(Received for publication, August 9, 1995; and in revised form, January 19, 1996)
From the
Human apolipoprotein (apo) B mediates the formation of neutral
lipid-containing lipoproteins in the liver and intestine. The
association of apoB with lipid is thought to be promoted by the
microsomal triglyceride transfer protein complex. We have reconstituted
lipoprotein assembly in an insect cell line that normally does not
support this process. Expression of human microsomal triglyceride
transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled
Sf-21 insect cells to secrete 60-fold more lipoprotein-associated
triacylglycerol than control cells. This dramatic effect demonstrates
that effective partitioning of triacylglycerol into the secretory
pathway requires an endoplasmic reticulum-associated neutral lipid
transporter (provided by MTP) and an apolipoprotein to shuttle the
lipid through the pathway. Expression of the human apoB48 gene in
insect cells resulted in secretion of the protein product. Including
both MTP subunits with apoB48 and oleic acid specifically increased
apoB48 secretion 8-fold over individual subunits alone. To assess
whether specific regions of apoB are necessary for MTP responsiveness,
nine apoB segments were expressed. These included
NH
-terminal segments as well as internal and COOH-terminal
regions of apoB fused with a heterologous signal sequence. ApoB
segments containing the NH
-terminal 17% of the protein were
secreted and responded to MTP activity; however, a segment containing
only the NH
-terminal 17% of the protein was not
significantly responsive to MTP. Segments lacking the NH
terminus were not MTP-responsive, and five of six of these
proteins were trapped intracellularly but, in certain cases, could be
rescued by fusion to apoB17. These results suggest that the NH
terminus of apoB is necessary but not sufficient for MTP
responsiveness.
The transport of water-insoluble lipids through the circulation
of all mammals is mediated by lipoprotein particles. Apolipoprotein B
(apoB) ()is an unusually large secretory protein (514 kDa)
that is required for the assembly and secretion of triacylglycerol-rich
lipoproteins from the liver and the intestine(1) . ApoB
production rates are highly variable in human populations. Clinically
important lipoprotein disorders such as familial combined
hyperlipidemia are often associated with apoB
overproduction(2) .
The microsomal triglyceride transfer protein (MTP) plays an essential role in the assembly of apoB-containing lipoproteins. MTP is a protein complex found in the endoplasmic reticulum (ER) lumen of liver and intestinal cells(3) . This complex primarily transfers neutral lipids between membranes in vitro(4) . MTP has two subunits: protein disulfide isomerase (PDI) and a 97-kDa subunit that possesses lipid transfer activity in vitro when coupled with PDI(5) . Mutations in the 97-kDa subunit of MTP have been found in patients with abetalipoproteinemia(6, 7, 8) . This condition leads to only trace amounts of apoB-containing lipoproteins in the plasma and a substantial decrease in plasma neutral lipid content.
Recent studies have shown that introduction of the 97-kDa subunit of MTP into cells that are nonhepatic and nonenteric in origin, enables these cells to secrete segments of apoB(9, 10) . ApoB secretion in these cases is increased when the cells are supplemented with oleic acid, a substrate for neutral lipid biosynthesis. The tissue-specific production of apoB-containing lipoproteins correlates with the tissue distribution of the 97-kDa subunit of MTP. These studies, coupled with our understanding of abetalipoproteinemia, reinforce the hypothesis that MTP promotes lipoprotein assembly and secretion by facilitating the coupling of apoB with lipid. The secretion of apoB and lipid appear to be interdependent in that apoB requires adequate neutral lipid for secretion and neutral lipid secretion requires apoB.
Studies of invertebrates (primarily insects) indicate that they have evolved an alternative mechanism for the release of neutral lipids from cells(11) . While adult insects have large triacylglycerol stores in their lipoprotein-producing fat body tissue, insects cannot target triacylglycerol into the secretory pathway. Insects fail to assemble lipoproteins intracellularly and instead secrete the protein component of their lipoprotein in a lipid poor form(12) . Mobilization of stored triacylglycerol occurs only after it is hydrolyzed to diacylglycerol. The diacylglycerol is released from the cell to the extracellular fluid, the hemolymph, where lipoprotein assembly takes place(13) . This assembly process requires an extracellular lipid transfer particle (14) which, like MTP, has been shown to possess a lipid transfer activity in vitro(15) .
