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An important aspect of inclusion-body myositis (IBM) vacuolated muscle fibers (VMF) is abnormal accumulation of amyloid- precursor protein (APP) epitopes and its product, amyloid- (A), and of phosphorylated tau (p-tau) in the form of paired helical filaments. Lipoprotein receptors and cholesterol are known to play an important role in APP processing, A production, and tau phosphorylation.

To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136;-150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa amino-terminal fragments (residues 1;-191) of apoE3 and apoE4 bound to the LDLR with approximately 100-fold greater affinity than the 22-kDa fragment of apoE2. The pK(a) values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pK(a) of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic alpha-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity.The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pK(a) of K146 significantly, but it increased the pK(a) of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR.

Apolipoprotein E7 (apoE7) (apoE3 E244K/E245K) is a naturally occurring mutant in humans that is associated with increased plasma lipid levels and accelerated atherosclerosis. It is reported to display defective binding to low density lipoprotein (LDL) receptors, high affinity binding for heparin, and like apoE4, preferential association with very low density lipoproteins (VLDL). There are two potential explanations for the preference of apoE7 for VLDL: lysine mutations, which occur in the major lipid-binding region (residues 244-272) of the carboxy-terminal domain of apoE7, could either directly determine the lipoprotein-binding preference or could interact with negatively charged residues in the amino-terminal domain, resulting in a domain interaction similar to that in apoE4 (interaction of Arg-61 with Glu-255), which is responsible for the apoE4 VLDL preference. To distinguish between these possibilities, we determined the binding preferences of recombinant apoE7 and two amino-terminal domain mutants, apoE7 (E49Q/E50Q) and apoE7 (D65N/E66Q), to VLDL-like emulsion particles. ApoE7 and both mutants displayed a higher preference for the emulsion particles than did apoE3, indicating that the carboxy-terminal lysine mutations in apoE7 are directly responsible for its preference for VLDL. Supporting this conclusion, the carboxy-terminal domain 12-kDa fragment of apoE7 (residues 192;-299) displayed a higher preference for VLDL emulsions than did the wild-type fragment. In addition, lipid-free apoE7 had a higher affinity for heparin than did apoE. However, when apoE7 was complexed with dimyristoylphosphatidylcholine or VLDL emulsions, the affinity difference was eliminated. In contrast to previous studies, we found that apoE7 does not bind defectively to the LDL receptor, as determined in both cell culture and solid-phase assays.We conclude that the two additional lysine residues in the carboxy-terminal domain of apoE7 directly alter its lipid- and heparin-binding affinities. These characteristics of apoE7 could contribute to its association with increased plasma lipid levels and atherosclerosis.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study. Membrane microsolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 micrograms/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic alpha-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta222-243), apo A-I (Delta190-243), apo E3 (Delta192-299) and apo E4 (Delta192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film. Overall, the results indicate that insertion of amphipathic alpha-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid efflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.

The stability and structure of human apolipoprotein (apo) E3 in aqueous solution were investigated by guanidine HCl denaturation and limited proteolysis. The guanidine HCl denaturation curve, as monitored by circular dichroism spectroscopy, was biphasic; the two transition midpoints occurred at 0.7 and 2.5 M guanidine HCl, indicating that there are stable intermediate structures in the unfolding of apoE. Limited proteolysis of apoE with five enzymes demonstrated two proteolytically resistant regions, an amino-terminal domain (residues 20-165) and a carboxyl-terminal domain (residues 225-299). The region between them was highly susceptible to proteolytic cleavage. Because of their similarity to the proteolytically resistant regions, the amino-terminal (residues 1-191) and carboxyl-terminal (residues 216-299) thrombolytic fragments of apoE were used as models for the two domains. Guanidine HCl denaturation of the carboxyl- and amino-terminal fragments gave transition midpoints of 0.7 and 2.4 M guanidine HCl, respectively. The results establish that the two domains identified by limited proteolysis correspond to the two domains detected by protein denaturation experiments. Therefore, the thrombolytic fragments are useful models for the two domains. The free energies of denaturation calculated from the denaturation curves of intact apoE or the model domains were approximately 4 and 8-12 kcal/mol for the carboxyl- and amino-terminal domains, respectively. The value for the carboxyl-terminal domain is similar to those of previously characterized apolipoproteins, whereas the value for the amino-terminal domain is considerably higher and resembles those of soluble globular proteins. These studies suggest that, in aqueous solution, apoE is unlike other apolipoproteins in that it contains two independently folded structural domains of markedly different stabilities: an amino-terminal domain and a carboxyl-terminal domain, separated by residues that may act as a hinge region.

