Search

To investigate the separate contributions of the lipolytic versus ligand-binding function of hepatic lipase (HL) to plasma lipoprotein metabolism and atherosclerosis, we compared mice expressing catalytically active wild-type HL (HL-WT) and inactive HL (HL-S145G) with no endogenous expression of mouse apoE or HL (E-KO x HL-KO, where KO is knockout). HL-WT and HL-S145G reduced plasma cholesterol (by 40 and 57%, respectively), non-high density lipoprotein cholesterol (by 48 and 61%, respectively), and apoB (by 36 and 44%, respectively) (p < 0.01), but only HL-WT decreased high density lipoprotein cholesterol (by 67%) and apoA-I (by 54%). Compared with E-KO x HL-KO mice, both active and inactive HL lowered the pro-atherogenic lipoproteins by enhancing the catabolism of autologous (125)I-apoB very low density/intermediate density lipoprotein (VLDL/IDL) (fractional catabolic rates of 2.87 +/- 0.04/day for E-KO x HL-KO, 3.77 +/- 0.03/day for E-KO x HL-WT, and 3.63 +/- 0.09/day for E-KO x HL-S145G mice) and (125)I-apoB-48 low density lipoprotein (LDL) (fractional catabolic rates of 5.67 +/- 0.34/day for E-KO x HL-KO, 18.88 +/- 1.72/day for E-KO x HL-WT, and 9.01 +/- 0.14/day for E-KO x HL-S145G mice). In contrast, the catabolism of apoE-free, (131)I-apoB-100 LDL was not increased by either HL-WT or HL-S145G. Infusion of the receptor-associated protein (RAP), which blocks LDL receptor-related protein function, decreased plasma clearance and hepatic uptake of (131)I-apoB-48 LDL induced by HL-S145G. Despite their similar effects on lowering pro-atherogenic apoB-containing lipoproteins, HL-WT enhanced atherosclerosis by up to 50%, whereas HL-S145G markedly reduced aortic atherosclerosis by up to 96% (p < 0.02) in both male and female E-KO x HL-KO mice. These data identify a major receptor pathway (LDL receptor-related protein) by which the ligand-binding function of HL alters remnant lipoprotein uptake in vivo and delineate the separate contributions of the lipolytic versus ligand-binding function of HL to plasma lipoprotein size and metabolism, identifying an anti-atherogenic role of the ligand-binding function of HL in vivo.

We examined the role of S-linked palmitoylation of human apolipoprotein (apo) B in the assembly and secretion of very low density lipoproteins using recombinant human apoB48. There are four free cysteine residues (Cys(1085), Cys(1396), Cys(1478), and Cys(1635)) within apoB48 that potentially can be palmitoylated. All four cysteine residues were substituted with serine by site-specific mutagenesis. The mutant protein was expressed in transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the stably transfected cells with iodopalmitic acid analog showed that the mutant apoB48 lacked palmitoylation. The lack of palmitoylation had little impact on the ability of apoB48 to assemble and secrete very low density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the mutant apoB48 protein from the endoplasmic reticulum to cis/medial Golgi. However, the mutant apoB48 lacking palmitoylation showed retarded movement toward the distal Golgi and increased association (>2-fold) with the membranes of the secretory compartments. A marginal decrease (by 15-20%) in secretion efficiency as compared with that of wild type apoB48 was also observed. These results suggest that lack of palmitoylation may influence the partitioning of apoB48 between microsomal membranes and microsomal lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.

Familial hypobetalipoproteinemia (FHBL), an autosomal co-dominant disorder, is associated with reduced plasma concentrations (<5th percentile for age and sex) of apolipoprotein (apo) B and beta-migrating lipoproteins. To date, only mutations in APOB encoding prematurely truncated apoB have been found in FHBL. We discovered a novel APOB gene mutation, namely R463W, in an extended Christian Lebanese FHBL kindred. Heterozygotes for R463W had the typical FHBL phenotype, whereas homozygotes had barely detectable apoB-100. The effect of the R463W mutation on apoB secretion was examined using transfected McA-RH7777 cells that expressed one of two recombinant human apoBs, namely B48 and B17. In both cases, the mutant proteins (B48RW and B17RW) were retained within the endoplasmic reticulum and were secreted poorly compared with their wild-type counterparts. Pulse-chase analysis showed that secretion efficiencies of B48RW and B17RW were, respectively, 45 and 40% lower than those of the wild-types. Substitution of Arg(463) with Ala in apoB-17 (B17RA) decreased secretion efficiency by approximately 50%, but substitution with Lys (B17RK) had no effect on secretion, indicating that the positive charge was important. Molecular modeling of apoB predicted that Arg(463) was in close proximity to Glu(756) and Asp(456). Substitution of Glu(756) with Gln (B17EQ) had no effect on secretion, but substitution of Asp(456) with Asn (B17DN) decreased secretion to the same extent as B17RW. In co-transfection experiments, the mutant B17RW showed increased binding to microsomal triglyceride transfer protein as compared with wild-type B17. Thus, the naturally occurring R463W mutant reveals a key local domain governing assembly and secretion of apoB-containing lipoproteins.

