apoE cDNA Research Articles

Human Apolipoprotein E Expression from Mouse Skeletal Muscle by Electrotransfer of Nonviral DNA (Plasmid) and Pseudotyped Recombinant Adeno-Associated Virus (AAV2/7)

Plasma apolipoprotein E (apoE) has multiple atheroprotective actions. However, although liver-directed adenoviral gene transfer of apoE reverses hypercholesterolemia and inhibits atherogenesis in apoE-deficient (apoE(-/-)) mice, safety considerations have revived interest in nonviral DNA (plasmid) and nonpathogenic adeno-associated viral (AAV) vectors. Here, we assess the effectiveness of these two delivery vehicles by minimally invasive intramuscular injection. First, we constructed AAV2-based expression plasmids harboring human apoE3 cDNA, driven by two muscle-specific promoters (CK6 and C5-12) and one ubiquitous promoter (CAG); each efficiently expressed apoE3 in transfected cultured C2C12 mouse myoblasts, although muscle-specific promoters were active only in differentiated multinucleate myotubes. Second, a pilot study verified that electrotransfer of the CAG-driven plasmid (p.CAG.apoE3) into tibialis anterior muscles, pretreated with hyaluronidase, of apoE(-/-) mice significantly enhanced (p < 0.001) local intramuscular expression of apoE3. However, in a 7-day experiment, the CK6- and C5-12-driven plasmids produced less apoE3 in muscle than did p.CAG.apoE3 (0.61 +/- 0.38 and 0.45 +/- 0.38 vs. 13.38 +/- 7.46 microg of apoE3 per muscle, respectively), but plasma apoE3 levels were below our detection limit (<15 ng/ml) in all mice and did not reverse the hyperlipidemia. Finally, we showed that intramuscular injection of a cross-packaged AAV serotype 7 viral vector, expressing human apoE3 from the CAG promoter, resulted in increasing levels of apoE3 in plasma over 4 weeks, although the concentration reached (1.40 +/- 0.35 microg/ml) was just below the threshold level needed to reduce the hypercholesterolemia. We conclude that skeletal muscle can serve as an effective secretory platform to express the apoE3 transgene, but that improved gene transfer vectors are needed to achieve full therapeutic levels of plasma apoE3 protein.

Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes

Fibroblast growth factor-1 (FGF-1) is secreted by astrocytes and stimulates apolipoprotein E (apoE)-HDL biogenesis by an autocrine mechanism to help in recovery from brain injury. In apoE-deficient mouse astrocytes, FGF-1 stimulated cholesterol biosynthesis without enhancing its release, indicating a signaling pathway independent of apoE biosynthesis upregulation. SU5402, an inhibitor of FGF receptor, inhibited FGF-1-induced phosphorylation of MEK, ERK, and Akt, as well as all the apoE-HDL biogenesis-related events in rat astrocytes. LY294002, an inhibitor of phosphatidylinositide 3-OH kinase (PI3K) and of Akt phosphorylation, inhibited apoE-HDL secretion but not cholesterol biosynthesis, whereas U0126, an inhibitor of MEK and of ERK phosphorylation, inhibited cholesterol biosynthesis but not apoE-HDL secretion. Increase of apoE-mRNA by FGF-1 was not influenced by either inhibitor. When rat apoE/pcDNA3.his was transfected to transformed rat astrocyte GA-1 cells that otherwise do not synthesize apoE (GA-1/25), FGF-1 did not influence apoE-mRNA, but did increase the apoE secretion and Akt phosphorylation that were suppressed by LY294002. Lipid biosynthesis was increased by FGF-1 in GA-1/25 cells and suppressed by U0126. FGF-1 upregulates apoE-HDL biogenesis by three independent signaling pathways. The PI3K/Akt pathway upregulates secretion of apoE/apoE-HDL, the MEK/ERK pathway stimulates cholesterol biosynthesis, and an unknown pathway enhances apoE transcription.