Hydrodynamic, chromatographic, and spectroscopic techniques were used to study the aqueous solution properties of the two structural domains of human apolipoprotein (apo) E3. An amino-terminal thrombolytic fragment of apoE (22 kDa, residues 1-191) and a carboxyl-terminal thrombolytic fragment of apoE (10 kDa, residues 216-299) were used as models for the two domains. Sedimentation equilibrium ultracentrifugation showed that apoE and the 10-kDa model domain self-associated predominantly as tetramers. The 22-kDa model domain was primarily monomeric. Molecular weights calculated from the weight average sedimentation and diffusion coefficients or from the sedimentation coefficients and Stokes radii were in agreement with the sedimentation equilibrium results. Derived frictional coefficients suggest larger axial ratios and/or more extensive hydration for the apoE and the 10-kDa domain tetramers as compared with the 22-kDa domain. Proteolysis of apoE followed by high performance liquid chromatography showed rapid production of free 22-kDa domain, whereas the free 10-kDa domain appeared as a tetramer late in the course of the hydrolysis. Assessment by circular dichroism demonstrated that both model domains and apoE had over 54% alpha-helical content, which changed little in a detergent (octyl-beta-D-glucopyranoside) or lipid (dimyristoylphosphatidylcholine) environment. In contrast to the circular dichroism results, apoE and the 10-kDa domain showed a marked blue shift in the fluorescence maximum in a lipid environment. The results suggest that the self-association of apoE in solution as a tetramer is mediated by the carboxyl-terminal domain and that the amino- and carboxyl-terminal domains do not associate with one another. The amino-terminal domain is most likely compact and globular, whereas the carboxyl-terminal domain is probably elongated. The isolated model domains appear to have structures that are similar to those of the domains in the intact protein.

Apolipoprotein (apo) E3-Leiden is a variant of apoE that is associated with dominant expression of type III hyperlipoproteinemia and that is defective in binding to the low density lipoprotein receptor. Therefore, the structure of apoE3-Leiden was investigated. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis apoE3-Leiden and its 22-kDa amino-terminal thrombolytic fragment migrated with a higher than normal apparent molecular weight. The structural abnormality of apoE3-Leiden was determined by sequencing its CNBr-, tryptic-, and Staphylococcus aureusV8 protease-generated peptides. In contrast to normal apoE3, which has a cysteine at residue 112, apoE3-Leiden does not contain any cysteine and has an arginine at position 112 (as does apoE4, which also completely lacks cysteine). The basis for the molecular weight difference was determined to be a seven-amino acid insertion that is a tandem repeat of residues 121-127 of normal apoE3, i.e.Glu-Val-Gln-Ala-Met-Leu-Gly, resulting in apoE3-Leiden having 306 amino acids rather than 299. The negatively charged glutamyl residues within the insertion compensates for the arginine substitution at residue 112; thus apoE3-Leiden focuses in the E3 position. The low density lipoprotein receptor binding activities of both intact apoE3-Leiden and its 22-kDa thrombolytic fragment were determined in an in vitroassay. Although apoE3-Leiden had only about 25% of normal binding activity, its 22-kDa thrombolytic fragment had nearly normal binding, suggesting that the carboxyl-terminal domain of apoE3-Leiden modulates the receptor binding function of its amino-terminal domain.

An apolipoprotein (apo) A-I variant that has a relative charge of -1 compared to normal apo-A-I on isoelectric focusing gels has been identified in five unrelated families as a result of screening a large number of individuals. The cause of the electrophoretic abnormality has been examined by analyzing the variant apo-A-I structure. The evidence suggests that a single amino acid, lysine 107, has been deleted in the variant apo-A-I of all affected individuals studied from these families, with the remainder of the variant apo-A-I sequence being unaffected. The deletion of this single basic amino acid residue is sufficient to account for the charge difference between the variant and normal apo-A-I as seen on isoelectric focusing gels. This variant, previously referred to as A-I-Marburg or A-I-Münster-2, can now be designated by the structural abnormality apo-A-I(Lys107—-0). The evidence from extensive pedigree analysis suggests the likelihood that the deletion mutant gene is allelic to the normal apo-A-I gene. At the same time, the kindred analyses have failed to yield a lipid abnormality that can be unequivocally related to the presence of this deletion mutant of apo-A-I. However, all subjects expressing apo-A-I(Lys107—-0) also express normal apo-A-I, so that any abnormality caused by the variant apo-A-I might be adequately compensated for by the normal apo-A-I. To examine directly the functional consequence of the lysine deletion, the isolated variant was tested in vitro for its ability to activate lecithin:cholesterol acyltransferase, the principal cholesterol-esterifying enzyme in plasma. It was found that apo-A-I(Lys107—-0) is deficient in its ability to activate lecithin:cholesterol acyltransferase, having only 40-60% of the cofactor activity of normal apo-A-I. The cofactor activity of the pro-apo-A-I component of the variant was also reduced to about 60% of either normal A-I or normal pro-apo-A-I. The functional defect is probably related to a disruption in the secondary and/or tertiary structure of the protein caused by the deletion of lysine 107 in the primary structure.