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (a) in vivo Triton WR1339 studies of apoB48 production as well as (b) ex vivo pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding or in vitro incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte de novo lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.

The peroxisome proliferator-activated receptor (PPAR) alpha agonist WY 14,643 increased the secretion of apolipoprotein (apo) B-100, but not that of apoB-48, and decreased triglyceride biosynthesis and secretion from primary rat hepatocytes. These effects resulted in decreased secretion of apoB-100-very low density lipoprotein (VLDL) and an increased secretion of apoB-100 on low density lipoproteins/intermediate density lipoproteins. ApoB-48-VLDL was also replaced by more dense particles. The proteasomal inhibitor lactacystin did not influence the recovery of apoB-100 or apoB-48 in primary rat hepatocytes, indicating that co-translational (proteasomal) degradation is of less importance in these cells. Treatment with WY 14,643 made the recovery of apoB-100 sensitive to lactacystin, most likely reflecting the decreased biosynthesis of triglycerides. The PPAR alpha agonist induced a significant increase in the accumulation of pulse-labeled apoB-100 even after a short pulse (2-5 min). There was also an increase in apoB-100 nascent polypeptides, indicating that the co-translational degradation of apoB-100 was inhibited. However, a minor influence on an early posttranslation degradation cannot be excluded. This decreased co-translational degradation of apoB-100 explained the increased secretion of the protein. The levels of apoB-48 remained unchanged during these pulse-chase experiments, and albumin production was not affected, indicating a specific effect of PPAR alpha agonists on the co-translational degradation of apoB-100. These findings explain the difference in the rate of secretion of the two apoB proteins seen after PPAR alpha activation. PPAR alpha agonists increased the expression and biosynthesis of liver fatty acid-binding protein (LFABP). Increased expression of LFABP by transfection of McA-RH7777 cells increased the secretion of apoB-100, decreased triglyceride biosynthesis and secretion, and increased PPAR alpha mRNA levels. These findings suggest that PPAR alpha and LFABP could interact to amplify the effect of endogenous PPAR alpha agonists on the assembly of VLDL.

The aim of this study was to investigate the types and characteristics of chylomicron precursors in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low fat diet, newly synthesized and immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized, and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.

Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipoproteins. The net production of apoB can be regulated by presecretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated. Subsequent incubation with rat hepatic cytosol led to proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay. GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast. Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between endoplasmic reticulum-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70.

In McA-RH7777 cells, the oleate-stimulated assembly and secretion of very low density lipoproteins (VLDL) was associated with enhanced deacylation of phospholipids, which was markedly decreased by inactivation of the cellular phospholipase A(2). Treatment of the cells with antagonists or antisense oligonucleotide of the Ca(2+)-independent phospholipase A(2) (iPLA(2)) significantly inhibited secretion of apoB100-VLDL and triglyceride. Similar inhibitory effect of the iPLA(2) antagonists was observed on apoB48-VLDL secretion, but secretion of high density lipoprotein particles (such as apoAI- and apoB48-high density lipoprotein) or proteins in general was unaffected. The iPLA(2) antagonist did not affect the synthesis of apoB100 or triglyceride, nor did it affect the activities of phospholipase D, phosphatidate phosphohydrolase, or microsomal triglyceride transfer protein. Inactivation of iPLA(2) resulted in impaired apoB100-VLDL assembly as shown by decreased apoB100-VLDL and triglyceride within the microsomal lumen, with concomitant increase in apoB100 association with the microsomal membranes. The inhibitory effect of iPLA(2) antagonists on apoB100-VLDL assembly/secretion could be abated by pretreatment of cells with oleate. Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of iPLA(2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA(2) activity.

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.