Cellular Sphingolipids Regulate Macrophage Apolipoprotein E Secretion

Macrophage-derived apolipoprotein E (apoE) in the vessel wall has important effects on the vessel-wall response to atherogenic injury. The current studies characterize a novel post-transcriptional pathway for the regulation of apoE secretion from macrophages. Treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA (to constitutively secrete a physiologic level of apoE) with sphingomyelinase led to a reduction of apoE secretion by nearly 50%. Increasing cellular ceramide by inhibiting ceramide degradation or by the direct treatment of cells with exogenous ceramide also reduced apoE secretion without a concomitant increase in cellular retention of newly synthesized apoE. Reducing cellular sphingomyelin (SM) by inhibiting its synthesis also reduced apoE secretion, but in this case, reduced apoE secretion was accompanied by increased cellular retention of apoE. The effect of sphingomyelin depletion to decrease apoE secretion and increase its cellular retention was dependent upon the presence of intact C-terminal amphipathic lipid-binding domains in apoE. ApoE expression also increased sphingomyelin secretion from macrophages, and this sphingomyelin was co-localized with apoE in secreted lipoprotein particles. The importance of sphingomyelin for apoE secretion and cell retention was confirmed using a Chinese hamster ovary model, in which cellular sphingolipids (both ceramide and sphingomyelin) are reduced secondary to absent serine palmitoyltransferase activity. Our results show that cellular sphingolipids, ceramide and sphingomyelin, have important effects on the post-transcriptional handling of nascent apoE by macrophages. Increased cellular ceramide reduces apoE secretion without increased cell retention, consistent with enhanced degradation of newly synthesized apoE. Reduction of cell SM also reduces apoE secretion, but this is associated with increased cellular retention of newly synthesized apoE. The dependence for this effect on the C-terminal domain of apoE suggests a model in which the SM content of intracellular membranes modulates the secretion of nascent apoE via the interaction with amphipathic lipid-binding domains.

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.

Cloning and expression of apolipoprotein E3 and its variant apoE2 and apoE4

In order to obtain three isoforms of apolipoprotein E (apoE), the cDNA encoding apoE3 was obtained by RT-PCR from normal human liver tissue. Site-directed mutagenesis was used to obtain the cDNAs encoding apoE2 and apoE4 isoforms. The 3 cDNAs were subcloned into vector pGEM-3Z and verified by DNA sequencing. The expression recombinant which can express the target protein as a (His) 6-tagged fusion was constructed by subcloning apoE cDNA into vector pT7-PL. The purified proteins were gained by Ni-NTA column. The SDS-PAGE results revealed the 6 His fusion proteins (apoE2, apoE3 and apoE4) were correctly expressed and purified successfully.

Baboon Lipoprotein(a) Binds Very Weakly to Lysine−Agarose and Fibrin Despite the Presence of a Strong Lysine-Binding Site in Apolipoprotein(a) Kringle IV Type 10

Human apolipoprotein(a) kringle IV type 10 [apo(a) KIV(10)] contains a strong lysine-binding site (LBS) that mediates the interaction of Lp(a) with biological substrates such as fibrin. Mutations in the KIV(10) LBS have been reported in both the rhesus monkey and chimpanzee, and have been proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and fibrin. To further the comparative analyses with other primate species, we sequenced a segment of baboon liver apo(a) cDNA spanning KIV(9) through the protease domain. Like rhesus monkey apo(a), baboon apo(a) lacks a kringle V (KV)-like domain. Interestingly, we found that the baboon apo(a) KIV(10) sequence contains all of the canonical LBS residues. We sequenced the apo(a) KIV(10) sequence from an additional 10 unrelated baboons; 17 of 20 alleles encoded Trp at position 70, whereas only two alleles encoded Arg at this position and thus a defective LBS. Despite the apparent presence of a functional KIV(10) LBS in most of the baboons, none of the Lp(a) in the plasma of the corresponding baboons bound specifically to lysine-Sepharose (agarose) even upon partial purification. Moreover, baboon Lp(a) bound very poorly to plasmin-modified fibrinogen. Expression of baboon and human KIV(10) in bacteria allowed us to verify that these domains bind comparably to lysine and lysine analogues. We conclude that presentation of KIV(10) in the context of apo(a) lacking KV may interfere with the ability of KIV(10) to bind to substrates such as fibrin; this paradigm may also be present in other non-human primates.

