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J. Biol. Chem., Vol. 279, Issue 38, 39757-39766, September 17, 2004
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¶**
From the
Department of Medicine, Atherosclerosis Research Unit and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294, the ¶Department of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, and ||Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received for publication, June 7, 2004 , and in revised form, July 13, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (HDL3) 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 HDL3. In the present study we have determined the stoichiometry of the lipid component of the apoB:931 and apoB:1000 particles. The secreted [3H]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 
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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The assembly of apoB-containing lipoproteins occurs cotranslationally, i.e. while the C-terminal portion is still being synthesized on the ribosome of the endoplasmic reticulum (ER), the N-terminal portion is translocated across the ER and is assembled as a small lipoprotein particle. Disulfide-dependent folding of portions of the N-terminal domain of apoB is required for its assembly into lipoprotein (7-9). In hepatocytes, the first step in apoB assembly involves the formation of a small particle in the high density lipoprotein (HDL) density range. It has been shown that the translation of the N-terminal 22-29% of apoB-100 is essential for the assembly of apoB-containing particles (8-12) and that microsomal triglyceride transfer protein (MTP) has a critical role in the assembly and secretion of apoB-containing lipoproteins (3, 13). However, the precise domain in the N-terminal region of apoB that is required for the initiation of particle assembly, the structural elements of the assembly competent domain, the lipid composition of this nascent particle, the mechanisms by which MTP transfers lipid to the nascent apoB, and the site(s) in the secretory pathway where this transfer occurs are not completely understood and remain controversial.
The full-length apoB, essentially the only protein component of the atherogenic LDL, has a pentapartite structure, NH2-1-1-2-2-3-COOH, the domains containing multiple amphipathic strands and the domains containing multiple amphipathic helices (14, 15). Because the amphipathic helixes of the 2 and 3 domains are mostly class A, the type found in exchangeable apolipoproteins (16-18), we have proposed that these two regions of apoB-100 represent flexible domains with reversible lipid affinity (19). The two amphipathic strand domains in apoB-100, 1 and 2, we have proposed form sheets that represent the irreversibly lipid-associated regions of apoB-100 (16-18). The 1 domain of apoB-100, i.e. the first 1000 amino acid residues of the mature protein, is a mixture of amphipathic strands and amphipathic helixes (15), has been proposed to be globular in structure (14, 19), and has sequence and amphipathic motif homologies to lamprey lipovitellin (LV) (20-23). Based on sequence homology between the N-terminal domain of apoB and LV, we proposed (19, 20) that formation of a LV-like lipid pocket is necessary for lipid transfer to apoB-containing lipoprotein particles. We suggested (19, 20) that initiation of particle assembly occurs when the 1 domain folds into a three-sided LV-like lipid-binding cavity, or alternatively, the lipid pocket is formed by association of the region of the 1 domain homologous to the A and B sheets of LV with D-like amphipathic sheet from MTP.
In this study, we report that, consistent with our previous study (24), a domain between amino acids 931 and 1000 of apoB-100 is critical for the initiation of particle assembly and formation of a lipid-containing particle. Lipid composition and the number of lipid molecules associated with the secreted apoB-containing particles demonstrated that within the 69 amino acid residues between apoB:931 and apoB:1000, the nature of particles is changed from a lipid-poor to a relatively lipid-enriched particle in the HDL3-like density range. The lack of a 1:1 molar ratio of apoB to MTP observed in this study supports a model in which the first 1000 amino acid residues of apoB are competent to complete the "lipid pocket" without a structural requirement for MTP. This nascent lipoprotein intermediate has a relatively constant Stokes diameter of 112 Å, a mean density of 1.21 g/ml, and has a maximum lipid-transporting capacity on the order of 70 molecules of lipid/particle, primarily phospholipids (PL). The surface to core lipid ratio of 4:1 supports a bilayer-like organization that is not responsive to the presence of oleate in the incubation medium.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Construction of Truncated ApoB Expression Plasmids—Truncated apoB cDNAs spanning nucleotides 1-2481, 1-2874, and 1-3081, respectively, of the full-length apoB-100 cDNA, were prepared from pB100L-L (10) as a PCR template and appropriate primers as described in detail previously (24). The amplified PCR products were cloned into the TOPO TA cloning vector and used to transform cells. Clones harboring the vector were selected and identified by restriction enzyme digestion and nucleotide sequencing of the entire open reading frames. Only clones with 100% correct sequence were used in these studies. The apoB fragments 2481 (apoB:800), 2874 (apoB:931), and 3081 bp (apoB:1000) were excised from the vector, extracted, purified, and ligated into the mammalian expression vector, the Moloney murine leukemia virus-based retrovirus LNCX (25) containing the neomycin phosphotransferase gene that confers G418 resistance for use as a selectable marker. The apoB expression vectors, pLNCB:800, pLNCB:931, and pLNCB: 1000, were used to transform cells, and clones harboring plasmids containing the apoB gene with the correct orientation were identified by restriction enzyme digestion and confirmed by nucleotide sequencing as described previously (24).
