Functional domains of apolipoprotein E and apolipoprotein B.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
T L Innerarity
Publications and source records attributed to T L Innerarity.
Structural relationship of human apolipoprotein B48 to apolipoprotein B100.
Although the complete amino acid sequence of human apolipoprotein (apo) B100 is known (4536 amino acids), the structure of apo B48 has not been defined. The objective of our study was to define the structure of apo B48 and its relationship to apo B100. Antibodies were produced against 22 synthetic peptides corresponding to sequences in human apo B100. The levels of immunoreactivity of the antipeptides to apo B100 and apo B48 were used to define the structural relationship between these two species of apo B. Six antibodies from sequences in the amino-terminal half of apo B100, including antipeptide 2110-2129, bound to both apo B100 and apo B48. 15 other apo B-specific antipeptides from sequences carboxyl-terminal to residue 2152 bound to apo B100, but not to apo B48. Immunoblots of cyanogen bromide digests of apo B100 and apo B48 with antipeptides 2068-2091 and 2110-2129 detected a 16-KD fragment (residues 2016-2151) in the apo B100 digest and a fragment of identical size in the apo B48 digest. Because apo B48 appears to contain the apo B100 cyanogen bromide fragment 2016-2151 and because an antiserum specific for the peptide 2152-2168 does not bind to apo B48, we conclude that apo B48 represents the amino-terminal 47% of apo B100 and that the carboxyl terminus of apo B48 is in the vicinity of residue 2151 of apo B100.
Amino Acids↗
Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments.
Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.
Apolipoprotein B-100↗
Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells.
The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.
Animals↗
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.
Animals↗
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.
Amino Acids↗
Type III hyperlipoproteinemia: a focus on lipoprotein receptor-apolipoprotein E2 interactions.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Lipoprotein-receptor interactions.
Explore the source record for details and available documents.
Animals↗
Cellular and molecular biology of lipoprotein metabolism: characterization of lipoprotein receptor-ligand interactions.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Fat feeding in humans induces lipoproteins of density less than 1.006 that are enriched in apolipoprotein [a] and that cause lipid accumulation in macrophages.
Formula diets containing lard or lard and egg yolks were fed to six normolipidemic volunteers to investigate subsequent changes in the composition of lipoproteins of d less than 1.006 g/ml and in their ability to bind and be taken up by receptors on mouse macrophages. Both formulas induced the formation of d less than 1.006 lipoproteins that were approximately 3.5-fold more active than fasting very low density lipoproteins (VLDL) in binding to the receptor for beta-VLDL on macrophages. Subfractionation of postprandial d less than 1.006 lipoproteins by agarose chromatography yielded two subfractions, fraction I (chylomicron remnants) and fraction II (hepatic VLDL remnants), which bound to receptors on macrophages. However, fraction I lipoproteins induced a 4.6-fold greater increase in macrophage triglyceride content than fraction II lipoproteins or fasting VLDL. Fraction I lipoproteins were enriched in apolipoproteins (apo) B48, E, and [a]. Fraction II lipoproteins lacked apo[a] but possessed apo B100 and apo E. The apo[a] was absent in normal fasting VLDL, but was present in the d less than 1.006 lipoproteins (beta-VLDL) of fasting individuals with type III hyperlipoproteinemia. The apo[a] from postprandial d less than 1.006 lipoproteins was larger than either of two apo[a] subspecies obtained from lipoprotein (a) [Lp(a)] isolated at d = 1.05-1.09. However, all three apo[a] subspecies were immunochemically identical and had similar amino acid compositions: all were enriched in proline and contained relatively little lysine, phenylalanine, isoleucine, or leucine. The association of apo[a] with dietary fat-induced fraction I lipoproteins suggests that the previously observed correlation between plasma Lp(a) concentrations and premature atherosclerosis may be mediated, in part, by the effect of apo[a] on chylomicron remnant metabolism.
Adult↗
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.
Animals↗
Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization.
Apolipoprotein (apo-) B is the ligand responsible for the receptor-mediated catabolism of low density lipoproteins, the principal cholesterol-transporting lipoproteins in plasma. The primary structure of the carboxyl-terminal 30 percent (1455 amino acids) of human apo-B (apo-B100) has been deduced from the nucleotide sequence of complementary DNA. Portions of the protein structure that may relate to its receptor binding function and lipid binding properties have been identified. The apo-B100 messenger RNA is about 19 kilobases in length. The apo-B100 gene is expressed primarily in liver and, to a lesser extent, in small intestine, but in no other tissues. The gene for apo-B100 is located in the p24 region (near the tip of the short arm) of chromosome 2.
Amino Acid Sequence↗
Apolipoprotein E: receptor binding properties.
The E apoprotein plays a central role in lipoprotein metabolism. It is a key protein determinant responsible for the recognition of lipoproteins by specific lipoprotein receptors. Structure-function studies are providing insights into the normal function of this apoprotein in cholesterol homeostasis and into the clinical abnormalities resulting from the occurrence of specific mutants.
