Isolation and characterization of the apolipoprotein E receptor from canine and human liver

Previous results have demonstrated that liver membranes possess two distinct lipoprotein receptors: a low density lipoprotein (LDL) receptor that binds lipoproteins containing either apolipoprotein (apo-) B or apo-E, and an apo-E-specific receptor that binds apo-E-containing lipoproteins, but not the apo-B-containing LDL. This study reports the isolation and purification of apo-B,E(LDL) and apo-E receptors from canine and human liver membranes. The receptors were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and were partially purified by DEAE-cellulose chromatography. The apo-B,E(LDL) receptor was isolated by affinity chromatography on LDL-Sepharose. The apo-E receptor, which did not bind to the LDL-Sepharose column, was then purified by using an HDLc (cholesterol-induced high density lipoprotein)-Sepharose affinity column and an immunoaffinity column. Characterization of the receptors revealed that the hepatic apo-B,E(LDL) receptor is similar to the extrahepatic LDL receptor with an apparent Mr = 130,000 on non-reducing sodium dodecyl sulfate-polyacrylamide gels. The apo-E receptor was found to be distinct from the apo-B,E(LDL) receptor, with an apparent Mr = 56,000. The purified apo-E receptor displayed Ca2+-dependent binding to apo-E-containing lipoproteins and did not bind to LDL or chemically modified apo-E HDLc. Antibodies raised against the apo-B,E(LDL) receptor cross-reacted with the apo-E receptor. However, an antibody prepared against the apo-E receptor did not react with the apo-B,E(LDL) receptor. The apo-E receptor also differed from the apo-B,E(LDL) receptor in amino acid composition, indicating that the apo-E receptor and the apo-B,E(LDL) receptor are two distinct proteins. Immunoblot characterization with anti-apo-E receptor immunoglobulin G indicated that the apo-E receptor is present in the hepatic membranes of man, dogs, rats, and mice and is localized to the rat liver parenchymal cells.     

Obligatory role of cholesterol and apolipoprotein E in the formation of large cholesterol-enriched and receptor-active high density lipoproteins

The formation of large cholesterol-enriched high density lipoproteins (HDL1/HDLc) from typical HDL3 requires lecithin:cholesterol acyltransferase activity, additional cholesterol, and a source of apolipoprotein (apo-) E. The present study explores the role of apo-E in promoting HDL1/HDLc formation and in imparting to these lipoprotein particles the ability to interact with the apo-B,E(low density lipoprotein (LDL] receptor. Incubation of normal canine serum with cholesterol-loaded mouse peritoneal macrophages resulted in the formation of HDL1/HDLc that competed with 125I-LDL for binding to the apo-B,E(LDL) receptors on cultured human fibroblasts. Cholesterol efflux from macrophages was necessary because incubation of normal canine serum with nonloaded macrophages did not cause HDL1/HDLc formation. However, cholesterol delivery to the serum was not sufficient to result in HDL1/HDLc formation. Apolipoprotein E had to be available. Incubation of apo-E-depleted canine serum with cholesterol-loaded J774 cells, a macrophage cell line that does not synthesize apo-E, demonstrated that no HDL1/HDLc formation was detected even in the presence of significant cholesterol efflux. However, addition of exogenous apo-E to the serum during the incubation with cholesterol-loaded J744 cells promoted the formation of large receptor-active HDL1/HDLc. The receptor binding activity of these particles produced in vitro correlated with the amount of apo-E incorporated into the HDL1/HDLc. Apolipoproteins A-I and C-III were ineffective in promoting HDL1/HDLc formation; thus, apo-E was unique in allowing HDL1/HDLc formation. These results demonstrate that when lecithin:cholesterol acyltransferase activity, cholesterol, and apo-E are present in serum, typical HDL can be transformed in vitro into large cholesterol-rich HDL1/HDLc that are capable of binding to lipoprotein receptors.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Binding of chylomicron remnants and beta-very low density lipoproteins to hepatic and extrahepatic lipoprotein receptors. A process independent of apolipoprotein B48

