Chylomicron remnant uptake in the livers of mice expressing human apolipoproteins E3, E2 (Arg158-->Cys), and E3-Leiden

Apolipoprotein E2 (apoE2) and apoE3-Leiden cause chylomicron remnant accumulation (type III hyperlipidemia). However, the degree of dyslipidemia and its penetrance are different in humans and mice. Remnant uptake by isolated liver from apoE-/- mice transgenic for human apoE2, apoE3-Leiden, or apoE3 was measured. In the presence of both LDL receptor (LDLR) and LDL receptor-related protein (LRP), remnant uptake was apoE3>E3-Leiden>E2 mice. Absence of LDLR reduced uptake in apoE3 and apoE3-Leiden-secreting livers but not in apoE2-secreting livers. LRP inhibition with receptor-associated protein reduced uptake in apoE3- and apoE2-secreting livers, but not in apoE3-Leiden-secreting livers, regardless of the presence of LDLR. Fluorescently labeled remnants clustered with LRP in apoE3-secreting livers only in the absence of LDLR, but clustered in livers that expressed apoE2 even in the presence of LDLR, and did not cluster with LRP in livers of apoE3-Leiden even in the absence of LDLR. Remnants were reconstituted with the three human apoE isoforms. Removal by liver of mApoe-/-/mldlr-/- mice expressing the human LDLR was slightly greater than removal in the previous experiments with apoE3>E2> E3-Leiden. Thus, in vivo, human apoE2 is cleared primarily by LRP, apoE3-Leiden is cleared only by the LDLR, and apoE3 is cleared by both.     

Heterozygosity for apolipoprotein E-4Philadelphia(Glu13----Lys, Arg145----Cys) is associated with incomplete dominance of type III hyperlipoproteinemia.

Apolipoprotein (apo) E-4Philadelphia is a double mutant of apoE in which residue 13 of the mature protein, glutamic acid (GAG), is replaced by lysine (AAG) and amino acid 145, arginine (CGT), is converted to cysteine (TGT). These mutations result in two restriction fragment length polymorphisms for the enzymes AvaI and BbvI, a smaller apparent molecular weight of apoE-4Philadelphia on sodium dodecyl polyacrylamide gels, and severe type III hyperlipoproteinemia (HLP) in a 24-year-old homozygous female (Lohse, P., Mann, W. A., Stein, E. A., and Brewer, H. B., Jr. (1991) J. Biol. Chem. 266, 10479-10484). In the current study, we have extended our analysis to include nine additional family members of the Philadelphia kindred spanning four generations. DNA and protein analysis demonstrated that the originally described propositus is a true homozygote for the epsilon-4Philadelphia allele and that six of the nine family members are heterozygous for the mutated allele and the normal epsilon-3 allele or, in one case, the epsilon-4 allele. Heterozygosity for apoE-4Philadelphia leads to the expression of a moderate form of type III HLP without clinical manifestations. These results are consistent with a dominant mode of inheritance of this dyslipoproteinemia. The simultaneous presence of unaffected individuals, heterozygotes, and a homozygote in the Philadelphia kindred makes it possible for the first time to demonstrate that the mutant apoE exhibits an incomplete or partial dominance of type III HLP. Heterozygosity for the normal epsilon-3 allele appears to have an influence on the expression of type III HLP, resulting in a phenotype intermediate between that of the two homozygous states.     

Apolipoprotein E2-Dunedin (228 Arg replaced by Cys): an apolipoprotein E2 variant with normal receptor-binding activity

Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg----Cys) invariably gives rise to dysbetalipoproteinemia, and when associated with obesity or a gene for hyperlipidemia, results in type III hyperlipoproteinemia. The association of the E2/2 phenotype with type IV/V hyperlipoproteinemia rather than type III hyperlipoproteinemia in identical twin brothers led us to investigate the primary structure of their apoE. Lipoprotein electrophoresis on agarose gels confirmed the presence of increased very low density lipoproteins (VLDL) and chylomicrons but little, if any, beta-VLDL, indicating that these subjects did not have dysbetalipoproteinemia. When the apoE from these twins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a system that can distinguish apoE2(158 Arg----Cys) from all other known apoE variants, it gave rise to two components. One had the unique mobility of apoE2(158 Arg----Cys), and one migrated in the position of the other variants of apoE (and normal apoE3), indicating that the brothers were heterozygous for apoE2(158 Arg----Cys) and a second apoE2 isoform. Cysteamine modification and isoelectric focusing showed that, like apoE2(158 Arg----Cys), the second apoE2 isoform also contained two cysteine residues. The structural mutation in the second apoE2 isoform was determined by peptide sequencing. Like normal apoE3, this variant had arginine at position 158, but differed from apoE3 by the substitution of cysteine for arginine at position 228. Total apoE isolated from the brothers had the same receptor-binding activity in a competitive binding assay as a 1:1 mixture of normal apoE3 and apoE2(158 Arg----Cys).(ABSTRACT TRUNCATED AT 250 WORDS)     

