LDL receptor binds newly synthesized apoE in macrophages. A precursor pool for apoe secretion.

There appear to be multiple post-translational sites for regulation of macrophage apolipoprotein (apo)E secretion, including the presence of a distinct cell surface pool of apoE. Cell surface proteoglycans have been shown to be involved in forming this pool. The current studies were designed to investigate the role of an additional cell surface site, i.e., the low density lipoprotein (LDL) receptor. Antiserum to the LDL receptor displaced apoE from the macrophage cell surface and into the medium during a 4 degrees C incubation from apoE-expressing J774 cells, from proteoglycan-depleted apoE-expressing J774 cells, and from human monocyte-derived macrophages. Similar results were obtained when purified monoclonal antibody to the LDL receptor was added to human monocyte-derived macrophages. J774 cells transfected to express an LDL receptor binding-defective mutant of apoE did not show a similar response to addition of LDL receptor antibody. Studies were conducted in which cells were pulse labeled for 30 min, followed by various periods of chase at 4 degrees C or 37 degrees C in the presence or absence of LDL receptor antibody. The results of these studies indicated that nascent macrophage-derived apoE binds to the LDL receptor, and that this apoE served as a precursor pool for apoE released into the medium.These studies establish a role for the LDL receptor in forming the cell surface pool of apoE and, along with data regarding the importance of proteoglycans, indicate that cell surface binding sites for nascent macrophage-derived apoE are heterogeneous. The heterogeneity of such sites could have implication for the size and turnover of this cell surface pool.     

Quantification and regulation of apolipoprotein E expression in rat Kupffer cells

Apolipoprotein E (apoE) is synthesized by a wide variety of cells including cells of the monocyte-macrophage lineage. In order to assess the quantitative significance of apoE synthesis in a mature tissue macrophage, apoE synthesis was compared in Kupffer cells and hepatocytes isolated from rat liver. Immunoreactive apoE synthesized by both cell types exhibited identical isoform patterns when examined by high-resolution two-dimensional gel analysis. ApoE synthesis was not detected in hepatic endothelial cells. Northern blot analysis using a rat apoE cDNA probe demonstrated a single mRNA species of approximately 1200 nucleotides in freshly isolated hepatocytes and Kupffer cells. The absolute content of apoE mRNA in each cell type was determined with a DNA-excess solution hybridization assay. The apoE mRNA content (pg/microgram RNA) for Kupffer cells and hepatocytes was 35.7 and 98.8, respectively. Accounting for cellular RNA content and the population size of each cell type in the liver, Kupffer cells were calculated to contain about 0.7% of liver apoE mRNA; hepatocytes account almost quantitatively for the remainder. These results suggest that Kupffer cells are not major contributors to the plasma apoE pool. After intravenous injection of bacterial endotoxin, apoE mRNA was decreased in freshly isolated Kupffer cells whereas whole liver showed no change in apoE mRNA. Endotoxin treatment had no effect on the apoE mRNA content in several peripheral tissues. These results indicate that apoE expression in vivo is differentially regulated by endotoxin in Kupffer cells as compared to hepatocytes or apoE-producing cells in peripheral tissues.     

Secreted apolipoprotein E reduces macrophage-mediated LDL oxidation in an isoform-dependent way

As an inflammatory cell, the macrophage produces various oxidizing agents, such as free radical species. These can modify LDL as a secondary effect and doing so may favor atherogenic processes. Any molecule able to counteract these reactions would be of much benefit, especially if secreted by the macrophage itself at the lesion site. Such is the case for apolipoprotein E (apoE), which has been shown to exert antioxidant properties in some studies, mostly in relation to Alzheimer's disease. In this study, we assessed the antioxidant potential of the various isoforms of apoE (E2, E3, and E4) using a metal-induced LDL oxidation system with exogenous recombinant apoE and an in vitro model of macrophage-mediated LDL oxidation. We found that all three isoforms had an antioxidant capacity. However, whereas apoE2 was the most protective isoform in the cell-free system, the opposite was observed in apoE-transfected J774 macrophages. In the latter model, cellular cholesterol efflux was found to be more important with apoE2, possibly explaining the larger quantity of oxidative indices observed in the medium. It is proposed that the antioxidant property of apoE results from a balance between direct apoE antioxidant capacities, such as the ability to trap free radicals, and potentially pro-oxidative indirect events associated with cholesterol efflux from cells. Our observations add to the therapeutic potential of apoE. However, they also suggest the need for more experiments in order to achieve careful selection of the apoE isoform to be targeted, especially in the perspective of apoE transgene use.     

Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface

Synthesis of apolipoprotein (apo)E in hepatocytes leads to both secretion and retention in cell surface pools. Inclusion of Brefeldin A to HepG2 cells prompted a rapid decrease of cell surface apoE to about 37% of control values after a 3-h incubation. The t(1/2) for this dynamic pool was estimated to be 15 min. In contrast, a stable fraction of apoE (t(1/2) > 20 h) was found in association with the extracellular matrix (ECM). Increased content of apoE on the ECM correlated with decreased binding of VLDL. Decreased apoE on the cell surface correlated with increased binding of VLDL to cells. Collectively, this suggests that glycosaminoglycan-bound apoE can occlude binding sites for apoE-containing lipoproteins on glycosaminoglycans. In solid-phase assays, heparin, suramin, and chondroitin sulfates A and B efficiently inhibited the binding of apoE to heparan sulfate proteoglycans, but were unable to displace apoE from this glycosaminoglycan. Finally, decreasing cell surface apoE with suramin subsequently decreased the apoE content on secreted apoB-containing lipoproteins without affecting the overall secretion of apoE or apoB to the extracellular medium.In summary, cell surface apoE comprises both dynamic fractions, which can be donated to newly secreted lipoproteins, and stable fractions, which may act to minimize the unproductive binding of lipoproteins to the ECM.     

Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid

Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease. We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods. Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P. (2000) Biophys. J. 78, 1606-1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4. In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers. Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate. We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mm) and to a lesser extent at submicellar concentrations (4 mm). The alpha-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine. These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.     

Cell surface protein of Pseudomonas (Hydrogenomonas) facilis

Intact cells of Pseudomonas facilis contain one major molecular weight class of protein that is exposed at the cell surface as revealed by lactoperoxidase-catalyzed iodination with (125)I. All molecular weight classes of protein in derived cell envelope preparations are apparently saturated by iodination by lactoperoxidase after prolonged sonic treatment. The molecular weight of the predominantly exposed protein in intact cells is approximately 16,000, which is the minimal molecular weight of a cell envelope protein that precipitates as a complex with phospholipid from extracts of P. facilis. The isolation of labeled phospholipoprotein (PLP) after labeling intact cells with (125)I corroborates previous experiments which suggested a surface location for the protein portion of the phospholipoprotein (P(PLP)). Solvent extraction of cells and immunological evidence, including studies with ferritin-coupled antibodies, indicate that P(PLP) is located at the cell surface and may also be within the cell envelope. These experiments suggest that P(PLP) is the major cell surface protein in P. facilis.     

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.     

Identification and characterization of a novel,intracellular isoform of fibroblast growth factor receptor-1 (FGFR-1)

A novel, low molecular weight, intracellular isoform of FGF receptor-1 (FGFR-1) was identified in embryonic chicken tissues using several antibodies specific for different domains of FGF receptors. This low molecular weight isoform differs from the previously characterized isoforms of FGFR-1 in that it contains little or no carbohydrate. Furthermore, in contrast to the other isoforms of FGFR-1, this novel isoform is located exclusively intracellularly. However, it is capable of binding ¹²⁵I-FGF-2 and it possesses intrinsic kinase activity. Pulse-chase experiments indicate that this isoform of FGFR-1 is not simply a precursor to glycosylated FGFR-1 since it can be detected long after the appearance of glycosylated FGFR-1 in the cells. These results suggest that the novel FGFR-1 isoform plays a role in regulating FGF activity distinct from cell surface, glycosylated FGFR-1. The possible roles of this FGFR-1 variant in FGF signaling are discussed. © 1996 Wiley-Liss, Inc.     