Cell Culture and Transfection—Clonal stable transformants of rat hepatoma McA-RH7777 cells expressing apoB:800 (B17.64), apoB:931 (B20.52), and apoB:1000 (B22.05) denoting amino acid residues 1-800, 1-931, and 1-1000, respectively, of the mature protein lacking the signal peptide, were generated as described in detail previously (24). We showed previously (24) that apoB:800, which lacks the entire B domain, was secreted as a lipid-poor aggregate, and apoB:1200, which contains 200 residues of the 1 domain was secreted as both lipid-rich and lipid-poor particles. Therefore, although the focus of this study was to determine the role of the domain between amino acids 931 and 1000 of apoB-100 in the initiation of particle assembly and formation of a lipid-containing particle, apoB:800-expressing cells as well as parental nontransfected McA-RH7777 and LNCX (neo)-transfected cells were used as controls. Cells were grown in DMEM containing 20% horse serum, 5% FBS, and 0.2 mg/ml G418, and medium was changed every 48 h. All experiments were conducted with 4-5-day-old cells as described previously (24), and the amounts of culture media used in all experiments were normalized for the expression and secretion level of truncated apoB proteins and total cell protein.
Metabolic Labeling Studies—Clonal stable transformants of McARH7777 cells, expressing apoB:800, apoB:931, and apoB:1000, were grown for 4 days in either 6-well dishes for immunoprecipitation studies or in 100-mm dishes for nondenaturing gradient gel electrophoresis (NDGGE) and immunoaffinity chromatography studies as described above. At the start of experiments, maintenance medium was removed, and monolayers were washed twice with phosphate-buffered saline (PBS) and serum-free DMEM containing [3H]glycerol or 0.4 mM [14C]oleic acid bound to 0.75% BSA was added. After the indicated incubation time, labeled conditioned medium was collected, preservative mixture at a final concentrations of 500 units/ml penicillin-G, 50 µg/ml streptomycin sulfate, 20 µg/ml chloramphenicol, 50 µg/ml leupeptin, 50 µg/ml pepstatin A, 1.3 mg/ml 
-amino caproic acid, 1 mg/ml EDTA, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 100 kallikrein-inactivating units of aprotinin/ml was added to prevent oxidative and proteolytic damage. The medium was centrifuged at 2,000 rpm for 30 min at 4 °C to remove broken cells and debris. The incorporation of [3H]glycerol or [14C]oleate into various lipid moieties of the secreted apoB-containing lipoproteins was determined by immunoprecipitation with polyclonal antibody to human apoB-100 or by NDGGE of labeled conditioned medium as described below. Cell monolayers were washed with cold PBS, scraped off the plate in PBS, and sonicated for determination of protein content by the method of Lowry et al. (26).
Lipoprotein Isolation—Cells were incubated overnight in 6 ml of serum-free DMEM with or without the labeled precursors. In studies without the labeled precursor, conditioned medium from four 100-mm dishes were combined; preservative mixture was added, and the medium was concentrated 4-fold using Centricon YM-30. The density of the concentrated conditioned medium was adjusted to 1.23 g/ml using solid KBr, and lipoproteins (d < 1.23 g/ml) were isolated by centrifugation for 45 h at 50,000 rpm. The lipoprotein fraction (d < 1.23 g/ml) and infranatant (d > 1.23 g/ml) were dialyzed and concentrated 5-6-fold. In metabolic labeling studies, medium from two 100-mm dishes were pooled, processed as above, and concentrated 2-fold. The density of the concentrated medium was adjusted to 1.23 g/ml and was subjected to ultracentrifugation as described above.
