Relation of the size and intracellular sorting of apoB to the formation of VLDL 1 and VLDL 2
2004; Elsevier BV; Volume: 46; Issue: 1 Linguagem: Inglês
10.1194/jlr.m400296-jlr200
ISSN1539-7262
AutoresPia Stillemark-Billton, Caroline B. Adiels, Jan Borén, Sven‐Olof Olofsson,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoIn this study, we tested the hypothesis that two separate pathways, the two-step process and an apolipoprotein B (apoB) size-dependent lipidation process, give rise to different lipoproteins. Expression of apoB-100 and C-terminally truncated forms of apoB-100 in McA-RH7777 cells demonstrated that VLDL particles can be assembled by apoB size-dependent linear lipidation, resulting in particles whose density is inversely related to the size of apoB. This lipidation results in a LDL-VLDL 2 particle containing apoB-100. VLDL 1 is assembled by the two-step process by apoB-48 and larger forms of apoB but not to any significant amount by apoB-41. The major amount of intracellular apoB-80 and apoB-100 banded with a mean density of 1.10 g/ml. Its formation was dependent on the sequence between apoB-72 and apoB-90. This dense particle, which is retained in the cell, possibly by chaperones or association with the microsomal membrane, is a precursor of secreted VLDL 1. The intracellular LDL-VLDL 2 particles formed during size-dependent lipidation appear to be the precursors of intracellular VLDL 1.We propose that the dense apoB-100 intracellular particle is converted to LDL-VLDL 2 by size-dependent lipidation. LDL-VLDL 2 is secreted or converted to VLDL 1 by the uptake of the major amount of triglycerides. In this study, we tested the hypothesis that two separate pathways, the two-step process and an apolipoprotein B (apoB) size-dependent lipidation process, give rise to different lipoproteins. Expression of apoB-100 and C-terminally truncated forms of apoB-100 in McA-RH7777 cells demonstrated that VLDL particles can be assembled by apoB size-dependent linear lipidation, resulting in particles whose density is inversely related to the size of apoB. This lipidation results in a LDL-VLDL 2 particle containing apoB-100. VLDL 1 is assembled by the two-step process by apoB-48 and larger forms of apoB but not to any significant amount by apoB-41. The major amount of intracellular apoB-80 and apoB-100 banded with a mean density of 1.10 g/ml. Its formation was dependent on the sequence between apoB-72 and apoB-90. This dense particle, which is retained in the cell, possibly by chaperones or association with the microsomal membrane, is a precursor of secreted VLDL 1. The intracellular LDL-VLDL 2 particles formed during size-dependent lipidation appear to be the precursors of intracellular VLDL 1. We propose that the dense apoB-100 intracellular particle is converted to LDL-VLDL 2 by size-dependent lipidation. LDL-VLDL 2 is secreted or converted to VLDL 1 by the uptake of the major amount of triglycerides. Immunoelectron microscopy (1Alexander C.A. Hamilton R.L. Havel R.J. Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar) and kinetic studies (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 3Swift L.L. Assembly of very low density lipoproteins in rat liver: a study of nascent particles recovered from the rough endoplasmic reticulum.J. Lipid Res. 1995; 36: 395-406Abstract Full Text PDF PubMed Google Scholar) indicate that VLDLs are assembled in two major steps (4Olofsson S-O. Asp L. Borén J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr. Opin. Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (187) Google Scholar, 5Olofsson S-O. Stillemark-Billton P. Asp L. The intracellular assembly of VLDL—a process that consists of two major steps that occur in separate cell compartments.Trends Cardiovasc. Med. 2000; 10: 338-345Crossref PubMed Scopus (105) Google Scholar). The first step occurs during the translation of apolipoprotein B (apoB) and gives rise to a premature particle (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 6Borén J. Graham L. Wettesten M. Scott J. White A. Olofsson S-O. The assembly and secretion of apoB 100-containing lipoproteins in Hep G2 cells. ApoB 100 is cotranslationally integrated into lipoproteins.J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar) we refer to as a primordial