Regulation of macrophage apoE secretion and sterol efflux by the LDL receptor

Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need.     

The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast.

NIL 8 hamster fibroblast cells were labeled by lactoperoxidase-catalyzed iodination. Their membranes were fractionated by sedimentation-rate and isopycnic zonal centrifugation. All the iodinated proteins except the very prominently labeled high molecular weight protein (greater than 200,000 daltons) were located in a fraction identified enzymically and compositionally as plasma membrane. The high molecular weight protein that was previously shown to be sensitive to virus transformation (Hynes, 1973) is concentrated in a very high density particle (rho equals 1.253-1.259) which contains mainly carbohydrate and protein and very low levels of lipid. 5'-nucleotidase was the only enzyme reproducibly demonstrated in this fraction, and electron micrographs revealed a predominantly amorphous morphology together with a few membraneous structures. The iodine label in this fraction was very sensitive to trypsinization prior to homogenization. All the available evidence indicates that this fraction is derived from the surface coat. Mitochondria, nuclei, and soluble protein were labeled to an insignificant extent. The presence of the iodinated surface proteins associated with the endoplasmic reticulum fraction is discussed in the light of these results.     

Fatty acids alter time dependent loss of apolipoprotein E expression by primary cultures of rat hepatocytes

Although the phenomenon of intracellular apolipoprotein E (apoE) degradation has been reported in other cell types, the fate of newly synthesized apoE in the liver is not well understood. In the present study, we examined the expression (the balance of synthesis, secretion, and degradation) of apoE in primary cultures of rat hepatocytes and compared it with albumin, a typical secretory protein. Synthesis and secretion of [(35)S]apoE was diminished in primary hepatocytes cultured for more than 2 days, in agreement with an observed decrease in apoE mRNA. Cells cultured for 1 day and labeled for up to 4 hours secreted total protein, apoE, and albumin, linearly. The apparent rates of synthesis for apoE and albumin were similar (1,158 vs. 1,334 dpm/mg/min) but rates of their secretion differed significantly (225 vs. 1,159 dpm/mg/min). Pulse-chase experiments indicated that cell-associated [(35)S]albumin was secreted without degradation, whereas significant quantities of newly synthesized apoE were degraded. The overall synthesis and secretion of total proteins, including secretion of apoE, was enhanced by oleic acid (1 mmol/L). However, this effect may not be limited to oleic acid because other fatty acids showed a similar effect on apoE mRNA abundance. In control cells, apoE was found to associate with high density lipoproteins predominantly, although the fraction associated with very low density lipoprotein was increased in hepatocytes incubated with oleic acid. Overall, the findings from this study suggest that the level of apoE expression by primary hepatocytes is dependent on the age of the culture. The study also indicates that the phenomenon of apoE degradation occurs in primary hepatocytes.     

Interaction with proteoglycans enhances the sterol efflux produced by endogenous expression of macrophage apoE.

Endogenous expression of apolipoprotein (apo)E in macrophages facilitates cholesterol efflux in the presence and absence of extracellular sterol acceptors. A proteoglycan-associated pool of apoE has also been described. The relationship between a proteoglycan-associated pool of apoE and enhanced cholesterol efflux was investigated in these studies. Inhibition of proteoglycan expression reduced cholesterol efflux from apoE-expressing cells ( J774E(+)) in the presence and absence of HDL, but did not do so from nonexpressing cells ( J774E(-)). The effect of proteoglycan depletion on sterol efflux from J774E(+) cells was confirmed by measuring differences in cell sterol mass, secreted sterol mass, and sterol efflux rates. Furthermore, apoE-containing particles secreted from proteoglycan-depleted J774E(+) cells were denser than those secreted from J774E(+) cells with intact proteoglycan expression. Also, in J774E(+) cells with intact proteoglycans, apoE particles isolated from the cell surface proteoglycan layer were denser than secreted particles. The apoE-lipid particles isolated from the cell surface proteoglycan layer had a lower lipid-to-apoE and cholesterol-to-apoE ratio compared with secreted particles. In distinction, proteoglycan depletion of J774E(-) cells did not reduce sterol efflux produced by the exogenous addition of apoE. These observations indicate that one mechanism by which endogenous expression of apoE facilitates effective cholesterol efflux from macrophages is related to its retention at the cell surface in a proteoglycan-associated pool. Further, our data suggest that apoE arrives at the cell surface in a relatively lipid-poor state, and that a proximate source of lipid available to the proteoglycan-bound apoE at the cell surface resides in the plasma membrane.     