The ability of apolipoprotein (apo-) B48 to interact with lipoprotein receptors was investigated using three different types of lipoproteins. First, canine chylomicron remnants, which contained apo-B48 as their primary apoprotein constituent, were generated by the hydrolysis of chylomicrons with milk lipoprotein lipase. These apo-B48-containing chylomicron remnants are deficient in apo-E and reacted very poorly with apo-E receptors on adult dog liver membranes and the low density lipoprotein (apo-B,E) receptors on human fibroblasts. Addition of normal human apo-E3 restored the receptor binding activity of these lipoproteins. Second, beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs were subfractionated into distinct classes containing apo-E along with either apo-B48 or apo-B100. Both classes bound to the apo-B,E and apo-E receptors. Their binding was almost completely mediated by apo-E, as evidenced by the ability of the anti-apo-E to inhibit the receptor interaction. Third, beta-VLDL from type III hyperlipoproteinemic patients were subfractionated by immunoaffinity chromatography into lipoproteins containing apo-E plus either apo-B48 or apo-B100. Both subfractions bound poorly to apo-B,E and apo-E receptors due to the presence of defective apo-E2. However, the residual binding of the apo-B48-containing and apo-B100-containing human beta-VLDL was inhibited by the anti-apo-E. After lipase hydrolysis, apo-B100 became a more prominant determinant responsible for mediating receptor binding to the apo-B,E receptor. By contrast, lipase hydrolysis did not increase the binding activity of the apo-B48-containing beta-VLDL. These results indicate that apo-B48 does not play a direct role in mediating the interaction of lipoproteins with receptors on fibroblasts or liver membranes.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis.     

Interaction of swine lipoproteins with the low density lipoprotein receptor in human fibroblasts.

HDLc, a cholesterol-rich lipoprotein that accumulates in the plasma of cholesterol-fed swine, was shown to resemble functionally human and swine low density lipoprotein in its ability to bind to the low density lipoprotein receptor in monolayers of cultured human fibroblasts. This binding occurred even though HDLc lacked detectable apoprotein B, which is the major protein of low density lipoprotein. After it was bound to the low density lipoprotein receptor, HDLc, like human and swine low density lipoprotein, delivered its cholesterol to the cells, and this, in turn, caused a suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, an activation of the cholesterol-esterifying system, and a net accumulation of free and esterified cholesterol within the cells. Swine HDLc, like human high density lipoprotein, did not bind to the low density lipoprotein receptor nor did it elicit any of the subsequent metabolic events. HDLc, like human low density lipoprotein, was incapable of producing a metabolic effect in fibroblasts derived from a subject with the homozygous form of familial hypercholesterolemia, which lack low density lipoprotein receptors. These results indicate that two lipoproteins that have been associated with athersclerosis--low density lipoprotein in humans and HDLc in cholesterol-fed swine--both can cause the accumulation of cholesterol and cholesteryl esters within cells through an interaction with the low density lipoprotein receptor.     

Defective hepatic lipoprotein receptor binding of beta-very low density lipoproteins from type III hyperlipoproteinemic patients. Importance of apolipoprotein E

Apolipoprotein (apo-) E2 and beta-migrating very low density lipoproteins (beta-VLDL) (which were isolated from type III hyperlipoproteinemic subjects) both demonstrated defective binding to apo-E and apo-B,E receptors on dog liver membranes and to apo-B,E low density lipoproteins (LDL) receptors on fibroblasts. The defective binding activity of the apo-E2 and beta-VLDL varied from very poor to nearly normal. The ability of the beta-VLDL to interact with hepatic apo-E receptors was enhanced by the addition of normal apo-E3 to the beta-VLDL. Furthermore, cysteamine treatment of the apo-E2 in beta-VLDL enhanced binding of the beta-VLDL to both apo-E and apo-B,E receptors. The importance of apo-E in mediating the receptor binding of beta-VLDL to these receptors was confirmed by using monoclonal antibodies. The residual binding activity of beta-VLDL to apo-E and apo-B,E receptors was inhibited by greater than 90% with anti-apo-E, while the addition of anti-apo-B had little effect. The apo-B in the beta-VLDL was capable of binding to apo-B,E receptors after the hydrolysis of the beta-VLDL triglycerides with milk lipoprotein lipase. Lipase treatment yielded, two subfractions of beta-VLDL. One fraction (d = 1.02 to 1.03 g/ml) was enriched with apo-B100; the other fraction (d less than 1.006 g/ml) was enriched with apo-B48 and apo-E2. Significantly increased amounts of the apo-B100-enriched fraction bound to apo-B,E receptors. Inhibition of this binding caused by the addition of anti-apo-B indicated that the binding activity of this subfraction was mediated by apo-B100. The apo-B48-enriched fraction did not show a significant increase in receptor binding, suggesting that apo-B48 does not bind to these receptors. In a control experiment, it was shown that triglyceride-rich VLDL, which contain normal apo-E3 and apo-B100, bind significantly to both liver apo-E receptors and fibroblast apo-B,E receptors. This binding activity was inhibited by greater than 90% with anti-apo-E. Lipase hydrolysis of the VLDL did not further enhance their receptor-binding activity. These results demonstrate that apo-E, and not apo-B, is the major determinant mediating the receptor-binding activity of cholesterol-rich beta-VLDL and triglyceride-rich VLDL.     