lipoprotein. Major amounts of lipid are added in the second step, resulting in bona fide VLDL (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 7Rustaeus S. Lindberg K. Borén J. Olofsson S-O. Brefeldin A reversibly inhibits the assembly of apoB containing lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1995; 270: 28879-28886Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Stillemark P. Borén J. Andersson M. Larsson T. Rustaeus S. Karlsson K-A. Olofsson S-O. The assembly and secretion of apolipoprotein-B48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 2000; 275: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar); a second precursor of VLDL, an apoB-free "lipid droplet" in the smooth endoplasmic reticulum (1Alexander C.A. Hamilton R.L. Havel R.J. Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar, 9Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficiency.J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar) whose assembly requires microsomal triglyceride transfer protein (MTP), may also be involved (10Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analyzing the role of microsomal triglyceride transfer protein in the liver with tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar). VLDLs are secreted in two forms: large, triglyceride-rich VLDL 1 and smaller, triglyceride-poor VLDL 2. Overproduction of VLDL 1 is linked to conditions such as insulin resistance and type II diabetes (11Taskinen M.R. Diabetic dyslipidaemia: from basic research to clinical practice.Diabetologia. 2003; 46: 733-749Crossref PubMed Scopus (657) Google Scholar). The lengths of C-terminally truncated forms of apoB-100 are inversely related to the amount of lipid in the lipoproteins they assemble; assembly with apoB-100 results in VLDL (12Yao Z.M. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins.J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar). This size-dependent lipidation of apoB does not fit the two-step model; in particular, it cannot explain why apoB-48 has the ability to assemble VLDL but apoB-40 lacks this ability (8Stillemark P. Borén J. Andersson M. Larsson T. Rustaeus S. Karlsson K-A. Olofsson S-O. The assembly and secretion of apolipoprotein-B48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 2000; 275: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In this study, we tested the hypothesis that two separate pathways, the two-step process and an apoB size-dependent lipidation process, give rise to different lipoproteins. Our results indicate that although apoB can assemble VLDL 1 once it reaches the size of apoB-48, the size-dependent process gives rise to LDL-VLDL 2 particles first when apoB-100 is reached. In contrast to apoB-48 (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar), the major intracellular form of apoB-100 is much denser than expected from the size/density relation, and it is retained in the secretory pathway. The sequence between apoB-72 and apoB-90 is essential for the formation of this intracellular particle, which is the precursor of the secreted VLDL 1. Immunoprecipitin, Eagle's minimal essential medium (with and without methionine), and Williams' medium E with Glutamax were from Life Technologies (Paisley, Scotland). Nonessential amino acids, glutamine, penicillin, and streptomycin were from ICN Biomedicals (Costa Mesa, CA). Fetal calf serum was from JRH Biosciences, and rabbit immunoglobulin was from DAKO. Brefeldin A was from Epicentre Technologies (Madison, WI). Collagenase type IV, methionine, sodium pyruvate, disodium carbonate, PMSF, pepstatin A, and leupeptin were from Sigma (St. Louis, MO). Trasylol (aprotinin) was from Bayer Leverkusen (Germany). N-Acetyl-Leu-Leu-norleucinal was from Boehringer Mannheim. Amplify, [35S]methionine-cysteine (Pro-mix), Rainbow molecular weight markers, and the ECL Western blot analysis system were from Amersham. Ready Safe was from Beckman (Fullerton, CA). Geneticin was from Duchefa. Antibodies to the chaperones binding protein (BiP), protein disulfide isomerase (PDI), glucose regulatory protein 94 (GRP94), and calreticulin were from Affinity BioReagents (Golden, CO). Primaria cell culture dishes were from Becton Dickinson Labware (Franklin Lakes, NJ). All enzymes for DNA work were from New England Biolabs (Beverly, MA). Dynabeads