ApoE-dependent sterol efflux from macrophages is modulated by scavenger receptor class B type I expression

Macrophages express a number of proteins involved in sterol efflux pathways, including apolipoprotein E (apoE) and scavenger receptor class B type I (SR-BI). We have investigated a potential interaction between these two sterol efflux pathways in modulating overall macrophage sterol flux. We utilized an experimental system in which we increased expression of each of these proteins to a high physiologic range in order to perform our evaluation. We show that in apoE-expressing cells, a 4-fold increase in SR-BI expression leads to reduction of sterol and phospholipid efflux. SR-BI-mediated reduction in sterol efflux was only observed in cells that expressed endogenous apoE. In J774 cells that did not express apoE, a similar increase in SR-BI level led to increased sterol efflux. The divergent response of sterol efflux after increased SR-BI was maintained in the presence of a number of structurally diverse extracellular sterol acceptors. Increased SR-BI expression also enhanced sterol efflux to exogenously added apoE. Investigation of a potential mechanism for reduced efflux in apoE-expressing cells indicated that SR-BI expression reduced macrophage apoE by accelerating the degradation of newly synthesized apoE. This led to decreased secretion of apoE and reduced the fraction of apoE sequestered on the cell surface. Thus, enhanced SR-BI expression in macrophages can reduce the cellular level and secretion of apoE by accelerating degradation of the newly synthesized protein. This reduction of endogenous apoE is accompanied by reduced sterol efflux from macrophages.     

Expression of dimeric and tetrameric acetylcholinesterase isoforms on the surface of cultured bovine adrenal chromaffin cells

Acetylcholinesterase is a highly polymorphic enzyme, which can be anchored to the cell surface through several different mechanisms. Dimeric (G2) acetylcholinesterase isoforms are attached by a glycosylphosphatidyl-inositol (GPI) linkage, whereas tetrameric (G4) forms are linked through a 20 kilodalton hydrophobic subunit. Although cells of haemopoietic origin contain large amounts of G2 GPI-linked acetylcholinesterase, most tissues express only trace amounts of this isoform. We examined the expression of acetylcholinesterase isoforms in cultured bovine adrenal medullary chromaffin cells. Two major isoforms (G2 and G4) were identified on the cell surface. The G2 isoform, which accounted for approximately half the cell-surface enzyme activity, was linked to the membrane through a GPI anchor. After treatment with diisopropylfluorophosphate to completely inhibit cellular acetylcholinesterase, the G4 isoform was found to be resynthesised and transported to the cell surface more rapidly than the G2 isoform. As the addition of GPI anchors is known to be a very rapid step, this finding suggested that the G2 and G4 isoforms might be transported to the cell surface by two different mechanisms. This conclusion was supported by results from subcellular fractionation experiments. The ratio of G4/G2 membrane-bound acetylcholinesterase varied between different subcellular fractions. The membrane-bound G2 isoform was greatly enriched in a high-speed “microsomal” fraction. G4 acetylcholinesterase is known to be actively secreted by chromaffin cells in culture. Although the G4 isoform was present on the cell surface, most of the secreted enzyme was derived from an intracellular pool. Thus, it is unlikely that the cell-surface G4 isoform contributes significantly to the pool of secreted enzyme. Instead, the expression of two different membrane-bound isoforms may provide a means by which chromaffin cells can target the enzyme to different locations on the cell surface. © 1994 Wiley-Liss, Inc.     