Human apolipoprotein (apo) E occurs as three common isoforms (apoE4, E3, and E2), all of which influence plasma cholesterol levels. Although both apoE4 and E3 bind with equal effectiveness to the low density lipoprotein receptor, they associate preferentially with different classes of plasma lipoproteins: apoE4 with very low density lipoproteins, apoE3 with high density lipoproteins. The primary structure of apoE3 differs from that of apoE4 at only a single site; apoE3 has its sole cysteine residue at position 112, while apoE4 contains arginine at position 112 and completely lacks cysteine. The present study investigated how this structural difference between apoE4 and E3 determines their distribution among plasma lipoproteins, and analyzed the role of the disulfide-linked heterodimer apoE-A-II (which apoE4 cannot form) in determining the distribution. Human plasma was incubated with 125I-labeled apoE, and lipoproteins were separated by agarose chromatography. Both apoE3 that had been reduced and alkylated with iodoacetamide and apoE3-A-II distributed with high density lipoproteins, indicating that a combination of an inherent property of the monomeric apoE3 structure and apoE-A-II formation account for distribution of apoE3 to the high density lipoproteins. Cysteamine modification of apoE3 resulted in an apoE4-like distribution, demonstrating that a positive charge at position 112 determined the apoE4 distribution and that the effect was not exclusively due to the presence of arginine at this position.(ABSTRACT TRUNCATED AT 250 WORDS)

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.

Human apolipoprotein (apo) E contains an amino- and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoform-specific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186-202 or 186-223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction.

Variant forms of apolipoprotein A-I (apo-A-I) have been shown to exist in the human population. One mutant form, referred to as apo-A-I-Münster-3, is one charge unit more basic than normal apo-A-I on isoelectric focusing gels. This variant has the same immunologic characteristics and molecular weight as normal apo-A-I. The apo-A-I-Münster-3 from subjects in three unrelated families (in two of which the trait has been shown to be transmitted as an autosomal co-dominant) has been analyzed by partial amino acid sequencing to define the cause of the electrophoretic abnormality. In the apo-A-I of family A, the abnormality was shown to occur in the smallest cyanogen bromide fragment, CB-2 (residues 87-112), and amino acid sequencing revealed asparagine instead of the usual aspartic acid at residue 103. Subjects with this mutant form have shown no signs of dyslipoproteinemia. The NH2-terminal cyanogen bromide fragment (CB-1, residues 1-86) from the apo-A-I of family B was shown to differ electrophoretically from normal CB-1, and amino acid sequencing revealed that a substitution of arginine for proline at residue 4 was responsible for this variant form. Analysis of the plasma lipids of one affected family B member demonstrated that the percentage of the total cholesterol that was esterified was somewhat lower than that normally observed. In a third family, family C, a variant having the same electrophoretic abnormality as the other two was determined to have an amino acid substitution at yet a different position. In this variant, histidine was found at residue 3 in the apo-A-I sequence, rather than the usual proline. In all three cases, the substitution could account for the electrophoretic abnormality. It is proposed that these three apo-A-I-Münster-3 variants be designated apo-A-I(Asp103—-Asn), apo-A-I(Pro4—-Arg), and apo-A-I(Pro3—-His), respectively, to indicate the substitution that accounts for the abnormality in isoelectric focusing gels.

Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, epsilon 4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid beta (beta/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic beta/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of beta/A4 peptide was saturable at 10(-4) M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound beta/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind beta/A4 peptide at pH < 6.6, whereas apoE3 bound beta/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the beta/A4 peptide. Binding of beta/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or beta/A4 peptide, and isoform-specific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.

Both apolipoprotein (apo) E2 and apoE-Leiden (tandem repeat of amino acids 121-127) are associated with type III hyperlipoproteinemia and bind defectively to low density lipoprotein receptors. Removing the carboxyl terminus of both variants (residues 192-299) increases receptor-binding activity, suggesting that the carboxyl terminus modulates activity. To identify the region(s) that modulated binding activity, we produced carboxyl-terminal truncations in apoE2 and apoE-Leiden (terminating at positions 191, 223, 244, and 272) and in apoE3 (terminating at positions 191, 223, and 244) and compared their receptor-binding activities as dimyristoylphosphatidylcholine (DMPC) discs. The results suggest that the entire carboxyl terminus up to residue 272, not a discrete smaller segment, is responsible for the modulation in apoE2. Intact apoE-Leiden and the 223 and 244 variants displayed similar activities (approximately 25% of apoE3's), but the 191 variant's activity was identical to that of intact apoE3. ApoE-Leiden and its truncated variants formed larger DMPC discs than did intact or truncated apoE3 or apoE2. These discs contained more apoE molecules than apoE3 discs, suggesting that the apparently normal binding activity of the apoE-Leiden 191 variant results from an increased number of apoE molecules and that the binding activity is actually defective. Direct comparison in a solid-phase assay revealed that the binding activity of the apoE-Leiden fragment was defective (51.4+/-9.4%). Thus, the defective binding of apoE-Leiden results from a direct effect of the seven amino acid repeat on receptor-binding activity rather than from an indirect effect operating through the carboxyl terminus as previously believed.