Antibodies, Monoclonal↗
Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.
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.
Amino Acid Sequence↗
Lipoproteins of special significance in atherosclerosis. Insights provided by studies of type III hyperlipoproteinemia.
In summary, the study of type III hyperlipoproteinemia has provided important insights into lipoprotein metabolism that have helped to elucidate several functional roles for apo E and have provided a better understanding of the mechanisms whereby specific lipoproteins may be atherogenic or anti-atherogenic. The molecular defect in type III hyperlipoproteinemia and dysbetalipoproteinemia is the presence of a mutant form of apo E, usually apo E2, that is defective in binding to both apo B,E(LDL) and apo E receptors. The receptor-defective apo E results in an impaired clearance of remnant lipoproteins (beta-VLDL). In addition, the abnormal apo E may impair the lipolytic processing of hepatic beta-VLDL through its involvement in lipid transfer or exchange processes. The accumulation of beta-VLDL may provide the most direct mechanism responsible for the accelerated atherosclerosis observed in type III hyperlipoproteinemia, a mechanism that involves the receptor mediated uptake of beta-VLDL by macrophages, which are then converted to arterial foam cells. Alterations in the HDL of patients with type III hyperlipoproteinemia further support the concept that HDL are anti-atherogenic. The increase in HDL-with apo E provides insight into the role of these cholesterol-enriched HDL in reverse cholesterol transport and in the cellular redistribution of cholesterol, processes whereby cholesterol deposition may be reversed. It should be stressed that both the accumulation of beta-VLDL and alterations in HDL (reduction in typical HDL and an increase in HDL-with apo E) are associated with accelerated atherogenesis in animals fed high levels of fat and cholesterol. Although valuable information has been gained concerning the mechanisms involved in type III hyperlipoproteinemia by the study of the disease, the clinical expression of this disorder is variable, ranging from hypocholesterolemia to marked hypercholesterolemia in subjects with the same molecular defect (E2/2). This variability in expression is more easily understood when one considers the various factors that can promote the hyperlipoproteinemia and when one considers the mechanisms of action whereby these factors may exacerbate the effects of the presence of an abnormal apo E. In most cases, development of type III hyperlipoproteinemia requires that a second event (a predisposing environmental factor or a second genetic defect) be associated with the primary genetic defect (an abnormal form of apo E).
Amino Acid Sequence↗
Receptor interactions controlling lipoprotein metabolism.
Lipoprotein receptors play a central role in lipoprotein metabolism and a major role in cholesterol homeostasis. The most completely characterized lipoprotein receptor is the LDL (low density lipoprotein) or apo-B,E(LDL) receptor. The apo-B,E(LDL) receptor is present on both hepatic and extrahepatic cells and is responsible for the metabolism of a major portion of plasma LDL. Binding and internalization of LDL particles by this receptor initiates a series of intracellular events, resulting in the regulation of cellular cholesterol metabolism. In addition to the apo-B on LDL interacting with the apo-B,E(LDL) receptor, the apo-E on apo-E-containing lipoproteins is also capable of interacting and regulating intracellular cholesterol metabolism. The liver has also been shown to contain a second distinct lipoprotein receptor that is specific for apo-E. This receptor has been demonstrated on hepatic membranes from humans, dogs, and swine and is referred to as the apo-E receptor. This receptor may be responsible for the clearance of chylomicron remnants from plasma by the liver and may participate in reverse cholesterol transport. Thus, apo-E is a major determinant in lipoprotein metabolism and cholesterol homeostasis. The receptor binding properties of apo-E are well characterized, and a series of structural variants, several with lipoprotein binding defects, have been identified. Studies of the binding activity of these receptor-defective apo-E variants have helped to define the receptor binding domain of apo-E.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
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.
Animals↗
Plasma lipoproteins: apolipoprotein structure and function.
Plasma lipoprotein metabolism is regulated and controlled by the specific apolipoprotein (apo-) constituents of the various lipoprotein classes. The major apolipoproteins include apoE, apoB, apoA-I, apoA-II, apoA-IV, apoC-I, apoC-II, and apoC-III. Specific apolipoproteins function in the regulation of lipoprotein metabolism through their involvement in the transport and redistribution of lipids among various cells and tissues, through their role as cofactors for enzymes of lipid metabolism, or through their maintenance of the structure of the lipoprotein particles. The primary structures of most of the apolipoproteins are now known, and various functional domains of these proteins are being mapped using selective chemical modification, synthetic peptides, and monoclonal antibodies. Furthermore, the establishment of structure-function relationships has been greatly advanced by the identification of genetically determined variants of specific apolipoproteins that are associated with a disorder of lipoprotein metabolism. Future studies will rely heavily on the use of recombinant DNA technology and site-specific mutagenesis to elucidate further the correlations between structure and function and the role of specific apolipoproteins in lipoprotein metabolism.
Apolipoprotein A-I↗