Contributions of the carboxyl-terminal helical segment to the self-association and lipoprotein preferences of human apolipoprotein E3 and E4 isoforms

To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.     

Apolipoprotein E-4Philadelphia (Glu13----Lys,Arg145----Cys). Homozygosity for two rare point mutations in the apolipoprotein E gene combined with severe type III hyperlipoproteinemia.

The molecular defect in a 24-year-old white female with severe type III hyperlipoproteinemia has been elucidated. The patient's apolipoprotein (apo) E migrated in the apoE-4 position on isoelectric focusing gels. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apoE-4 variant had a smaller apparent molecular weight than apoE-4(Cys112----Arg). Sequence analysis of DNA amplified with the polymerase chain reaction revealed two nucleotide substitutions in the proband's apoE gene. A C to T mutation converted arginine (CGT) at position 145 of the mature protein to cysteine (TGT) thus creating the apoE-2 variant. A second G to A substitution at amino acid 13 led to the exchange of lysine (AAG) for glutamic acid (GAG), thereby adding 2 positive charge units to the protein and producing the apoE-5 variant. Computer analysis of the apoE-4Philadelphia gene revealed that the G to A mutation in exon 3 resulted in the loss of an AvaI restriction enzyme site. The second mutation, a C to T substitution in the fourth exon of the apoE gene, eliminated a cleavage site for the enzyme BbvI. Using these restriction fragment length polymorphisms as well as DNA sequence analysis we have demonstrated that the patient is homozygous for both point mutations in the apoE gene.     

Molecular cloning of a human apolipoprotein E variant: E5 (Glu3----Lys3)

Two types of apoE alleles from a genomic DNA library of a subject with the apoE2/E5 phenotype have been cloned. One of these alleles encodes an apoE isoprotein having cysteine, arginine, and cysteine at positions 112, 145, and 158 of mature apoE isoproteins, respectively, indicating an epsilon apoE2 allele. The other allele encodes a different isoprotein, having cysteine, arginine, and arginine at each of the above positions, respectively. In addition, this allele has a base substitution (G----A) which causes the substitution of lysine for glutamic acid at position 3 near the NH2-terminus of the mature apoE. This allele is a new variant, the epsilon apoE5 allele.     

Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain

Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.     

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2)

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.     

Identification of human apolipoprotein E variant gene: apolipoprotein E7 (Glu244,245----Lys244,245)

Apolipoprotein E (apoE) is one of the protein moieties of the human serum lipoproteins. Three major isoforms of apoE (apoE2, apoE3, and apoE4) and minor variant isoforms (apoE1, apoE5, and apoE7) have been detected by isoelectric focusing. In this study we have cloned the apoE7 gene from a patient with the apoE3/E7 phenotype associated with hypertriglyceridemia and diabetes mellitus. DNA sequencing revealed that the apoE7 gene has two base substitutions (G----A) changing Glu244,245----Lys244,245, compared with the apoE3 gene. The replacement of the two amino acids is consistent with the result of isoelectric focusing of the apoE7 isoprotein, which shifts to four positively charged units compared with the apoE3 isoprotein.     

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

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

Rare variant of apolipoprotein E (Arg136 -->Ser) in two normolipidemic individuals

Through the analysis of the common apolipoprotein (apo) E gene polymorphism in large Caucasian population study with the PCR and subsequent restriction analysis, we have identified carriers of mutant allele Arg136-->Ser. Both of them (71-years-old female and her 43-years-old son) have normal lipid parameters. We suggest that Arg136-->Ser mutation in apoE is not necessarily connected with elevated lipid levels in all cases. Furthermore, so far unidentified factors (environmental and/or genetic) are important for the development of lipid metabolism disorders in apoE Arg136-->Ser mutation carriers.     

The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4相似文献   

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Structural and functional variations in human apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.     

