Apolipoprotein E: far more than a lipid transport protein.

First recognized as a major determinant in lipoprotein metabolism and cardiovascular disease, apolipoprotein (apo) E has emerged as an important molecule in several biological processes not directly related to its lipid transport function, including Alzheimer's disease and cognitive function, immunoregulation, and possibly even infectious diseases. ApoE is a polymorphic protein arising from three alleles at a single gene locus. The three major isoforms, apoE4, apoE3, and apoE2, differ from one another only by single amino acid substitutions, yet these changes have profound functional consequences at both the cellular and molecular levels. ApoE3 seems to be the normal isoform in all known functions, while apoE4 and apoE2 can each be dysfunctional. Isoform (allele)-specific effects include the association of apoE2 with the genetic disorder type III hyperlipoproteinemia and with both increased and decreased risk for atherosclerosis and the association of apoE4 with increased risk for both atherosclerosis and Alzheimer's disease, impaired cognitive function, and reduced neurite outgrowth; isoform-specific differences in cellular signaling events may also exist. Functional differences in the apoE isoforms that affect (or did affect) survival before the reproductive years probably account, at least in part, for the allele frequencies of the present day.

Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis.

The differential effects of overexpression of human apolipoprotein (apo) E3 on plasma cholesterol and triglyceride metabolism were investigated in transgenic rabbits expressing low (<10 mg/dL), medium (10 to 20 mg/dL), or high (>20 mg/dL) levels of apoE3. Cholesterol levels increased progressively with increasing levels of apoE3, whereas triglyceride levels were not significantly affected at apoE3 levels up to 20 mg/dL but were markedly increased at levels of apoE3 >20 mg/dL. The medium expressers had marked hypercholesterolemia (up to 3- to 4-fold over nontransgenics), characterized by an increase in low density lipoprotein (LDL) cholesterol, while the low expressers had only slightly increased plasma cholesterol levels. The medium expressers displayed an 18-fold increase in LDL but also had a 2-fold increase in hepatic very low density lipoprotein (VLDL) triglyceride production, an 8-fold increase in VLDL apoB, and a moderate decrease in the ability of the VLDL to be lipolyzed. However, plasma clearance of VLDL was increased, likely because of the increased apoE3 content. The increase in LDL appears to be due to an enhanced competition of VLDL for LDL receptor binding and uptake, resulting in the accumulation of LDL. The combined hyperlipidemia of the apoE3 high expressers (>20 mg/dL) was characterized by a 19-fold increase in LDL cholesterol but also a 4-fold increase in hepatic VLDL triglyceride production associated with a marked elevation of plasma VLDL triglycerides, cholesterol, and apoB100 (4-, 9-, and 25-fold over nontransgenics, respectively). The VLDL from the high expressers was much more enriched in apoE3 and markedly depleted in apoC-II, which contributed to a >60% inhibition of VLDL lipolysis. The combined effects of stimulated VLDL production and impaired VLDL lipolysis accounted for the increases in plasma triglyceride and VLDL concentrations in the apoE3 high expressers. The hyperlipidemic apoE3 rabbits have phenotypes similar to those of familial combined hyperlipidemia, in which VLDL overproduction is a major biochemical feature. Overall, elevated expression of apoE3 appears to determine plasma lipid levels by stimulating hepatic VLDL production, enhancing VLDL clearance, and inhibiting VLDL lipolysis. Thus, the differential expression of apoE may, within a rather narrow range of concentrations, play a critical role in modulating plasma cholesterol and triglyceride levels and may represent an important determinant of specific types of hyperlipoproteinemia.

Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes.