Apolipoprotein (apo-) E3, when combined with the phospholipid dimyristoylphosphatidylcholine (DMPC), binds avidly to apo-B,E (low density lipoprotein) receptors on human fibroblasts. Apolipoprotein E2 isolated from type III hyperlipoproteinemic subjects, which differs from apo-E3 by the presence of cysteine instead of arginine at residue 158, possesses only about 1% of the receptor binding activity of apo-E3. Modification of apo-E2 with cysteamine, which converts the cysteine at position 158 to a positively charged lysine analogue, activates receptor binding approximately 13-fold. In the present experiments, thrombin was used to cleave apo-E2 into two fragments (Mr = 22,000 and Mr = 10,000). The larger fragment, which has been shown to possess the receptor binding domain, displayed binding activity up to 12-fold greater than intact apo-E2 or equivalent to apo-E2 treated with cysteamine. When the Mr = 22,000 fragment was modified with cysteamine and combined with DMPC, receptor binding was further enhanced, attaining the level of activity of normal apo-E3 X DMPC, a 100-fold increase over apo-E2 X DMPC binding. When the cysteamine modification was reversed by incubation with beta-mercaptoethanol, the Mr = 22,000 fragment retained most of its binding activity. However, when the same sample was tested 24 h later, the level of binding activity dropped significantly. The receptor binding of apo-E2-containing beta-very low density lipoproteins could also be activated by cysteamine treatment, with the same retention of enhanced binding activity occurring after the reversal of the modification. These results indicate that apo-E2 can attain full binding activity by the removal of the carboxyl-terminal one-third of the molecule and the addition of a positive charge at residue 158 of the molecule. The retention of enhanced binding after the reversal of the cysteamine modification indicates that the enhanced binding is probably due to conformational changes induced in the binding domain (and maintained by the phospholipid) and not merely to the presence of the positive charge at residue 158.

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL) in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg+/0) and homozygous (hHLTg+/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg+/0) and 75% (hHLTg+/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE-/-) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that the huBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both the huBTg,hHLTg+/0 mice and the apoE-/-,hHLTg+/0 mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE-/- mice. The postheparin plasma HL activities were similar in the apoE-/- and the apoE-/-,HL-CAT+/0 mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in the apoE-/-,hHLTg+/0 mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of both apoE-/-,hHLTg+/0 and apoE-/-,HL-CAT+/0 mice. Plasma cholesterol and apoB-containing lipoprotein levels were approximately 60% lower in apoE-/-,HL-CAT+/0 mice than in apoE-/- mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.

The gene for human apolipoprotein (apo-) E was isolated from a human genomic library constructed in the cosmid shuttle vector pCV108. The transient expression of the apo-E gene was examined in cultured mammalian cells 48 h following calcium phosphate-mediated gene transfer. The expression of the cloned human apo-E gene, which contained between 0.7 and 29 kilobases of 5'-flanking DNA, was not restricted to human cells or to cultured cells derived from tissues that have been shown to synthesize apo-E. Several independent mouse L cell stable transfectants with the human apo-E gene integrated into their genome were selected on the basis of G418 resistance, which is conferred by the selectable gene marker in the cosmid vector. The levels of human apo-E mRNA found in the stable transfected mouse L cells ranged from undetectable to a level comparable to that found in the human liver. The size of the apo-E mRNA observed in the stable transfectants was identical to that found in the liver, indicating that the mouse L cells were capable of correctly processing the human apo-E gene transcripts. The integrated human apo-E genes had not undergone major rearrangements or deletions during transfer, and the level of apo-E mRNA found in the different stable transfectants correlated directly with the number of integrated copies of the human apo-E gene. The stable transfected L cells secreted authentic human apo-E into the medium. The secreted protein interacted specifically with antibodies to human plasma apo-E and had an apparent Mr = 35,000 to 36,000, which is slightly larger than that of plasma apo-E. The secreted human apo-E was associated with lipid (presumably phospholipids), floated at d approximately 1.09 g/ml, and bound with high affinity to the apo-B,E(LDL) receptor on fibroblasts.

The cardinal pathological features of Alzheimer disease are depositions of aggregated amyloid beta protein (A beta) in the brain and cerebrovasculature. However, the A beta is found in a soluble form in cerebrospinal fluid in healthy individuals and patients with Alzheimer disease. We postulate that sequestration of A beta precludes amyloid formation. Failure to sequester A beta in Alzheimer disease may result in amyloidosis. When we added A beta to cerebrospinal fluid of patients and controls it was rapidly sequestered into stable complexes with transthyretin. Complexes with apolipoprotein E, which has been shown to bind A beta in vitro, were not observed in cerebrospinal fluid. Additional in vitro studies showed that both purified transthyretin and apolipoprotein E prevent amyloid formation.

