Clinical chemistry of common apolipoprotein E isoforms

Apolipoprotein E plays a central role in clearance of lipoprotein remnants by serving as a ligand for low-density lipoprotein and apolipoprotein E receptors. Three common alleles (apolipoprotein E₂, E₃ and E₄) give rise to six phenotypes. Apolipoprotein E₃ is the ancestral form. Common apolipoprotein E isoforms derive from nucleotide substitutions in codons 112 and 158. Resulting cysteine-arginine substitutions cause differences in: affinities for low-density lipoprotein and apolipoprotein E receptors, low-density lipoprotein receptor activities, distribution of apolipoprotein E among lipoproteins, low-density lipoprotein formation rate, and cholesterol absorption. Accompanying changes in triglycerides, cholesterol and low-density lipoprotein may promote atherosclerosis development. Over 90% of patients with familial dysbetalipoproteinaemia have apolipoprotein E₂/E₂. Apolipoprotein E₄ may promote atherosclerosis by its low-density lipoprotein raising effect. Establishment of apolipoprotein E isoforms may be important for patients with diabetes mellitus and several non-atherosclerotic diseases. Apolipoprotein E phenotyping exploits differences in isoelectric points. Isoelectric focusing uses gels that contain pH4–7 ampholytes and urea. Serum is directly applied, or prepurified by delipidation, lipoprotein precipitation or dialysation. Isoelectric focusing is followed by immunofixation/protein staining. Another approach is electro- or diffusion blotting, followed by protein staining or immunological detection with anti-apolipoprotein E antibodies and an enzyme-conjugated second antibody. Apolipoprotein E genotyping demonstrates underlying point mutations. Analyses of polymerase chain reaction products are done by allele-specific oligonucleotide probes, restriction fragment length polymorphism, single-stranded conformational polymorphism, the primer-guided nucleotide incorporation assay, or denaturating gradient gel electrophoresis. Detection with primers that either or not initiate amplification is performed with the amplification refractory mutation system. Disparities between phenotyping and genotyping may derive from isoelectric focusing methods that do not adequately separate apolipoprotein E posttranslational variants, storage artifacts or faint isoelectric focusing bands.     

In vitro metabolism of apolipoprotein E

Apolipoprotein E plays a major role in the uptake of chylomicrons and of very-low-density lipoprotein (VLDL) remnants by the liver. It has also been clearly demonstrated that apolipoprotein E rapidly and spontaneously exchanges between lipoproteins. To assess whether all lipoprotein-bound apolipoprotein E is available to participate in spontaneous transfer and/or exchange, the present study followed the fate of radiolabeled apolipoprotein E in an in vitro system. The results show that in vitro, apolipoprotein E can be considered as having both a spontaneously exchangeable pool and a nonexchangeable pool. Based upon specific radioactivity data, only a limited amount of apolipoprotein E originating in VLDL or in high-density lipoproteins (HDL) was capable of in vitro exchange with that in other lipoprotein fractions. Lipolysis of VLDL triacylglycerol by milk lipoprotein lipase, however, resulted in complete transfer of VLDL apolipoprotein E mass and radioactivity to HDL, supporting the potential for transformation of exchangeable apolipoprotein to a transferable pool in vivo. The results of these studies indicate that during the course of lipoprotein metabolism, conformational changes occur which alter the accessibility of apolipoprotein E. Such dynamic heterogeneity may have implications for the regulation of lipoprotein metabolism.     

Apolipoproteins and atherosclerosis. Apolipoprotein E and apolipoprotein(a) as candidate genes of premature development of atherosclerosis

Apolipoprotein E (apoE) is a plasma lipoprotein which plays a basic role in the degradation of particles rich in cholesterol and triglycerides. It is able to bind to LDL receptors, but also to receptors for chylomicron remnants. There are three major apoE isoforms, E2, E3, and E4. Their role in lipoprotein metabolism is related to their affinity for receptors. Allele E3 is predominant and apoE3 affects metabolism of lipoproteins in a standard way. When compared to allele E3, allele E2 is associated with lower LDL levels, whereas allele E4 with higher LDL levels. This has an impact on the progression of atherosclerosis. Allele E2 exhibits a protective role, whereas allele E4 is associated with a high risk factor. Lipoprotein(a) [Lp(a)] is a plasma lipoprotein, consisting of apolipoprotein(a), linked by a covalent bond with the LDL particle. Increased Lp(a) levels are associated with an increased incidence of diseases based on atherosclerosis, namely the ischemic heart disease. Another effect of Lp(a) is its competition with plasminogen, resulting in a decrease of fibrinolysis and thrombogenic activity. ApoE and Lp(a) are independent risk factors for premature development of atherosclerosis and therefore can be considered as candidate genes of premature atherosclerosis.     

Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans

Amino acid precursors labelled with stable isotopes have been successfully used to explore the metabolism of the apolipoproteins of HDL. Some methodological and mathematical modelling problems remain, mainly related to amino acid recycling in a plasma protein such as apolipoprotein A-I with a long residence time (the reciprocal of the fractional catabolic rate) of 4-5 days. Apolipoprotein A-I, apolipoprotein E, and apolipoprotein A-IV in triglyceride-rich lipoproteins (containing chylomicrons, VLDL, and remnants) exhibit more complex kinetics. The small amounts of apolipoprotein A-I and of apolipoprotein A-IV in the triglyceride-rich lipoproteins have a residence time similar to that of the apolipoprotein A-I of HDL. In contrast, the apolipoprotein E in triglyceride-rich lipoproteins has been found to have an average residence time of 0.11 days. Diets low in saturated fat and cholesterol, which lower HDL levels, do so by decreasing the secretion of apolipoprotein A-I, with apolipoprotein A-II kinetics unaffected. Individuals with impaired glucose tolerance have a decreased residence time of apolipoprotein A-I but no change in secretion rate or in apolipoprotein A-II kinetics. This suggests a link between insulin resistance and the risk of atherosclerosis. In heterozygous familial hypercholesterolemia, both the fractional catabolic rate and the secretion rate of apolipoprotein A-I are increased, resulting in no change in the plasma level. Stable isotope studies have strengthened the evidence that triglyceride enrichment of HDL increases its catabolism Laboratory.     

Apolipoprotein E Bethesda. Isolation and partial characterization of a variant of human apolipoprotein E isolated from very low density lipoproteins

A variant of apolipoprotein E, denoted E Bethesda, has been identified in the plasma of a 72-year-old woman with type III hyperlipoproteinemia. An offspring of the proband also has this variant and type III hyperlipoproteinemia. Apolipoprotein E Bethesda was isolated by preparative isoelectrofocusing followed by preparative SDS-polyacrylamide gel electrophoresis from the very low density lipoproteins of the proband's son. The purity and the identity of the preparation were analyzed by analytical SDS-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by immunochemical analysis. Apolipoprotein E Bethesda migrates in the E 1 position and its electrophoretic mobility is not affected by neuraminidase treatment. The protein is shifted to the E3 position after cysteamine treatment. The amino acid composition revealed the presence of two cysteine residues. These data support the concept that the apolipoprotein E Bethesda allele is derived from a mutation of the E2 or E2* allele.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Intracellular metabolism of triglyceride-rich lipoproteins

Over the past 10 years, many advances have been made in our understanding of the intravascular metabolism of triglyceride-rich lipoproteins. It is now known that the complex extracellular interactions of triglyceride-rich lipoprotein-associated apolipoprotein E, lipoprotein lipase and hepatic lipase with heparan sulfate proteoglycans and lipoprotein receptors facilitate the hepatocellular uptake of triglyceride-rich lipoproteins. Recent studies have also revealed that the intracellular fate of internalized triglyceride-rich lipoproteins is highly complex. The dissociation of triglyceride-rich lipoprotein components within intracellular endosomal compartments involves the recycling of apolipoprotein E, whereas the remaining lipid core associated with apolipoprotein B is susceptible to lysosomal degradation. Apolipoprotein E recycling is an important newly discovered feature of lipoprotein metabolism, and will be discussed in the context of its intracellular transport mechanisms and cholesterol efflux. Current concepts concerning its potential relevance with regard to lipoprotein metabolism and atherosclerosis will also be discussed.     

Determination of human apolipoprotein E by electroimmunoassay.