Immunoprecipitation—After an overnight (17-20 h) incubation with serum-free medium and [3H]glycerol or 0.4 mM [14C]oleic acid bound to 0.75% BSA, the labeled apoB-containing lipoproteins secreted into the conditioned medium were immunoprecipitated under nondenaturing conditions (27-29) using monospecific polyclonal antibody to human apoB-100 coupled to protein G-Sepharose CL-4B as described previously (27, 28). The beads were washed six to seven times until background count was obtained in the wash and then extracted for lipids as described below.
NDGGE—In metabolic labeling studies, aliquots of total medium and d < 1.23 g/ml lipoprotein fraction were run on 4-20% NDGGE; gels were stained and the bands corresponding to apoB:931 and apoB:1000, identified by their Stokes diameter and immunoblotting of a duplicate gel, were excised and analyzed for lipids described below. The incorporation of [3H]glycerol or [14C]oleic acid into total lipids of intact apoB-containing lipoproteins was also determined by NDGGE of the labeled conditioned medium and autoradiography.
Isolation of Truncated ApoB-containing Particles by Immunoaffinity Chromatography—Affinity-purified monospecific polyclonal antibody to human apoB-100 was coupled to the cross-linked agarose activated with N-hydroxysuccinimide (Affi-Gel 10). After exhaustive washing of the Affi-Gel with cold, double-distilled water, the antibody solution was added to the gel slurry (2 mg of protein/ml of gel), and the mixture was gently shaken for 2-3 h at room temperature. The supernatant was removed, and the remaining active sites were blocked. The gel was then washed and equilibrated with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl and 1.5 mg/ml EDTA as described previously (30). The column was packed with the gel flanked with two layers of Sephadex G-25, a 25-ml bottom layer to minimize the exposure time of lipoproteins to dissociating agent and a 3-ml protective top layer, as described previously (30).
Stable transformants of McA-RH7777 expressing apoB:931 or apoB: 1000 were grown in 100-mm dishes in serum-containing DMEM for 4 days. Monolayers were washed three times with PBS to remove residual serum and were incubated for 24 h in serum-free DMEM with or without 0.4 mM oleic acid bound to 0.75% BSA. The conditioned medium from 10 plates was pooled, and preservatives were added. The medium was centrifuged for 30 min at 2000 rpm to remove broken cells and debris. The conditioned medium was concentrated 10-fold and applied to the immunoaffinity column. The unretained fraction was eluted with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl and 1.5 mg/ml EDTA, and the retained fraction was eluted with 5 ml of 4.5 M NaSCN as described previously (30). After addition of preservative mixture, the retained fraction was concentrated and analyzed for the mass of PL, TAG, and cholesteryl ester.
Lipid Analysis of Isolated Truncated ApoB-containing Particles—Radioactive lipids associated with the immunoprecipitated apoB-containing particles were extracted from protein G with chloroform/methanol (2:1). In NDGGE studies, the bands corresponding to labeled apoB:931-or apoB:1000-containing particles were excised from the gel and homogenized using a Dounce homogenizer. SDS was added to the final concentration of 0.2%, and the volume of the homogenate was adjusted to 5 ml with distilled water. The homogenate was incubated at 37 °C for 2 h to dissociate the lipids from the polyacrylamide gel and then lyophilized. Total lipids were extracted from the lyophilized homogenate in a total volume of 10 ml of chloroform/methanol (2:1); complete extraction was assessed by counting the final gel homogenate. Total labeled lipids extracted from immunoprecipitated and gel isolated apoB-containing lipoproteins were washed by the Folch method (31) as described previously (32). The final washed extracts were dried under a nitrogen stream, dissolved in small volume of ether, and applied to TLC plate. The bands corresponding to PL, DAG, and TAG, identified by comparison to known standards, were visualized with iodine; each band was scraped off the plate, placed in a counting vial, and quantified by liquid scintillation counting. In immunoaffinity chromatography studies, the concentrated retained fraction from the anti-apoB immunosorber containing unlabeled apoB:1000-containing particles was analyzed for the mass of TAG and cholesteryl esters by gas chromatography (33) and for the mass of PL according to the micro-method of Gerlach and Deuticke (34).