Type III hyperlipoproteinemia (HLP) is a genetic disorder characterized by accumulation of remnant lipoproteins in the plasma and development of premature atherosclerosis. Although receptor binding-defective forms of apolipoprotein (apo) E are the common denominator in this disorder, a number of apparent paradoxes concerning its pathogenesis still exist. However, studies in transgenic animals are resolving the mechanisms underlying this disorder. PARADOX I: Defective apoE (commonly apoE2) is essential but not sufficient to cause overt type III HLP. In fact, most apoE2 homozygotes are hypolipidemic. Studies in apoE2 transgenic models have demonstrated the impact of other genes or hormones in converting the hypolipidemia to hyperlipidemia. PARADOX II: Among apoE2 homozygotes, men are more susceptible than women to type III HLP. Transgenic studies have shown that estrogen affects both LDL receptor expression and lipolytic processing, explaining the resistance of women to this disorder until after menopause. PARADOX III: ApoE deficiency is associated with hypercholesterolemia, whereas the type III HLP phenotype is characterized by both hypercholesterolemia and hypertriglyceridemia. The hypercholesterolemia is caused by impaired receptor-mediated clearance, whereas the hypertriglyceridemia is caused primarily by impaired lipolytic processing of remnants and increased VLDL production associated with increased levels of apoE. PARADOX IV: ApoE2 is associated with recessive inheritance of this disorder, whereas other defective apoE variants are associated with dominant inheritance. Determinants of the mode of inheritance are the differential binding of apoE variants to the LDL receptor versus the HSPG/LRP complex and the preference of certain apoE variants for specific lipoproteins. Thus, the pathogenesis of this sometimes mysterious disorder has been clarified.

Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia.

The molecular mechanisms of hypertriglyceridemia (HTG), a common lipid metabolic disorder in humans, often of genetic origin, are not well understood. In studying the effect of apolipoprotein (apo) E on the metabolism of triglyceride-rich lipoproteins, we found that expressing high plasma levels of human apoE3 in transgenic mice lacking endogenous mouse apoE caused HTG. These transgenic animals had 3-fold higher plasma triglyceride levels, higher very low density lipoproteins (VLDL), and lower high density lipoproteins than did nontransgenics. Removing one or both low density lipoprotein receptor alleles in the apoE3-overexpressing mice caused severe HTG (8-11-fold over nontransgenics) and increased VLDL and decreased low and high density lipoproteins, and apoE3-enriched VLDL were markedly depleted in apoC-II. At least two mechanisms could explain HTG associated with apoE3 overexpression: stimulated VLDL triglyceride production and impaired VLDL lipolysis. The apoE3 mice with HTG had a 50% increase in hepatic VLDL triglyceride production. Furthermore, overexpression of apoE (E2, E3, or E4) in cultured hepatocytes (McA-RH7777 cells) correlated positively with secretion of VLDL into the medium. However, apoE3 overexpression-associated HTG was only partially explained by VLDL overproduction, as lipoprotein lipase-mediated VLDL lipolysis was also decreased 20-86% depending on apoE3 levels, most likely by displacing or masking apoC-II on the particles. In human subjects, HTG correlated positively with increased VLDL triglyceride and plasma and VLDL apoE levels. However, plasma and VLDL apoE correlated negatively with VLDL apoC-II levels and lipoprotein lipase-mediated VLDL lipolysis. Thus, optimal expression of apoE is crucial for normal metabolism of triglyceride-rich lipoproteins, and overexpression and/or accumulation of apoE may contribute to HTG by stimulating VLDL triglyceride production and by impairing VLDL lipolysis. The apoE3-overexpressing mice will be useful for studying the pathophysiology of this disorder.

The domain structure of human receptor-associated protein. Protease sensitivity and guanidine HCl denaturation.