1. Apolipoprotein E ("arginine-rich" polypeptide) was isolated from delipidized human very low density lipoproteins by agarose column chromatography in the presence of 6 M guanidine-hydrochloride. 2. An electroimmunoassay ("rocket" electrophoresis) is described for quantitative determination of human serum apolipoprotein E. Purified apolipoprotein E was used for the preparation of monospecific antisera and standardization of assay. This sensitive, specific, rapid (time required for the completion of the assay is 5 h) and precise (the within- and between-assay coefficients of variation are 5 and 8%, respectively) assay is applicable to measurement of apolipoprotein E in whole serum and density classes. The results correlated well with those obtained by radial immunodiffusion (r = 0.85). 3. Serum apolipoprotein E levels of normal subjects and hyperlipoproteinemic phenotypes IIa, IIb and IV were the same (10 to 16 mg/100 ml). In contrast, patients with type III and V hyperlipoproteinemias had markedly elevated serum apolipoprotein E levels )27 and 25 mg/100 ml, respectively). The apolipoprotein E in serum of normolipidemic subjects was equally distributed among three major lipoprotein density classes: d less than 1.030 g/ml (27%), d 1.030-1.063 g/ml (36%)and d 1.063-1.21 g/ml (37%).     

Apolipoprotein M--a novel player in high-density lipoprotein metabolism and atherosclerosis

PURPOSE OF REVIEW: Apolipoprotein M is a recently described apolipoprotein predominantly associated with high-density lipoprotein, but also found in chylomicrons, very low-density lipoproteins, and low-density lipoprotein. The purpose is to review recent information on the unusual structural properties of apolipoprotein M and its possible role in formation of pre-beta high-density lipoprotein and reverse cholesterol metabolism. RECENT FINDINGS: Apolipoprotein M is a lipocalin having a coffee filter-like structure with a hydrophobic ligand-binding pocket. Mature apolipoprotein M retains its signal peptide, which serves as a hydrophobic anchor. In mice, silencing of expression in the liver with siRNA led to disappearance of pre-beta high-density lipoprotein and appearance of unusually large high-density lipoproteins. This suggests that apolipoprotein M is important for the formation of pre-beta high-density lipoprotein and reverse cholesterol transport. In accordance with this idea, hepatic overexpression of apolipoprotein M with an adenovirus in low-density lipoprotein-receptor deficient mice led to an approximately 70% reduction of atherosclerosis. In addition to the liver, apolipoprotein M is also expressed in the kidney. Kidney-derived apolipoprotein M binds to megalin, a member of the low-density lipoprotein-receptor family, which interacts with many lipocalins in renal tubuli. Apolipoprotein M is excreted in the urine of mice with a kidney-specific megalin deficiency but not in the urine of normal mice, suggesting megalin-mediated uptake of apolipoprotein M in the tubular epithelium of normal mice. SUMMARY: Apolipoprotein M is a novel apolipoprotein with unusual structural features that appears to play important roles in high-density lipoprotein metabolism and prevention of atherosclerosis.     

Characterization and metabolic fate of two very-low-density lipoprotein subfractions separated by heparin-sepharose chromatography

Human very-low-density lipoproteins (VLDL) have been separated into two discrete subfractions by heparin-Sepharose chromatography. The retained fraction relative to the unretained fraction is characterized by an increased cholesterol ester/triacylglycerol ratio and an increased ratio of apolipoprotein E relative to apolipoprotein C. We have subfractionated VLDL from type IV hyperlipoproteinemic subjects and characterized these subfractions with respect to (i) composition and (ii) the metabolic fate of apolipoprotein B of each subfraction. The unretained fraction accounted for an average of 42% of total VLDL in type IV subjects. A similar distribution was obtained with VLDL from Type III subjects; however, only 25% of normal VLDL is in the unretained fraction. The apolipoprotein E/apolipoprotein C ratio was 2-8-fold higher in the retained fraction. The distribution of apolipoprotein E isomorphs and the individual C apolipoproteins were similar in each fraction. Retained and unretained fractions were labelled with 125I and/or 131I and injected simultaneously into miniature pigs. Apolipoprotein B of retained fractions was catabolized at a greater rate (fractional catabolic rate = 0.98 h-1 vs. 0.54 h-1, n = 7, P less than 0.05) compared to unretained fractions. These results are consistent with the concept that reduced content of C apolipoproteins in VLDL is correlated with enhanced uptake by perfused rat livers. Apolipoprotein B from retained fractions was converted to intermediate-density lipoproteins (IDL) at a greater rate, and apolipoprotein B from both fractions were converted to low-density lipoproteins (LDL). Although the unretained fraction may be the precursor of the retained fraction, the possibility exists that each fraction is largely synthesized and catabolized independently.     