Formation of cholesterol- and apoprotein E-enriched high density lipoproteins in vitro

The delivery of cholesterol to canine serum or plasma altered the distribution of cholesterol and apoproteins in subclasses of high density lipoproteins (HDL). In these experiments, two in vitro systems were employed. The first system used cholesterol-celite particles to deliver cholesterol to canine plasma during 4-h incubations. When the cholesterol distribution in the lipoproteins was analyzed by Geon-Pevikon electrophoresis, an increase in cholesterol content was found in the slower migrating subclasses of HDL (HDL1 and HDLc). A large increase in apoprotein E (apo-E) was also observed in the lipoproteins. Densitometric analysis of lipid-stained, 4 to 30% gradient acrylamide gels of canine plasma after incubation with cholesterol-celite revealed that the concentration of the major high density lipoproteins (HDL3) decreased, and the concentration of subclasses of HDL-with apo-E (HDL1 and HDLc) increased 2- to 5-fold. In the second system, cholesterol-loaded mouse peritoneal macrophages released cholesterol to HDL in an incubation medium containing 10 to 20% canine serum. The HDL1 and HDLc, which demonstrated slower electrophoretic mobility as determined by Geon-Pevikon block electrophoresis, became enriched in cholesterol and cholesteryl esters. Gradient gel electrophoresis showed substantial increases in these subclasses of HDL-with apo-E. The cholesterol-loaded mouse peritoneal macrophages synthesized and secreted apo-E into the medium. When L-[35S]methionine was used as a precursor, 65 to 90% of the 35S-labeled protein associated with the lipoproteins in the 1.02 to 1.09 density range was immunoprecipitated with antibody directed against rat apo-E. Gradient gel electrophoresis of density fractions demonstrated the presence of HDL1 and HDLc as the major lipoproteins. In addition, when canine 125I-HDL3 (primarily apo-A-I-containing HDL) were added to canine serum and incubated with cholesterol-loaded macrophages, the appearance of HDL1 and HDLc was associated with a marked increase in the 125I label in these newly formed, cholesteryl ester-rich lipoproteins. There was a corresponding marked reduction in the 125I-HDL3 in the serum. Similar results were observed using human HDL3 and human serum.     

Characterization of the estrogen-induced lipoprotein receptor of rat liver

The ethinyl estradiol-induced lipoprotein receptor of rat liver was purified and characterized. Liver membranes were prepared from ethinyl estradiol-treated rats, solubilized, and subjected to DEAE chromatography. A fraction with a high specific activity for low density lipoprotein (LDL) binding was isolated and used to immunize mice. Hybridomas were prepared from their spleen cells, and a clone that secreted an IgG antibody, which cross-reacted with an ethinyl estradiol-induced protein of the same molecular weight as the bovine adrenal LDL receptor, was expanded. This antibody, designated P1B3, immunoprecipitated the induced lipoprotein receptor. P1B3 was used to purify the receptor, and a polyclonal antibody was raised against the pure protein. This antibody recognized a protein of similar molecular weight in rat liver, adult dog liver, and human skin fibroblasts, thus demonstrating that the induced rat lipoprotein receptor was related to the LDL receptor of other species. This receptor is present in normal rat liver, and its content is reduced by feeding an atherogenic diet, but not by feeding a diet containing 0.5% cholesterol. Moreover, cholestyramine supplementation of the diet did not induce the receptor on liver membranes. The polyclonal antibody could prevent the binding of LDL to liver membranes from control or ethinyl estradiol-treated rats. It decreased chylomicron remnant binding to membranes from ethinyl estradiol-treated membranes, but did not affect chylomicron remnant binding to liver membranes of untreated rats, a result compatible with the existence of a distinct receptor for these latter particles. The amount of LDL receptor-independent, specific remnant binding was the same in both control and ethinyl estradiol-treated rats. This is consistent with the concept that the remnant receptor is not regulated by this treatment. Based on the above, we conclude that the ethinyl estradiol-induced lipoprotein receptor of rat liver is biochemically and immunologically similar to the LDL receptor of other species. It is present on the liver of normal adult rats and could account for LDL as well as beta VLDL and HDLc removal. Although it may contribute to chylomicron remnant removal, there appears to be a second unrelated receptor or process which recognizes this lipoprotein.     

Interaction of canine and swine lipoproteins with the low density lipoprotein receptor of fibroblasts as correlated with heparin/manganese precipitability.

