INTRODUCTION

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Apolipoprotein (apo)¹ B100 is a large, hydrophobic polypeptide consisting of 4536 amino acids (1, 2). In humans, apoB100 serves as a backbone for the assembly of triacylglycerol-enriched very low density lipoproteins (VLDL) (3). Assembly of VLDL requires coordinate biosynthesis of apoB100 and lipid components (4, 5). The association with lipid may occur during apoB100 translation and its translocation across the endoplasmic reticulum (ER), a process facilitated by the microsomal triglyceride transfer protein (MTP) (6). The sequence elements within apoB that are responsible for lipid recruitment have not been precisely defined. Mutational studies with C-terminal-truncated apoB variants have shown that the amount of core lipid in the resulting lipoprotein particles is positively correlated with the apoB polypeptide length (7-11), suggesting that the lipid recruiting ability is a function of the number of hydrophobic sequences. However, when abundant lipid is available, assembly of VLDL can be achieved by utilizing C-terminal-truncated apoB forms (e.g. apoB48) (12-14). Thus, both the length of apoB polypeptide and sufficient lipid supply are important determinants in the assembly and secretion of VLDL.

Despite the absence of information concerning specific sequence elements involved in VLDL assembly, several experimental observations suggest that the N-terminal 17% of apoB100 (i.e. apoB17) is critical in lipoprotein formation. Transfection studies have shown that although the C-terminal-truncated apoB forms that contain the autologous N terminus of apoB100 are usually secretion-competent (7, 11, 13), recombinant apoB variants that lack the N-terminal sequences of apoB are often secreted poorly (13, 15). Several studies have demonstrated that apoB17 has relatively low ability to associate with neutral lipids (7, 16). Secondary structure analysis has predicted that apoB17 is composed mainly of amphipathic -helices (2, 16) and may form a globular structure owing to the existence of concentrated disulfide bonds (17). It is not clear why the region of apoB with low lipid binding ability is essential for efficient assembly of lipid into a lipoprotein.

Of eight disulfide linkages within human plasma apoB, six are located within the N-terminal 500 amino acids (17). Recently, involvement of the concentrated disulfide bonds within the N terminus of apoB in lipoprotein assembly and secretion has been examined. Using dithiothreitol to disrupt disulfide bonding, Shelness and Thornburg (18) have observed that formation of the N-terminal disulfide bonds, which occurs within 1 min of translation, is required for the initiation of MTP-dependent lipid recruitment and the secretion of lipoproteins. It has been postulated that folding of the N-terminal region, presumably mediated through disulfide bonding, is essential for lipoprotein assembly (19). Considering the intrinsic nature of protein folding and the role of disulfide bonds in stabilizing native protein conformation, we hypothesized that the N-terminal six disulfide bonds play a crucial role in VLDL assembly and secretion.

In the current studies, we have directly examined the function of six disulfide bonds using two truncated forms of apoB, apoB50 that has the ability to form VLDL and apoB18 that does not associate with neutral lipids. In these proteins, the cysteine residues were selectively substituted with alanine. Our results demonstrate that the fourth disulfide bond (between Cys-7 and Cys-8) is most important for apoB50 to assemble VLDL. However, the requirement for disulfide bonds is less manifest in apoB18. The present results suggest that an interaction between the disulfide-bonded domain and the downstream lipid binding sequences may play an important role in the post-translational stability of apoB and in VLDL assembly and secretion.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- Restriction and DNA modification enzymes and endoglycosidase H were purchased from New England Biolabs. ProMix^TM (a mixture of [³⁵S]methionine and [³⁵S]cysteine, 1000 Ci/mmol), horseradish peroxidase-conjugated antibody to mouse immunoglobulin G, and the enhanced chemiluminescence (ECL^TM) reagents for immunoblotting were obtained from Amersham Pharmacia Biotech. Protease inhibitors MG132 and N-acetyl-leucyl-leucyl-norleucinal and sheep anti-human apoB antiserum were obtained from Boehringer Mannheim. Monoclonal antibodies 1D1 and MB19 were gifts of R. Milne and Y. Marcel (Ottawa Heart Institute), S. Young (Gladstone Institute), and L. Curtiss (Scripps Research Institute). Monoclonal antibody LRB220 for rat apoB was a gift of L. Wong (Louisiana State University). Protein A-Sepharose CL-4B beads were obtained from Pharmacia LKB Biotechnology Inc.