Studies in different liver-derived cells in culture indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the ubiquitin-proteasome pathway is a major mechanism for the degradation. The proteasomal degradation of apoB-100 was postulated to be an intrinsic property of the protein that occurs even in the presence of optimal amounts of lipids supplied to the cell. We examined apoB-100 and apoB-48 biogenesis in CaCo2, a human colon carcinoma cell line. To our surprise, apoB-100 and apoB-48 were quantitatively secreted by CaCo2 cells; essentially none of the newly synthesized apoB was degraded before secretion in a 2-h period whether the cells were cultured on filter or on plastic. Furthermore, although ubiquitin immunoreactivity was readily detected in the intracellular apoB isolated from HepG2 cells, little or no ubiquitin was detectable in the intracellular apoB from CaCo2 cells. The amounts of free ubiquitin and total and non-apoB ubiquitinated proteins were comparable in HepG2 and CaCo2 cells, indicating that CaCo2 cells have the necessary machinery for tagging ubiquitin chains onto cellular proteins for proteasomal degradation. Incubation in lipoprotein-deficient serum did not induce apoB degradation, but the addition of a microsomal triglyceride transfer protein inhibitor led to apoB degradation in CaCo2 cells. Finally, similar proportions of apoB polypeptide in isolated microsomes from CaCo2 and HepG2 cells were accessible to exogenously added trypsin, indicating that the mere exposure of apoB nascent chains to the cytosolic compartment is insufficient to cause the proteasomal degradation. Therefore, the intracellular degradation of apoB is not an intrinsic property of the protein, and the phenomenon is neither universal nor inevitable. The unconditional use of apoB as a paradigm for intracellular protein degradation is not warranted.

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. The co-localization of apolipoprotein (apo) B and proteoglycans within lesions has suggested that retention is due to lipoprotein interaction with these highly electronegative glycoconjugates. Both apoB100- and apoB48-containing lipoproteins, i.e. low density lipoproteins (LDLs) and chylomicron remnants, are atherogenic. This suggests that retention is due to determinants in the initial 48% of apoB. To test this, the interaction of an apoB fragment (apoB17), and apoB48- and apoB100- containing lipoproteins with heparin, subendothelial matrix, and artery wall purified proteoglycans was studied. ApoB100-containing LDL from humans and human apoB transgenic mice and apoB48-containing LDLs from apoE knockout mice were used. Despite the lack of the carboxyl-terminal 52% of apoB, the apoB48-LDL bound to heparin-affinity gel as well as did apoB100-LDL. An NH2-terminal fragment containing 17% of full-length apoB was made using a recombinant adenovirus; apoB17 bound to heparin as well as did LDL. Monoclonal antibodies against the NH2-terminal region of apoB decreased apoB100 LDL binding to heparin, whereas antibodies against the LDL receptor-binding region did not alter LDL-heparin interaction. The role of the NH2-terminal region of apoB in LDL interaction with matrix molecules was also assessed. Media containing apoB17 decreased LDL binding to subendothelial matrix by 42%. Moreover, removal of the apoB17 by immunoprecipitation abrogated the inhibitory effect of these media. Antibodies to the NH2-terminal region decreased LDL binding to matrix and dermatan sulfate proteoglycans. Purified apoB17 effectively competed for binding of LDL to artery derived decorin and to subendothelial matrix. Thus, despite the presence of multiple basic amino acids near the LDL receptor-binding domain of LDL, the NH2-terminal region of apoB is sufficient for the interaction of lipoproteins with glycoconjugates produced by endothelial and smooth muscle cells. The presence of a proteoglycan-binding site in the NH2-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.

The initial assembly of apolipoprotein B100 (apoB) into lipoprotein particles occurs cotranslationally. To examine steps required to initiate this process, the intracellular folding and assembly of the amino-terminal 28% of apoB (apoB28) was examined using several criteria including nonreducing gel electrophoresis, sensitivity to dithiothreitol (DTT)-mediated reduction, and buoyant density gradient centrifugation. In hepatoma cells, after a 1-min pulse with radiolabeled amino acids, labeled apoB28 migrated during gel electrophoresis in the folded position and was resistant to reduction in vivo with 2 mM DTT. A similar rate and extent of folding was observed in Chinese hamster ovary cells, a microsomal triglyceride transfer protein (MTP)-negative cell line that can neither lipidate nor efficiently secrete apoB28. Amino-terminal folding of apoB28 was essential for its subsequent intracellular lipidation as apoB28 synthesized in hepatoma cells under reducing conditions remained lipid poor (d > 1.25 g/ml) and was retained intracellularly. Upon DTT removal, reduced apoB28 underwent a process of rapid (t1/2 approximately 2 min) post-translational folding followed by a slower process of MTP-dependent lipidation. As with the cotranslational assembly pathway, post-translational lipidation of apoB28 displayed a strict dependence upon amino-terminal folding. We conclude that: 1) folding of the amino-terminal disulfide bonded domain of apoB is achieved prior to the completion of translation and is independent of MTP and events associated with buoyant lipoprotein formation and 2) domain-specific folding of apoBs amino-terminal region is required to initiate MTP-dependent lipid transfer to nascent apoB in the hepatic endoplasmic reticulum.