Stimulation of Vascular Smooth Muscle Cell Proliferation and Migration by Apolipoprotein(a) Is Dependent on Inhibition of Transforming Growth Factor-β Activation and on the Presence of Kringle IV Type 9

Elevated plasma concentrations of lipoprotein(a) are a risk factor for the development of a variety of atherosclerotic disorders. Despite intensive study, the mechanisms by which lipoprotein(a) promotes these disorders remain to be unequivocally defined. It has been demonstrated that lipoprotein(a), through its unique constituent apolipoprotein(a) (apo(a)), stimulates vascular smooth muscle cell (SMC) migration and proliferation. These effects arise from the ability of apo(a) to inhibit the formation of active transforming growth factor beta (TGF-beta) from its latent precursor, which in turn is caused by the ability of apo(a) to decrease the formation of plasmin from its precursor plasminogen. We utilized a battery of recombinant apo(a) variants that represent systematic deletions of the various domains in the molecule to further probe the mechanism underlying the effect of apo(a) on SMC responses. All recombinant apo(a) variants that contained kringle IV type 9 were able to stimulate SMC proliferation and migration and to decrease the formation of active TGF-beta; conversely all recombinant apo(a) variants lacking kringle IV type 9 had no effect on these parameters. The kringle IV type 9-dependent effects of apo(a) on SMC proliferation required the presence of plasminogen, suggesting for the first time that this kringle mediates the ability of apo(a) to inhibit pericellular plasmin formation.

Oleic Acid Modulates the Post-translational Glycosylation of Macrophage ApoE to Increase Its Secretion

There has been increasing interest in a potential role for fatty acids in adversely affecting organismal substrate utilization and contributing to the cardiovascular complications in insulin resistance. Fatty acids have already been implicated in regulating the expression of a number of genes in resident cells of the vessel wall. In the current studies, we evaluated a potential role for fatty acids in the regulation of macrophage apoE expression. Incubation in oleic acid increased the synthesis and secretion of apoE by human monocyte-derived macrophages. Part of this stimulation was mediated at a post-translational locus. Oleic acid increased the secretion of apoE from macrophages that constitutively expressed a human apoE3 cDNA. Incubation in palmitic acid decreased apoE secretion from these cells. The effect of oleic acid on apoE secretion could not be accounted for by the known effect of fatty acid on cellular sterol, because incubation in oleic acid did not suppress the degradation of nascent apoE. Incubation in oleic acid for at least 6 h was required to observe an effect on apoE secretion. Oleic acid altered the glycosylation pattern of cellular and secreted apoE, with a loss of the most heavily sialylated isoform. Oleic acid had no effect on the glycosylation of interleukin 6 secreted from macrophages. Elimination of apoE glycosylation, by substitution of threonine 194 with alanine, eliminated oleic acid-mediated stimulation of apoE secretion. These results indicate that oleic acid increases apoE secretion from macrophages at a locus involving post-translational glycosylation.

What do we know about apolipoprotein E and the prevention of cardiovascular disease?