Apolipoprotein composition of bovine lipoproteins isolated by gel filtration chromatography

1. Bovine lipoproteins were isolated from plasma by gel filtration and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. Bovine triglyceride-rich lipoproteins contained a novel low mol. wt protein Mr = 22,000 and low mol. wt proteins that may be analogous to non-ruminant apolipoproteins A-I, A-IV, and E. 3. Apolipoprotein C appeared to be a minor constituent of bovine triglyceride-rich lipoproteins. 4. Triglyceride-rich lipoproteins contained two high mol. wt proteins of approx. Mr = 220,000 and 290,000. 5. The predominant bovine low density lipoprotein apolipoprotein was approx. Mr = 290,000, however, greater then 25 proteins were often observed between Mr = 110,000 and 370,000. 6. Bovine high density lipoprotein contained proteins analogous to apolipoprotein A-I and C apolipoproteins. 7. Differences in apolipoprotein profiles between non-lactating and lactating cows were not apparent.     

Avian apolipoprotein A-V binds to LDL receptor gene family members

Apolipoprotein A-V (apoA-V) affects plasma triglyceride (TG) levels; however, the properties of apoA-V that mediate its action(s) are still incompletely understood. It is unclear how apoA-V, whose plasma concentration is extremely low, can affect the pronounced TG differences observed in individuals with various apoA-V dysfunctions. To gain novel insights into apoA-V biology, we expanded our previous studies in the chicken to this apolipoprotein. First, we characterized the first avian apoA-V, revealing its expression not only in liver and small intestine but also in brain, kidney, and ovarian follicles and showing its presence in the circulation. Second, we demonstrate directly that galline apoA-V binds to the major LDL receptor family member (LR) of the laying hen and that this interaction does not depend on the association of the apolipoprotein with lipid or lipoproteins. We propose that a direct interaction with LRs may represent a novel, additional mechanism for the modulation of TG levels by apoA-V.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Apolipoprotein A-IV protein polymorphism: frequency and effects on lipids,lipoproteins, and apolipoproteins among Mexican-Americans in Starr County,Texas

Summary Apolipoprotein A-IV phenotypes were determined by reprobing immunoblots initially typed for the apolipoprotein E polymorphism on a representative sample of Mexican-Americans from South Texas. Typings on 331 individuals gave frequency estimates of 0.928, 0.066, 0.003, and 0.003 for alleles 1, 2, 3, and 4, respectively. To evaluate the effects of this polymorphic variability on lipid-related measures, mean levels between phenotypes were tested for equality following adjustment for age, sex, and body mass index. Analyses of levels of cholesterol, triglycerides, total high density lipoprotein, and its subfractions, low density lipoprotein, alpha and beta lipoproteins and apolipoproteins A-I, A-II, B, C-II, C-III, and E demonstrate that the A-IV genetic variability contributes minimally to normal variation of these quantitative factors in the population. Examination of the rare types, however, indicates the possibility of large metabolic effects whose follow-up may be useful for elucidating the metabolic roles of apolipoprotein A-IV.     

Structure-function relationships of apolipoprotein A-I: a flexible protein with dynamic lipid associations

PURPOSE OF REVIEW: Apolipoprotein A-I is the major structural protein of HDL. Its physicochemical properties maintain a delicate balance between maintenance of stable lipoproteins and the ability to associate with and dissociate from the lipid transported. Here we review the progress made in the last 2-3 years on the structure-function relationships of apolipoprotein A-I, including elements related to the ATP binding cassette transporter A1. RECENT FINDINGS: Current evidence now supports the so-called 'belt' or 'hairpin' models for apolipoprotein A-I conformation when bound to discoidal lipoproteins. In-vivo expression of apolipoprotein A-I mutant proteins has shown that both the N- and C-terminal domains are important for lipid association as well as for the esterification reaction, particularly binding of cholesteryl esters and formation of mature alpha-migrating lipoproteins. This property is apparently quite distinct from the activation of the enzyme lecithin cholesterol acyl transferase, which requires interaction with the central helix 6. The interaction of apolipoprotein A-I with the ATP binding cassette transporter A1 has been shown to require the C-terminal domain, which is proposed to mediate the opening of the helix bundle formed by lipid-free or lipid-poor apolipoprotein A-I and allow its association with hydrophobic binding sites. SUMMARY: Significant progress has been made in the understanding of the molecular mechanisms controlling the folding of apolipoprotein A-I and its interaction with lipids and various other protein factors involved in HDL metabolism.     