Preparation of ApoB Expression Plasmids Containing Cys-to-Ala Substitution-- Selective Cys-to-Ala substitutions were introduced into cDNA constructs encoding apoB18 (7) or apoB50. We first created an expression plasmid for B50 (pB50L-L) in which the 3' end of the apoB50 coding sequences contained an MluI site at nucleotide 7011 of the apoB cDNA that was introduced by site-specific mutagenesis as described previously (20). To create Cys-to-Ala substitutions, a 2.45-kb EcoRI-HindIII fragment was excised from pB100L-L (prepared in pCMV5) (11) by RecA-assisted restriction endonuclease (RARE) cleavage using two oligonucleotides, RARE-1 (5'-ccgcggccgcataggccactagtgaattcgggcgggctgagtgccctcggttgctgc-3') and RARE-2 (5'-gcaaggatttttcccagacagtgtcaacaaagctttgtactgggttaatggtcaagttcc-3'). RARE-1 protects an EcoRI site within the polylinker of pCMV5 that flanked the 5' end of the apoB cDNA (11, 20), whereas RARE-2 protects the HindIII site at nucleotide 2279 of the apoB cDNA. The RARE cleavage was performed as described in detail (21), except that both EcoRI methylase and AluI methylase were used together. After RARE cleavage, the 2.45-kb EcoRI-HindIII fragment was inserted into the pZero-1 vector (Invitrogen) to generate a subclone designated pZero-2.45kb that was then subjected to site-directed mutagenesis using the Morph^TM System (5 Prime  3 Prime, Inc.). To create pZeroC1-4, pZeroC5-8, and pZeroC9-12 in which 4 cysteines (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12, respectively) were changed to alanines, oligonucleotides designated Cys-1 to Cys-12 shown in Table I were used. To create pZeroC1-12 in which all 12 cysteines (i.e. Cys-1 to Cys-12) were changed to alanines, a fragment encompassing Cys-1 to Cys-4 mutations was excised from pZeroC1-4 by digestion with EcoRI (in the polylinker of pCMV5) and Bsu36I (nucleotide 449 of the apoB cDNA) and ligated to pZeroC5-8 that had been digested with the same enzymes to generate pZeroC1-8. Then a XmnI-HindIII fragment (nucleotides 1547-2279 of the apoB cDNA) containing Cys-9 to Cys-12 mutations was excised from pZeroC9-12 and used to replace the identical sequence in pZeroC1-8 to generate the resulting pZeroC1-12. To create pZeroC5-6 and pZeroC7-8 in which Cys-5 to Cys-6 and Cys-7 to Cys-8, respectively, were changed to alanines, an EcoRI-BclI fragment containing Cys-5 and Cys-6 mutations from pZeroC5-8 and an EcoRI-BclI fragment containing control sequences from the subclone pZero-2.45 kb were swapped. The mutated 2.45-kb EcoRI-HindIII fragments containing various Cys-to-Ala substitutions were inserted into pB50L-L after RARE cleavage with oligomers RARE-1 and RARE-2 to create apoB50 or into pCMV5 to create apoB18 expression plasmids. Sequences encompassing the Cys-to-Ala substitutions were verified in each plasmid by DNA sequencing with an ABI 373A DNA sequencer (Perkin-Elmer) using oligonucleotides shown in Table I. The resulting plasmids were purified by centrifugation twice in a a CsCl gradient before transfection experiments.

Cell Culture and Transfection-- McA-RH7777 cells were cultured in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum. Stable transformants expressing recombinant apoB were generated using McA-RH7777 cells according to the previously described method (7).