AbstractApolipoprotein E (ApoE) serves as a ligand for low‐density lipoprotein (LDL) receptor and LDL receptor–related protein and plays an important role in the clearance of triglyceride‐rich lipoproteins. The links between ApoE and cardiovascular disease has been shown in animal models and numerous epidemiological studies. Deletion of the APOE gene in mice resulted in high plasma cholesterol and spontaneous atherosclerosis. Atheroma formation in APOE‐deficient mice can be prevented by expression of ApoE through various genetic transfer techniques. These effects may be independent of the effect on cholesterol levels, suggesting a direct antiatherogenic effect of ApoE. In humans, the APOE genotypes are important determinants of LDL‐C and were highest in subjects with the ϵ4 allele, intermediate in ϵ3 subjects, and lowest in ϵ2 subjects. Targeting APOE expression or ApoE levels represents an attractive means of reversing or preventing atherosclerosis. Infusion of recombinant adenovirus containing ApoE, intramuscular injection of plasmid vector encoding for APOE3 cDNA, or infusion of recombinant ApoE in animal models have all resulted in reduction in cholesterol and prevention of atherosclerosis. The use of peptide models of ApoE in animals has also shown some potential in reducing atherosclerosis. However, the short half‐lives of recombinant ApoE or its peptide models are a limitation to their usefulness. New modalities for drug delivery that allow sustained delivery of ApoE, such as the multivesicular liposome depot‐delivery system (DepoFoam) may pave the way for the use of ApoE as a means of therapy in atherosclerosis prevention. Interactions between APOE genotypes and response to lipid‐lowering therapy is another area of focus when considering the role of ApoE in the prevention of atherosclerosis. Drug Dev. Res. 62:200–206, 2004. © 2004 Wiley‐Liss, Inc.

Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.

To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA.

We report on systemic delivery and long-term biological effects of apolipoprotein E (apoE) obtained by intramuscular (i.m.) plasmid DNA injection. ApoE plays an important role in lipoprotein catabolism and apoE knock-out mice develop severe hypercholesterolemia and diffuse atherosclerosis. We have injected apoE-deficient mice with 80 microg of a plasmid vector (pCMV-E3) encoding the human apoE3 cDNA under the control of the CMV promoter-enhancer in both posterior legs. Local expression of the transgene was demonstrated throughout 16 weeks. Human apoE3 recombinant protein reached 0.6 ng/ml serum level. After i.m. injection of pCMV-E3 expression vector the mean serum cholesterol concentrations decreased from 439 +/- 57 mg/dl to 253 +/- 99 mg/dl (P < 0.05) 2 weeks after injection and persisted at a significantly reduced level throughout the 16 weeks observation period (P < 0.005). Serum cholesterol was unaffected and reached an absolute level of 636 +/- 67 mg/dl in control groups. Finally, injection of pCMV-E3 into apoE-deficient mice resulted in a redistribution of cholesterol content between lipoprotein fractions, with a marked decrease in VLDL, IDL and LDL cholesterol content and an increase in HDL cholesterol. These results demonstrate that severe hypercholesterolemia in apoE-deficient mice can be effectively reversed by i.m. DNA injection, and indicate that this approach could represent a useful tool to correct several hyperlipidemic conditions resulting in atherosclerosis.

Sterols and inhibitors of sterol transport modulate the degradation and secretion of macrophage ApoE: requirement for the C-terminal domain

Macrophage-derived apoE, produced in the vessel wall, may have important effects during atherogenesis. The production of apoE by macrophages can be regulated at a transcriptional level by cellular differentiation state, cytokines and sterol loading. In addition, there are post-transcriptional and post-translational loci for regulation. We have recently identified an intermediate density cell membrane fraction in which the degradation of apoE can be modulated by sterols. Suppressing degradation of apoE in this fraction by pre-incubating cells in sterols led to enhanced apoE secretion. In this report we demonstrate that the suppressive effect of sterols on the degradation of newly synthesized apoE in this fraction depends on the presence on its C-terminal domain, by studying a macrophage cell line transfected to express a mutant form of apoE in which amino acids beyond amino acid 202 were deleted. In addition, two modulators of cellular sterol transport, progesterone and U1866A, inhibited the degradation of full-length apoE. In contrast, incubation of cells in the acyl-CoA:cholesterol acyltransferase inhibitor S58035 did not influence apoE degradation. As would be predicted based on the results of degradation assays, U1866A, but not S58035, increased the secretion of apoE from a cell line transfected to constitutively express full-length apoE cDNA. The effect of U1866A on apoE degradation, like the effect of sterol, required the presence of the apoE C-terminal domain. Our results indicate that alteration of intracellular sterol homeostasis by pre-incubation in sterols or by drugs that modify the subcellular transport of sterol, modulates the susceptibility of apoE to degradation and that this modulation requires the presence of C-terminal lipid binding domains.