Regulation of macrophage ApoE expression and processing by extracellular matrix

Macrophage-derived apoE in the vessel wall has important effects on atherogenesis in vivo, making it important to understand factors that regulate its expression. Vessel wall macrophages are embedded in an extracellular matrix produced largely by arterial smooth muscle cells and endothelial cells. In this series of studies, we evaluated the influence of extracellular matrix on macrophage apoE expression. Subendothelial matrix, fibronectin, or collagen I stimulated macrophage apoE gene expression and apoE synthesis. Adhesion of macrophages to a polylysine substrate had no effect. An increase in apoE synthesis after plating on fibronectin could be observed by 2 h and was inhibited by blocking antibodies to the alpha(5)beta(1) integrin receptor for fibronectin. Fibronectin also regulated the post-translational processing of newly synthesized macrophage apoE by inhibiting its degradation. The increment in apoE resulting from suppressed degradation was retained in the cell-fibronectin monolayer in a pool that was resistant to release by exogenous high density lipoprotein subfraction 3. These observations establish a new pathway for the regulation of macrophage apoE expression in the vessel wall. The composition of the extracellular matrix changes after vessel wall injury and in response to locally produced cytokines and growth factors. The evolving composition of this matrix will, therefore, be important for regulating apoE expression and processing by vessel wall macrophages.     

Dissociation of apolipoprotein E oligomers to monomer is required for high-affinity binding to phospholipid vesicles

The apolipoprotein apoE plays a key role in cholesterol and lipid metabolism. There are three isoforms of this protein, one of which, apoE4, is the major risk factor for Alzheimer's disease. At micromolar concentrations all lipid-free apoE isoforms exist primarily as monomers, dimers, and tetramers. However, the molecular weight form of apoE that binds to lipid has not been clearly defined. We have examined the role of self-association of apoE with respect to interactions with phospholipids. Measurements of the time dependence of turbidity clearance of small unilamellar vesicles of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) upon addition of apoE show that higher molecular weight oligomers bind poorly if at all. The kinetic data can be described by a reaction model in which tetramers and dimers of apoE must first dissociate to monomers which then bind to the liposome surface in a fast and reversible manner. A slow but not readily reversible conformational conversion of the monomer then occurs. Prior knowledge of the rate constants for the association-dissociation process allows us to determine the rate constant of the conformational conversion. This rate constant is isoform dependent and appears to correlate with the stability of the apoE isoforms with the rate of dissociation of the apoE oligomers to monomers being the rate-limiting process for lipidation. Differences in the lipidation kinetics between the apoE isoforms arise from their differences in the self-association behavior leading to the conclusion that self-association behavior may influence biological functions of apoE in an isoform-dependent manner.     

Glycosylation of human apolipoprotein E. The carbohydrate attachment site is threonine 194

The glycosylation of human apolipoprotein (apo) E was examined with purified plasma apoE and apoE produced by transfected cell lines. The carbohydrate attachment site of plasma apoE was localized to a single tryptic peptide (residues 192-206). Sequence analysis and amino sugar analysis of this peptide derived from asialo-, monosialo-, or disialo-apoE indicated that the carbohydrate moiety is attached only to Thr194 in monosialo- and disialo-apoE and that asialo-apoE is not glycosylated. Mammalian cells that normally do not express apoE were transfected with human apoE plasmid expression vectors to test the utilization of potential carbohydrate attachment sites and the role of apoE glycosylation in secretion. Site-specific mutants of apoE, designed to eliminate or alter glycosylation sites, were expressed in HeLa cells by acute transfection. Apolipoprotein E(Thr194----Ala) was secreted exclusively as the asialo isoform, confirming that Thr194 is the site of carbohydrate attachment in these cells and indicating that glycosylation of apoE is not essential for secretion. Apolipoprotein E(Thr194----Asn,Gly196----Ser), which introduces a potential site for N-glycosylation at position 194, was secreted with a higher apparent molecular weight than native, O-glycosylated apoE. Studies with tunicamycin indicated that this apoE was N-glycosylated at Asn194. Stably transfected cell lines expressing human apoE were prepared from wild-type Chinese hamster ovary (CHO) cells and from CHO ldlD cells, which are defective in glycosylation. The transfected wild-type cells secreted multiply sialylated apoE. The transfected ldlD cells also secreted high levels of apoE even in the absence of glycosylation, which confirms that glycosylation is not essential for secretion of apoE.     

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.