Immunoblot Analysis—The isolated apoB-containing particles were run on 4-12% SDS-PAGE (35) or on 4-20% NDGGE. After electrophoresis, proteins were detected by Western blot analysis (36) using biotinylated antibody to human apoB-100 as described previously (24).
Calculation of Number of Lipid Molecules/Particle—Calculations were made essentially as described by Carraway et al. (37). The number of lipid molecules/apoB particle was calculated from percent composition of PL, DAG, and TAG determined by metabolic labeling and TLC analysis or by mass determination of PL, TAG, and cholesteryl ester in the retained fraction from the anti-apoB immunosorber. The equation for calculating lipid to apoB molar ratios of apoB:1000-containing particles was derived by using the calculated molecular mass of 111,375 Da for nonglycosylated apoB:1000 based on amino acid sequence using DNAMAN program and particle density of 1.21 g/ml, determined by density gradient ultracentrifugation, followed by NDGGE and immunoblotting as described previously (24). In a few experiments where apoB:931-containing particles were studied, the calculated molecular mass of 103,610 Da and particle density of 1.26 g/ml were used.
Electron Microscopy—For electron microscopy, concentrated retained fraction from anti-apoB immunosorber was dialyzed against ammonium acetate buffer (2.6 mM, pH 7.4) and negatively stained with 2% sodium phosphotungstate as described previously (38). Samples were examined in a JEOL 100C electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The apoB:1000 expressing cells secreted a major particle with an apparent Sd of 112 Å and trace amounts of a smaller particle with an Sd of 95 Å (24). In comparison to apoB:931-containing particles, a considerably larger fraction of apoB: 1000 was recovered in d < 1.23 g/ml, suggesting that the apoB:1000-containing particles are relatively lipid-rich (24). Only the larger, apparently monodisperse, apoB:1000-containing particle floated (24).
To determine the relative content of labeled lipids in individual truncated apoB-containing particles, cells were incubated with 0.4 mM [14C]oleic acid bound to 0.75% BSA, and aliquots of conditioned media, normalized for the expression level of truncated apoB proteins, were subjected to NDGGE followed by immunoblotting or autoradiography. As shown in Fig. 1A, we did not detect any human or rat apoB-containing particles in the conditioned medium of parental McA-RH7777 cells (lane 1) and LNCX (neo)-transfected cells (lane 2) by immunoblotting with antibody to human apoB-100, indicating that the antibody to human apoB-100 used in this study is monospecific and does not cross-react with endogenous rat apoB-containing particles. We have demonstrated previously (24) that apoB:800 is secreted into the medium, but as shown in Fig. 1A, lane 3, it fails to form lipoprotein particles. In contrast, apoB:931- and apoB:1000-expressing cells secreted distinct apoB-containing particles (Fig. 1A, lanes 4 and 5). Autoradiography of a duplicate gel demonstrated that neither of the two apoB:931-containing particles with an Sd of 110 and 96 Å contained substantial radioactivity (Fig. 1B, lane 4). In contrast, the band corresponding to the apoB:1000-containing particle with an Sd of 112 Å demonstrated an 8-fold greater level of radioactivity (Fig. 1B, lane 5) than that associated with the 110-Å apoB:931 particle (Fig. 1B, lane 4). The smaller apoB:1000 particle did not display any radioactivity. Similar results were obtained when studies were carried out using [3H]glycerol as the lipid precursor (data not shown).
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5-fold higher than that in apoB:931-containing particles (data not shown). It can be seen from the gel in Fig. 1B that a substantial band of radioactivity appears at the top of each of the lanes. The top bands present in the conditioned medium of parental McARH7777 cells (Fig. 1B, lane 1) and LNCX (neo)-transfected cells (lane 2) almost certainly represent endogenous lipoprotein particles, presumably LDL. However, the bands present in approximately the same positions in Fig. 1B, lanes 3-5, are more prominent. Furthermore, substantially more radioactivity was consistently observed in the top band from secreted apoB:931 particles (Fig. 1B, lane 4) than from either secreted poorly expressed apoB:800 or well expressed apoB:1000 particles (lanes 3 and 5).
