Efficient glycosylation site utilization by intracellular apolipoprotein B. Implications for proteasomal degradation

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) may control the secretion of apoB-containing lipoproteins. In one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation behavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-terminal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide cleavage, all of the sites were glycosylated irrespective of cell type, labeling time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn residues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-linked glycosylation occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2. 5-fold increase in intracellular apoB but failed to cause accumulation of an unglycosylated form. These results indicate that 1) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2) despite its large size and affinity for lipid, delivery of misassembled apoB to the proteasome requires retrograde translocation from the ER lumen to cytosol.     

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.     

A novel nontruncating APOB gene mutation,R463W,causes familial hypobetalipoproteinemia

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.     

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.     

Missense mutations in APOB within the betaalpha1 domain of human APOB-100 result in impaired secretion of ApoB and ApoB-containing lipoproteins in familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the alpha-helical domain within the N terminus of apoB. Thus, proper folding of the alpha-helical domain of apoB-100 is essential for efficient secretion.     

Characterization of a mutant form of human apolipoprotein B (Thr26_Tyr27del) associated with familial hypobetalipoproteinemia

We have previously identified a deletion mutant of human apoB [apoB (Thr26_Tyr27del)] in a subject with primary hypobetalipoproteinemia. The present study determined the effect of Thr26_Tyr27del mutation on apoB secretion using transfected McA-RH7777 cells. Transient or stable transfection of apoB-48 containing the Thr26_Tyr27del mutation showed drastically reduced secretion of the mutant as compared to wild-type apoB-48. No lipoproteins containing the mutant apoB-48 were secreted into the medium. Incubation of transfected cells in a lipid-rich medium in the presence of cycloheximide showed rapid turnover of cell-associated mutant apoB-48 as compared to that of wild-type apoB-48. Immunofluorescence experiments showed that the mutant apoB-48 was mostly localized in the endoplasmic reticulum. Treatment with the proteasomal inhibitor MG132 markedly attenuated the turnover of cell-associated mutant apoB-48, whereas treatment with inhibitors of autophagosomal/lysosomal function (e.g. 3-MA or ammonium chloride) had no effect. Taken together, these results indicated that the defective secretion of the Thr26_Tyr27del mutant was associated with increased intracellular degradation of apoB through the proteasome-dependent pathway.     

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.     

Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes

The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.     

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.     

Human placenta secretes apolipoprotein B-100-containing lipoproteins

Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very early during pregnancy in the placenta. To examine whether the human placenta produces lipoproteins, we examined apoB and microsomal triglyceride transfer protein (MTP) mRNA expression in placental biopsies. ApoB and MTP are mandatory for assembly and secretion of apoB-containing lipoproteins. Both genes were expressed in placenta and microsomal extracts from human placenta contained triglyceride transfer activity, indicating expression of bioactive MTP. To detect lipoprotein secretion, biopsies from term placentas were placed in medium with [(35)S]methionine and [(35)S]cysteine for 3-24 h. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB-immunoprecipitation. (35)S-labeled apoB-100 was recovered in d approximately 1.02-1.04 g/ml particles (i.e. similar to the density of plasma low density lipoproteins). Electron microscopy of negatively stained lipoproteins secreted from placental tissue showed spherical particles with a diameter of 47 +/- 10 nm. These results demonstrate that human placenta expresses both apoB and MTP and consequently synthesize and secrete apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel pathway of lipid transfer from the mother to the developing fetus.     

The complete cDNA and amino acid sequence of human apolipoprotein B-100

We have determined the complete sequence of apolipoprotein (apo) B-100 cDNA. It is 14.1 kilobases in length and codes for a 4563-amino acid protein, including a 27-amino acid signal peptide and a 4536-amino acid mature protein. Further, we identified 2366 residues of apoB-100 by direct sequence analysis of apoB-100 tryptic peptides. The mature peptide is characterized by high hydrophobicity (0.916 kcal/residue) and predicted beta-sheet content (21%). Dot matrix analysis revealed the presence of many long internal repeats in apoB-100. The mature peptide contains 25 cysteine residues, 12 of which are in the N-terminal 500 residues. Twenty potential N-linked glycosylation sites were identified, of which 13 were proven to be glycosylated, and 4 were found not to be glycosylated by direct analysis of tryptic peptides. Our findings on apoB structure provide a basis for future experimentation on the role of apoB-100-containing lipoproteins in atherosclerosis.     

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

Pluronic L81 affects the lipid particle sizes and apolipoprotein B conformation

The chylomicron assembly has been proposed to involve the core expansion of apolipoprotein B (apoB)-containing primordial lipoproteins by fusing with triglyceride-rich lipid droplets. We examined the effects of an inhibitor of chylomicron secretion, Pluronic L81, on triolein-phosphatidylcholine emulsions and low density lipoproteins (LDL) which were used for the models of lipid droplets and primordial lipoproteins, respectively. We showed by dynamic light scattering that the sizes of lipid emulsions and LDL were increased in the presence of Pluronic L81. The binding of apoB-100 to lipid emulsions was enhanced by Pluronic L81. CD and fluorescence lifetime measurements revealed that Pluronic L81 altered the secondary structure of apoB-100 with an increased local hydration. The proper hydrophilic-to-hydrophobic balance of Pluronic L81 is important for these actions. It is proposed that Pluronic L81 inhibits the secretion of chylomicrons by leading the excess core expansion of the primordial lipoproteins and the conformational modification of apoB.     

Influence of peroxisome proliferator-activated receptor alpha agonists on the intracellular turnover and secretion of apolipoprotein (Apo) B-100 and ApoB-48

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.     

Assembly and secretion of very low density lipoproteins containing apolipoprotein B48 in transfected McA-RH7777 cells. Lack of evidence that palmitoylation of apolipoprotein B48 is required for lipoprotein secretion

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.     

Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B

Previous studies demonstrated that structural perturbation of the alpha(1) domain of apolipoprotein B (apoB) blocked the initiation of lipoprotein assembly. We explored the hypothesis that this domain may interact with the inner leaflet of the endoplasmic reticulum membrane in a manner that may nucleate microsomal triglyceride transfer protein-dependent lipid sequestration. ApoB-17 (amino-terminal 17% of apoB), which contains most of the alpha(1) domain, was expressed stably in rat hepatoma cells and recovered from medium in lipid-poor form. On incubation with phospholipid vesicles composed of 1-myristol-2-myristoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-gylycero-3-phosphocholine, apoB-17 underwent vesicle binding and was recovered in the d < 1.25 g/ml gradient fraction. To determine whether vesicle binding is disrupted by the same structural perturbations that block lipoprotein assembly in vivo, apoB-17 was subjected to partial and complete chemical reduction. Although normally a soluble peptide, mild reduction of apoB-17 caused its precipitation, suggesting that hydrophobic, solvent-inaccessible domains within the alpha(1) domain of apoB are stabilized by intramolecular disulfide bonds. In contrast to apoB-17 chemically reduced in vitro, forms of apoB-17 bearing pairwise cysteine-to-serine substitutions were recovered in soluble form from transiently transfected COS-1 cell extracts. Although individual disruption of disulfide bond 2 or 4 in apoB-28 and apoB-50 was previously shown to block lipoprotein assembly in vivo, these alterations had no impact on the ability of apoB-17 to bind to phospholipid vesicles in vitro or on its capacity to form recombinant lipoprotein particles. These results suggest that while the vesicle/lipid-binding property of the alpha(1) domain may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of alpha(1) domain function is perturbed by disruption of native disulfide bonds. -- DeLozier, J. A., J. S. Parks, and G. S. Shelness. Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B. J. Lipid Res. 2001. 42: 399--406.     

Regulation of intestinal apolipoprotein B synthesis and secretion by Caco-2 cells. Lack of fatty acid effects and control by intracellular calcium ion

To investigate the mechanism of control of intestinal apolipoprotein B (apoB) secretion, we studied the effects of fatty acids and calcium ionophores on the human intestinal model cell line Caco-2. Although treatment with various fatty acids (18:1w9, 18:2w6, and 20:5w3) complexed to bovine serum albumin resulted in a dramatic redistribution of apoB-100 from the low density and high density lipoproteins to the very low density lipoprotein fraction, there was no effect of any of the fatty acids on the overall rate of total apoB (apoB-100 and apoB-48) secretion. Treatment of differentiated monolayers with calcium ionophores A23187 or ionomycin caused dose-specific increases (125% at 1 microM) in the accumulation of total apoB, but not apoA-I, in conditioned medium as measured by specific immunoassays. Incubation studies with 35S-labeled Caco-2 apoB,E-containing low density lipoprotein particles revealed that treatment with ionomycin over a broad concentration range had no effect on the reuptake of secreted apoB-100. The effect on A23187 on total apoB secretion was blocked by prior chelation of medium calcium and was significantly enhanced by the addition of calcium (up to 50 mM) to the medium. The effect of A23187 was significantly blunted by treatment with the calmodulin antagonist trifluoperazine (10 microM). The time course of A23187 action on Caco-2 apoB secretion required at least 6 h to occur. In contrast to the concentration of apoB in the medium, cellular apoB content was not influenced by treatment with ionophore. Pulse-chase experiments demonstrated a significant reduction in the synthesis-secretion interval for apoB-100 and apoB-48 after 24 h of exposure to ionomycin. Neither fatty acid treatment nor stimulation with ionophore affected the ratio of apoB-100 to apoB-48 produced by the cells. These findings with calcium ionophores implicate the involvement of calcium ion in the mechanism of intestinal apoB secretion. A role for calcium-dependent processes in apoB production raises the possibility that, rather than fatty acid flux, calcium-evoked or calcium-dependent hormones may be important regulators of apoB secretion.     

ADP-ribosylation factor 1 and its activation of phospholipase D are important for the assembly of very low density lipoproteins

The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low density lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells. Transient expression of ARF-1(T31N), a GDP-restrictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) VLDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the second step of VLDL assembly) and without altering total protein production or biosynthesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.     

Microsomal triglyceride transfer protein is essential for hepatic secretion of apoB-100 and apoB-48 but not triglyceride

Despite a complete lack of microsomal triglyceride transfer protein (MTP), L35 rat hepatoma cells secrete triglyceride-containing lipoproteins, albeit at a rate 25% of that of parental FAO hepatoma cells, which express high levels of MTP. The inability to express MTP was associated with a complete block in the secretion of both apolipoprotein (apo)B-100 and apoB-48. Stable expression of a MTP transgene restored the secretion of both apoB-100 and apoB-48 in L35 cells, indicating that MTP is essential for the secretion of both forms of apoB. Treatment with the MTP inhibitor BMS-200150 reduced the secretion of triglyceride by 70% in FAO cells, whereas the inhibitor did not affect the secretion of triglycerides by L35 cells. Thus, in the presence of the MTP inhibitor, both cell types secreted triglycerides at similar rates. Essentially, all of the triglycerides secreted by L35 cells were associated with HDL containing apoA-IV and apoE but devoid of apoB-100 or apoB-48. These results suggest that these triglyceride-containing lipoproteins are assembled and secreted via a pathway that is independent of both apoB and MTP. Our findings support the concept that apoB and MTP co-evolved and provided a means to augment the secretion of triglyceride through the formation of lipoproteins containing large hydrophobic cores enriched with triglycerides.