were from Dynal Biotech (Oslo, Norway). All chemicals for SDS-PAGE and alkaline phosphatase-conjugated goat anti-rabbit and rabbit anti-mouse immunoglobulins were from Bio-Rad (Hercules, CA). McA-RH7777 cells were cultured in the presence of oleic acid as described (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). The cultures were fed daily and split twice weekly. They were pulse-labeled with [35S]methionine-cysteine (Pro-mix) and chased in culture medium supplemented with 10 mM methionine as described (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Treatment with brefeldin A and inhibition of MTP were carried out as described (13Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S-O. The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells.J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Briefly, McA-RH7777 cells were pretreated with brefeldin A (15 min), pulse-labeled (30 min), and chased (30 min) in the presence of brefeldin A. During the chase, initiated nascent chains were converted to full-length apoB, but brefeldin A blocked further processing to VLDL. In the next step, apoB-100 was allowed to form VLDL during a 180 min chase in the presence of oleic acid but in the absence of brefeldin A. Control cells were compared with cells in which MTP was irreversibly inhibited. The carboxyl-terminal 18% and 28% of apoB-100 were amplified by PCR with the following oligonucleotides: 5′-CGACGCGTCCTAGGTTCTGACGTCCG-3′ and 5′-GGGGTACCCTATCTTCTCAGCTTTGAAGACACTTCT-3′ [B-50+(72–90)] and 5′-CCATCGATCTAGGTTCTGACGTCCGTGT-3′ and 5′-GGGGTACCCTAGAGGATGATAGTAAGTTCTCCTG-3′ [B-41+(72–100)]. PCR fragments were digested with MluI-KpnI [B-50+(72–90)] or ClaI-KpnI [B-41+(72–100)] and ligated into the apoB-100 L-L plasmid (14Yao Z. Blackhart B.D. Johnson D.F. Taylor S.M. Haubold K.W. McCarthy B.J. Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs.J. Biol. Chem. 1992; 267: 1175-1182Abstract Full Text PDF PubMed Google Scholar). The resulting plasmids encode the amino-terminal 50% of apoB-100 fused to the carboxyl-terminal 18% of apoB-100 [B-50+(72–90)] or the amino-terminal 41% of apoB-100 fused to the carboxyl-terminal 28% of apoB-100 [B-41+72–100]. ApoB-41 was obtained by digesting the apoB-100 L-L plasmid with ClaI-KpnI and ligating the purified fragment with the following oligonucleotide: 5′-CGATTGATAGTAAGGTAC-3′. The plasmids were transfected into McA-RH7777 cells with Tfx50 (Promega) and maintained in medium containing 600 μg/ml geneticin. Approximately 3–4 weeks after transfection, colonies were picked and maintained in selective culture medium. Stable clones were selected by PCR of cDNA with the following oligonucleotides: 5′-CGACGCGTCCTAGGTTCTGACGTCCG-3′ and 5′-CCTGTTGTTCCCAGTGGTA-3′ [B-50+(72–90)] and 5′-CCATCGATCTAGGTTCTGACGTCCGTT-3′ and 5′-CCTGTGTGTTCCCAGTGGTA-3′ [B-41+(72–100)]. The RNA was converted to cDNA by reverse transcriptase PCR (Taqman; Applied Biosystems), and the PCR products from both DNA and cDNA were compared on an agarose gel. McA-RH7777 cells were also transfected with carboxyl-terminal-truncated human apoB-53, apoB-72, and apoB-80 (from Zemin Yao, University of Ottawa Heart Institute, Ottawa, Canada) using DOSPER liposomal transfection reagent (Boehringer Mannheim). The cells were maintained in culture medium containing 800 μg/ml geneticin. Approximately 3–4 weeks after transfection, colonies were picked and maintained in selective culture medium. Two weeks before experiments, geneticin was omitted from the culture medium. Mouse hepatocytes were isolated essentially as described for rat hepatocytes (15Sjöberg A. Oscarsson J. Boström K. Innerarity T.L. Edén S. Olofsson S-O. Effects of growth hormone on apolipoprotein-B (apoB) messenger ribonucleic acid editing, and apoB 48 and apoB 100 synthesis and secretion in the rat liver.Endocrinology. 