The binding of native rabbit beta-very low density lipoproteins (beta-VLDL) to the low density lipoprotein receptor-related protein (LRP) requires incubation with exogenous apolipoprotein (apo) E. Inclusion of a mixture of the C apolipoproteins in the incubation inhibits this binding. In the present study, the ability of the individual C apolipoproteins (C-I, C-II, and C-III) to block binding of beta-VLDL to the LRP was examined by measuring cholesteryl ester formation in mutant fibroblasts that lack low density lipoprotein receptors or by measuring binding to the LRP using ligand blotting. In each assay, both apoC-I and apoC-II inhibited binding; apoC-I was the more effective inhibitor. Apolipoprotein C-III had no effect on binding activity, regardless of its sialylation level. Binding of human apoE to rabbit beta-VLDL in the absence or presence of human apoC-I, apoC-II, and monosialo-apoC-III was also determined, by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these studies are consistent with a mechanism in which exogenous human apoE displaces the endogenous apoE and the beta-VLDL particle becomes enriched with apoE (by 4.2-fold in this study). At this higher apoE content, the beta-VLDL bound to the LRP. Inclusion of apoC-I, apoC-II, or apoC-III in the incubation mixture resulted in a differential displacement of apoE from the beta-VLDL; however, at the concentrations examined, only apoC-I and apoC-II were capable of displacing sufficient apoE to abolish binding to LRP.

A reproducible and quantitative subfractionation of human high density lipoproteins (HDL) by heparin-Sepharose affinity chromatography has been developed. Two elution methods (A and B) were used to subfractionate HDL(2) (d 1.063-1.125 g/ml) or total HDL (d 1.063-1.21 g/ml). Method A separated HDL(2) into three subclasses, each with distinct chemical properties and in vitro metabolic characteristics. The first subclass, referred to as HDL(2)-without E, passed through the affinity column unretarded and represented approximately 85% of the HDL(2) lipoprotein protein. HDL(2)-without E contained the A-I, A-II, and C apoproteins which characterize typical HDL. The second subclass eluted from the column (7-10% of the protein) contained, in addition to the A-I and A-II apoproteins, the E and (E-A-II) apoproteins, and was designated as HDL(2)-with E. The B apoprotein was the major protein component of the third subclass eluted from the column (beta lipoproteins). The beta subclass accounted for approximately 3-8% of the HDL(2) protein and was similar to Lp(a) in composition and size. Method B further subdivided the beta subclass into two fractions (beta(1) and beta(2)) with slightly different electrophoretic mobilities. The various heparin-Sepharose subclasses were further characterized by their ability to compete with (125)I-labeled low density lipoproteins (LDL) for binding to cell surface receptor sites of fibroblasts. By virtue of the presence of the E apoprotein, HDL(2)-with E competed effectively with (125)I-labeled LDL for binding to the cell surface receptors, whereas HDL(2)-without E were ineffective in competing with LDL. The beta subclass possessed binding capability similar to that of LDL. Subfractionation of HDL by heparin-Sepharose affinity column chromatography provides an attractive alternative to methods based solely on ultracentrifugation, in that it subfractionates HDL into subclasses with differing apoprotein contents that impart distinct metabolic characteristics to each class.-Weisgraber, K. H., and R. W. Mahley. Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography.

The apolipoprotein E (apoE) type 4 allele (APOE4) is a susceptibility gene for late-onset familial and sporadic Alzheimer disease. ApoE is found in some neurofibrillary tangle-bearing neurons, one of the major pathologic hallmarks of the disease. Neurofibrillary tangles contain paired helical filaments formed from hyperphosphorylated microtubule-associated protein tau. In vitro, tau binds avidly to apoE3, but not to apoE4, forming a bimolecular complex. Tau phosphorylated with a brain extract does not bind either isoform. ApoE3 binds to the microtubule-binding repeat region of tau, which is also the region that is thought to cause self-assembly into the paired helical filament. Binding studies with fragments of ApoE demonstrate that the tau-binding region of apoE3 corresponds to its receptor-binding domain and is distinct from the region that binds lipoprotein particles or beta/A4 peptide. Isoform-specific interactions of apoE with tau may regulate intraneuronal tau metabolism in Alzheimer disease and alter the rate of formation of paired helical filaments and neurofibrillary tangles.

Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity (approximately equal to 9.3 micrograms of G.R. LDL per ml were required to displace 50% of 125I-labeled normal LDL, vs. approximately equal to 3.0 micrograms of normal LDL per ml). Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated with an abnormal lipid composition or structure of the LDL: the chemical and physical properties of the particles were normal, and partial delipidation of the LDL did not alter receptor binding activity. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients.

