The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells

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.     

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2;-8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2;-5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver.     

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.     

The assembly and secretion of ApoB 100-containing lipoproteins in Hep G2 cells. ApoB 100 is cotranslationally integrated into lipoproteins.

The possibility that apoB 100 is cotranslationally translocated to the endoplasmic reticulum lumen and integrated into lipoproteins has been investigated. ApoB 100 nascent polypeptides were shown to be secreted from pulse-labeled Hep G2 cells after treatment with puromycin and chase for 1 or 2 h in the presence of puromycin and cycloheximide. These nascent polypeptides banded during sucrose gradient ultracentrifugation between the position of the high (HDL) and the low (LDL) density lipoproteins, revealing an inverse relationship between the length of the polypeptide and the density of the fraction. ApoB 100 occurred in the position of LDL and very low density lipoproteins (VLDL). Electronmicroscopy studies of the apoB-containing particles from the gradient indicated an increase in size with increasing length of the polypeptide. Furthermore, labeling studies indicated that the triglyceride load increased with the length of the polypeptide. An inverse relationship between the size of C-terminally truncated apoB polypeptides and the density of the assembled lipoproteins was also observed in experiments with transfected minigenes coding for apoB 41, apoB 29, and apoB 23. These proteins appeared on HDL particles. Pulse-chase experiments indicated that 80-200-kDa apoB nascent polypeptides on particles with HDL density, with time, were converted into larger polypeptides on lighter particles, to be fully replaced by apoB 100 on LDL-VLDL particles. The formation of these LDL-VLDL particles could be blocked by cycloheximide. Sixty-five percent of pulse-labeled apoB nascent polypeptides present in the microsomal fraction was released by sodium carbonate treatment, and 77% of these polypeptides could be recovered on the immature particles (banding between HDL and LDL) after sucrose gradient ultracentrifugation. Pulse-chase experiments indicated that these nascent polypeptides, on the immature lipoproteins, had the capacity to be precursors for all the apoB 100-containing LDL and VLDL particles formed in the cell. The obtained results indicate that a major portion of the apoB nascent polypeptides in the cell form lipoproteins cotranslationally during the translocation to the lumen of the endoplasmic reticulum.     

Microsomal membrane-associated apoB is the direct precursor of secreted VLDL in primary cultures of rat hepatocytes

Brefeldin A (BFA) added to primary cultures of rat hepatocytes, at a concentration of 0.2 microg/ml, prevented the assembly of newly synthesized apolipoprotein B (apoB) into mature, secretory VLDL but did not prevent the secretion of apoB as denser particles (HDL apoB), or of albumin. The unassembled apoB remained associated with the membranes of the cellular microsomal fraction. There was no effect of BFA on the removal of apoB from the lumen of these vesicles. VLDL apoB formed only a minor component of the total apoB in the microsomal lumen. Higher (5 microg/ml) concentrations of BFA were required to prevent the secretion of HDL apoB and albumin. Under these conditions apoB accumulated in the microsomal lumen, as well as in the membranes of these vesicles. Again, apoB VLDL formed only a minor proportion of the total lumenal apoB. ApoB-48 VLDL and apoB-100 VLDL assembly could be restored by removing BFA from the medium. This reactivation of VLDL assembly was accompanied by an increased removal of apoB from the microsomal membranes, but there was no detectable increase in the small quantity of VLDL apoB that was recovered from the microsomal lumen. In the absence of BFA, during pulse-chase experiments the pattern of change in the specific radioactivity of microsomal membrane apoB was similar to that of the secreted VLDL apoB whereas that of the lumenal apoB resembled that of the secreted HDL apoB. The results suggest that membrane-associated apoB is the main direct precursor of secreted VLDL apoB in primary cultures of rat hepatocytes and that VLDL assembly does not involve primarily microsomal lumenal apoB as an intermediate.     

The degradation of apolipoprotein B100: multiple opportunities to regulate VLDL triglyceride production by different proteolytic pathways

Very low density lipoproteins (VLDL) are a major secretory product of the liver. They serve to transport endogenously synthesized lipids, mainly triglycerides (but also some cholesterol and cholesteryl esters) to peripheral tissues. VLDL is also the precursor of LDL. ApoB100 is absolutely required for VLDL assembly and secretion. The amount of VLDL triglycerides secreted by the liver depends on the amount loaded onto each lipoprotein particle, as well as the number of particles. Each VLDL has one apoB100 molecule, making apoB100 availability a key determinant of the number of VLDL particles, and hence, triglycerides, that can be secreted by hepatic cells. Surprisingly, the pool of apoB100 in the liver is typically regulated not by its level of synthesis, which is relatively constant, but by its level of degradation. It is now recognized that there are multiple opportunities for the hepatic cell to intercept apoB100 molecules and to direct them to distinct degradative processes. This mini-review will summarize progress in understanding these processes, with an emphasis on autophagy, the most recently described pathway of apoB100 degradation, and the one with possibly the most physiologic relevance to common metabolic perturbations affecting VLDL production. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Modulation of apolipoprotein B-100 mRNA editing: effects on hepatic very low density lipoprotein assembly and intracellular apoB distribution in the rat