1992; 130: 3356-3364Crossref PubMed Scopus (0) Google Scholar). The liver was perfused first with Hanks' balanced salt solution containing 0.6 mM EGTA, 20 mM HEPES, and 10 mM sodium hydrogen carbonate without calcium or magnesium, pH 7.4 (7–8 min, 40–50 ml/min) and then with Williams' medium E with Glutamax supplemented with penicillin (50,000 IU/l), streptomycin (50 mg/l), 0.28 mM sodium ascorbate, 0.1 μM sodium selenite, and 400 mg/l collagenase type IV (8–10 min, 40 ml/min). The cells were filtered through a 250 μm nylon filter and a 100 μm cell strainer and washed by centrifugation at 50 g three times for 1 min each at 4°C in Williams' E medium with Glutamax containing glucose (3 g/l), insulin (Actrapid; Novo Nordisk; 28.6 U/mg), sodium ascorbate, sodium selenite, and the antibiotics described above. The cells were seeded (70,000 cells/cm2) in 10 ml of medium in Primaria cell culture dishes. After 4 h, the medium was replaced with fresh medium, and the cells were cultured for 13 h before use. Control experiments with cells from human apoB-100 transgenic mice demonstrated that the secretion of apoB-100 VLDL was stable for at least 24 h after the initiation of the experiment but started to decline after 3 days in culture and had nearly ceased by 4 days. This is in agreement with previous observations (16Boren J. Lee I. Zhu W. Arnold K. Taylor S. Innerarity T.L. Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100.J. Clin. Invest. 1998; 101: 1084-1093Crossref PubMed Google Scholar). To investigate the influence of the LDL receptor (LDLR) on the secretion of pre-VLDL, human apoB-100 transgenic mice were crossed with LDLR-null (LDLR −/−) mice (16Boren J. Lee I. Zhu W. Arnold K. Taylor S. Innerarity T.L. Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100.J. Clin. Invest. 1998; 101: 1084-1093Crossref PubMed Google Scholar). Sucrose gradient ultracentrifugation of lipoproteins was performed as described (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Before centrifugation, the conditioned medium or microsomal extract was supplemented with 0.1 mM leupeptin, 1 mM PMSF, 1 mM pepstatin A, 5 mM N-acetyl-Leu-Leu-norleucinal, and 100 Kallikrein inhibitory units (KIU)/ml aprotinin. The luminal content of the microsomes was extracted by the deoxycholate-carbonate method (13Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S-O. The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells.J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) or the carbonate method (17Fujiki Y. Hubbard A.L. Fowler S. Lazarow P. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1383) Google Scholar) as modified (18Boström K. Wettesten M. Borén J. Bondjers G. Wiklund O. Olofsson S-O. Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells.J. Biol. Chem. 1986; 261: 13800-13806Abstract Full Text PDF PubMed Google Scholar). Virtually all of the apoB-100 and apoB-48 can be extracted from McA-RH7777 microsomes by the deoxycholate-carbonate method (13Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S-O. The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells.J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Thus, the amount of primordial particles containing apoB-100 and apoB-48 in the deoxycholate-carbonate extract provides an estimate of the total intracellular pool. ApoB was isolated by immunoprecipitation and SDS-PAGE (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 19Wettesten M. Boström K. Bondjers G. Jarfeldt M. Norfeldt P-I. Carrella M. Wiklund O. Borén J. Olofsson S-O. Pulse-chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2.Eur. J. Biochem. 1985; 149: 461-466Crossref PubMed Scopus (32) Google Scholar). Immunoblotting was carried out as described (8Stillemark P. Borén J. Andersson M. Larsson T. Rustaeus S. Karlsson K-A. Olofsson S-O. The assembly and secretion of apolipoprotein-B48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 2000; 275: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Specific immunoglobulins were isolated from rabbit anti-human apoB antiserum by affinity chromatography on LDL immobilized to Sepharose. The specific immunoglobulins were eluted with 3 M sodium thiocyanate from the affinity column and coupled to Dynabeads (M-450 Tosylactivated; Dynal Biotech). To isolate apoB-containing primordial lipoproteins, we extracted the luminal content from microsomes (13Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S-O. The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells.J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Density fractions containing the primordial particles were isolated by gradient ultracentrifugation and immunoaffinity purified by incubation with human apoB antibodies coupled to Dynabeads for 2 h at 4°C in PBS, pH 