Human apolipoprotein B48 (apoB48) and apoB15 (the NH2-terminal 48 and 15% of apoB100, respectively) were translated in vitro from their respective mRNAs using a rabbit reticulocyte lysate and microsomes derived from rat liver or dog pancreas. Synthesis of phosphatidylcholine and triacylglycerols was reconstituted in freshly isolated microsomes by the addition of precursors of these glycerolipids (acylcoenzyme A, glycerol 3-phosphate, and CDP-choline) before, during, or after translation. Assembly of apoB15 and apoB48 with newly synthesized phospholipids and triacylglycerols was favored by active, co-translational lipid synthesis. Moreover, translocation of apoB48 but not B15 into the microsomal lumen was increased in the presence of co-translational lipid synthesis. When apoB48 was translated in vitro, approximately 50% of apoB48 was buoyant at a density of <1.10 g/ml in the lumen of liver microsomes only when lipid synthesis was reconstituted during translation. Microsomal triacylglycerol transfer protein has been proposed to be essential for lipidation and/or translocation of apoB48. However, apoB48 was translocated into the lumen of dog pancreas microsomes in which the activity of the microsomal triacylglycerol transfer protein was not detectable. These data indicate that (i) apoB15 and apoB48 bind newly synthesized phosphatidylcholine during translocation; (ii) apoB48 but not apoB15 associates co-translationally with triacylglycerols; (iii) translocation of apoB48 but not apoB15 is stimulated by lipid synthesis; (iv) assembly of buoyant apoB48-containing lipoproteins can be reconstituted in vitro in the presence of active lipid synthesis; and (v) even in microsomes lacking microsomal triacylglycerol transfer protein activity, apoB48 is translocated into the lumen.

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.

We studied the structural requirements of apolipoprotein (apo) B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB). Recombinant h-apoB48, like endogenous rat apoB48 (r-apoB48), was secreted as VLDL in addition to high density lipoproteins (HDL) by transfected cells, indicating that the N-terminal 48% of apoB contains sequences sufficient for VLDL assembly. Truncation of the C terminus of h-apo-B48 to -B42 or -B37 had little effect on the ability of apoB to assemble VLDL, whereas truncation to -B34 or -B29 markedly diminished or abolished VLDL formation. None of the truncations affected the integration of apoB into HDL. To determine whether the ability to assemble VLDL is governed by apoB length or by sequences beyond apoB29, we created chimeric proteins that contained human apoA-I and a segment derived from between the C-terminal 29 and 34%, 34 and 37%, or 37 and 42% of apoB100. The resulting chimeras, namely AI/B29-34, AI/B34-37, and AI/B37-42, were secreted by the transfected cells as lipoproteins with buoyant density (d < 1.006 g/ml), electrophoretic mobility (pre-beta), and size characteristics of human plasma VLDL. The chimeras could assemble discrete VLDL particles devoid of endogenous r-apoB100, and could actively recruit triglycerides and phospholipids into the lipoproteins. However, these chimeras were secreted inefficiently. Pulse-chase analysis showed that less than 5% of the newly synthesized AI/B proteins were secreted, and more than 70% was degraded intracellularly. Degradation of the chimeras could be blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, but the treatment did not enhance their secretion. Protease protection analysis of microsomes isolated from transfected cells indicated that >65% of AI/B chimeras (compared with <25% of r-apoB100) were inaccessible to exogenous trypsin. These data suggest that the recruitment of large quantities of triglycerides during VLDL formation is not governed simply by apoB length, but is mediated by short hydrophobic sequences ranging from 152 to 237 amino acids (3-5%) of apoB. The existence of multiple such hydrophobic sequences within apoB48 may facilitate efficient assembly of hepatic VLDL particles.