Endogenously expressed apolipoprotein E has different effects on cell lipid metabolism as compared to exogenous apolipoprotein E carried on triglyceride-rich particles.

Apolipoprotein E (apoE) on model triglyceride-rich particles (TGRP) increases triglyceride (TG) utilization and cholesteryl ester (CE) hydrolysis, independent of its effect on enhancing particle uptake. We questioned whether, under physiological concentrations, endogenously expressed apoE has similar effects on cellular lipid metabolism as compared to exogenous apoE. J774 macrophages, which do not express apoE, were engineered to express endogenous apoE by transfection of human apoE3 cDNA expression constructs (E(+)) or control vectors (E(-)) into the cells. To compare the effects of exogenous apoE and endogenous apoE on TGRP uptake, cells were incubated with or without apoE associated with (3)H-cholesteryl ether-labeled TGRP. Exogenous apoE enhanced TGRP uptake in both E(-) and E(+) cells. E(-) cells displayed significantly higher TGRP uptake than E(+) cells. Sodium chlorate, which inhibits cell proteoglycan synthesis, markedly diminished differences in TGRP uptake between E(-) and E(+) cells, suggesting that endogenous apoE-proteoglycan interaction contributes to differences in uptake between the two cell types. Particle uptake by the LDL receptor, by the LDL receptor related protein, or by scavenger receptors were similar between E(-) and E(+) cells indicating that endogenous apoE expression does not have a general effect on endocytic pathways. Exogenous apoE carried on TGRP stimulated TG utilization and CE hydrolysis in both cell types. However, TG utilization and CE hydrolysis were not affected by endogenous apoE expression. In conclusion, macrophage expression of apoE has very different effects on TGRP metabolism than exogenously supplied apoE. The fluorescence microscopy results in this study showing that exogenous apoE and endogenous apoE were confined in separate cellular compartments support the hypothesis that these differences resulted from distinct intracellular trafficking pathways followed by exogenous apoE bound to TGRP as compared to endogenous cell-expressed apoE.

Complete atherosclerosis regression after human ApoE gene transfer in ApoE-deficient/nude mice.

The apolipoprotein E (apoE)-deficient mouse is a relevant animal model of human atherosclerosis. Although the prevention of atherosclerosis development has been documented after somatic gene transfer into animal models, regression of lesions remains to be demonstrated. Thus, we used this genetically defined mouse model nn the nude background to show atherosclerosis regression. ApoE-deficient nude mice were infected with 5 x 10(8) or 10(9) plaque-forming units of a first-generation adenovirus encoding human apoE cDNA. The secretion of human apoE resulted in a rapid decrease of total cholesterol, which normalized the hypercholesterolemic phenotype within 14 days (from 600+/-100 to <100 microg/mL). Transgene expression was observed during a period of >4 months, with a normalization of cholesterol and triglyceride levels during 5 months. At that time, we successfully reinjected the recombinant adenovirus and observed the appearance of the human protein as well as the correction of lipoprotein phenotype. In mice killed 6 months-after the first infection, we observed a dose-dependent regression of fatty streak lesions in the aorta. We showed sustained expression of a transgene with a first-generation adenoviral vector and a correction of dyslipoproteinemia phenotype leading to lesion regression. These data demonstrate that somatic gene transfer can induce plaque regression.

Apolipoprotein E Participates in the Regulation of Very Low Density Lipoprotein-Triglyceride Secretion by the Liver

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe-/- mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe-/- mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe-/- mice (3.4 +/- 0.5 mM) were reduced when compared with apoe-/- mice (12.6 +/- 1.4 mM) but still elevated when compared with wild type control values (1.9 +/- 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/apoe-/- mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of (3)H-labeled triglycerides synthesized from [(3)H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe-/- or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/apoe-/- and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.