Canine HDL1 and canine and swine HDLc were fractionated into several lipoprotein subpopulations by heparin/manganese precipitation. The ability of the various subfractions of HDL1 or HDLc to compete with 125I-labeled low density lipoproteins (LDL) for binding and degradation by human fibroblasts was compared. The HDL1 or HDLc which precipitated at the lowest concentration of heparin (a concentration which precipitates LDL) were the most effective in competing with 125I-LDL for binding, internalization, and degradation. A striking characteristic of these lipoproteins was the occurrence of a prominence of the arginine-rich apoprotein. The HDL1 or HDLc subfractions which were not precipitated by heparin/managanese lacked detectable arginine-rich apoprotein and did not compete significantly with the 125I-LDL for binding and degradation. Furthermore, the lipid to protein ratio differed in the precipitable and nonprecipitable lipoproteins, with those which were most efficiently bound and degraded containing more cholesterol. Specific lipoprotein interaction with heparin and with the cell surface receptors may occur by a common mechanism; namely, through a positively charged region on the lipoprotein surface which may reside with the B and arginine-rich apoproteins.     

The levels of apolipoprotein-E in hypercholesterolemic rat serum.

The levels of apolipoprotein-C (apo-E) in serum and isolated liproproteins from diet-induced hypercholesterolemic, and to some extent hypertriglycerdemic rats were measured by electroimmunoassay. The hypocholesterolemia was accompanied by a mild hypertriglyceridemia. The apo-E was increased by 60% in the hypercholesterolemic serum with a 5- and 50-fold increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) respectively. However, the proportion of apo-E in nascent VLDL isolated from the hepatic Golgi apparatus of hypercholesterolemic rats was significantly decreased. In control serum, 40--50% of the apo-E is found in the density greater than 1.21 g/ml fraction, although this is at least partially due to ultracentrifugation. The aproprotein is absent from the density greater than 1.21 g/ml fraction from hypercholesterolemic serum, suggesting that it is bound more firmly to the lipoprotein complex. It is concluded that the large increases in apo-E in the VLDL and LDL density ranges of serum from hypercholesterolemic rats may in part be accounted for by the utilization of apo-E normally found at higher densities.     

Normalization of receptor binding of apolipoprotein E2. Evidence for modulation of the binding site conformation

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.     

Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins

Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.     

Inhibition of apolipoprotein A-I gene expression by obesity-associated endocannabinoids

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and 2-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG2 and the intestinal cell line Caco-2. Apo A-I protein secretion was suppressed nearly 50% by anandamide and 2-arachidonoylglycerol in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG2 cells suppressed apo A-I promoter activity, while in Caco-2 cells, exogenous expression of both CBR1 and CBR2 could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G2 cells could be inhibited by CBR1 antagonist AM251 but not by AM630, a selective and potent CBR2 inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

Peripheral synthesis and isoform distribution of dog apoprotein E. An in vivo approach

Purified dog plasma apo-E is composed of four major isoforms with the following pI values: 5.40, 5.31, 5.26, and 5.22. Treatment with neuraminidase suggests that the multiple forms are due to progressive sialation. The acidic isoforms (pI = 5.22 and less), rarely detectable in plasma lipoprotein samples (except in the d less than 1.05 g/ml fraction of cholesterol-fed dogs), are present in high concentrations in the interstitial fluid high density lipoprotein fraction I (HDLI) of cholesterol-fed dogs, a lipoprotein recently described (Dory, L., Boquet, L.M., Hamilton, R.L., Sloop, C.H., and Roheim, P.S. (1985) J. Lipid Res. 26, 519-527). Apo-Es0 (the most basic isoform) is a major constituent of the plasma d less than 1.05 g/ml fraction, but it is usually only a minor component of other plasma or interstitial fluid lipoproteins. This is likely a reflection of the prolonged residence time of a specific lipoprotein species in this density range, resulting in more extensive desialation. Peripheral apo-E synthesis has been measured under in vivo conditions using a hindlimb cannulation of the lymphatics and collection of prenodal peripheral lymph. Following injection of [3H]leucine or [35S]methionine into the dorsal skin of the toes, the specific activity of the interstitial fluid apo-E far exceeded that of plasma apo-E at all time points examined. Incorporation was measurable 30 min after the isotope injection and peaked at 150 min in control dogs and between 120-150 min in cholesterol-fed dogs. The rate of peripheral synthesis of apo-E in cholesterol-fed dogs appeared to be twice that of control dogs. The newly synthesized and secreted apo-E preferentially associated with the disc-shaped HDLI of the interstitial fluid; less than 15% of the apo-E-associated radioactivity was recovered in the d less than 1.05 g/ml fraction, despite the fact that this fraction contains well over 50% of the total interstitial fluid apo-E mass. The newly secreted, HDLI-associated apo-E can be converted by neuraminidase into apo-Es0.     

Low-density-lipoprotein binding by mast-cell granules. Demonstration of binding of apolipoprotein B to heparin proteoglycan of exocytosed granules.

To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.     

The receptor-binding domain of human apolipoprotein E. Binding of apolipoprotein E fragments

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.