A site-specific C to U editing reaction modifies nuclear apolipoprotein B100 (apoB100) mRNA, producing apolipoprotein B48 in the mammalian small intestine. This reaction is mediated by a multicomponent enzyme complex, which contains a catalytic subunit, Apobec-1. We have used gene targeting to disrupt mouse apobec-1 in order to establish its requisite importance in apoB mRNA editing and also, in view of its widespread tissue distribution in rodents, as a preliminary indication of other potential roles. Both heterozygous (apobec-1+/-) and homozygous (apobec-1-/-) gene-targeted mice appear healthy and fertile with no alterations in serum cholesterol or triglyceride concentrations. The apobec-1+/- mice demonstrated reduced levels of hepatic apoB mRNA editing. By contrast, levels of small intestinal apoB mRNA editing were indistinguishable in wild-type and apobec-1+/- animals, suggesting that Apobec-1 is expressed in limited quantities in the liver but not in the small intestine. The apobec-1-/- mice lacked detectable levels of Apobec-1 mRNA, expressed only unedited apoB mRNA in all tissues, and contained no apoB48 in their serum, demonstrating that there is no functional duplication of this gene.

BFA inhibited in a dose dependent way the assembly of apoB-48 very low density lipoprotein (VLDL) but allowed a normal rate of biosynthesis of the apolipoprotein and of the assembly of the dense ("high density lipoprotein (HDL)-like") apoB-48 particle (apoB-48 HDL). The inhibition of the assembly of apoB-48 VLDL occurred at BFA levels that allowed a major secretion of both transferrin and apoB-48 HDL. The assembly of apoB-100 containing lipoproteins was also inhibited by BFA but could be reactivated by a 30-60 min chase in the absence of BFA, which agreed with the time that was estimated to be needed to restore the secretory pathway (approximately 60 min). Also the assembly of apoB-48 VLDL was reversible. Both apoB-48 and apoB-100 that was labeled in the presence of BFA assembled VLDL after removal of the BFA. Both apoB-100 and apoB-48 were associated with the membrane pellet of the microsomes. Virtually all (122 +/- 30%) of the membrane associated pulse-labeled apoB-48 remained in the membrane after a 180-min chase in the presence of BFA, compared to only 21 +/- 2% in normal cells (mean +/- S.D., n = 4). The corresponding figures for apoB-100 was 40 +/- 7% in BFA-treated cells and 9 +/- 7% in normal cells (mean +/- S.D., n = 4). Pulse-chase experiments with BFA offered conditions to selectively follow the turnover of membrane-associated apoB-100. Such experiments indicated that this apoB-100 pool is a precursor to VLDL.

Apolipoprotein (apo) B-100 is the major protein component in low density lipoprotein (LDL); it contains the binding domain for the LDL receptor and the attachment site for apolipoprotein(a) in lipoprotein(a). ApoB-48 is colinear with the amino-terminal half of apoB-100 and misses the part of the molecule required for LDL receptor interaction and lipoprotein(a) formation. ApoB-48 mRNA is produced by the editing of apoB-100 mRNA, a process by which the codon CAA for Gln-2153 is changed to UAA, an in-frame stop codon. We used the cloned catalytic component of the rat apoB mRNA-editing enzyme (REPR) to construct a replication-defective recombinant adenoviral vector containing REPR cDNA (AvREPR) and a control vector (Av1LacZ4) containing a beta-galactosidase cDNA to investigate the effect of REPR gene delivery in C57BL/6 mice. Intravenous injection of AvREPR in mice resulted in efficient transduction of liver cells, where REPR mRNA and protein were overexpressed, reaching a peak at 7 and 12 days, returning toward control levels at 39 days after AvREPR administration. ApoB mRNA editing activity in liver extracts showed changes parallel to those of REPR mRNA expression; the proportion of edited apoB mRNA in the total hepatic apoB mRNA increased from approximately 60% to more than 90% at the peak of REPR expression. The proportion of plasma apoB-100 in AvREPR-transduced animals decreased from approximately 50% to < 10% of total plasma apoB concentration. Plasma very low density lipoproteins were polydisperse in control animals with an average diameter of 54.9 +/- 20.6 nm (uninjected control) and 54.7 +/- 16.8 nm (Av1LacZ4-treated), respectively. They became much smaller (average diameter 39.3 +/- 12.7 nm) and more uniform in size at day 12 following AvREPR administration. On the same day, the normal plasma LDL (26.2-25.5 nm) was almost completely eliminated in treated animals. Adenovirus-mediated transfer of the REPR cDNA is an efficient method to reduce plasma apoB-100 and normal LDL production.