Density Ultracentrifugation of Medium ApoBs-- Cells were cultured in Dulbecco's modified Eagle's medium containing 20% serum and 0.4 mM oleate for 10 h. The conditioned media were fractionated into 12 fractions in a sucrose density gradient (12). Total apoBs in each fraction were recovered by immunoprecipitation using a polyclonal anti-apoB antibody (Boehringer Mannheim) that recognized both human and rat apoBs. After separation by electrophoresis on a 5% polyacrylamide gel containing 0.1% SDS (SDS-PAGE), human apoBs were visualized by immunoblotting using antibody 1D1 and rat apoB using antibody LRB220.

Metabolic Labeling of ApoBs-- Cells were pulse-labeled with [³⁵S]methionine/cysteine for 1 h and chased for up to 4.5 h in the presence of 0.4 mM oleate and 20% serum (13). Cell and medium apoBs were immunoprecipitated using the polyclonal antibody (Boehringer Mannheim) and were analyzed by SDS-PAGE. The recovery of apoB with the polyclonal antibody was 80-90% for human apoB and 70-80% for rat apoB.

Northern Blot Analysis-- Northern blot analysis was performed as described previously (22) using a SacI-SacI fragment (nucleotides 3194-3835) of the human apoB cDNA as a probe.

Microsomal Membrane Isolation, Protease Digestion, and Characterization of Lumenal Lipoprotein Particles-- Cells were cultured in Dulbecco's modified Eagle's medium (20% serum ± 0.4 mM oleate) for 6 h. The cells (combined from three 10-cm dishes) were disrupted by ball-bearing homogenization in a lysis buffer (10 mM Tris, pH 7.4, 250 mM sucrose, 0.1 mM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 20 µg/ml N-acetyl-leucyl-leucyl-norleucinal) containing 20 mM N-ethylmaleimide. The intact microsomes isolated from the post-nuclear supernatant were treated with 0.1 M sodium carbonate, pH 11.3, for 30 min in the presence of protease inhibitors and 20 mM N-ethylmaleimide. The microsomal membranes and lumenal contents were separated by centrifugation at 400,000 × g for 30 min (23). The membrane pellets were dissolved in sample buffer and analyzed by SDS-PAGE under reducing or nonreducing conditions. The lumenal contents (0.8 ml) were diluted to 5 ml with phosphate-buffered saline containing 12.5% (w/v) sucrose and subjected to sucrose gradient ultracentrifugation (12).

Trypsinization of ApoB in Permeabilized Cells-- Permeabilization and trypsinization of transfected cells were performed as described (24).

Endoglycosidase H Digestion-- Endoglycosidase H (New England Biolabs) digestion was performed according to the manufacturer's instructions. The reaction products were separated by SDS-PAGE and visualized by immunoblot analysis using antibody 1D1 (25).

Other Assays-- Protein was quantified by the method of Lowry (26) using bovine serum albumin as a standard.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Generation of ApoB Variants-- Involvement of disulfide bonds in apoB-lipoprotein production was tested using two truncated apoB variants, namely B18 and B50, that contained three levels of Cys-to-Ala substitutions (Fig. 1A). First, all 12 cysteine residues within the N-terminal 500 amino acids of B100 were substituted with alanines (i.e. C1-12 in Fig. 1B). Second, 4 cysteine residues were selectively mutated in tandem (i.e. C1-4, C-5-8, and C9-12 in Fig. 1B). Third, a single disulfide bond was eliminated by mutating one pair of cysteines (i.e. C5-6 and C7-8 in Fig. 1B). The mutant B18 or B50 proteins were all expressed in McA-RH7777 cells as demonstrated by immunoblot analysis (Fig. 1C).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of the Cys-to-Ala substitution mutants. A, the top thin line represents human B100. Positions of 25 cysteines are indicated above the line, and the number of amino acids (a.a.) of B100 are indicated below the line. The bottom thick lines, with a.a. indicated at the right end, represent two C-terminal-truncated apoB variants, B18 and B50. Epitopes of monoclonal antibodies 1D1 and MB19 (25) are shown. B, position of cysteines in the N-terminal 500 amino acids of B100 (control) and the position of Cys-to-Ala substitution in mutant apoBs (Cx-y, where x and y denote the position of mutated cysteine residues). C, immunoblot analysis (using antibody 1D1 recognizing the epitope at position 401-582) of the expressed control (control) or mutant B18 or B50 forms (Cx-y) containing various Cys-to-Ala substitutions in transiently transfected McA-RH7777 cells. Mock, transfection without apoB expression plasmids.

