Cholesteryl ester transfer protein: friend or foe?
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Apolipoprotein A-I↗
Biomedical subjects
R J Havel
Publications and source records attributed to R J Havel.
Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor.
We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca(2+)-dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is the most prominent 35S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.
Amino Acid Sequence↗
Identification, characterization, cloning, and expression of apolipoprotein C-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins.
We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver. The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.
Amino Acid Sequence↗
Prolonged postprandial responses of lipids and apolipoproteins in triglyceride-rich lipoproteins of individuals expressing an apolipoprotein epsilon 4 allele.
The postprandial responses of apo B48, B100, E and lipids in triglyceride-rich lipoproteins (TRL) to a meal containing one-third of daily energy (39% fat calories) were compared in normolipidemic young men with apo E3/3 and apo E4/3 phenotypes. After the two groups consumed a diet rich in polyunsaturated fat for 15-29 d, their postabsorptive concentrations of TRL triglycerides, apo B48, and apo B100 were virtually identical, but their postprandial responses differed. In both groups, TRL apo B48 increased at 3 h but returned to postabsorptive values at 6 h only in the apo E3/3 group; in the apo E4/3 group the concentration of apo B48 at 6 h was 80% higher than postabsorptive values. TRL apo B100 also increased at 3 h in the two groups and fell to post-absorptive values at 6 h in the apo E3/3 group but remained 51% higher than postabsorptive concentrations in the apo E4/3 group; this response was closely coupled to that of TRL cholesterol and apo E. These observations suggest that clearance of intestinal and hepatogenous TRL remnants is impaired in young men with an apo E4/3 phenotype.
Adult↗
The polymeric immunoglobulin receptor is the major calmodulin-binding protein in an endosome fraction from rat liver enriched in recycling receptors.
Rat liver endosomes contain one major high-affinity calmodulin-binding protein (CaMBP) that now has been identified as the polymeric immunoglobulin receptor (pIgR). In isolated endosomes pIgR was enriched in the receptor-recycling compartment (RRC); lesser enrichment was found in 'early' endosome (CURL) and much less in 'late' endosome fractions (multivesicular bodies, MVB). The distribution of the major CaMBP, shown by Western blotting or by overlay with I125-calmodulin in the isolated fractions, was consistent with rapid accumulation of I125-immunoglobulin A (IgA) in RRC and CURL after intravenous injection into rats. The receptor was also found in sinusoidal plasma membranes but not in cell fractions containing apical (bile canalicular) or lateral plasma membrane domains of the hepatocyte. The interaction of pIgR with calmodulin was shown by direct binding assays and by affinity chromatography. Thus, calmodulin is the first cytoplasmic protein shown to interact with the pIgR. We postulate that calmodulin regulates pIgA trafficking in rat liver. In addition, the receptor recycling fraction emerges as an endosomal subcompartment involved in pIgA transport via pIgR.
Adult↗
Sepsis increases endocytosis of endotoxin into hepatocytes.
BACKGROUND: Chylomicrons bind endotoxins and accelerate their clearance from plasma to the liver. This results in reduced mortality from septic shock in a rodent model. We hypothesized that the clearance of the LPS-chylomicron (LPS-CM) complex by hepatocytes is due to receptor-mediated endocytosis and that sepsis up-regulates this process. METHODS: Three groups of Sprague-Dawley rats; (1) control; (2) pretreated with 10 micrograms/kg LPS 24 hours before treatment; and (3) pretreated with 17-alpha-ethinyl estradiol (EE, 5 mg/kg subcutaneously for 3 days), were infused with labeled I125-LPS alone or with I125-LPS bound to chylomicron. Livers were removed 2.5, 15, and 30 minutes after LPS injection, and hepatic endosomes were isolated from the liver homogenates by serial ultracentrifugation in sucrose gradients. RESULTS: The injection of I125-LPS-CM complexes resulted in higher levels of endosomal I125-LPS in all groups, as compared with I125-LPS alone. In addition, the endosomal uptake of I125-LPS was markedly increased by both LPS and EE pretreatments. CONCLUSIONS: These data strongly suggest a primary role for receptor-mediated endocytosis in the increased clearance of LPS when bound to chylomicron. In addition, exposure to LPS appears to increase the accumulation of LPS in endosomes by a mechanism similar to that of EE, which is known to up-regulate receptor-mediated lipoprotein uptake. This endogenous pathway for the catabolism of endotoxins may provide a teleological explanation for the hypertriglyceridemia observed during sepsis.