Carboxyl-terminal domain of human apolipoprotein E: expression, purification, and crystallization.

Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.

Molecular cloning of the cDNA encoding the carboxy-terminal domain of chimpanzee apolipoprotein(a): An asp57 --> asn mutation in kringle IV-10 is associated with poor fibrin binding

Insight into the structural features of human lipoprotein(a) [Lp(a)] which underlie its functional implication in fibrinolysis may be gained from comparative studies of apo(a). Indeed, cloning of rhesus monkey apo(a) has shown that a Trp72 --> Arg mutation in the lysine-binding site (LBS) of KIV-10 leads to loss of lysine-binding properties of the rhesus Lp(a) particle. Consequently, comparative studies of apo(a) sequences in different Old World monkey species should further our understanding of the molecular role of Lp(a) in the fibrinolytic process. In contrast to other Old World monkeys, including rhesus monkey, cynomolgus, and baboon, the chimpanzee exhibits an elevated level of Lp(a) and a distinct isoform distribution as compared to humans [Doucet et al. J. Lipid Res. (1994) 35, 263-270]. Clearly then, the chimpanzee is an interesting animal model for study of the structure, function, and potential pathophysiological roles of Lp(a). We have cloned and sequenced the region of chimpanzee apo(a) cDNA spanning KIV-3 to the stop codon. The global organization of this region is similar to that of human apo(a) with the presence of KV, which is absent in rhesus monkey apo(a). Nucleotide sequence comparison indicates a variation of 1.4% between chimpanzee and man and 5.1% between chimpanzee and rhesus monkey. The differences concerned single base changes. An Asp57 --> Asn mutation was detected in KIV-10; this residue is critical to the LBS of KIV-10 in human apo(a). To verify that the Asp57 --> Asn substitution was specific to apo(a), we have also cloned the cDNA-encoding plasminogen, which exhibited an Asp at the corresponding position in kringle IV. Using an in vitro binding assay, we have demonstrated that chimpanzee Lp(a) exhibits poor lysine-specific interaction with both intact and plasmin-degraded fibrin as compared to its human counterpart. We propose that the Asn57 substitution in KIV-10 of chimpanzee apo(a) is responsible for this property. Chimpanzee Lp(a) therefore represents an appropriate particle with which to explore the potential effects of Lp(a) on the fibrinolytic system, such as the inhibition of plasminogen activation or inhibition of t-PA activity.

Molecular cloning of the cDNA encoding the carboxy-terminal domain of chimpanzee apolipoprotein(a): an Asp57 --> Asn mutation in kringle IV-10 is associated with poor fibrin binding.

Insight into the structural features of human lipoprotein(a) [Lp(a)] which underlie its functional implication in fibrinolysis may be gained from comparative studies of apo(a). Indeed, cloning of rhesus monkey apo(a) has shown that a Trp72 --> Arg mutation in the lysine-binding site (LBS) of KIV-10 leads to loss of lysine-binding properties of the rhesus Lp(a) particle. Consequently, comparative studies of apo(a) sequences in different Old World monkey species should further our understanding of the molecular role of Lp(a) in the fibrinolytic process. In contrast to other Old World monkeys, including rhesus monkey, cynomolgus, and baboon, the chimpanzee exhibits an elevated level of Lp(a) and a distinct isoform distribution as compared to humans [Doucet et al. J. Lipid Res. (1994) 35, 263-270]. Clearly then, the chimpanzee is an interesting animal model for study of the structure, function, and potential pathophysiological roles of Lp(a). We have cloned and sequenced the region of chimpanzee apo(a) cDNA spanning KIV-3 to the stop codon. The global organization of this region is similar to that of human apo(a) with the presence of KV, which is absent in rhesus monkey apo(a). Nucleotide sequence comparison indicates a variation of 1.4% between chimpanzee and man and 5.1% between chimpanzee and rhesus monkey. The differences concerned single base changes. An Asp57 --> Asn mutation was detected in KIV-10; this residue is critical to the LBS of KIV-10 in human apo(a). To verify that the Asp57 --> Asn substitution was specific to apo(a), we have also cloned the cDNA-encoding plasminogen, which exhibited an Asp at the corresponding position in kringle IV. Using an in vitro binding assay, we have demonstrated that chimpanzee Lp(a) exhibits poor lysine-specific interaction with both intact and plasmin-degraded fibrin as compared to its human counterpart. We propose that the Asn57 substitution in KIV-10 of chimpanzee apo(a) is responsible for this property. Chimpanzee Lp(a) therefore represents an appropriate particle with which to explore the potential effects of Lp(a) on the fibrinolytic system, such as the inhibition of plasminogen activation or inhibition of t-PA activity.