We have studied the consequences of alterations to hepatic apoB mRNA editing on the biosynthesis and intracellular distribution of newly synthesized apoB variants together with their mass distribution in nascent Golgi very low density lipoproteins (VLDL). Radiolabeled liver membrane fractions were prepared from control or hypothyroid animals and separated by discontinuous sucrose gradient centrifugation. Hepatic apoB-100 synthesis in these groups accounted for 93-100% of total newly synthesized apoB species of Golgi fractions recovered from the sucrose gradients (G1 and G2). The analogous fractions isolated from the livers of hyperthyroid (treated with 3,3',5-triiodo-L-thyronine, T3) animals revealed that newly synthesized apoB-100 accounted for only 46 +/- 10% (G1) and 24 +/- 11% (G2), respectively, of total newly synthesized apoB. ApoB-100 mass in nascent Golgi VLDL from control and hypothyroid G1 fractions represented 70-78% total apoB as determined by Western blot analysis. By contrast, Golgi VLDL from hyperthyroid animals contained predominantly (greater than 78%) apoB-48 as the apoB species. Electron microscopy revealed that the morphology and size distribution of hyperthyroid G1 VLDL were similar to particles isolated from control animals. Thus, despite a profound reduction in the proportion of apoB-100 mRNA species containing an unmodified codon (CAA, B-GLN) at position 2153 in hyperthyroid animals (6 +/- 1% vs 50-61% in control and hypothyroid animals) apoB-100 biosynthesis was detectable in a defined membrane fraction isolated by discontinuous sucrose gradient centrifugation. However, no apoB-100 synthesis was detectable in liver samples prepared by Polytron disruption in Triton-containing buffers. These data suggest that effective hepatic VLDL assembly and secretion in the T3-treated rat continues despite a profound reduction in apoB-100 biosynthesis and implies that apoB-48 contains the requisite domains to direct this process, a situation analogous to that in the intestine.     

Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent,and its lipidation to very low density lipoprotein occurs post-ER

Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL). Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs. Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23. Moreover, ER exit of apoB was inhibited by dominant-negative Sar1. Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER. Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs. VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells. We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.     

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.     

Metabolic behavior of hepatic VLDL and plasma LDL apoB-100 in African green monkeys

Recently, evidence has accumulated suggesting that significant amounts of plasma low density lipoproteins (LDL) may be derived by direct production. These plasma very low density lipoprotein (VLDL)-independent sources include the production and secretion of LDL-like particles directly by the liver, and/or a small pool of nascent precursor particles that are converted rapidly to LDL. The current studies were designed to test the hypothesis that hepatic VLDL represent a rapidly turning over precursor pool to plasma LDL in African green monkeys. Livers from African green monkeys were perfused with serum-free medium containing [3H]leucine or 3H-labeled amino acids for 4-6 hr. Hepatic [3H]VLDL and autologous plasma 125I-labeled LDL were injected simultaneously into recipient animals and density gradient ultracentrifugation and gel filtration were used to characterize the distribution of 3H and 125I radioactivity at selected times after injection. These studies show that 4 to 66% of the injected dose of hepatic VLDL [3H]apoB-100 was metabolized extremely rapidly into particles that resembled the recipient's plasma LDL by size and density. Based on the kinetic model developed to describe the metabolic behavior of hepatic VLDL [3H]apoB-100, the estimated maximal pool size of hepatic VLDL apoB-100 in these animals was very small (0.042 and 0.112 mg) and represented, at best, approximately 10% of the average plasma VLDL apoB-100 mass found in cholesterol-fed African green monkeys. In addition, the radiolabeled hepatic LDL appear to be metabolized similarly to plasma LDL. That is, the rapid conversion of hepatic VLDL as well as the direct production of hepatic particles within the LDL density range appear to contribute to plasma LDL. Metabolic heterogeneity was also seen within the LDL class. The more buoyant subfraction (LDL1) had a higher turnover rate than the more dense subfraction (LDL2) and hepatic VLDL-derived [3H]LDL1 had a slower final rate of plasma disappearance than the plasma-derived 125I-labeled LDL1 in most animals. The results from these studies suggest that a small pool of hepatic VLDL can be converted very rapidly to plasma LDL and may contribute significantly to the large plasma pool of LDL seen in cholesterol-fed African green monkeys. This pathway may be analogous to the pathway in some human subjects in which a portion of human plasma VLDL is converted rapidly into LDL without passing through a delipidation cascade, often referred to as direct LDL production.     