The 39-kDa receptor-associated protein (RAP), a specialized chaperone for endocytic receptors of the low density lipoprotein receptor gene family, is a triplicate repeat sequence (residues 1-100, 101-200, and 201-323, respectively), with the three repeats having different functional roles. The goal of the present study was to use a combination of protease sensitivity and guanidine denaturation analyses to investigate whether human RAP correspondingly contained multiple structural domains. Protease sensitivity analysis using six proteolytic enzymes of varying specificity showed that RAP has two protease-resistant regions contained within repeat 1 (residues 15-94) and repeat 3 (residues 223-323). Guanidine denaturation analysis showed that RAP has two phases in its denaturation, an early denaturation transition at 0.6 M guanidine HCl, and a broad second transition between 1.0 and 3.0 M guanidine HCl. Analysis of the denaturation of the individual repeats showed that, despite the similarity in sequence and protease sensitivity between repeats 1 and 3, repeat 1 was a stable structure, with a sharp transition midpoint at 2.4 M guanidine HCl, while repeat 3 was relatively unstable, with a transition midpoint at 0.6 M guanidine HCl. Repeat 2 had a denaturation profile almost identical to that of repeat 3. Denaturation analysis of the contiguous repeats 1 and 2 (residues 1-210) indicated that repeats 1 and 2 probably interact to form one structural domain represented by the broad transition, while repeat 3 constitutes a separate domain represented by the early transition. A two-domain model of RAP three-dimensional structure is proposed that integrates both structural and functional information, in which a helical segment from repeat 2 interacts with the known three-helix bundle of repeat 1 to form a four-helix bundle structural domain, while repeat 3 forms the other structural domain.

Apolipoprotein E2 reduces the low density lipoprotein level in transgenic mice by impairing lipoprotein lipase-mediated lipolysis of triglyceride-rich lipoproteins.

Apolipoprotein (apo) E2 is often associated with low levels of low density lipoprotein (LDL) cholesterol and high levels of plasma triglycerides in humans. Mice expressing apoE2 also have low LDL levels. To evaluate the possible role of the LDL receptor in the cholesterol-lowering effect of apoE2, we bred transgenic mice expressing low levels of apoE2 with LDL receptor-null mice (hE2(+/0), LDLR-/-). Even in the absence of the LDL receptor, plasma total and LDL cholesterol levels decreased progressively with increasing levels of plasma apoE2. At plasma apoE2 levels >20 mg/dl, LDL cholesterol was approximately 45% lower than in LDLR-/- mice. Thus, the LDL cholesterol-lowering effect of apoE2 is independent of the LDL receptor. In contrast, plasma triglyceride levels increased (mostly in very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL)) progressively as apoE2 levels increased. At plasma apoE2 levels >20 mg/dl, triglycerides were approximately 150% higher than in LDLR-/- mice. Furthermore, in apoE-null mice (hE2(+/0), mE-/-), apoE2 levels also correlated positively with plasma triglyceride levels, suggesting impaired lipolysis in both hE2(+/0),LDLR-/- and hE2(+/0),mE-/- mice. Incubating VLDL or IDL from the hE2(+/0),LDLR-/- or the hE2(+/0),mE-/- mice with mouse postheparin plasma inhibited lipoprotein lipase-mediated lipolysis of apoE2-containing VLDL and IDL by approximately 80 and approximately 70%, respectively, versus normal VLDL and IDL. This observation was confirmed by studies with triglyceride-rich emulsion particles, apoE2, and purified lipoprotein lipase. Furthermore, apoE2-containing VLDL had much less apoC-II than normal VLDL. Adding apoC-II to the incubation partially corrected the apoE2-impaired lipolysis in apoE2-containing VLDL or IDL and corrected it completely in apoE2-containing emulsion particles. Thus, apoE2 lowers LDL cholesterol by impairing lipoprotein lipase-mediated lipolysis of triglyceride-rich lipoproteins (mostly by displacing or masking apoC-II). Furthermore, the effects of apoE2 on both plasma cholesterol and triglyceride levels are dose dependent and act via different mechanisms. The increase in plasma cholesterol caused by apoE2 is due mostly to impaired clearance, whereas the increase in plasma triglycerides is caused mainly by apoE2-impaired lipolysis of triglyceride-rich lipoproteins.

Genetic factors precipitating type III hyperlipoproteinemia in hypolipidemic transgenic mice expressing human apolipoprotein E2.