Characterization of the sheep apolipoprotein E ( ApoE) gene and allelic variations of the ApoE gene in scrapie Suffolk sheep

Apolipoprotein E (ApoE) plays a central role in lipid transport and is suggested to be involved in neuronal repair. Human ApoE ε4 allele is known as a risk factor for Alzheimer's disease, and an association of the human ApoE genotype with the human prion disease, Creutzfeldt–Jakob disease, is suggested, albeit controversial. We analyzed the sheep ApoE gene to determine whether any association between the sheep ApoE genotype and the sheep prion disease, scrapie, existed. The sheep ApoE cDNA contained an open reading frame (ORF) consisting of 948 base pairs (bp) that encoded 316 amino acids (aa). The sheep ApoE gene was composed of four exons separated by three introns, and the ORF was encoded by three exons, designated exons 2, 3, and 4. Nucleotide sequence analysis also showed the presence of one G/T nucleotide polymorphism in the ORF that resulted in an Ala/Ser amino-acid substitution at codon 258. PCR-restriction fragment length polymorphism analysis of genomic DNA showed the presence of three sheep ApoE genotypes that were the result of the homologous and heterologous combinations of the two alleles. We analyzed the sheep ApoE genotypic and the allelic frequencies in scrapie and control Suffolk sheep, but they did not significantly differ from those in the control sheep, even though PrP genotype-matched populations were compared. The ApoE genotype appeared not to be associated with the progression of the disease when looking at the age at death. These results indicated that in Suffolk sheep, none of the ApoE genotypes was associated with scrapie.

Endogenous apoE expression modulates HDL3 binding to macrophages

We have previously shown that expression of a human apoE cDNA in J774 macrophages enhances cholesterol efflux to HDL3. We have also shown that endogenous apoE expression produces a cell surface pool of apoE associated with proteoglycans. In this series of experiments, we first demonstrate the presence of a cell surface proteoglycan-associated apoE pool in human monocyte-derived macrophages. We then examine the hypothesis that endogenous expression of apoE modulates HDL3 binding to macrophages, thereby, accounting for enhanced cholesterol efflux to HDL3, specifically examining a role for the cell surface pool. Enhanced binding of apoE-free human HDL3 to apoE-expressing macrophages, compared to non-expressing macrophages, was observed at 37°C and 4°C. The enhanced binding was not due to apoE secreted into the medium, as determined by experiments utilizing conditioned medium from apoE-secreting cells. Removal of the cell surface pool of apoE, however, substantially reduced the incremental HDL3 binding produced by apoE expression. Cellular cholesterol mass measurements demonstrated that experimental conditions that reduced HDL3 binding to apoE-expressing macrophages, did not substantially reduce cholesterol efflux to HDL3. In summary, our results document a clear role for cell surface pool of apoE in modulating HDL3 interaction with macrophages. The enhanced binding, however, does not appear to be a major mechanism contributing to the increased cholesterol efflux to HDL3, which results from endogenous macrophage expression of apoE.—Lin, C-Y., M. Lucas, and T. Mazzone. Endogenous apoE expression modulates HDL3 binding to macrophages.