Substituting 12 Cysteines with Alanines Impairs ApoB50 Secretion-- We first determined the effect of 12 Cys-to-Ala substitutions on apoB secretion. We used B50 as a model since it has the ability to form VLDL. The secreted control B50 from stable cells (cultured in exogenous oleate) was associated with particles of d  1.01 g/ml (corresponding to VLDL) and of d = 1.09-1.13 g/ml (corresponding to high density lipoproteins) (Fig. 2A). Like the oleate-induced assembly and secretion of B48-VLDL (12-14), secretion of B50 as VLDL was observed only when the culture medium was supplemented with oleate (data not shown).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Effect of 12 Cys-to-Ala substitutions on the secretion of apoB50. A, density gradient profiles of secreted control B50 and B50C1-12 and secreted endogenous rat B100 and B48. Conditioned media were fractionated into 12 fractions. Human apoB was detected with antibody 1D1 and rat apoB with antibody LRB220. W.t.McA, non-transfected McA-RH7777 cells. B, Northern blot analysis of the human B50 mRNA in transfected cells. Human B100 mRNA (~14,000 bases) isolated from HepG2 cells is used as a size marker. Position of 28 S ribosomal RNA is indicated. C, pulse-chase analysis of apoB secretion. Cells were pulse labeled with [35S]methionine/cysteine for 1 h and chased for the indicated time. ApoBs from the cells and medium samples were immunoprecipitated, resolved by SDS-PAGE, and subjected to fluorography (top). Radioactivity associated with 35S-B50 was quantified by scintillation counting (bottom). Results are expressed as percent of the initial radiolabeled protein recovered at the end of chase.

Secretion of ApoB50 Is Impaired by Selected Cys-to-Ala Substitution-- We next determined the effect of selected Cys-to-Ala substitution using three B50 mutants, namely Cys-1-4, Cys-5-8 and Cys-9-12. These mutants were all expressed normally in transfected cells (Fig. 3A, Cell). Although B50C1-4 and B50C9-12 were secreted as efficiently as the control B50 (Fig. 3A, Medium) on both high density lipoproteins and VLDL particles (Fig. 3B), B50C5-8 was not detectable in the medium. Thus, disulfide bonds involving Cys-1-4 and Cys-9-12 are not essential for apoB-lipoprotein assembly and secretion. Notably, expression of B50C5-8 affected endogenous apoB secretion as VLDL (Fig. 3C), which was similar to that observed in B50C1-12 cells. Total secretion of endogenous rat B100 was reduced by 60-80% in B50C5-8 cells as compared with B50C1-4 or B50C9-12 cells (determined by scanning densitometry of the immunoblots in Fig. 3C).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effect of selected Cys-to-Ala substitution on the secretion of apoB50. A, immunoblots of control and mutant (C1-4, C5-8, and C9-12) human B50s in cells and media. B, density profiles of secreted control and mutant (C1-4, C9-12) human B50s from stably transfected cells. The atypical bands below B50 in B50C1-4 were only present in that cell line and were not observed in others. C, density profiles of secreted endogenous rat B100 and B48 from indicated B50s transfected cells. The experiments were performed essentially as described in Fig. 2A.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of single disulfide bond mutation on the secretion of apoB50. A, immunoblots of control B50 and mutants B50C5-6 and B50C7-8 in cells and media. B, density profiles of secreted B50s from stably transfected cells. C, immunoblots of endogenous rat B100 and B48 in fractionated media from indicated B50-transfected cells.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Pulse-chase analysis of secretion of mutant apoB50s with selected Cys-to-Ala substitution. A, stably transfected cells expressing control B50 or mutant B50C5-8 were pulse-labeled with [35S]methionine/cysteine for 1 h and chased for up to 4.5 h. ApoBs from the cell and medium samples were immunoprecipitated at indicated times and were analyzed by SDS-PAGE (top). Radioactivity associated with 35S-B50 was quantified by scintillation counting (bottom) as described in Fig. 2C. B, stable cell lines expressing individual B50 variants were pulse-labeled for 1 h and chased for 2 h. Results are expressed as the percent of the initial radiolabeled B50s recovered in medium and in cells at the end of the 2-h chase. The dashed line indicates the secretion efficiency of control B50. Total, the sum of radioactivity associated with cell and medium B50s.