Several factors are hypothesized to precipitate or exacerbate type III hyperlipoproteinemia (HLP) in humans. Among such factors are those that directly overload remnant lipoprotein production or disrupt removal pathways, including an increased ratio of apolipoprotein (apo) E2 to normal apoE, overproduction of apoB-containing lipoproteins, and decreased LDL receptor activity. Hypolipidemic apoE2-transgenic mice bred onto an apoE-null background had dramatically higher plasma total cholesterol (192 +/- 26 mg/dL for males, 203 +/- 40 mg/dL for females) and triglyceride (295 +/- 51 mg/dL for males, 277 +/- 58 mg/dL for females) levels than apoE2 mice with endogenous mouse apoE. Thus, eliminating normal apoE in the presence of apoE2 (thereby increasing the relative abundance of the defective ligand) can convert a hypolipidemic to a hyperlipidemic phenotype. Hypolipidemic apoE2 transgenic mice overexpressing human apoB had moderate remnant accumulation compared with apoE2-only or apoB-only transgenic mice, indicating that overproduction of apoB-containing lipoproteins in the presence of apoE2 can augment remnant production. Hypolipidemic apoE2 transgenic mice bred-onto an LDL receptor-null background had markedly higher plasma total cholesterol (288 +/- 51 mg/dL for males, 298 +/- 73 mg/dL for females) and triglyceride (356 +/- 72 mg/dL for males, 317 +/- 88 mg/dL for females) levels than apoE2-only mice, and remnant accumulation increased even in apoE2 mice with a heterozygous LDL receptor-knockout background (compared with apoE2-only mice), suggesting that reducing or eliminating a major receptor-mediated remnant-removal pathway in the presence of apoE2 can also precipitate a hyperlipidemic phenotype. In all cases where either lipoprotein remnant production or removal pathways were severely stressed, increased remnant accumulation was apparent. As judged by the chemical characteristics of the remnant lipoproteins, the lipoprotein phenotype was quite similar to that of human type III HLP, especially in the apoE2-expressing mice with no endogenous apoE or LDL receptors, and thus these mice represent improved models of the disorder.

Apolipoprotein E2 transgenic rabbits. Modulation of the type III hyperlipoproteinemic phenotype by estrogen and occurrence of spontaneous atherosclerosis.

Transgenic rabbits were produced that expressed high plasma levels (30-70 mg/dl) of human apolipoprotein (apo) E2(Cys-158), an apoE variant associated with the human genetic disorder type III hyperlipoproteinemia (HLP). Male transgenic rabbits fed normal chow had up to 8-fold (289 +/- 148 mg/dl) and 15-fold (697 +/- 452 mg/dl) increases in plasma total cholesterol and triglycerides, respectively, compared with nontransgenic males. Female transgenic rabbits had only a modest hyperlipidemia (total cholesterol, 140 +/- 46 mg/dl; total triglycerides, 174 +/- 66 mg/dl). Both sexes displayed the hallmarks fo type III HLP: beta-migrating very low density lipoproteins (beta-VLDL) (intestinal and hepatic remnant lipoproteins) and significantly increased VLDL and intermediate density lipoproteins. Apolipoprotein E2-containing VLDL particles were cleared from teh circulation more slowly and were more resistant to lipoprotein lipase-mediated lipolysis than normal VLDL. Only females had increased high density lipoproteins (HDL) (40%), which were shifted from typical small HDL to larger HDL1. Plasma apoE2 was predominantly associated with beta-VLDL in males and with HDL in females. To ascertain reasons for the phenotypic gender difference, we treated male transgenic rabbits with 17alpha-ethinyl estradiol. Estrogen treatment for 10 days dramatically decreased total cholesterol (73%) and triglycerides (89%) and converted beta-VLDL to pre-beta-migrating VLDL. Concomitantly, lipoprotein lipase and hepatic lipase activities increased by 90%, low density lipoprotein receptor activity was stimulated significantly, apoE2 was redistributed to HDL, and HDL were converted to HDL1. Conversely, ovariectomy in female transgenic rabbits significantly increased total cholesterol (75%), triglycerides (117%), and beta-VLDL, while decreasing lipoprotein lipase and hepatic lipase activities by 35% and redistributing apoE2 to the beta-VLDL. Thus, estrogen status appears to be responsible for much of the gender difference of the lipoprotein phenotype, mainly by modulating both lipase and low density lipoprotein receptor activities. Furthermore, transgenic rabbits fed normal chow for 11 months developed fatty streaks, and some had more advanced atherosclerotic lesions, especially around the aortic arch and proximal abdominal aorta. The lesions were more extensive in males, roughly correlating with the magnitude of the hyperlipidemia. Therefore, high plasma levels of human apoE2 in transgenic rabbits result in a type III HLP phenotype, in which males have both more severe hyperlipidemia and more extensive atherosclerosis than females.