The mechanisms by which apolipoprotein B-100 (apoB-100) and apoB-48 assemble lipoproteins have been studied in the McA-RH7777 cell line. After incubation for 2 h with 360 microM oleic acid, the McA-RH7777 cells secreted apoB-100 mainly on very low density lipoprotein (VLDL) particles, as judged from sucrose gradient and sequential ultracentrifugation. ApoB-48 was secreted on both VLDL and on denser, high density lipoprotein (HDL)-like lipoproteins. Both apoB-48 and apoB-100 occurred on VLDL particles in the luminal content of the total microsomal fraction. In addition, both proteins were present on denser particles, in particular, those that banded in the HDL region. The denser particles containing apoB-48 were secreted from the cells, whereas those containing apoB-100 were retained in the cell and degraded. Pulse-chase experiments showed that apoB-100 on VLDL was present in the secretory pathway already after a labeling period as short as 3 min. Thus, this particle was the first apoB-100-containing lipoprotein that could be detected in the microsomal fraction of the cell. The assembly of labeled apoB-100 VLDL was acutely (within min) and completely blocked by cycloheximide, if the cycloheximide was added after the pulse labeling period. If, on the other hand, a 15-min chase was introduced after the labeling period, there was no effect of cycloheximide on the assembly of apoB-100 VLDL. A 15-min chase is enough to allow the pulse-labeled apoB nascent polypeptides to be completed to apoB-100. These results indicated that the assembly of apoB-100-containing VLDL is dependent upon ongoing protein biosynthesis during the time when the nascent polypeptide elongate. After this period, the assembly and secretion of the particles are independent of ongoing protein biosynthesis. The first apoB-48-containing particle seen in the luminal content of the microsomal fraction was the HDL-like particle. Pulse-chase experiments as well as experiments with different lengths of the radioactive pulse indicated that the formation of apoB-48-containing VLDL was delayed compared with the formation of the apoB-48-containing HDL-like particle and also in relation to the assembly of the different apoB-100-containing particles, including VLDL. After a 30-min pulse with [35S]methionine followed by a 120-min chase the secretory pathway of the cell was depleted of almost all lipoproteins except the HDL-like apoB-48-containing particle.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein B (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoproteins. Recent studies have revealed that in both hepatic and nonhepatic cells a large percentage of newly synthesized apoB polypeptides engage in transmembrane interactions with the endoplasmic reticulum (ER). These apoB-membrane interactions have been implicated in the processes of lipoprotein assembly and regulation. To identify domains of apoB that are responsible for its transmembrane localization, overlapping 300 amino acid segments of human apoB48 (amino-terminal 48% of apoB) and two control proteins, human complement component C3 and mouse CD4, were appended to the amino-terminal 77 amino acids of a soluble secretory precursor protein and expressed in COS-1 cells. While the integral membrane protein CD4 conferred predictable transmembrane orientation on the hybrid protein, as evidenced by its partial protease accessibility in intact microsomes, all of the apoB-containing proteins and the soluble secretory control, C3, were fully protease-resistant, consistent with their complete translocation into the ER. To determine if conformational properties of apoB are responsible for its transmembrane interactions with the ER, proteins containing the entire amino-terminal approximately 50% of apoB (apoB50) were expressed in COS-1 cells. Irrespective of whether targeting and translocation initiation were directed by a heterologous signal peptide or the native apoB signal peptide, apoB50 appeared to undergo complete membrane translocation into a protease-inaccessible compartment. These results demonstrate that the amino-terminal 50% of apoB lacks autonomous signals or properties that can fully block ER membrane translocation or promote any other form of stable transmembrane assembly in nonhepatic cells.

Mammalian small intestine secretes a truncated apolipoprotein B (apoB48) species as a result of tissue-specific post-transcriptional RNA editing. The human liver, by contrast, contains only unedited apoB mRNA and secretes only apoB100. We have recently isolated a cDNA clone from rat small intestine which encodes an apoB mRNA editing protein, REPR (Teng, B., Burant, C.F., and Davidson, N.O. (1993) Science 260, 1816-1819). The current study demonstrates that homogenates of Xenopus oocytes expressing REPR confer editing ability upon S100 extracts prepared from human liver when tested on a synthetic apoB RNA template in vitro. Transfection of REPR into HepG2 cells resulted in editing of endogenous apoB mRNA and the appearance of an apoB48-like protein in the media. Extracts prepared from these transfected cells edit mammalian apoB RNA templates when incubated alone and with enhanced efficiency in the presence of chicken intestinal S100 extracts. The results suggest that human liver expresses factor(s) which are critical to apoB mRNA editing and which allow functional complementation of REPR in vivo.