When the top bands shown in Fig. 1B, lanes 3-5, were excised from the gel, eluted, and their protein content analyzed by SDS-PAGE, immunoblotting, and densitometry, the appropriate size apoB fragment was seen for each construct, but the amount of apoB:931 recovered (Fig. 1C, lane 4) was considerably greater than that for either apoB:800 (Fig. 1C, lane 3) or apoB:1000 (Fig. 1C, lane 5). Protein eluted from the apoB:1000 major particle (Sd 112 Å) is shown in Fig. 1C, lane 1, and that eluted from the top band of LNCX (neo) is shown in Fig. 1C, lane 2. Our interpretation of these results is that both apoB:800 and apoB:931 form similar types of secreted particles as follows: (i) particles that contain bulk lipid but are unstable and aggregate, probably as dimers; (ii) particles that are lipid-poor but are stable and monomeric. In contradistinction, the apoB: 1000 construct contains only 69 more amino acid residues than the unstable apoB:931 particle. However, although the major secreted apoB:1000 particle contains bulk lipid, unlike the bulk lipid-containing particles secreted by apoB:800 and apoB:931, this major particle is stable and monomeric.
The relative content of labeled lipids in individual truncated apoB-containing particles was further evaluated by incubation of cells with [3H]glycerol or [14C]oleic acid, isolation of secreted apoB-containing lipoproteins by immunoprecipitation or NDGGE, and lipid analysis as described under "Experimental Procedures." The results shown in Table I demonstrate that, after normalization for the expression level and secretion rate of truncated apoB proteins, the apoB:1000-containing particles contain at least four times as much labeled lipid as the apoB: 931-containing particles. The lipid content of apoB:800 was not determined by NDGGE because it does not undergo particle formation. The above results clearly show that a portion, or perhaps all, of the 69 amino acid residues between apoB:931 and apoB:1000 are necessary for the formation of a stable, bulk lipid-containing particle that floats in the HDL3 density range.
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We used immunoprecipitation as the first step in determining the lipid composition of the secreted apoB-containing particles. Because apoB:931 was secreted as mostly lipid-poor particles with a mean density of 1.26 g/ml, their lipid composition could not be accurately determined. Results of the lipid composition of metabolically labeled apoB:1000-containing particles isolated by immunoprecipitation with monospecific polyclonal anti-human apoB-100 are shown in Table II. ApoB:1000-containing particles secreted by control cells contained 57% PL and 33% TAG, and those secreted by the oleate-supplemented cells contained a higher content of TAG (58%) and a lower content of PL (39%) (Table II). The calculated stoichiometries of PL, DAG, and TAG molecules per apoB:1000 were 36, 8, and 19, respectively, in the absence of oleate and 22, 2, and 31, respectively, in the presence of oleate (Table II). The surface to core lipid ratios of apoB:1000 particles were 2.3 and 0.78 in the absence and presence of oleic acid, respectively (Table II).
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4:1 and was not affected by oleate supplementation.
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4:1 and thus supports the results obtained with metabolic labeling and NDGGE (Table III). These results indicate that the lipid pocket created by the first 1000 amino acid residues of apoB-100 has a maximum capacity of 70 molecules of lipid, primarily PL, that is not responsive to oleate supplementation of cells.
