg/dl. The functional dependences of K(a)(VLDL), IC(50) and apoE content in VLDL (both fractional and absolute) and in serum on TG content in the whole concentration range studied were fitted to a saturation model. For all five parameters, the mean half-maximum values TG(1/2) were in the range 52-103 mg/dl. The efficiency of protein-protein interactions is suggested to differ in normolipidemic vs. HTG-VLDL and apoE content and/or protein density on VLDL surface may be the primary determinant(s) of the increased binding of HTG-VLDL to the LDL receptor. ApoCs may compete with apoE for the binding to the VLDL lipid surface as plasma triglyceride content increases. The possible competition of VLDL with LDL for the catabolism site(s) in vivo, when plasma TG increases, could explain the atherogenic action of TG-rich lipoproteins. Moreover, the 'dual action' hypothesis on anti-atherogenic action of apoE-containing high density lipoproteins (HDL) in vivo is suggested: besides the well-known effect of HDL as cholesteryl ester catabolic outway, the formation of a transient complex of apoE-containing discs appearing at the site of VLDL TG hydrolysis by lipoprotein lipase with VLDL particles proposed in our preceding paper promotes the efficient uptake of TG-rich particles; in hypertriglyceridemia due to the diminished HDL content this uptake seems to be impaired which results in the increased accumulation of the remnants of TG-rich particles. This explains the observed increase in cholesterol and triglyceride content in VLDL and LDL, respectively, due to the CETP-mediated exchange of cholesteryl ester and triglyceride molecules between these particles.

Structural organization of lipid phase and protein-lipid interface in apolipoprotein-phospholipid recombinants: influence of cholesterol.

The complexes of individual human plasma apolipoproteins (apo) A-I, E and A-II with dipalmitoylphosphatidylcholine (DPPC) in the absence or in the presence of cholesterol (Chol) were prepared with initial DPPC/Chol/protein weight ratio as 3:0.15:1. ApoA-I/DPPC/Chol complexes with different protein content (initial DPPC/apoA-I weight ratios were changed from 10.5:1 to 2.6:1) but with a fixed initial DPPC/Chol weight ratio of 20:1 were also prepared. The complexes were isolated by gel-filtration and characterized by size and composition. ApoA-I- and apoA-II-complexes had the same size (80-84 A) and the complexes became more heterogeneous upon Chol inclusion; apoE-complexes were larger (97-100 A) and more homogeneous and Chol addition had no effect on their hydrodynamic properties. Chol seems to be excluded partially in the following manner for isolated complexes with different apo's: A-II > E > A-I. The possible existence of two lipid regions in the complexes differing in lipid dynamics - the lipid shell in the vicinity of apolipoprotein (boundary lipid) opposite to the remaining part of the lipid bilayer - has been studied by absorbance and fluorescence spectroscopy with cis-parinaric acid (cis-PA) and trans-parinaric acid (trans-PA) embedded into the complexes. Their application is based on a strong preference of trans-PA for solid lipid while cis-PA distributes more equally between co-existing fluid and solid lipid regions (Sklar et al. (1979) Biochemistry 18, 1707-1716). (1) For apoA-I-complexes, the partition of cis-PA between water and lipid phase at temperatures below and above the transition temperature of DPPC (T(t)) was insensitive to Chol and temperature, while partition of trans-PA into the lipid phase of Chol-containing complex was increased at high temperature and decreased at low temperature. These results seem to be related to trans-PA redistribution between Chol-rich and protein-rich lipid domains, the latter being more disordered at T < T(t) and more immobilized at T > T(t) compared to the bulk bilayer; cis-PA localizes preferentially in boundary lipid. This hypothesis was directly confirmed by measurements of energy transfer between apoA-I tryptophanyls and probe molecules. (2) The relative response of trans-PA fluorescence intensity to temperature-induced phase transition of DPPC in apoA-I/DPPC/Chol complexes was decreased as a function of apolipoprotein content in a non-monotonic fashion with a transition midpoint at a mol ratio DPPC/A-I of 250:1, probably indicating two different modes of apolipoprotein/DPPC interaction in different sized complexes. (3) The comparative study of lipid dynamics in apoA-I-, apoE- and apoA-II-containing complexes with temperature response to phospholipid phase transition with fluorescence parameters such as intensity and anisotropy of cis-PA and trans-PA revealed the presence of boundary lipid in all three complexes without Chol. In contrast to apoA-I-containing complexes, in apoA-II/DPPC/Chol complexes, trans-PA seems to move preferentially into boundary lipid and cis-PA to distribute between two different regions probably as a result of more ordering action induced by apoA-II compared to apoA-I on the nearest phospholipid molecules in Chol-containing complexes; the apoE action on trans-PA and cis-PA distribution could be intermediate. Based on these results, the degree of Chol exclusion from the boundary lipid region for complexes with different apo's increasing in the order A-II > E > A-I can be suggested. Different Chol distributions between two lipid regions in the complexes seems not to be a function of complex size, but rather is an inherent property of the particular apolipoprotein molecule.