Hypolipidemic and hyperlipidemic phenotypes in transgenic mice expressing human apolipoprotein E2.

Transgenic mice were produced that expressed different plasma levels (3-60 mg/dl) of human apolipoprotein (apo) E2(Arg158 --> Cys), which is associated with the recessive form of human type III hyperlipoproteinemia (HLP). In transgenic mice fed a normal chow diet, low levels of apoE2 (<10 mg/dl) did not significantly alter the lipid phenotype. Mice expressing intermediate levels of apoE2 (10-30 mg/dl) had a 50-60% decrease in total cholesterol compared with nontransgenic mice. The decrease was almost entirely due to a reduction in high density lipoprotein (HDL) cholesterol. These hypolipidemic apoE2 transgenic mice were cross-bred with human apoB transgenic mice, which have increased total cholesterol and low density lipoprotein (LDL) levels. The apoE2/apoB double transgenics revealed that expression of apoE2 on the background of human apoB overexpression resulted in a substantial decrease in LDL and HDL cholesterol and a corresponding accumulation of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL). Thus, the double transgenics had a lipid phenotype resembling human type III HLP. In contrast to the hypolipidemic mice, mice expressing high levels of apoE2 (>50 mg/dl) were hyperlipidemic. The VLDL and IDL in these mice were significantly increased and cholesterol-enriched and had other characteristics of remnant lipoproteins. Upon agarose gel electrophoresis, the VLDL and IDL from both intermediate and high expressers migrated more slowly toward the beta position compared with the pre-beta-mobility of nontransgenic mouse VLDL and IDL. Thus, depending on plasma apoE2 levels, the expression of human apoE2 in the transgenic mice leads to either a hypolipidemic or hyperlipidemic phenotype. This animal model provides the opportunity to study the factors that cause hypolipidemia and those that precipitate the hyperlipidemia of type III HLP.

Co-expression of cholesteryl ester transfer protein and defective apolipoprotein E in transgenic mice alters plasma cholesterol distribution. Implications for the pathogenesis of type III hyperlipoproteinemia.

Despite the definite etiologic link between apolipoprotein (apo) E mutations and type III hyperlipoproteinemia (HLP), it is not clear what additional factors are involved in the development of florid hyperlipidemia and how to explain the wide variability in the expression of the hyperlipidemic phenotype in carriers of receptor binding-defective apoE variants. The present study was designed to determine whether the overexpression of cholesteryl ester transfer protein (CETP), a plasma protein that transfers cholesteryl esters from the high density lipoproteins (HDL) to the very low density lipoproteins (VLDL) and whose activity is increased in hyperlipidemic states, plays a role in the development of hyperlipidemia and beta-VLDL accumulation in type III HLP. We produced double-transgenic mice that co-expressed high levels of simian CETP and either high or low levels of a human receptor binding-defective apoE variant, apoE(Cys-142). We previously reported that apoE(Cys-142) high-expresser mice showed spontaneous hyperlipidemia and accumulation of beta-VLDL, whereas the low-expresser mice showed only a modest increase in VLDL cholesterol. Co-expression of CETP induced a massive transfer of cholesteryl esters from the HDL to the VLDL in both lines of double-transgenic mice. As a result, HDL cholesterol and apoA-I levels were reduced to about 50% of normal, VLDL cholesterol increased 2.5-fold, and the cholesteryl ester content of VLDL reached values similar to those observed in human beta-VLDL. The ratio of defective to normal apoE in VLDL was unaffected by CETP co-expression and was higher in animals expressing high apoE levels. Finally, in spite of an increased accumulation of beta-VLDL in the high-expresser mice, the VLDL of the low-expresser mice maintained pre-beta mobility upon co-expression of CETP. The results of this study demonstrate that the ratio of defective to normal apoE on the VLDL, rather than the cholesteryl ester content of VLDL, is the major factor determining the development of severe hyperlipidemia and the formation and accumulation of beta-VLDL in type III HLP.