Requirement of Disulfide Bonds Is Manifest Less in ApoB18-- The role of disulfide bonds in apoB secretion was also tested using B18. When only two pairs of cysteine residues or either pair within Cys-5-8 (i.e. Cys-5-6 or Cys-7-8) were mutated, there was no appreciable difference in B18 secretion (Fig. 6A). In contrast, secretion of B50C5-8 was totally impaired, and the level of B50C7-8 was markedly reduced as compared with B50C5-6 (Fig. 6A). Therefore, formation of disulfide bonds within Cys-5-8 is essential for B50 to assemble into lipoproteins, but it is not as important for B18. However, secretion of B18 was abolished by mutation of all six disulfide bonds (C1-12, Fig. 6A), suggesting that severe alteration of the N-terminal folding impedes apoB secretion regardless of its lipid association.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Comparison between mutant apoB18 and apoB50. A, immunoblots of control and mutant (C1-12, C5-8, C5-6 and C7-8) B18 or B50 in the media of transfected cells. B, endoglycosidase H digestion of control B50, B50C1-12, control B18, and B18C1-12. The apoB proteins were immunoprecipitated from cell lysate and subjected to endoglycosidase H digestion as described under "Experimental Procedures." Positions of potential N-glycosylation sites (circles) and the glycosylated residues (shaded circles) within B50 or B18 are shown in the top panel. a.a., amino acids.

Translocation of apoB50 Is Not Affected by Cys-to-Ala Substitution-- Protease protection assay using isolated microsomes was performed to determine if Cys-to-Ala substitution has any effect on B50 translocation. In Fig. 7A, top shows quantification of the proportion of total apoB that is protected from limited proteolysis in the absence or presence of trypsin, whereas the bottom shows representative immunoblots. Recombinant B50, either control or mutant (C1-12), was almost fully protected from trypsin (i.e. 83% of control B50 and 87% of B50C1-12 were protected), suggesting that disruption of disulfide bonding does not affect apoB translocation. Endogenous B48 was also insensitive to trypsin, but B100 was only partially protected (~50% of total). The complete resistance of protein disulfide isomerase to trypsin confirmed the integrity of the microsomes (Fig. 7A). Translocation of apoB was also examined using permeabilized cells in which trypsin digestion was performed in situ. There was no difference in the degree of protection from trypsin digestion of control or mutant B50s (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Translocation and post-translational degradation of apoB. A, trypsin digestion of microsome-associated apoB. Microsomes prepared from cells expressing control B50 or B50C1-12 were subjected to limited proteolysis with trypsin (50 µg/ml) in the presence or absence of 0.5% Triton X-100 (TX-100). Top, percent of human B50, endogenous rat B100, B48, and protein disulfide isomerase (PDI) that were resistant to trypsin digestion. The results were analyzed by scanning densitometry of the immunoblots shown in the bottom. B, effect of MG132 on apoB degradation. Cells expressing control B50 or B50C1-12 were permeabilized by digitonin for 10 min and incubated with (+) or without () MG132 in a cytoskeletal buffer (24) for 2 h. Cells were solubilized, and cellular apoBs were analyzed by immunoblotting. Top, endogenous rat apoBs. Middle, control B50. Bottom, B50C1-12.