Overexpression of human apolipoprotein (apo) C-III in the plasma of transgenic mice results in hypertriglyceridemia, with up to a 20-fold elevation in plasma triglyceride. Nearly all of the triglyceride accumulates in the d < 1.006 g/ml lipoprotein fraction, which consists predominantly of apoB48-containing particles having a low apoE:apoB48 ratio in contrast to normal mice. The transgenic and nontransgenic d < 1.006 g/ml lipoproteins are similar in size, and they are equivalent substrates for lipoprotein lipase in vitro. Total apoB100 levels are similar in transgenic and normal plasma, but apoB48 levels are increased in transgenic mice. The transgenic d < 1.006 g/ml particles are poor competitors for the binding of low density lipoproteins to the low density lipoprotein receptor in vitro, which is corrected by the addition of exogenous apoE. The rate of clearance of labeled chylomicron remnants in apoC-III-transgenic mice was about half that in nontransgenic mice. The lipoprotein alterations are accompanied by up to a 5-fold increase in circulating nonesterified fatty acids, which may be the cause of fatty livers and increased liver triglyceride production also observed in the transgenic mice. These observations indicate that the primary defect leading to hypertriglyceridemia in apoC-III overexpressers is an impaired clearance of apoB48 remnants due to apoE insufficiency. Therefore, transgenic mice that overexpressed human apoE were cross-bred with the apoC-III overexpressers. Transgenic progeny that produced both human apoE and human apoC-III had normal levels of plasma triglyceride and normal amounts of apoB48 remnants. Thus, our studies suggest that a function of apoC-III is to modulate the apoE-mediated clearance of lipoproteins, and that the concentration of apoC-III relative to apoE is a key determinant of triglyceride levels in plasma.

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.

Apolipoprotein B (apoB) 48 mRNA is the product of a C----U conversion of the first base of the codon CAA for Gln-2153 in apoB-100 mRNA, changing it to an in-frame stop codon (UAA). Methods for measuring the ratio of apoB-48 to apoB-100 mRNA that have been authenticated with standard mixtures of the two apoB mRNA species have not been described. Using RNA mixtures consisting of known proportions of in vitro transcripts of apoB-100 and apoB-48, we directly compared four different assays. We found that a procedure based on the polymerase chain reaction, cDNA cloning, and oligonucleotide colony hybridization was the most sensitive and accurate assay. Total RNAs isolated from adult rat small intestine, adult liver, Day 15 and term placentas, and term fetal membranes were found to contain apoB mRNA in the following relative amounts: 100, 59.8, 0.9, 6.96, and 1087, respectively. They all contained both apoB-48 and apoB-100 mRNAs, with the former constituting 95.8, 59.2, 4.6, 1.3, and 0.8%, respectively, of the apoB mRNA. We examined the ontogeny of apoB-48 mRNA biogenesis in the liver and intestine in the rat prenatally on Days 17, 19, and 20 of gestation, and postnatally on Days 1, 3, 7, 13, 20, 24, and 37 after birth. Slot-blot hybridization demonstrated that apoB mRNA showed a peak at birth (Days 1-3 in the liver and Days 1-7 in the small intestine) and then decreased on Days 7 (in the liver) and 13 (in the intestine) before it increased again on Day 20 toward the adult level. Quantitation of the ratio of apoB-48 to apoB-100 mRNA at the different time points showed that RNA editing became highly competent prenatally on Day 19 of gestation in the small intestine, but postnatally on Day 24 after birth in the liver. The asynchronous nature for this developmentally regulated process in the liver and small intestine of the rat has implications for the mechanism of RNA editing and lipid homeostasis in this animal.

The present study characterizes the substructural organization of apolipoprotein B-100 (B-100) of human plasma low density lipoproteins (LDL) and its relation to B-74 and B-26 of LDL and B-48 of chylomicrons and very low density lipoproteins. LDL were digested with human kallikrein and thrombin to yield two major fragments: K1 (Mr 410,000) and K2 (Mr 145,000) and T1 (Mr 385,000) and T2 (Mr 170,000), respectively. The antigenic sequences, Mr, and amino acid compositions of K1 and K2 were identical to those of plasma B-74 and B-26; B-26 and K2 had identical NH2-terminal sequences and correspond to the NH2-terminal region of B-100. K1 was further degraded by kallikrein to give K3 (Mr 235,000) and K4 (Mr 170,000); these peptides correspond immunochemically to two newly discovered plasma LDL peptides B-44 and B-30 and are assigned as complementary fragments of B-74. The thrombin cleavage fragments, T1 and T2, did not correspond to B-74 and B-26. Neither kallikrein nor thrombin generated a fragment from B-100 corresponding to B-48 in chylomicrons. However, B-48 showed antigenic homology with B-26 and to the of B-74 adjoining B-26, indicating that its structure is represented in the NH2-terminal half of B-100. The structural studies further clarify the relatedness among the B-100, B-74, B-26, and B-48 polypeptides and should now make possible the delineation of the functional domains mediating the interactions of apolipoprotein B in the circulation and arterial wall.