A 37-kDa glycoprotein has been described recently, whose synthesis is dramatically increased after injury of the rat sciatic and optic nerves. Cells in the nerve sheath, distal to the site of injury, produce and secrete large amounts of this protein, so that by 3 weeks after injury, it represents 2-5% of the total soluble extracellular protein in the regenerating sciatic nerve sheath, although it fails to accumulate in damaged optic nerve. Results presented here reveal extensive homology between the 37-kDa nerve injury-induced protein and a well-studied serum protein, apolipoprotein E (apoE), that is involved in lipid and cholesterol metabolism and that has been shown recently to be present in adult and developing rat astroglia. Both proteins have identical isoelectric focusing points and similar molecular masses. Antibodies raised against the 37-kDa protein recognize apoE and anti-apoE serum crossreacts with the 37-kDa protein. Sequence data for two 14 amino acid stretches of the 37-kDa protein match identical regions of apoE. These data suggest that the 37-kDa protein is identical to serum apoE and that it could have similar functions to the latter. In the nervous system, for example, it may be involved in the mobilization and reutilization of lipid in the repair, growth, and maintenance of myelin and axonal membranes, both during development and after injury.

Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein.

Apolipoprotein (apo) E, an important protein involved in cholesterol transport in the plasma, binds with high specificity and high affinity to the apoB, E (low density lipoprotein) receptor. Several lines of evidence have indicated that key basic residues in the vicinity of residues 140-160 of apoE are important in mediating binding to the receptor. Furthermore, apoE variants exhibiting defective receptor binding are associated with the genetic lipid disorder type III hyperlipoproteinemia. To determine whether other basic amino acids in this region of apoE also affect receptor binding activity, site-specific mutagenesis of apoE in a bacterial expression system was undertaken. This system had been used successfully to produce apoE3 that was structurally and functionally equivalent to human plasma apoE3. Variants of apoE in which neutral amino acids were substituted for basic residues at positions 136, 140, 143, and 150 were produced. The variants all displayed defective binding; their activity ranged from 9 to 52% of normal (a range similar to that seen with naturally occurring variants of human apoE). In addition, to determine whether the conformation of this region is important for receptor binding, we designed variants in which proline was substituted for leucine 144 or alanine 152. Both variants were defective, exhibiting 13 and 27% of normal binding, respectively. In contrast, a double mutant in which arginine was substituted for serine 139 and alanine for leucine 149 displayed slightly enhanced receptor binding activity. These studies confirm that the middle of the apoE molecule is important in receptor binding and indicate that only certain amino acid substitutions in this region interfere with receptor binding activity.

This study was undertaken to determine if apolipoprotein (apo) E-containing lipoproteins and their receptors could provide a system for lipid transport and cholesterol homeostasis in the brain, as they do in other tissues. To accomplish this goal, the lipoproteins in human and canine cerebrospinal fluid (CSF) were characterized, and rat brain and monkey brain were examined for the presence of apoB,E(LDL) receptors. Apolipoprotein E and apoA-I were present in human and canine CSF, but apoB could not be detected. Apo-lipoprotein E and apoA-I were both present on lipoproteins with a density of approximately 1.09 to 1.15 g/ml. In human CSF, the lipoproteins were primarily spherical (approximately 140 A), whereas in canine CSF the lipoproteins were a mixture of discs (200 × 65 A) and spheres (approximately 130 A). Apolipoproteins E and A-I were contained primarily in separate populations of lipoproteins. Although the apoE of CSF was more highly sialylated than plasma apoE, the apoE-containing lipoproteins in canine CSF competed as effectively as canine plasma apoE HDLc for binding of 125I-LDL to the apoB,E(LDL) receptors on human fibroblasts. The presence of apoB,E(LDL) receptors in both rat and monkey brain was demonstrated by immunocytochemistry. Astrocytes abutting on the arachnoid space and pial cells of the arachnoid itself, both of which contact CSF, expressed apoB,E(LDL) receptors. Relatively few receptors were present in the cells of the gray matter of the cortex. Receptors were more prominent on the astrocytes of white matter and in the cells of the brain stem. The expression of apoB,E(LDL) receptors by brain cells and the presence of apoE- and apoA-I-containing lipoproteins in CSF suggest that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues.

The apolipoprotein E4 (APOE4) allele is associated with an early age of onset of the nonfamilial form of Alzheimer disease (AD) and with increased beta protein amyloid deposition in the brain. These two observations may both arise from an effect of the apoE family of proteins on the rate of in vivo amyloidogenesis. We report here that apoE3, the common apoE isoform, is an in vitro amyloid nucleation inhibitor at physiological concentrations. A significant delay in the onset of amyloid fibril formation by the beta-amyloid protein of AD (beta 1-40) was observed at a low apoE3 concentration (40 nM), corresponding to an apoE3/beta protein molar ratio of 1:1000. The inhibitory activity of a proteolytic fragment of apoE3, containing the N-terminal 191 amino acids, is comparable to the native protein, whereas the C-terminal fragment has no activity. ApoE4 is equipotent or slightly less potent than apoE3, which may be due to its inability to form a disulfide dimer, since the apoE3 dimer is a significantly more potent nucleation inhibitor than apoE4. Neither apoE3 nor apoE4 inhibits the seeded growth of amyloid or affects the solubility or structure of the amyloid fibrils, indicating that apoE is not a thermodynamic amyloid inhibitor. We propose that the linkage between the APOE4 allele and AD reflects the reduced ability of APOE4 homozygotes to suppress in vivo amyloid formation.