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ApoB:1000-containing Lipoproteins Are Shown by Electron Microscopy to Represent Relatively Monodisperse Particles with Irregular Shapes—ApoB:1000-containing particles secreted by stable transformant of McA-RH7777 cells were isolated by immunoaffinity chromatography on an immunosorber with affinity-purified, monospecific polyclonal antibody to human apoB-100 as described above. The retained fraction from immunosorber was analyzed by negative stain EM (Fig. 4). Fig. 4A shows that this preparation consists of HDL-sized particles. The particles differ from typical HDL in two ways: 1) they are asymmetric in shape, and 2) their edges are somewhat fuzzy in appearance. Fig. 4B represents an enlargement of the boxed region in Fig. 4A, and it is clear from this image that the majority of the particles displays an elongated shape, with dimensions of 100 x 150 Å. In a few views, e.g. view 6, the particles have a spheroidal appearance. Fig. 4C shows enlargements of six of the images circled in Fig. 4B, suggesting that the particles tend to have a pear-like taper along their long axis. A molecular graphics image of the model for apoB:1000, described in our concurrent study (48), is displayed for comparison at the same magnification as the EM images in Fig. 4C. This model, whose dimensions are 80 x 100 x 130 Å, is oriented so that its long axis is parallel (Fig. 4C, upper model) and perpendicular (lower model) to the plane of the figure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 domain of apoB-100, i.e. the first 1000 amino acid residues of the mature protein, and LV, we proposed that formation of an LV-like lipid pocket is necessary for lipid transfer to apoB-containing lipoprotein particles. We suggested that initiation of particle assembly occurs when the 1 domain folds into a three-sided LV-like lipid-binding cavity, or alternatively, the lipid pocket is formed by association of the region of the 1 domain homologous to the A and B sheets of LV with D-like amphipathic sheet from MTP (19, 20).
In our earlier study using McA-RH7777 cells (24), we showed the following. (i) ApoB:800 (B17.64), missing the B domain, did not form a particle and was secreted as a lipid-poor aggregate. (ii) ApoB:931 (B20.52), containing most, but not all, of the B domain was secreted as mostly lipid-poor particles with a constant diameter of 110 Å across a wide range of densities and a mean density of 1.25 g/ml or greater, denser than the classical HDL density range of 1.063-1.21 g/ml. (iii) ApoB:1000 (B22.05), containing the entire B domain, was secreted as a relatively lipid-rich particle and had a diameter that remained constant at 112 Å across a wide range of densities and a mean density of 1.21 g/ml, within the HDL3 density range of 1.125-1.21 g/ml. (iv) ApoB:1200 (B26.46), containing a significant number of the amphipathic strands located in the 1 domain, was secreted as two particles as follows: a large relatively lipid-rich particle with a diameter that increased with decreasing density, ranging from 118 to 127 Å, and a mean density of 1.197 g/ml, within the HDL3 density range and a small relatively lipid-poor particle with a diameter of 99 Å, and a mean non-HDL density of 1.24 g/ml or greater. Based on the results, we suggested that the 69-amino acid residues between apoB: 931 and apoB:1000 are necessary for the formation of HDL3-like lipoprotein particles. Because small amounts of MTP were detected in apoB:1000-containing particles by immunoblotting with antibody to the bovine MTP 97-kDa subunit, we suggested that surrogate D amphipathic strands of MTP may complete the LV-like lipid pocket formed by the first 1000 amino acid residues of apoB-100 (24).
In the present study, we have shown that apoB:1000 forms stable particles without the 1:1 molar ratio of MTP to apoB required if MTP was necessary as an integral structural component of the lipid pocket. In a concurrent study, using the program MACAW, we were able to identify local sequence homology (20.1% identity and 39.6% similarity) between residues 1 and 1000 of human and mouse apoB and lamprey, frog, and chicken LV; beyond residue 1000 the homology drops off suddenly (48). Based on this homology, we have proposed an all-atom model for the formation of the lipid pocket by the first 1000 amino acid residues of apoB-100 for the initiation of lipoprotein particle assembly that supports our experimentally derived results. In this model (Fig. 5A), a portion of a nonhomologous loop from one of the two amphipathic sheets of the lipid pocket, i.e. residues 667-746, forms an amphipathic helical hairpin that connects A to B without the structural requirement for MTP. This hairpin bridge spans the third side of the pyramidal lipid pocket allowing the charged residues in the turn region to form complementary salt bridges with the charged residues at the C-terminal portion of the B sheet. Four salt bridges are formed from this arrangement as follows: Arg997-Glu720, Glu998-His719, Asp999-Lys718, and Arg1000-Asp717 (Fig. 5A). For a detailed description of this model, see Ref. 48.