Animals↗
Simultaneous quantification of apolipoproteins B-100, B-48, and E separated by SDS-PAGE.
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Animals↗
Dissection of steps mediating the hepatic uptake and catabolism of chylomicron remnants in rodents--removal from the blood and endocytosis into hepatocytes.
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Animals↗
Isolation and characterization of two distinct species of human very low density lipoproteins lacking apolipoprotein E.
We have isolated two fractions of very low density lipoprotein particles in human plasma that lack apolipoprotein (apo) E by combined anti-apoE and heparin affinity chromatography of whole plasma followed by ultracentrifugation. The two fractions are distinguished by their ability to bind to heparin. Each of these fractions, designated "B" particles to distinguish them from very low density lipoproteins that contain apoE ("B,E" particles), comprises an appreciable fraction of total particles in very low density lipoproteins of normolipidemic and hypertriglyceridemic subjects. The heparin-unbound B particles, which have been reported previously by others, are larger and have negligible affinity for low density lipoprotein receptors. The heparin-bound B particles are smaller and do bind to low density lipoprotein receptors, albeit with much lower affinity than B,E particles. No differences in accessibility to limited protease digestion were found between apoB-100 in the two types of B particles. Our data indicate that a substantial fraction of human very low density lipoproteins lacks apoE, the principal ligand for lipoprotein receptors that mediate the terminal catabolism of these lipoproteins. Whereas the B particles that fail to bind to heparin are likely to represent a form of nascent lipoprotein, the origin of those B particles that bind to heparin remains to be determined.
Adult↗
Management of primary hyperlipidemia.
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Aged↗
Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.
Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.
Analysis of Variance↗
Chylomicron remnants: hepatic receptors and metabolism.
Recent studies support a sequential model for hepatic processing and catabolism of chylomicron remnants. Removal of chylomicron remnants by the liver involves interaction with several macromolecules on the cell surface, including the LDL receptor and heparan-sulfate-bound hepatic lipase and apolipoprotein E. Endocytosis of chylomicron remnants into hepatocytes is mediated by apolipoprotein E and normally occurs through the LDL receptor. Interaction of chylomicron remnants with the LDL receptor-related protein, or another endocytic receptor with similar properties, occurs slowly, requiring modification of surface-bound chylomicron remnants, and serves as a backup mechanism that is utilized primarily when LDL receptors are deficient or down-regulated.
Animals↗
Influence of diets rich in saturated and omega-6 polyunsaturated fatty acids on the postprandial responses of apolipoproteins B-48, B-100, E, and lipids in triglyceride-rich lipoproteins.
The effects of diets rich in saturated fatty acids (SFA) (total polyunsaturated fatty acids [PUFA] [g]/total SFA [g][P/S ratio], 0.2) or omega-6 PUFA (P/S ratio, 1.3) on the postprandial response of triglyceride-rich lipoproteins (TRL) was determined in normolipidemic young men. After 15 and 29 days of diet intervention, the postabsorptive concentrations of apolipoprotein (apo) B-48 and apoB-100 were higher in the SFA group than in the PUFA group, but the absolute increase in apoB-48 was similar 3 hours after a challenge meal containing one third of daily energy and returned to postabsorptive values at 6 hours; this response was closely coupled to that of TRL triglycerides. In both groups, the percent increase in TRL apoB-48 and triglycerides was greater after the PUFA meal than after the SFA meal. The concentration of TRL apoB-100 also increased at 3 hours in both diet groups but returned to postabsorptive values at 6 hours only in those fed the PUFA diet; in the SFA group, apoB-100 remained high at 6 hours and fell below postabsorptive values only 9 hours after the meal. This apoB-100 response was affected primarily by the fatty acid composition of the diet and not by that of the challenge meal. The postprandial response of apoB-100 was closely coupled to that of cholesterol and apoE. These observations suggest that in healthy young men, neither the fatty acid composition of the diet nor that of the challenge meal affects the clearance of chylomicron remnants after a fat-containing meal. By contrast, the postprandial accumulation of hepatogenous TRL is prolonged in individuals fed a diet rich in SFA.
Adult↗
Effects of dietary cholesterol and fat saturation on plasma lipoproteins in an ethnically diverse population of healthy young men.