Cytosolic Proteasome Degrades a Fraction of Mutant ApoB50-- Post-translational degradation of newly synthesized apoB can occur in the cytosol by the proteasome (27, 28) and/or within the ER (29). We treated the cells with MG132 (Fig. 7B) and found that MG132 had no effect on post-translational degradation of endogenous B100 (Fig. 7B, top) or control B50 (Fig. 7B, middle). Scanning densitometry revealed that irrespective of MG132 treatment, ~65% (in control B50 cells) or ~35% (in B50C1-12 cells) of B100 and ~40% of control B50 were recovered after incubation with MG132. Thus, post-translational degradation of B100 or control B50 is not catalyzed by the proteasome. The recovery of B50C1-12 increased from 9% without MG132 to 17% with MG132 (Fig. 7B, bottom), suggesting that a fraction of B50C1-12 was probably degraded post-translationally by the proteasome, and the majority of B50C1-12 was degraded in the ER. The enhanced degradation of B100 and B50 in B50C1-12 cells compared with that in control B50 cells will be discussed below.

Mutant ApoB50 C1-12 Is Associated with Lipid-poor Particles within the Microsomes-- We next examined whether the mutant B50s were membrane-bound or lumenal in nature. Immunoblotting under reducing condition (Fig. 8A, top) showed that at steady state, control B50 was associated with both membrane and lumenal fractions (control lanes). In contrast, B50C1-12 was associated almost exclusively with membrane fraction (C1-12 lanes). Treatment with oleate had little effect on the distribution of B50 (either control or mutant) between membrane and lumen. The absence of B50C1-12 in the microsomal lumen was also apparent when the lumenal content was fractionated by density ultracentrifugation (Fig. 8B). Some studies have suggested that the membrane-bound apoB may serve as the precursor of lumenal lipoprotein-associated apoB (12, 29). We assessed possible protein-protein interaction of membrane-associated B50 using nonreducing SDS-PAGE (Fig. 8A, top). Mutant B50C1-12 migrated slower than control B50 during electrophoresis (compare control lanes with C1-12 lanes), suggesting a less restrained configuration of the mutant protein. Notably, several bands of high molecular mass representing covalent complexes that reacted with antibody 1D1 were observed in microsomal membranes of control B50 cells. These bands were intensified when the cells were treated with exogenous oleate. However, the covalent complexes were not present in B50C1-12 cells.

View larger version (45K): [in this window] [in a new window]   Fig. 8.   Analysis of apoB50 in the membrane and lumenal fractions of microsomes. A, stably transfected cells were cultured in Dulbecco's modified Eagle's medium (20% serum) ± 0.4 mM oleate (OA) for 6 h. Microsomes were isolated from cell homogenates in the presence of 20 mM N-ethylmaleimide and separated into membrane and lumenal content fractions. Control B50 and B50C1-12 in the microsomal membrane and the lumenal content were resolved by SDS-PAGE under nonreducing (top) or reducing (bottom) conditions and detected by antibody 1D1. B, density profile of control and mutant B50 in the lumenal content fractionated by sucrose density ultracentrifugation. C, immunoblots of control B50 and B50C1-12 using antibody 1D1 or MB19 under reducing or nonreducing condition. D, immunoblots of control B50 and indicated B50 mutants (C1-4, C5-8, C5-6, C7-8, C9-12) using antibody 1D1 or MB19 under nonreducing conditions.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Protein folding and conformational stabilization by disulfide bonds during translation and translocation into the ER is re-garded as a crucial step for the sorting of many secretory proteins. Using site-directed mutagenesis to selectively eliminate disulfide bond formation within B50, we have observed that not all, but only the fourth pair of cysteines (Cys-7 and Cys-8) is essential for the formation and secretion of B50-VLDL (Fig. 4B) and for efficient B50 secretion (Fig. 5B). Mutation at other disulfide bonds has less or no effect, whereas elimination of all six disulfide bonds (B50C1-12) almost abolishes B50 secretion as lipoproteins. The decreased secretion of mutant B50 is not caused by reduced protein expression nor is it attributable to impaired translocation across the ER membrane. Rather, the failure to secrete mutant B50 results from a defect at the post-translational level, leading to degradation of the secretion-incompetent proteins. It is most likely that the inefficiency of lipid recruitment impairs secretion of the mutant proteins. The specific requirement for the fourth disulfide bond in B50 is striking, which suggests that the failed assembly and secretion of mutant B50-VLDL may not be simply the consequence of misfolding of the N terminus of apoB. In fact, mutation at cysteine residues other than Cys-7 and Cys-8 equally impairs proper folding of apoB (as determined by the reduced affinity to MB19 shown in Fig. 8D) but has less effect on B50 secretion. Therefore, it is possible that the fourth disulfide bond plays an important functional role in lipid assembly other than a structural role in folding. However, the possibility that mutation at Cys-7 and Cys-8 may affect the correct formation of other disulfide bonds within apoB has not been ruled out.