The beta-very low density lipoproteins (beta-VLDL) that accumulate in type III hyperlipoproteinemic subjects can be divided into two fractions (fraction I and fraction II), which differ in size, lipid composition, and the type of apolipoprotein B (apo-B) present in the particles. The apo-B48-containing particles (fraction I) are of intestinal origin, while apo-B100-containing particles (fraction II) are derived from the liver. Both fractions contain a defective form of apo-E referred to as apo-E2. Intravenous infusion of heparin into two subjects with type III hyperlipoproteinemia resulted in the complete removal of fraction II particles from density less than 1.006 g/ml, while fraction I particles remained at this density. In vitro studies confirmed that fraction I particles did not change density when subjected to hydrolysis with lipoprotein lipase, while fraction II particles shifted to the intermediate density lipoprotein range (approximately equal to 1.02 g/ml). When the beta-VLDL were hydrolyzed by lipoprotein lipase in the presence of density greater than 1.21 g/ml lipoprotein-deficient plasma, the addition of normal apo-E (apo-E3), but not apo-E2, resulted in a shift of fraction II particles to the low density lipoprotein (LDL) range (approximately equal to 1.05 g/ml). Fraction I particles did not undergo a shift to this higher density, supporting previous observations that apo-B48-containing particles are not converted to LDL. The demonstration that apo-B100-containing particles in type III hyperlipoproteinemic subjects could be converted to particles with the density of LDL suggests that apo-E plays a role in the normal conversion of VLDL to LDL. The mutant form of apo-E (apo-E2) found in the beta-VLDL from type III hyperlipoproteinemic subjects appears to impede this conversion, whereas the addition of normal apo-E (apo-E3) allows the processing to occur.

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.

Seven distinct heparin-binding sites have been demonstrated on human apolipoprotein (apo) B-100 by using a combination of digestion with cyanogen bromide or Staphylococcus aureus V-8 protease and heparin-Sepharose affinity chromatography. Based on fragment analysis, the approximate boundaries of the seven binding sites are as follows: site A, residues 5-99; site B, residues 205-279; site C, residues 875-932; site D, residues 2016-2151; site E, residues 3134-3209; site F, 3356-3489; and site G, residues 3659-3719. In sites E and F, two short regions enriched in basic amino acids have been identified, and it is likely that they are responsible for a major portion of the heparin-binding properties of these sites. The relative binding affinity of each of the seven sites was estimated in two ways. First, the affinity was assessed in a ligand blot assay using a 125I-labeled high-reactive heparin subfraction. Second, apoB-100 fragments generated by cyanogen bromide or S. aureus V-8 protease were separated into low- and high-affinity fractions by gradient salt elution of a heparin-Sepharose column. The distribution of the seven binding sites in the two fractions was determined in an immunoblotting assay using antibodies specific to each site, i.e. antibodies raised against synthetic peptide sequences found within each of the seven sites. The results of these two approaches demonstrate that site E and, to a somewhat lesser extent, site F bind to heparin with the highest affinity. Based on the analogy with apoE, in which the high-affinity heparin-binding site coincides with the domain of the protein that interacts with apoB,E (low density lipoprotein) receptors, the results of this study indicate that site E and site F, either singly or in combination, might constitute the receptor binding domain of apoB-100.

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.

Familial defective apolipoprotein (apo) B-100 is a recently described genetic disorder that appears to result from a mutation in the apoB-100 gene. This disorder is characterized by hypercholesterolemia resulting from elevated plasma concentrations of low density lipoprotein LDL. The disorder was first detected in three members of one family. The LDL from affected subjects binds defectively (approximately 30% of normal) to LDL receptors, retarding the clearance of LDL from plasma. In the present study, two other members of the affected family were found to possess abnormal LDL. In addition, abnormal LDL with a similar binding defect were found in a second, unrelated family. In both families, the defect is transmitted over three generations as an autosomal codominant trait and all affected members are heterozygotes. Since there is only one apoB-100 molecule per LDL particle, the abnormal LDL in heterozygous subjects is made up of two populations of particles: one that has normal binding activity to receptors and one that binds defectively. To localize the mutation in apoB-100, the binding of five apoB-100-specific monoclonal antibodies to abnormal LDL was assessed in a solid-phase RIA. Only antibody MB47, whose epitope is between residues 3350 and 3506, distinguished abnormal LDL from normal LDL isolated from control subjects with normal lipid levels; MB47 bound with a higher affinity (by approximately 60%) to abnormal LDL. In every individual with abnormal LDL, the MB47 antibody bound with a higher affinity. The convenience of this assay will facilitate screening of large populations to determine the frequency of this disorder.