Inhibition of apolipoprotein B secretion by taurocholate is controlled by the N-terminal end of the protein in rat hepatoma McArdle-RH7777 cells

Bile salts (BS) inhibit the secretion of apolipoprotein B (apoB) and triacylglycerol (TG) in primary rat, mouse and human hepatocytes and in mice in vivo. We investigated whether lipidation of apoB into a lipoprotein particle is required for this inhibitory action of BS. The sodium/taurocholate co-transporting polypeptide (Ntcp) was co-expressed in McArdle-RH7777 (McA-RH7777) cells stably expressing the full-length human apoB100 (h-apoB100, secreted as TG-rich lipoprotein particles) or carboxyl-truncated human apoB18 (h-apoB18, secreted in lipid-free form). The doubly transfected cell lines (h-apoB/r-Ntcp) effectively accumulated taurocholic acid (TC). TC incubation decreased the secretion of endogenous rat apoB100 (-50%) and h-apoB18 (-35%), but did not affect secretion of rat apoA-I. Pulse-chase experiments (35S-methionine) indicated that the impaired secretion of radiolabeled h-apoB18 and h-apoB100 was associated with accelerated intracellular degradation. The calpain protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) partially inhibited intracellular apoB degradation but did not affect the amount of either h-apoB18 or h-apoB100 secreted into the medium, indicating that inhibition of apoB secretion by TC is not due to calpain-dependent proteasomal degradation. We conclude that TC does not inhibit apoB secretion by interference with its lipidation, but rather involves a mechanism dependent on the N-terminal end of apoB.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.     

Apolipoprotein B-containing lipoprotein particle assembly: lipid capacity of the nascent lipoprotein particle

We previously proposed that the N-terminal 1000-residue betaalpha(1) domain of apolipoprotein B (apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin. In support of this "lipid pocket" hypothesis, we demonstrated that apoB:1000 (residues 1-1000) is secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with high density lipoprotein 3 (HDL(3)) density. In contrast, apoB:931 (residues 1-931), missing only 69 residues of the sequence homologous to lipovitellin, was secreted as a particle considerably more dense than HDL(3). In the present study we have determined the stoichiometry of the lipid component of the apoB:931 and apoB:1000 particles. The secreted [(3)H]glycerol-labeled apoB:1000 particles, isolated by nondenaturing gradient gel electrophoresis, contained 50 phospholipid (PL) and 11 triacylglycerol (TAG) molecules/particle. In contrast, apoB:931 particles contained only a few molecules of PL and were devoid of TAG. The unlabeled apoB:1000 particles, isolated by immunoaffinity chromatography, contained 56 PL, 8 TAG, and 7 cholesteryl ester molecules/particle. The surface to core lipid ratio of apoB:1000-containing particles was approximately 4:1 and was not affected by oleate supplementation. Although very small amounts of microsomal triglyceride transfer protein (MTP) were associated with apoB:1000 particles, it never approached a 1:1 molar ratio of MTP to apoB. These results support a model in which (i) the first 1000 amino acid residues of apoB are competent to complete the lipid pocket without a structural requirement for MTP; (ii) a portion, or perhaps all, of the amino acid residues between 931 and 1000 of apoB-100 are critical for the formation of a stable, bulk lipid-containing nascent lipoprotein particle, and (iii) the lipid pocket created by the first 1000 residues of apoB-100 is PL-rich, suggesting a small bilayer type organization and has a maximum capacity on the order of 50 molecules of phospholipid.     

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures.

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.     

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular mechanisms regulating apoB-containing lipoprotein assembly and secretion in primary hamster hepatocytes

We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o-phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post-translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB-containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates.     