Susceptibility to diet-induced atherosclerosis in transgenic mice expressing a dysfunctional human apolipoprotein E(Arg 112,Cys142).

Transgenic mice expressing apolipoprotein (apo) E(Cys 142), a human defective variant of apo E, have elevated levels of plasma cholesterol, triglycerides, and very-low-density lipoproteins (VLDL); beta-VLDL, the biochemical hallmark of the human genetic disease type III hyperlipoproteinemia (HLP), is also present in these mice. This study was designed to determine whether these type III HLP mice have an increased susceptibility to spontaneous or diet-induced atherosclerosis. Three 4-month-old male transgenic mice and three male nontransgenic littermates were assessed for the presence of atherosclerotic lesions in the proximal aorta. No lipid-stained microscopic lesions were visible in the aortas of nontransgenic mice, whereas minimal lesions were observed on the aortic valve stumps of transgenic mice. To magnify the effect of the mutant apo E on the susceptibility of the transgenic animals to atherosclerosis, 8 transgenic and 8 nontransgenic mice were fed a synthetic diet containing 1% cholesterol, 16% fat, and 0.5% cholic acid for 3 months. The diet induced an increase in plasma cholesterol level in both transgenic and nontransgenic mice. However, the increase in plasma cholesterol level in the transgenic mice was all in the VLDL fraction, whereas in nontransgenic mice it was due to increases in both VLDL and high-density lipoprotein (HDL) fractions. Plasma triglyceride levels fell in both groups of mice. After 3 months on the diet, there were compositional changes in the VLDL of both groups, characterized mainly by higher cholesteryl ester content, that resulted in beta-migration on agarose gel electrophoresis. Despite similar VLDL lipid compositions, the extent of atherosclerosis differed markedly in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered lipoprotein metabolism in transgenic mice expressing low levels of a human receptor-binding-defective apolipoprotein E variant.

Transgenic mouse lines were produced that expressed low levels of a receptor-binding-defective variant of human apolipoprotein (apo) E, apoE(Arg112, Cys142). In transgenic mice, the human apoE was produced only by the kidney, whereas endogenous mouse apoE was produced mainly by the liver. The plasma concentration of the transgenic protein was about half that of endogenous apoE. The expression of transgenic apoE did not affect total plasma cholesterol and triglyceride levels, but the distribution of the human variant differed from that of endogenous apoE in the intermediate size and density range, where the transgenic protein accumulated selectively. Immunoblots of agarose gels of lipoprotein fractions showed that the transgenic protein occurred primarily on large alpha-migrating particles (HDL1). This phenomenon was not observed in transgenic mice expressing normal human apoE-3, which distributed like endogenous apoE, suggesting that the defective apoE variant perturbed HDL1 metabolism. In mice fed a high-fat, high-cholesterol diet, the transgenic apoE associated primarily with the apoB-containing lipoproteins. A significantly higher increase in very low density lipoprotein cholesterol was observed in fat-fed transgenics compared to fat-fed nontransgenic mice, suggesting a metabolic perturbation of apoB-containing lipoproteins. Thus, the receptor-binding-defective variant, apoE(Arg112, Cys142), expressed at low levels by the kidney, alters lipoprotein metabolism in transgenic mice, presumably by interfering with apoE-mediated removal of the lipoproteins from circulation.

Type III hyperlipoproteinemic phenotype in transgenic mice expressing dysfunctional apolipoprotein E.