Unexpectedly, mutation at Cys-7 and Cys-8 also impairs assembly and secretion of endogenous VLDL (Figs. 2A, 3C, and 4C). Intracellular degradation of endogenous B100 and B48 are markedly increased in cells transfected with mutant B50. Expression of misfolded proteins inside the ER has been shown to induce expression of some molecular chaperones that assist both the folding and degradation processes (33, 34). Considering that lipid assembly is a general process for both endogenous and exogenous apoBs, expression of mutant B50 lacking the functional disulfide bond at Cys-7 and Cys-8 may trigger a stress response that typically affects lipid assembly. This would then generate unstable apoB molecules that are more susceptible to degradation. The nature of this stress response is not known. Identification of molecular chaperones or protease systems that are induced in cells transfected with mutant B50 may provide an explanation for the enhanced degradation of endogenous apoBs.

Another important observation made in the current study is the oleate-induced covalent complex formation of apoB in the microsomal membrane (Fig. 8). Direct interaction of apoB with MTP and other ER resident proteins, including BiP/grp78 and calnexin, has been recently demonstrated (35, 36). Our results are the first demonstration that complex formation is sensitive to mutations at the N-terminal cysteines of apoB. The concept of a high-order association of ER resident proteins forming a matrix or a gel inside the ER lumen led to a new paradigm that newly synthesized proteins form complexes of heterogeneous sizes with each other or with ER molecular chaperones to acquire more intrachain disulfide bonds until they are fully oxidized and released from the aggregates (37, 38). Vesicular stomatitis virus G proteins and procollagen chains are a few of the examples demonstrating complex formation with protein folding catalysts such as BiP/grp78 and protein disulfide isomerase before their maturation (38, 39). Similar biochemical events may also occur during apoB-lipoprotein assembly, involving transient protein-protein interactions until the apoB polypeptide achieves a certain degree of lipidation to become secretion-competent.

The current studies have emphasized that interfering with folding of the N terminus of apoB in general has less effect on the secretion of B18 than on B50. Although secretion of apoB18 is abolished when the six pairs of cysteines are mutated, its secretion is relatively resistant to one- or two-pair cysteine mutations. Thus, the stringent requirement for the disulfide bonding appears to be by and large dependent on MTP-mediated lipid assembly. On the other hand, gross disruption of disulfide bonding affects both lipid recruitment and protein secretion. On the basis of these observations, we postulate that correct folding and lipid association may represent two interdependent biochemical events that, in concert with the ER quality control system, regulate the assembly and secretion of VLDL. (a) Proper folding of a critical region involving the fourth disulfide bond might signal a lipid recruitment sensor to initiate VLDL assembly. This forward-signaling event (that is, appropriate folding of the N terminus precedes lipid assembly by acting as an acceptor for MTP) has been suggested by studies with dithiothreitol-treated HepG2 cells (19). (b) The downstream lipid binding sequences, after acquiring adequate lipid load might in turn signal the ER quality control to release the secretion-competent particle. This reverse-signaling event has been suggested by studies with oleate-treated Sf-21 cells transfected with apoB, MTP, and protein disulfide isomerase (40).