Arg343 in human surfactant protein D governs discrimination between glucose and N-acetylglucosamine ligands

Surfactant protein D (SP-D), one of the members of the collectin family of C-type lectins, is an important component of pulmonary innate immunity. SP-D binds carbohydrates in a calcium-dependent manner, but the mechanisms governing its ligand recognition specificity are not well understood. SP-D binds glucose (Glc) stronger than N-acetylglucosamine (GlcNAc). Structural superimposition of hSP-D with mannose- binding protein C (MBP-C) complexed with GlcNAc reveals steric clashes between the ligand and the side chain of Arg343 in hSP-D. To test whether Arg343 contributes to Glc > GlcNAc recognition specificity, we constructed a computational model of Arg343-->Val (R343V) mutant hSP-D based on homology with MBP-C. Automated docking of alpha-Me-Glc and alpha-Me-GlcNAc into wild-type hSP-D and the R343V mutant of hSP-D suggests that Arg343 is critical in determining ligand-binding specificity by sterically prohibiting one binding orientation. To empirically test the docking predictions, an R343V mutant recombinant hSP-D was constructed. Inhibition analysis shows that the R343V mutant binds both Glc and GlcNAc with higher affinity than the wild-type protein and that the R343V mutant binds Glc and GlcNAc equally well. These data demonstrate that Arg343 is critical for hSP-D recognition specificity and plays a key role in defining ligand specificity differences between MBP and SP-D. Additionally, our results suggest that the number of binding orientations contributes to monosaccharide binding affinity.     

Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions.

We describe sensitive new approaches for detecting and quantitating protein-lipid interactions using analytical ultracentrifugation and continuous size-distribution analysis [Schuck (2000) Biophys. J.78, 1606-1619]. The new methods were developed to investigate the binding of human apolipoprotein E (apoE) isoforms to size-fractionated lipid emulsions, and demonstrate that apoE3 binds preferentially to small lipid emulsions, whereas apoE4 exhibits a preference for large lipid particles. Although the apparent binding affinity for large emulsions is similar (Kd approximately 0.5 micro m), the maximum binding capacity for apoE4 is significantly higher than for apoE3 (3.0 and 1.8 amino acids per phospholipid, respectively). This indicates that apoE4 has a smaller binding footprint at saturation. We propose that apoE isoforms differentiate between lipid surfaces on the basis of size, and that these differences in lipid binding are due to a greater propensity of apoE4 to adopt a more compact closed conformation. Implications for the role of apoE4 in blood lipid transport and disease are discussed.     

Defective VLDL metabolism and severe atherosclerosis in mice expressing human apolipoprotein E isoforms but lacking the LDL receptor

Differences in affinity of human apolipoprotein E (apoE) isoforms for the low density lipoprotein receptor (LDLR) are thought to result in the differences in lipid metabolism observed in humans with different APOE genotypes. Mice expressing three common human apoE isoforms, E2, E3, and E4, in place of endogenous mouse apoE were used to investigate the relative roles of apoE isoforms in LDLR- and non-LDLR-mediated very low density lipoprotein (VLDL) clearance. While both VLDL particles isolated from mice expressing apoE3 and apoE4 bound to mouse LDLR with affinity and Bmax similar to VLDL containing mouse apoE, VLDL with apoE2 bound with only half the Bmax. In the absence of the LDLR, all lines of mice expressing human apoE showed dramatic increases in VLDL cholesterol and triglycerides (TG) compared to LDLR knockout mice expressing mouse apoE. The mechanism of the hyperlipidemia in mice expressing human apoE isoforms is due to impairment of non-LDL-receptor-mediated VLDL clearance. This results in the severe atherosclerosis observed in mice expressing human apoE but lacking the LDLR, even when fed normal chow diet. Our data show that defects in LDLR independent pathway(s) are a potential factor that trigger hyperlipoproteinemia when the LDLR pathway is perturbed, as in E2/2 mice.     

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

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

Rare variant of apolipoprotein E (Arg136-->Cys) in a subject with normal lipid values

During the screening of apolipoprotein (apo) E gene polymorphism with PCR and subsequent restriction analysis, we have identified a female carrier with a mutant allele Arg136-->Cys. This proband had normal lipid parameters and no history of coronary artery disease (CAD). We did not confirm the previously described connection between apo E Arg136-->Cys mutation and elevated lipid levels. In the case of this mutation, other factors (environmental and/or genetic) are important for the development of lipid metabolism disorders.