Composition and structural and functional properties of discoidal and spherical phospholipid-apoE3 complexes.

To model the common structural unit in the system of reverse cholesterol transport, we studied the composition, structure, and physicochemical properties of complexes generated between dipalmitoylphosphatidylcholine (DPPC) or palmitoyllinoleoylphosphatidylcholine (PLPC) and apoE3 in the absence and in the presence of cholesterol (Chol); the data were compared with similar experiments using apoA-I, the major proteins of high-density lipoproteins. The conformation and organization of lipid-binding domains of apoE3 within the complexes were calculated by computer modeling. The transition temperatures of DPPC within discoidal complexes with mean diameters of 116 A (GGE) or 148 A (EM) were higher for complexes versus liposomes both in the absence and in the presence of Chol. Association of apoE3 with DPPC resulted in a more structured state of the apolipoprotein molecule versus the soluble apolipoprotein; this state was characterized by parallel orientation of alpha-helixes of apoE3 and DPPC acyl chains. Substrate efficiency of the apoE3-PLPC-Chol complexes in the lecithin-cholesterol acyltransferase (LCAT) reaction expressed as Vmax/Km was 0.5 mole cholesteryl esters/h per 1 microM. The transformation of discoidal apoE3-DPPC-Chol complexes into spherical particles was induced by LCAT and accumulation of cholesteryl esters was approximately 62% of the total cholesterol. Parallel orientation of phospholipid acyl chains with helical segments disappeared in these particles. Discoidal apoE3-DPPC complexes incorporated unesterified cholesterol released from Chol-loaded J774 macrophages. The data support the concept that association of apoE3 and apoA-I with phospholipids is qualitatively similar due to similar orientation of helical repeats in the C-terminal domains of apoE3 and apoA-I.

Apolipoprotein E self-association in solution studied by non-radiative energy transfer.

The self-association of human apolipoprotein E (apoE), isolated from plasma very low density lipoproteins, was studied at apoE concentrations less than 0.6 microM by non-radiative energy transfer. ApoE was separately labeled with a fluorescent donor group i.e. dansyl chloride (apoE/D) and with an acceptor i.e. fluorescein isothiocyanate (apoE/F). Mixed apoE/D:apoE/F complexes were prepared either by incubation or the donor- and of the acceptor-labeled apoE or by renaturation during dialysis of the apoE/D:apoE/F mixture pre-denatured by addition of guanidine hydrochloride or by treatment with sodium cholate. The efficiency of energy transfer E at an equimolar ratio of the donor to acceptor and a ratio of 1.9 mol fluorescein/mol protein amounted to 29.2 +/- 2.6% (n = 3). The E value increased linearly with increasing acceptor fraction in the mixture. The state of self-association of apoE as tetramers within this concentration range was confirmed by cross-linking experiments with a water-soluble bifunctional reagent. This approach can be applied to the study of protein-protein interactions in apolipoprotein-phospholipid recombinants.

The significance of apolipoprotein E structure to the metabolism of plasma triglyceride-rich lipoproteins.

In this paper we analyse the structural organization of human apolipoprotein E (apoE) at the surface of triglyceride (TG)-rich lipoproteins, in relation to the metabolic pathway of these particles. ApoE acts as a receptor-binding ligand at the surface of chylomicrons and VLDL (very low density lipoproteins). The degree of exposure of apoE at the surface of lipoproteins and its affinity for the receptor both determine the uptake and catabolism of these lipoproteins. ApoE and/or apoB100, the major apolipoprotein constituent of LDL, contribute to the interaction of lipoproteins with five different cellular receptors: 1) the low density lipoprotein (LDL) receptor; 2) the LDL receptor-related protein (LRP); 3) the macrophage receptor for hypertriglyceridemic VLDL; 4) the scavenger receptor; 5) the VLDL receptor. The degree of exposure of apoE at the surface of normo- and hyperlipidemic VLDL can modulate their uptake by the LDL receptor. Normolipidemic VLDL are poorly recognized by the LDL receptor whereas hypertriglyceridemic VLDL are cleared more efficiently through this pathway. On the other hand, the extent of apoE self-association, which is dependent upon the degree of hydrolysis of the TG-rich particles, can control their interaction with the LDL-receptor related protein. The lateral organization of apoE at the surface of TG-rich particles, its interaction with other apoproteins and its extent of self-association might therefore be important factors in the clearance of these lipoproteins. Finally, structural defects of apoE might result in an impaired interaction of apoE-containing lipoproteins with these receptors and lead to the development of atherogenic dyslipidemias.