Specificity of lipid incorporation is determined by sequences in the N-terminal 37 of apoB

The N-terminal 17% of apolipoprotein B (apoB-17) is secreted lipid-poor while apoB-41 particles are secreted with a triacylglycerol (TAG)-rich core. Thus, the sequence between apoB-17 and apoB-41 is necessary for the assembly of TAG-rich lipoproteins. To delineate this region, C127 cells were permanently transfected to secrete the N-terminal 29, 32.5, or 37% of apoB. Density gradient centrifugation showed that secreted apoB-29, apoB-32.5, and apoB-37 had peak densities of 1.25, 1.22, and 1.16 g/mL and percent lipid of particle weights of 30, 37, and 49%, respectively. Calculated anhydrous particle diameters were: apoB-29 = 81 A, apoB-32.5 = 88 A, and apoB-37 = 101 A. Immunoprecipitated particles labeled with [(3)H]oleate showed that, as apoB length increased from apoB-29 to apoB-32.5 and apoB-37, the number of TAG (core) molecules per apoB particle increased almost 16-fold from 8 to 32 to 124, while phospholipids and diacylglycerols (surface lipids) increased only slightly from 71 to 87 to 97 molecules, respectively. Thus, sequences in the C-terminus of apoB-29 bind phospholipids and diacylglycerols, sequences between apoB-29 and apoB-32.5 augment TAG binding and sequences between apoB-32.5 and apoB-41 account for the marked incorporation of TAG at a rate of approximately 1 TAG per 2 amino acids. Cryoelectron micrographs of isolated apoB-37 particles revealed mostly spherical particles of approximately 110 A (11.0 nm) with an electron lucent center, consistent with these particles having a TAG core. We suggest that the predicted amphipathic beta-sheets beginning at apoB-29, starts to preferentially recruit core lipids into apoB and propose that the consistent presence of DAG in the secreted particles may have a role in fission of the nascent lipoprotein particles from the endoplasmic reticulum membrane.     

Apolipoprotein B metabolism and the distribution of VLDL and LDL subfractions

Apolipoprotein B (apoB) metabolism was investigated in 20 men with plasma triglyceride 0.66-2.40 mmol/l and plasma cholesterol 3.95-6. 95 mmol/l. Kinetics of VLDL(1) (S(f) 60-400), VLDL(2) (S(f) 20-60), IDL (S(f) 12-20), and LDL (S(f) 0;-12) apoB were analyzed using a trideuterated leucine tracer and a multicompartmental model which allowed input into each fraction. VLDL(1) apoB production varied widely (from 5.4 to 26.6 mg/kg/d) as did VLDL(2) apoB production (from 0.18 to 8.4 mg/kg/d) but the two were not correlated. IDL plus LDL apoB direct production accounted for up to half of total apoB production and was inversely related to plasma triglyceride (r = -0.54, P = 0.009). Percent of direct apoB production into the IDL/LDL density range (r = 0.50, P < 0.02) was positively related to the LDL apoB fractional catabolic rate (FCR). Plasma triglyceride in these subjects was determined principally by VLDL(1) and VLDL(2) apoB fractional transfer rates (FTR), i.e., lipolysis. IDL apoB concentration was regulated mainly by the IDL to LDL FTR (r = -0.71, P < 0.0001). LDL apoB concentration correlated with VLDL(2) apoB production (r = 0.48, P = 0.018) and the LDL FCR (r = -0.77, P < 0. 001) but not with VLDL(1), IDL, or LDL apoB production. Subjects with predominantly small, dense LDL (pattern B) had lower VLDL(1) and VLDL(2) apoB FTRs, higher VLDL(2) apoB production, and a lower LDL apoB FCR than those with large LDL (pattern A). Thus, the metabolic conditions that favored appearance of small, dense LDL were diminished lipolysis of VLDL, resulting in a raised plasma triglyceride above the putative threshold of 1.5 mmol/l, and a prolonged residence time for LDL. This latter condition presumably permitted sufficient time for the processes of lipid exchange and lipolysis to generate small LDL particles.     

Disulfide bonds are required for folding and secretion of apolipoprotein B regardless of its lipidation state

Apolipoprotein (apo) B-100, an essential protein for the assembly and secretion of very low density lipoproteins depends on lipid binding (lipidation) for its secretion. Seven of its 8 disulfides are clustered within the N-terminal 21%. The role of these disulfides in the secretion of lipidated or unlipidated truncated forms of apoB was studied in C127 cells expressing apoB-17, apoB-29, or apoB-41. These cells do not express microsomal triglyceride transfer protein yet secrete apoB-41 on triacylglycerol-rich lipoproteins while apoB-29 and apoB-17 are secreted with little or no lipid, respectively. Dithiothreitol utilized in pulse-chase studies prevented the cotranslational formation of disulfides and when added posttranslationally reduced native disulfides. As a result, the secretion of reduced apoB forms was blocked and they were retained in the cells. Reduced apoB polypeptides were rescued following removal of dithiothreitol, as they underwent post-translational disulfide bonding, attained their mature form, and were subsequently secreted. Together the data suggest that in C127 cells the formation of native disulfides is critical for the folding and secretion of apoB independent of its length, its requirement for lipidation or microsomal triglyceride transfer protein expression. Therefore, these cells provide an appropriate model to study the folding of apoB in great detail.