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4:1. The x-ray crystal structure of lamprey LV (39) suggests a lipid pocket containing 27 molecules of PL and 11 molecules of TAG per LV monomer, a surface to core lipid ratio of 2:1. To put these ratios in perspective, the surface/core lipid ratio of spheroidal HDL3 particles is 1.5:1 (40), supporting the concept that the lipid in the apoB:1000-containing particles is in the form of a bilayer assembly, rather than in the form of a mixed micelle assembly like HDL3. This is further supported by our recent molecular model of an apoB:1000 lipid pocket (48). In this model, based upon the depth of the pocket (40 Å), similar to the thickness of the hydrophobic core of a phospholipid bilayer, we have suggested it is likely that lipids form an asymmetric bilayer assembly, containing a neutral lipid lens as described previously (20) in the nascent lipid pocket. A minimum of 48 palmitoyloleoylphosphatidylcholine molecules were manually docked into the lipid pocket of the model for the 1 domain of apoB (Fig. 5B), a number in excellent agreement with the experimental number of 50 phospholipids/particle found in this study.
Negative stain electron microscopy of isolated apoB:1000-containing particles showed that these particles differ from typical HDL in the following two ways: 1) they are asymmetric in shape, and 2) their edges are fuzzy in appearance. The majority of the particles displays an elongated shape, with dimensions of 100 x 150 Å, and tends to have a pear-like taper along their long axis. A molecular graphics image of the model for apoB:1000 described in our recent paper (48), with dimensions of 80 x 100 x 130 Å, displays close similarity to the EM images of apoB:1000-containing particles.
Biochemical studies have suggested that assembly of apoB-100 into a lipoprotein particle occurs co-translationally (2, 3) and requires the activity of MTP (3, 13). However, little is known about the exact mechanisms by which apoB is assembled into a TAG-rich lipoprotein. One often quoted mechanism for the physical assembly of lipid particles containing apoB is the budding oil droplet model (1). In this model, the N-terminal portion of apoB is embedded in the inner monolayer of the ER membrane, where it nucleates an oil droplet from the super-saturated rough ER membranes. Upon completion of apoB synthesis, this oil droplet is detached from the bilayer to form the nascent lipoprotein. One weakness of this model is that an extensive search by electron microscopy for inner rough ER membrane blebs in the liver microsomal preparation has failed (1). Furthermore, thermodynamic considerations make it unlikely that lipoproteins assemble through the wholesale remodeling or dismantling of membrane bilayers.
An alternative model for the initiation of apoB assembly has been suggested by Small and co-workers (37). These investigators have suggested that the formation of the "primordial" lipoprotein particle by a multistep process involves the initial recruitment of PL by the N-terminal region followed by incorporation of core lipids directed by the presence of sheets at and beyond apoB-29 (37). Our results demonstrating that apoB:1000-containing particles are PL-rich and contain a few TAG molecules are consistent with the model proposed by Small and co-workers (37).
Our results demonstrating that apoB:931 (B20.52) does not contain the structural elements required to initiate lipoprotein assembly and contains only a few molecules of PL, whereas apoB:1000 (B22.05) forms a relatively lipid-enriched HDL3-like particle with a surface to core lipid ratio of 4:1, are contrary to recent studies by Shelness et al. (41). In the latter study, which used COS cells, it was shown that the 50-amino acid domain between amino acids 862 and 912 specifies the formation of particles and initiates lipoprotein assembly and that the secreted apoB:931-containing particles have a peak density of 1.20 g/ml and contain 34 molecules of surface lipids and 49 molecules of core lipids, predominantly TAG (41). They suggested (41) that apoB-containing lipoproteins are initially formed as small, dense emulsion particles, with a surface to core lipid ratio of 
1:1, where apoB inserts itself into a saturated membrane surface and desorbs lipid as a preformed core-containing lipoprotein, a model consistent with the budding oil droplet mechanism (1).