The interaction of human serum albumin in the native and fully reduced states with apolipoprotein E, serum amyloid protein and very low density lipoproteins from human plasma.

1. Complex formation in a solution of apolipoprotein E (apoE) isolated from human plasma very low density lipoproteins (VLDL) and human serum albumin (HSA) in both native and fully reduced states was studied. The existence of a kinetically unstable complex of apoE and native albumin was shown. The complex became more stable with the reduction of the S--S links in the albumin molecules capable of forming aggregates under these conditions. 2. The interaction between native HSA as opposed to a fully reduced one and isolated VLDL particles was more pronounced, probably, due to the existence of amphipathic alpha-helical regions. 3. Dissociation of the serum amyloid protein (SAP) oligomeric form in solution and the interaction of the protein with fully reduced HSA owing to the provision with the additional hydrophobic surface was shown. ApoE displaced SAP from the complex with fully reduced albumin. 4. It is suggested that the ability of the apolipoprotein to interact with albumin is determined by internal stability of the molecular structure of the latter and the complexes detected in vitro may be a new transport form of apolipoproteins in lipid-free form in serum. It is assumed that competitive interactions in the HSA-SAP-apoE system may be involved in the development of secondary amyloidosis.

[Interaction of serum albumin with apoprotein E and very low density lipoproteins in human blood]. / Vzaimodeistviesyvorotochnogo al'bumina s apobelkom E i lipoproteidami ochen' nizkoi plotnosti plazmy krovi cheloveka.

The complex formation between apolipoprotein E (apoE) isolated from human plasma very low density lipoproteins (VLDL) and human serum albumin (HSA) in both native and fully reduced states has been studied. Using measurements of fluorescence anisotropy and fluorescence intensity of the fluorescein-labelled apoE, the elution profiles of the apoE/HSA complex and the kinetics of the protein cross-linking within the complex by a water-soluble bifunctional reagent, the existence of a kinetically unstable complex of apoE with native albumin has been shown. The complex became more stable after the reduction of the S-S links in the albumin molecules capable of forming aggregates under these conditions. The interaction between native HSA as opposed to the fully reduced one and the isolated VLDL particles was far more pronounced, apparently due to the existence of amphipathic alpha-helical regions. It is suggested that the ability of the apolipoprotein to interact with albumin is determined by the inner stability of the albumin molecular structure and that the complexes detected in vitro may be regarded as a new transport form of apolipoproteins in a lipid-free form in the serum.

[The competition of serum amyloid protein and apoprotein E for binding with human serum albumin]. / Konkurentsiia syvorotochnogo amiloidnogo belka i apobelka E za sviazyvanie s syvorotochnym al'buminom cheloveka.

The relationship between the concentration of serum amyloid protein (SAP) isolated from human serum and the parameters of the protein elution during gel-filtration and alos with the efficiency of Ca(2+)-dependent SAP binding with sepharose 4B was studied. The dissociation of the SAP oligomeric form in solution and the interaction of the protein with human serum albumin with fully reduced S--S bridges due to the introduction of the additional hydrophobic surface was shown. Apoprotein E isolated from human plasma very-low-density lipoproteins replaced SAP in the complex with albumin.

[The immunochemical detection of apoprotein dissociation from particles of very low-density lipoproteins in human blood plasma]. / Immunokhimicheskoe obnaruzhenie otdeleniia apobelkov ot chastits lipoproteinov ochen' nizkoi plotnosti plazmy krovi cheloveka.

The dissociation of very-low-density lipoprotein (VLDL) apoproteins was studied using immunochemical approaches. The analysis of monospecific antibody binding to apo E, C-II and C-III on VLDL surface showed low apoprotein accessibility for the antibodies while the accessibility of apo C-II and C-III in solution was complete. Lipoprotein preparation dilution resulted in increasing of apo E and C-II accessibility. It was suggested that apoprotein dissociation led to apoprotein cluster dissolving on VLDL surface and higher antigen determinant accessibility. The findings confirmed previous theoretical analysis of apoprotein dissociation.

Quaternary structure of apolipoprotein E in solution: fluorimetric, chromatographic and immunochemical studies.

Self-association and stability in solution of apolipoprotein E (apoE), isolated from human plasma very low density lipoproteins, were studied in the nanomolar concentration range. Equilibrium denaturation of fluorescein-labelled apoE (induced by guanidine hydrochloride) was studied by measurement of fluorescence anisotropy, total fluorescence emission intensity, the shift in wavelength of maximal fluorescence emission, and gel-chromatographic behaviour. The protein denaturation was reversible, displayed biphasic behaviour, and was dependent on the apoE concentration. As measured by fluorescence anisotropy, the kinetics of apoE denaturation in the presence of 6M denaturant were heterogeneous, and the contribution of the long-lived component increased with the apoprotein concentration. The results are in agreement with the following scheme: Oligomer (in aged preparations) in equilibrium with tetramer in equilibrium with native or partially denatured monomer in equilibrium with fully denatured monomer. It is suggested that self-association of individual apoE molecules in solution is due to their lipid-binding domains, and leads to additional stabilization of apoprotein structure. Monoclonal antibody 3D12F11 of the IgG1 subclass bound with high affinity to apoE (Kd = 3.5 +/- 0.5 nM), and had no effect on apoprotein binding to heparin-Sepharose or the apoprotein-induced destabilization of liposomes formed from dipalmitoyl-phosphatidylcholine. This indicates that the epitope to the antibody is localized outside the heparin- and lipid-binding sites of the apoprotein molecule.