The three major isoforms of human apolipoprotein E (apo-E2, -E3, and -E4) are coded for by three alleles (epsilon 2, epsilon 3, and epsilon 4) which have a common genetic locus. Previously, we demonstrated that E2, E3, and E4 differ in primary structure from one another at two substitution sites, site A (residue 112) and site B (residue 158). At sites A/B, apo-E2, -E3, and -E4 contain cysteine/cysteine, cysteine/arginine, and arginine/arginine, respectively. We demonstrated that the substitution of cysteine for arginine at site B is at least partly responsible for the defective binding of apo-E2 to human fibroblast low density lipoprotein receptors, compared to the normal binding activity of apo-E3 or -E4. Subjects with the genetic disorder type III hyperlipoproteinemia are phenotypically homozygous for apo-E2, but the binding activity of apo-E to the fibroblast receptor differs considerably from one type III individual to another. We therefore undertook a partial comparative sequence analysis of apo-E2 from three type III subjects whose apo-E displayed this heterogeneity. The subject with the poorest binding apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cysteine was found at sites A and B. The subject with the most active apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cystine was found at site A and at a new site (site C, residue 145). The epsilon 2 allele specifies a protein that has arginine at site B (residue 158); the epsilon 2 allele specifies a protein that has arginine at site C (residue 145). Therefore, the two alleles differ from one another by cysteine/arginine interchanges at two positions, sites B and C. The third subject, whose apo-E2 displayed binding activity intermediate between the activities of the other two, was genotypically heterozygous, having one epsilon 2 allele and one epsilon 2 allele. The intermediate binding activity of apo-E2 from this subject resulted from having a mixture of severely defective apo-E (specified by epsilon 2) and slightly defective apo-E (specified by epsilon 2).

To identify the domain of apolipoprotein E (apo-E) involved in binding to low density lipoprotein (LDL) receptors on cultured human fibroblasts, apo-E was cleaved and the fragments were tested for receptor binding activity. Two large thrombolytic peptides (residues 1-191 and 216-299) of normal apo-E3 were combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) and tested for their ability to compete with 125I-LDL for binding to the LDL (apo-B,E) receptors on human fibroblasts. The NH2-terminal two-thirds (residues 1-191) of apo-E3 was as active as intact apo-E3 . DMPC, while the smaller peptide (residues 216-299) was devoid of receptor-binding activity. When apo-E3 was digested with cyanogen bromide (CNBr) and the four largest CNBr fragments were combined with DMPC and tested, only one fragment competed with 125I-LDL for binding to cultured human fibroblasts (CNBr II, residues 126-218). This fragment possessed binding activity similar to that of human LDL. The 125I-labeled CNBr II . DMPC complex also demonstrated high affinity, calcium-dependent saturable binding to solubilized bovine adrenal membranes. The binding of CNBr II . DMPC was inhibited by 1,2-cyclohexanedione modification of arginyl residues or diketene modification of lysyl residues. In addition, the CNBr II had to be combined with DMPC before it demonstrated any receptor-binding activity. Pronase treatment of the membranes abolished the ability of this fragment to bind to the apo-B,E receptors. This same basic region in the center of the molecule has been implicated as the apo-B,E receptor-binding domain not only by this study but also by other studies showing that 1) natural mutants of apo-E that display defective binding have single amino acid substitutions at residues 145, 146, or 158; and 2) the apo-E epitope of the monoclonal antibody 1D7, which inhibits apo-E binding, is centered around residues 139-146.

To investigate the potential of monoclonal antibodies as probes to determine the receptor-binding domain of apolipoprotein E (apo-E), five apo-E antibodies were tested to see if any of them inhibited 125I-apo-E3 . dimyristoylphosphatidylcholine binding to apo-B,E receptors on cultured fibroblasts. Only one of the five antibodies, referred to as 1D7, was found to inhibit binding, blocking greater than 90% of the receptor-binding activity of apo-E3 dimyristoylphosphatidyl-choline. The 1D7 Fab fragments were also effective inhibitors. The 1D7 bound to a Mr = 22,000 NH2-terminal thrombolytic fragment of apo-E (residues 1-191) and to a 93-residue cyanogen bromide fragment of apo-E (residues 126-218). The four noninhibitory antibodies bound only to the NH2-terminal thrombolytic fragment. These results suggested that the 1D7 epitope is contained between residues 126 and 191, and that the epitopes of the other antibodies are not contained in this region. The use of synthetic apo-E fragments, which cover various lengths of the sequence from residues 129-169, and human apo-E variants with substitutions at residues 145, 146, or 158, narrowed the location of the 1D7 epitope to residues 139-169 and, most likely, to the immediate vicinity of residues 140-150. It is of interest that 1D7 was found to bind to the same region of apo-E that has been implicated as the receptor-binding domain in receptor-binding studies using human apo-E variants and apo-E3 fragments.

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.

The structure of a new naturally-occurring azulene, 1-stearoyloxymethylene-4-methyl-7-isopropenylazulene, obtained from an acetone extract of the blue mushroomLactarius indigo, has been determined by chemical and spectral methods.