Transgenic mice were prepared that expressed a dysfunctional apo E variant, apo E (Arg-112, Cys-142), which is associated with dominant inheritance of type III hyperlipoproteinemia (type III HLP) in humans. Among eight founder mice, plasma apo E (Arg-112, Cys-142) levels varied 100-fold and directly correlated with plasma cholesterol and triglyceride levels. On a normal chow diet, mice expressing high levels (> 70 mg/dl) of the dysfunctional apo E had grossly elevated plasma lipids, with cholesterol levels of up to 410 mg/dl and triglyceride levels of up to 1,210 mg/dl. Upon agarose electrophoresis, plasma from these mice demonstrated beta-very low density lipoproteins (beta-VLDL). Mice expressing low (< 2.5 mg/dl) or intermediate (21 mg/dl) levels of the apo E variant had much less severe hyperlipidemia and did not have beta-VLDL. Although the transgenic mouse beta-VLDL were enriched in cholesteryl esters compared with normal mouse VLDL, they were not as cholesterol enriched as human beta-VLDL from type III HLP subjects. Transgenic mouse beta-VLDL injected into normal mice were cleared from plasma at a significantly slower rate than normal mouse VLDL, demonstrating the impaired catabolism of beta-VLDL. Thus, transgenic mice expressing high levels of the dysfunctional apo E (Arg-112, Cys-142) variant have many characteristics of the human type III HLP phenotype and appear to be a suitable animal model for this disorder.

Preferential association of apolipoprotein E Leiden with very low density lipoproteins of human plasma.

Apolipoprotein (apo) E Leiden is a rare variant of human apoE characterized by defective receptor binding and associated with dominant transmission of type III hyperlipoproteinemia. In heterozygotes, apoE Leiden is present in higher concentrations in both total plasma and very low density lipoproteins (VLDL) than the other apoE allele product. In the present study we analyzed cell expression and plasma lipoprotein association of apoE Leiden to determine whether the unequal concentration of the two apoE allele products could be explained by differences in secretion rate from the hepatocyte or by preferential association with VLDL. We transfected the rat hepatoma cell line McA-RH7777 with apoE Leiden or normal human apoE3, and studied their secretion and media distribution. In pulse-chase experiments, the secretion of apoE Leiden was comparable to that of both human apoE3 and rat endogenous apoE, approaching 100% in 90 min. In similar transfection experiments, secreted apoE Leiden was significantly less glycosylated than normal apoE3 (21.7% vs. 36.6%, P < 0.005, n = 4), a finding also noted for apoE Leiden in human plasma. In in vitro incubation experiments, apoE Leiden showed a markedly higher preference for VLDL of normolipidemic human plasma when compared to both apoE3 (2.6-fold, P < 0.001) and apoE4 (1.6-fold, P < 0.001). These results suggest that the accumulation of apoE Leiden in VLDL derives from a high affinity of the mutant protein for the VLDL. This enrichment in defective apoE probably exacerbates impairment of VLDL removal from the circulation, thus contributing to the dominant transmission of type III hyperlipoproteinemia.

The role of apolipoprotein E genetic variants in lipoprotein disorders.

Apolipoprotein E plays a central role in lipoprotein metabolism by serving as a ligand for the binding of lipoproteins to lipoprotein receptors. Both common and rare variants of apoE have been described. The common variants apoE2 and apoE4 have a significant impact on interindividual variation of lipid and lipoprotein levels in normal subjects. The common variant apoE2 and more than half a dozen rare variants are defective in binding to the low-density lipoprotein (LDL) receptor, and all are causally associated with the lipid disorder type III hyperlipoproteinaemia (HLP). The mode of inheritance of the disorder can be either dominant or recessive, depending on the particular mutation(s) in apoE, although the mechanisms involved are not fully understood. The common variant apoE4 and other rare variants have been reported to be associated with a variety of other lipoprotein disorders, but a causal link has not been established.

Synthesis and secretion of apolipoprotein E occur independently of synthesis and secretion of apolipoprotein B-containing lipoproteins in HepG2 cells.