The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of approximately 6 kDa was isolated from a tryptic digest of apo-B-100. The sequence of the first 22 amino acids of MDB-18 was determined by Edman degradation. A 15-residue peptide corresponding to this sequence was synthesized by the solid-phase method and was utilized to develop a sequence-specific polyclonal antibody. On immunoblot analysis, the antibody recognized both intact apo-B-100 and apo-B-48. In addition, preincubating the antibody with the synthetic peptide abolished the recognition of both apo-B-100 and apo-B-48. These data are interpreted as indicating that there is an amino acid sequence homology between apo-B-100 and apo-B-48. Since the MDB-18 peptide is located in the carboxyl region of the B-100 molecule, we propose apo-B-100 and apo-B-48 share a common carboxyl region sequence.

Immunochemical studies have demonstrated that apoprotein B-100 and apoprotein B-48 share some antigenic determinants, but whether they are products of the same gene has remained uncertain. Utilizing a specific mouse monoclonal antibody, MB19, we recently characterized a common form of genetic polymorphism that was expressed in apo-B-100 (Young, S. G., Bertics, S. J., Curtiss, L. K., Casal, D. C., and Witztum, J.L. (1985) Proc. Natl. Acad. Sci. U.S.A., in press). Antibody MB19 binds different allotypes of apo-B-100 (MB19(1) and MB19(2] with high and low affinities, respectively. Compared to a rabbit antiserum against human low density lipoprotein, which detects 100% of apo-B mass in all individuals, antibody MB19 detects 100% of apo-B with allotype MB19(1) but less than 10% of apo-B with allotype MB19(2). Western blots demonstrate that MB19 binds to both apo-B-100 and apo-B-48. To determine if apo-B-48 and apo-B-100 from the same individual express the same polymorphism, chylomicrons and very low density lipoproteins were isolated from 23 subjects in whom the allotypes of apo-B-100 were known. Delipidated apoproteins were separated electrophoretically and then transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with 125I-MB19 (to detect the polymorphism) and 131I-antiserum to human apo-B (to quantitate total apo-B transferred to nitrocellulose membranes). Apo-B-100 and apo-B-48 bands were removed and the ratio of 125/131 counts in each band was calculated. In all 23 subjects studied, the same MB19 polymorphism was present in both apo-B-100 and apo-B-48. This observation provides strong evidence that both apoproteins are products of the same gene.

Apolipoprotein (apo) B occurs in two forms, apoB100 (512 kDa) and apoB48 (240 kDa); both are derived from the same gene. A novel mechanism involving editing of the apoB mRNA causes the formation of apoB48; the first base of codon 2153 is changed from cytosine to uracil, converting a glutamine codon to a premature stop codon. To identify the apoB mRNA sequence elements recognized by the apoB mRNA editing mechanism, two apoB cDNA fragments (354 and 63 base pairs) with codon 2153 near their centers were inserted into a high expression vector of another secreted apolipoprotein, apoE. The resulting vectors, pHEB-354 and -63, were transfected into Chinese hamster ovary cells, HepG2 cells, and apoB48-producing CaCo-2 cells. The secreted chimeric apolipoproteins (apoEB354 and apoEB63) were analyzed for premature truncation, and the mRNA was analyzed for the presence of an edited base. The pHEB-354 construct produced a truncated protein only in CaCo-2 cells, whereas pHEB-63 produced no truncated protein in any of the three cell types. The mRNA was converted to cDNA and amplified by the polymerase chain reaction technique. Differential hybridization of the polymerase chain reaction products with CAA (Gln) and TAA (Stop) specific probes detected an edited base only in cDNA from CaCo-2 cells transfected with pHEB-354, in agreement with the protein analysis. We conclude that the nucleotide sequence of the apoB cDNA insert in pHEB-354 contains sufficient information to be edited in CaCo-2 cells. In these cells, a cryptic polyadenylation site was activated in the edited pHEB-354 mRNA. As a result, CaCo-2 cells transfected with pHEB-354 produced a short, edited pHEB-354 mRNA and a long, unedited pHEB-354 mRNA. Chinese hamster ovary cells transfected with pHEB-354 or CaCo-2 cells transfected with pHEB-63 produced only a full length transcript. Amplification of the pHEB-354 cDNA using 3'-primers upstream and downstream of the poly(A) addition site and hybridization with the TAA probe confirmed these results. This unusual mRNA editing apparently occurs before polyadenylation, probably in the nucleus.

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.