The individual effects of dietary cholesterol and fat saturation on plasma lipoprotein concentrations were determined in an ethnically diverse population of normolipidemic young men (52 Caucasian, 32 non-Caucasian). The experimental diets contained approximately 200 or 600 mg/d of cholesterol, 36-38% of calories as fat, and high or low proportions of saturated and polyunsaturated fat (polyunsaturated/saturated fat ratio approximately 0.8 vs 0.3). At the lower cholesterol intake, the high saturated fat diet had only a modest effect on LDL cholesterol in Caucasians (+ 6 mg/dl-1) and none in non-Caucasians. 600 mg cholesterol with high saturated fat led to a substantial mean increase in LDL cholesterol, which was significantly greater in Caucasian than in non-Caucasian subjects (+ 31 mg/dl vs 16 mg/dl, P < 0.005). 600 mg cholesterol with increased polyunsaturated fat gave a mean LDL increase of 16 mg/dl, lower than found when the same high cholesterol intake was coupled with increased saturated fat. Variation in cholesterol rather than the proportions of saturated and polyunsaturated fat had the most influence on LDL-cholesterol levels. Among non-Caucasians it was the only significant factor.
Adult↗
Quantification of apolipoproteins B-100, B-48, and E in human triglyceride-rich lipoproteins.
We have developed a procedure to quantify apolipoprotein (apo) B-100, apoB-48, and apoE in human triglyceride-rich lipoproteins. This procedure permits delipidation of small amounts of triglyceride-rich lipoproteins without appreciable losses, and quantification of these apolipoproteins in samples containing as little as 10 micrograms of protein. Delipidated triglyceride-rich lipoproteins are subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis, and the mass of apolipoproteins is estimated after densitometric scanning and volume integration of Coomassie blue-stained bands. The chromogenicities of apoB-100 and apoB-48 are virtually identical, and twofold lower than that of apoE. The standard curve for each apolipoprotein follows a power function over a wide protein range, permitting quantification of as little as 0.2 microgram of apoB-48 and as much as 30 micrograms of apoB-100 from a single application of triglyceride-rich lipoproteins to the gels. This method is suitable for routine use in studies of the intestinal and hepatic contributions to triglyceride-rich lipoproteins and their responses to postprandial lipemia.
Apolipoprotein B-100↗
"Healthy Dubec" -design of a joint Czech-American community project for the reduction of cardiovascular and cerebrovascular disease (adapted from American experience).
The project is a combination of individual and community-based intervention which adapted and modified methods and techniques originally used in The Three-City Community Study and The Stanford Five-City Project in a small community outside Prague. The goal is to reduce cardiovascular and cerebrovascular risk factors by primary and secondary prevention, using a community-based approach. 61.5% of examined population with the low risk score during the baseline survey was exposed to intervention by community-based methods. 38.5% of adult population was included in intervention activities within the framework of secondary prevention in high risk groups. There are described: the method of community oriented intervention and the intervention oriented on high risk groups - nutrition intervention, quit smoking activities, stress control, physical activity promotion.
Adolescent↗
Effect of the 39-kDa receptor-associated protein on the hepatic uptake and endocytosis of chylomicron remnants and low density lipoproteins in the rat.
The low density lipoprotein (LDL) receptor-related protein, which serves as the cell surface receptor for several proteins including alpha 2-macroglobulin-protease complexes, has been proposed to be a candidate hepatocytic receptor for chylomicron remnants through its recognition of apolipoprotein E. We have studied the effect of two ligands for this receptor, activated alpha 2-macroglobulin and the recently described 39-kDa protein that copurifies with the LDL receptor-related protein (receptor-associated protein), on the uptake and endocytosis of chylomicrons in intact rats and in isolated, perfused rat livers. Both of these ligands were rapidly taken up and endocytosed into the liver of rats. Chylomicrons from normal rats were injected in vivo and chylomicrons from estradiol-treated rats that were enriched in apolipoprotein E were added to liver perfusates. Prior administration of amounts of activated alpha 2-macroglobulin that saturated hepatic uptake mechanisms did not inhibit hepatic uptake or endocytosis of endogenously labeled cholesteryl esters of chylomicron particles in vivo or in perfused livers over a period of 15 min. By contrast, prior administration of saturating amounts of the receptor-associated protein (expressed in bacteria as a fusion protein with glutathione S-transferase) reduced hepatic uptake by about 30% and virtually abolished endocytosis. The receptor-associated protein inhibited hepatic uptake of human LDL in vivo by 70% and endocytosis by 81%. Our results show that receptor-associated protein-sensitive processes predominate in the overall pathways by which chylomicron remnants are endocytosed by rat liver. Furthermore, they show that the receptor-associated protein binds to and inhibits the function of the LDL receptor as well as the LDL receptor-related protein.
Animals↗