The synthesis and secretion of apolipoprotein (apo) E, a major protein component of very low density lipoproteins, were examined in the human hepatoma cell line HepG2 under metabolic conditions known to stimulate lipogenesis and the production of apoB-containing lipoproteins. When HepG2 cells were incubated in the presence of fetal bovine serum (5 or 10%) or canine chylomicron remnants (5 or 10 micrograms of protein), the secretion of triglycerides and cholesteryl esters of lipoproteins of d less than 1.063 g/ml increased, as determined by the incorporation of [14C]acetate. Determination of the distribution of apoE among media lipoproteins by agarose column chromatography showed that the majority of secreted apoE was associated with large lipoproteins when cells were incubated with fetal bovine serum. However, immunoblot analysis of total media apoE revealed that incubating cells with or without the lipogenic factors had no effect on the amount of apoE secreted. Pulse-chase and continuous labeling experiments demonstrated that the synthesis and secretion of apoE did not vary under the different metabolic conditions, even though there was a 5-fold increase in apoB secretion in response to increased lipogenesis. Neither apoE nor apoB mRNA levels responded to the lipogenic stimuli. We conclude that the synthesis and secretion of apoE are independent of the production of apoB-containing lipoproteins in HepG2 cells.

The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.

Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.

Two apolipoprotein E5 variants illustrate the importance of the position of additional positive charge on receptor-binding activity.

Apolipoprotein (apo) E polymorphism has a significant effect on plasma cholesterol and low density lipoprotein cholesterol concentrations. The association of two apoE5 isoforms with elevated plasma low density lipoprotein cholesterol levels in two unrelated subjects led us to investigate the primary structures and receptor-binding properties of their apoE. Cysteamine modification and isoelectric focusing demonstrated that the apoE5 isoform from subject 1 did not contain cysteine but that the apoE5 isoform from subject 2 contained one residue of cysteine. The structural mutation in the apoE5 isoform of subject 1 was determined by peptide sequencing. Like apoE4, this variant had arginine at position 112 but differed from apoE4 by the substitution of arginine for proline at position 84. When purified and subjected to a competitive binding assay, this apoE5(84 Pro----Arg, 112 Cys----Arg) variant had the same receptor-binding activity as normal apoE3. Because subject 2 was of Japanese descent and her apoE5 contained one cysteine residue, we suspected that it would contain the lysine-forglutamic acid mutation at position 3 that has been described previously in Japanese subjects. This was confirmed by directly sequencing the first 10 amino acid residues of her apoE. When subjected to the competitive binding assay, the total apoE from subject 2, which consisted of approximately equal amounts of normal apoE3 and apoE5(3 Glu----Lys), had a binding activity of 188%, confirming the previously reported enhanced binding of this variant. These results demonstrate that the enhancement of receptor-binding activity of more basic isoforms of apoE depends on the position at which additional positively charged amino acids are incorporated.

Genetic defects in lipoprotein metabolism. Elevation of atherogenic lipoproteins caused by impaired catabolism.

Certain proteins (called apolipoproteins B and E) on the surface of lipoprotein particles are responsible for mediating the binding of cholesterol-rich particles to specific lipoprotein receptors on the surface of cells and represent a major pathway controlling blood cholesterol levels. Three important disorders of lipoprotein metabolism, which provide insights into the molecular mechanisms responsible for the elevation of specific atherogenic lipoproteins, are the following: (1) Type III hyperlipoproteinemia results from specific mutations in apolipoprotein E that prevent the normal binding of chylomicron remnants and very-low-density lipoprotein remnants to lipoprotein receptors. Patients with this disorder who have elevated levels of these remnant lipoproteins develop atherosclerosis. (2) Familial defective apolipoprotein B-100 results from a single amino acid substitution in apolipoprotein B that prevents low-density lipoprotein from binding normally to the low-density lipoprotein receptor and elevates plasma cholesterol levels. (3) Familial hypercholesterolemia, which results in elevated levels of plasma low-density lipoprotein and premature atherosclerosis, is caused by a variety of mutations in the low-density lipoprotein receptor that interfere with the normal binding of lipoproteins to this receptor. These observations not only provide insights into the mechanisms responsible for normal lipoprotein metabolism, but also highlight the potential role of specific lipoproteins in atherogenesis.