We are not sure of the reasons for the observed differences in density and lipid composition of apoB-containing particles, especially apoB:931 (B20.5), in our study and the study by Shelness et al. (41). We speculate that this discrepancy may be due, in part, to the fact that we used hepatically derived cells and Shelness et al. (41) used nonhepatic COS cells that do not normally secrete lipoproteins and hence may have a different profile of the numerous chaperones known to be involved in apoB maturation (2) than that in hepatic McA-RH7777 cells. Consistent with our results, McLeod et al. (42) showed that only C-terminal truncations with a size greater than apoB-23 are able to assemble a neutral lipid core. Our present results, supported by several other studies (9-11, 37, 43), suggest rather unambiguously that the N-terminal 931-amino acid domain of apoB-100 does not have the structural and functional elements necessary for the assembly of apoB into a nascent lipoprotein particle.
Although the mechanism by which apoB:1000 acquires PL is not known, our study indicates that gradual lipid transfer into an apoB-containing particle during biosynthesis requires translocation of a critical length of the apoB sequence, i.e. the 1 domain (residues 1-1000), necessary for creation of a competent lipid pocket. Consistent with this hypothesis, several studies (44-46) have suggested that the acquisition of lipid occurs stepwise along the secretory pathway, and it has been proposed that the initial step in the assembly of apoB-containing lipoproteins involves the recruitment by the N-terminal domain of apoB of surface lipids, primarily PL (47).
Based on the results presented in this paper and the hairpin-bridge mechanism for the formation of the lipid pocket described in our concurrent study (48), we suggest the following model for the initiation of lipoprotein particle assembly. The lipid pocket formed by apoB:1000 begins to recruit PL and some TAG co-translationally to form a primordial lipoprotein particle. The protein, MTP, together with the C domain of apoB: 1000, serves as a shuttle to deliver lipids into the lipid pocket upon translation of apoB to and beyond residue 1000. Co-translation of the amphipathic strands in the 1 domain provides a mechanism for stabilization of the particle beyond the nascent apoB:1000 particle. When the particle reaches a critical size, the salt bridges holding the hairpin-bridge in place break, and apoB undergoes conformational change. As the hairpin-bridge is unlocked, A and B sheets separate; lipids are added to the flexible basal opening of the lipid pocket, and the hydrophobic helices of helix-turn-helix associate with the growing particles. Further addition of lipids cause the formation of a V-shaped pocket between A and B sheets. This open conformation of apoB would allow its association with a much larger surface than the protein itself. We suggest that this is the conformation that apoB assumes in LDL, intermediate density lipoprotein, and VLDL. In support of this model, preliminary results from our ongoing studies have shown that the larger and more buoyant apoB:1200 particles (mean density of 1.19 g/ml) (24) contain 51 PL, 8 DAG, and 27 TAG molecules/particle, a surface to core lipid ratio of 2:1 (data not shown).
In summary, we have demonstrated that a portion, or perhaps all, of amino acid residues between 931 and 1000 of apoB-100 are critical for initiation of apoB lipoprotein assembly. Based on experimentally derived results and molecular modeling, we propose that initiation of particle assembly occurs when 1 domain (amino acid residues 1-1000) folds into a three-sided LV-like lipid-binding cavity to form the lipid pocket without the structural requirement of MTP. This hydrophobic cavity is subsequently loaded with PL and forms a particle with lipid composition and surface to core lipid ratio consistent with the LV lipid pocket. Lipid composition, total number of lipid molecules/particle, and the particle peak density of apoB:1000-containing particles are not responsive to oleic acid supplementation of cells indicating that the lipid pocket formed by the N-terminal 1000 residues of apoB-100 has a fixed lipid capacity on the order of 50 PL for a total stoichiometry of 70 lipid molecules.
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** To whom correspondence should be addressed. Tel.: 205-975-2159; Fax: 205-975-8079; E-mail: ndashti{at}uab.edu.
1 The abbreviations used are: apo, apolipoprotein; BSA, bovine serum albumin; CE, cholesteryl ester; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; HDL, high density lipoprotein; LDL, low density lipoprotein; LV, lipovitellin; MTP, microsomal triglyceride transfer protein; NDGGE, nondenaturing gradient gel electrophoresis; PL, phospholipids; PBS, phosphate-buffered saline; TAG, triacylglycerol; VLDL, very low density lipoprotein; PVDF, polyvinylidene difluoride.
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