Apolipoprotein A-I-containing lipoproteins with pre-beta electrophoretic mobility

Among the apoA-I-containing lipoproteins isolated by selected-affinity immunosorption from human serum and plasma, we have identified a subpopulation which, unlike the bulk of high density lipoproteins, has pre-beta electrophoretic mobility. This pre-beta subpopulation can be observed directly in fresh plasma by immunoelectrophoresis. It contains phospholipid and free and esterified cholesterol, but protein constitutes 90% of its mass. Apolipoprotein A-I is the predominant apolipoprotein in this subpopulation; apolipoprotein A-II and the B lipoproteins are not detected. The protein moiety of this subpopulation exhibits markedly lower helicity than that of high density lipoproteins isolated by ultracentrifugation.     

Apolipoprotein A-II: beyond genetic associations with lipid disorders and insulin resistance

PURPOSE OF REVIEW: Apolipoprotein A-II, the second major HDL apolipoprotein, was often considered of minor importance relatively to apolipoprotein A-I and its role was controversial. This picture is now rapidly changing, due to novel polymorphisms and mutations, to the outcome of clinical trials, and to studies with transgenic mice. RECENT FINDINGS: The -265 T/C polymorphism supports a role for apolipoprotein A-II in postprandial very-low-density lipoprotein metabolism. Fibrates, which increase apolipoprotein A-II synthesis, significantly decrease the incidence of major coronary artery disease events, particularly in subjects with low HDL cholesterol, high plasma triglyceride, and high body weight. The comparison of transgenic mice overexpressing human or murine apolipoprotein A-II has highlighted major structural differences between the two proteins; they have opposite effects on HDL size, apolipoprotein A-I content, plasma concentration, and protection from oxidation. Human apolipoprotein A-II is more hydrophobic, displaces apolipoprotein A-I from HDL, accelerates apolipoprotein A-I catabolism, and its plasma concentration is decreased by fasting. Apolipoprotein A-II stimulates ATP binding cassette transporter 1-mediated cholesterol efflux. Human and murine apolipoprotein A-II differently affect glucose metabolism and insulin resistance. A novel beneficial role for apolipoprotein A-II in the pathogenesis of hepatitis C virus has been shown. SUMMARY: The hydrophobicity of human apolipoprotein A-II is a key regulatory factor of HDL metabolism. Due to the lower plasma apolipoprotein A-II concentration during fasting, measurements of apolipoprotein A-II in fed subjects are more relevant. More clinical studies are necessary to clarify the role of apolipoprotein A-II in well-characterized subsets of patients and in the insulin resistance syndrome.     

Cholesterol from Degenerating Nerve Myelin Becomes Associated with Lipoproteins Containing Apolipoprotein E

Apolipoprotein E is synthesized and secreted by rat sciatic nerve consequent to several types of injury. It has been proposed that endoneurial apolipoprotein E, in analogy to its role in systemic cholesterol transport, is involved in the salvage and reutilization of myelin cholesterol during degeneration and regeneration. To test this hypothesis, nerve lipids were prelabeled via intraneural injection of [3H]acetate. Four weeks later the nerves were crushed. From 1 to 12 weeks later, crushed nerves were examined for extracellular lipoprotein-bound cholesterol label. By 2 weeks after injury, 10% of the endoneurial lipid label was in a soluble form that was releasable into incubation medium. This released fraction was enriched in labeled cholesterol, and its labeled lipid composition was constant, in contrast to the changing distribution of label in the nerve with time after injury. On a KBr gradient, the released lipid label cofractionated with the released apolipoprotein E at densities similar to that of lipoproteins. These data indicate that at least some myelin cholesterol in injured nerve becomes associated with apolipoprotein E-containing lipoproteins and thus is available for reutilization via the hypothesized model.