[Preparation and characterization of monoclonal antibodies to human apolipoprotein E]. / Poluchenie i kharakteristika monoklonal'nykh antitel protiv apolipoproteina E cheloveka.

Monoclonal antibodies to human apolipoprotein E (apoE) were prepared and characterized. Antibodies of 3D12F11 clone were shown to be specific to apoE and belong to IgG1 subclass. Dissociation constant of antigen-antibody complex in solution was determined as 3.5 +/- 0.5 nM. The antibodies interacted neither heparin- nor lipid-binding sites of human apoE molecule. The obtained antibodies may be useful for metabolic and structural investigations of apoE as well as for clinical studies.

[Supermolecular organization of apolipoprotein E in solution]. / Nadmolekuliarnaia organizatsiia apolipoproteina E v rastvore.

The aggregation behaviour and stability in solution of apolipoprotein E (apoE) isolated from human blood plasma very low density lipoproteins were investigated. The equilibrium denaturation of fluorescein-labeled apoE by guanidine-hydrochloride determined by anisotropy and overall intensity of fluorescence, shift of the emission spectrum maximum and gel-chromatographic behaviour was characterized by reversibility, biphasity, apoE concentration dependence and the existence of native structure of the apoE monomer. The contribution of the long-living component to the kinetic dependence of fluorescence anisotropy in the presence of the 6 M denaturant increased with an increase in apoE concentration. The data obtained fit into the following scheme: oligomer (upon aging of the preparation) in equilibrium tetramer in equilibrium native monomer in equilibrium denaturated monomer. The presence in the tetrameric structure of apoE of two domains is postulated; one of those is formed by lipid-binding fragments during aggregation of individual molecules of apoE. Monoclonal antibody 3D12F11 (subclass IgG1) showed a high affinity for the apoE (Kd = 3.5 +/- 0.5 nM) without any effect on the apoprotein binding to heparin-Sepharose and apoE-induced destruction of dipalmitoylphosphatidylcholine liposomes. It is concluded that the 3D12F11 epitope is localized outside heparin- and lipid-binding sites of the apoprotein molecule.

Localization of apolipoprotein E in normal and atherosclerotic human aorta.

To elucidate the role of apolipoprotein E (apo E) in atherogenesis, we have investigated the localization of apo E in normal and atherosclerotic aortas as well as in other tissues of 32 post-mortem individuals. Using double immunofluorescence it has been found that normal intima of individuals older than 20 years and some adolescents contained immunoreactive material that reacted with poly- and monoclonal antibodies to apo E. A staining pattern of apo E differed from that of apolipoprotein B, the latter being seen in normal intima of each child older than 7 years. Apo E was present extracellularly in lipid streaks and atheromatous plaques, where its staining was particularly intensive around the necrotic zone of plaques. Some macrophages in the plaques of 4 aortas exhibited apo E-positive staining, while aortic endothelial and smooth muscle cells never contained apo E. Apo E-positive staining was not found in the majority of vessel cells, it was always, however, observed in other types of cells including hepatocytes. Kupffer cells, spleen macrophages and cerebral astrocytes. Our findings indicate that only some macrophages in human aorta may be responsible for the production of apo E that can participate in reverse cholesterol transport. At the same time, apo E accumulation in the aortic wall may promote the development of atherosclerosis.

[Heparin-sepharose affinity chromatography and reduction conditions as a method of a selective process for separate isoforms of apolipoprotein A]. / Affinnaia khromatografiia na geparin-sefaroze v redutsiruiushchikh usloviiakh kak sposob izbiratel'nogo obogashcheniia po otdel'nym izoformam apolipoproteina E.

Gel permeation chromatography of VLDL apoproteins on Sepharose CL-6B in denaturing conditions and affinity chromatography on heparin-sepharose 4B in the presence of reducing agent dithiothreitol were used for preparative isolation of apolipoprotein E of high purity from human plasma VLDL. Sequential elution of apolipoprotein E from affinity column using increasing ionic strength solutions (0.4 M NaCl and 1.0 M NaCl) permitted to obtain "high affinity" apo E preparation with increased relative apoprotein isoform content with the highest positive charge.