7.4. The precipitate was washed three times with PBS and incubated for 5 min at 95°C in sample buffer to release the proteins. The solubilized precipitate was subjected to SDS-PAGE with 10% gels and blotted with antibodies to BiP, calreticulin, GRP94, PDI, and immunoglobulins from nonimmunized rabbit or mouse. Sucrose gradient ultracentrifugation was used to investigate the lipoproteins secreted by McA-RH7777 cells transfected with apoB-41, apoB-53, apoB-72, and apoB-80 as well as the species secreted by endogenous apoB-100 and apoB-48. Purified human lipoproteins were used to characterize the gradient. The most buoyant VLDL particles (VLDL 1) were confined to the two top fractions (11 and 12). The total VLDL fraction (VLDL 1 and VLDL 2) banded in the four top fractions (9Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficiency.J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar, 10Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analyzing the role of microsomal triglyceride transfer protein in the liver with tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar, 11Taskinen M.R. Diabetic dyslipidaemia: from basic research to clinical practice.Diabetologia. 2003; 46: 733-749Crossref PubMed Scopus (657) Google Scholar, 12Yao Z.M. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins.J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar). LDL was present in fractions 5–9, and HDL was present in fractions 3–6. Only a small amount of apoB-41 was secreted as VLDL 1. All other apoB species examined assembled substantial amounts of VLDL 1. Thus, 7.5% of the total secreted pool of apoB-41 was recovered with VLDL 1; the corresponding figure for apoB-48 was 47%. This indicates that the ability to assemble VLDL 1 increases severalfold between apoB-41 and apoB-48 (Fig. 1A). Thus, linear elongation of apoB-41 to apoB-48 allowed the formation of VLDL 1. A chimera consisting of apoB-41 fused to the carboxyl terminus of apoB-100 [B-41+(72–100)] corresponding in length to apoB-69 failed to assemble VLDL 1, demonstrating that the ability to assemble VLDL 1 is not just a matter of the size of apoB.Fig. 1Lipoprotein secretion by McA-RH7777 cells cultured in the presence of oleic acid. Wild-type cells and cells transfected with constructs encoding human apolipoprotein B (apoB)-41, apoB-53, apoB-72, and apoB-80 were labeled with [35S]methionine-cysteine for 120 min. The conditioned medium was recovered and fractionated by sucrose gradient ultracentrifugation. ApoB was recovered from each fraction by immunoprecipitation and SDS-PAGE. A: Patterns obtained with apoB-41 through apoB-100 [triangles represent density (g/ml); squares represent dpm]. B: Density of secreted non-VLDL 1 lipoproteins in relation to the length of apoB. The densities of the chimeras B-41+(72–100) (triangles) and B-50+(72–90) (squares) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ApoB-41 through apoB-100 were secreted on a denser non-VLDL 1 particle whose density was inversely proportional to the length of apoB (Fig. 1B). In the case of apoB-100, this particle, which we refer to as LDL-VLDL 2, banded in the LDL and VLDL 2 density regions. These results indicate that particles with a density of LDL-VLDL 2 were formed by size-dependent lipidation of apoB-100. They also indicate that the non-VLDL 1 particles assembled by truncated forms of apoB are analogs of the LDL-VLDL 2 particles formed by apoB-100. We refer to them as LDL-VLDL 2 analogs. To determine if the assembly of apoB size-dependent particles was dependent on the linear elongation of apoB or only on the size of the protein, we created a chimera consisting of apoB-50 and the carboxyl terminus of apoB-100 [B-50+(72–90)] and corresponding in length to apoB-68. The density of the assembled particle decreased, as would be expected from a linear increase from apoB-50 to apoB-68 (Fig. 1B). However B-41+(72–100) failed to acquire the expected density of apoB-69 (Fig. 1B), which indicates that the size-dependent lipidation is not just a matter of the size of apoB. Because these results were based on stable transfections, the cell background (selected clones) might have influenced the results. If so, apoB-48 and apoB-100 lipoproteins would also be influenced. We therefore used endogenous lipoproteins as an "internal" standard. In all of the experiments reported here, the lipoproteins formed by apoB-48 and apoB-100 had the expected density, both in the secretory pathway and after secretion (data not shown). Analysis of the contents of the secretory pathway revealed that all apoB species assembled apoB size-dependent particles with a density that corresponded to the secreted particle (Fig. 2A). The size of apoB was inversely related to the density of the non-VLDL 1 particle, except for apoB-80 and apoB-100, which formed particles of similar density (Fig. 2B). The only VLDL 1 species that showed a significant intracellular pool was apoB-100-containing VLDL (Fig. 2A).Fig. 2Lipoprotein assembly by McA-RH7777 cells cultured in the presence of oleic acid. Wild-type cells and cells transfected with constructs encoding human apoB-41, apoB-53, apoB-72, and apoB-80 were labeled as described for Fig. 1. The cells were recovered, and the total microsomal fraction was isolated and extracted. The luminal contents were fractionated by sucrose gradient ultracentrifugation, and apoB was recovered from each fraction by immunoprecipitation and SDS-PAGE. A: Patterns obtained with apoB-41 through apoB-100 [triangles represent density (g/ml); squares represent dpm]. B: Density of non-VLDL 1 lipoproteins in relation to the length of apoB. The denser particles formed by apoB-80 (circle), apoB-100 (triangle), and the chimera B-50+(72–90) (square) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ApoB-80 and apoB-100 were also present in a third form in the secretory pathway (Fig. 2A). This form of apoB banded with a density of 1.10 g/ml, did not follow the relation between the length of apoB and the density of the particle (Fig. 2A, B), and could not be detected in significant amounts in the culture medium (Fig. 1A). This particle, which we refer to as the dense B-80/B-100 intracellular particle, must be taken into account as an intracellular precursor in the assembly of VLDL 1. To further investigate this, we carried out a pulse-chase experiment in which the cells were pulse-labeled for 10 min. ApoB-100 radioactivity peaked after a 20–30 min chase (data not shown) (2Borén J. Rustaeus S. Olofsson S-O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). We therefore chased the cells for 20, 30, 60, and 180 min and followed the turnover of radioactivity in the dense apoB-100 intracellular particle, LDL-VLDL 2, and VLDL 1. Radiolabeled apoB-100 VLDL in the medium reached a plateau at 180 min (data not shown), indicating that the maximal amount of apoB-100 had been secreted. We next analyzed the medium for radioactive apoB-100 in LDL-VLDL 2 and VLDL 1 after a 180 min chase as a measure of the maximal secretion of radiolabeled apoB-100. The results (Fig. 3)demonstrate that the decay in the dense apoB-100 intracellular particle must be taken into account to explain the secretion of radiolabeled VLDL 1. The radioactivity in intracellular VLDL 1 and LDL-VLDL 2 was small compared with that in secreted VLDL 1 or in the dense apoB-100 intracellular particle. Moreover, the turnover of intracellular LDL-VLDL 2 and VLDL 1 could not explain the increase in apoB-100 VLDL 1 in the medium. Thus, intracellular VLDL 1 and LDL-VLDL 2 behaved as intermediate pools between the dense apoB-100 intracellular particle and the secreted apoB-100-containing lipoproteins. Not all of the apoB-100 radioactivity in the dense apoB-100 intracellular particle could be recovered in the medium, indicating that a portion of this dense particle is degraded. The turnover of apoB-100 in intracellular LDL-VLDL 2 and VLDL 1 was very similar. To further investigate the relation between these two apoB-100-containing lipoproteins, we carried out a second pulse-chase experiment (Fig. 4). Radioactive apoB-100 was detected in LDL-VLDL 2 by the end of the labeling period, indicating that these particles are assembled relatively close to the translation of apoB-100. LDL-VLDL 2 particles were assembled before VLDL 1, and the turnover kinetics of the two lipoproteins were consistent with a precursor-product relationship. A precursor-product relationship between LDL-VLDL 2 and VLDL 1 is further supported by the observation that LDL-VLDL 2 analogs were the p
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