No molecular explanation exists for the contrasting mechanisms of triacylglycerol mobilization that have evolved within the animal kingdom. We hypothesized that the ability of vertebrates to partition triacylglycerol into the secretory pathway is dependent upon ER retention of their neutral lipid transfer activity. If this hypothesis is correct, then providing invertebrate cells with an intracellular neutral lipid transfer activity should enable them to directly secrete triacylglycerol from their intracellular stores.
Sf-21 cells (from the fall armyworm Spodoptera frugiperda) are invertebrate cells that do not produce lipoprotein particles. We have utilized these cells to assess the requirements for the secretion of triacylglycerol and apoB. Although they accumulate high levels of intracellular triacylglycerol, Sf-21 cells release little triacylglycerol into the media. Here we show that expression of human MTP and the intestinal form of human apoB (apoB48) in Sf-21 cells is sufficient to confer upon them the ability to efficiently partition triacylglycerol into the secretory pathway.
The role of specific
apoB sequences in the responsiveness to MTP has not been elucidated.
COOH-terminal apoB truncations result in a decreased capacity to bind
lipid, but do not eliminate apoB's ability to form
lipoproteins(16, 17, 18) . However, the
potential role of the NH terminus of apoB in mediating MTP
responsiveness is undefined. A segment of the NH
terminus
of apoB is thought to undergo pausing during translocation across the
ER membrane (19, 20, 21) . Thus, this region
might be essential in the initial combination of protein with
triacylglycerol that occurs during lipoprotein assembly. We therefore
investigated the possibility that the NH
-terminal region is
essential for apoB's responsiveness to MTP. We report that the
NH
terminus of apoB is required for MTP responsiveness.
However, by itself the NH
terminus is not MTP-responsive,
indicating that it is necessary but not sufficient for MTP
responsiveness. The current work further demonstrates that the
targeting of triacylglycerol into the secretory pathway is dependent
upon expression of an ER-associated neutral lipid transporter. The work
also suggests that the evolutionary differences in animal lipid
secretion are related to the secretion or retention of the
organism's neutral lipid transfer activity.
Generation of additional recombinant baculoviruses encoding regions of apoB represented in Fig. 11, was carried out as follows. Initially, a plasmid was constructed to contain an entire apoB100 minigene. Two oligonucleotides, 5`-GAT CCG CGG CCG CAT AGG CCA CTA GTG-3` and 5`-AAT TCA CTA GTG GCC TAT GCG GCC GCG-3`, were synthesized and annealed to generate the ``Bam-Eco'' polylinker containing (from the 5` end) BamHI, SacII, NotI, Sfi I, SpeI, and EcoRI sites. The polylinker was phosphorylated with polynucleotide kinase (U.S. Biochemical Corp.) prior to use. The Bam-Eco linker was ligated with a 2.5-kb EcoRI-BamHI fragment of apoB cDNA from pB18 (24) and pCMV5 that had been digested with BglII. This resulted in pB18LII. pB18LII was digested with BstEII (+1359 of the apoB cDNA) and KpnI (in the linker region of pCMV5). This product was ligated to a BstEII-KpnI fragment from pB100 (24) that was generated by a complete digestion with KpnI and a partial digestion (at +1359 of apoB cDNA) with BstEII. The resulting plasmid is pB100LII.
Figure 11:
Apolipoprotein B constructs used in this
study. The figure represents nine apoB constructs that were
expressed by recombinant baculoviruses during the course of this study.
The construct names reflect the region of apoB they encompass on a
centile basis. For example, apoB-17 contains the
NH-terminal 17% of apoB, while apoB-33-46 contains
sequences between 33% of full-length apoB and 46% of the protein. The
three NH
-terminal constructs all utilize the apoB signal
sequence (black box), while the internal and COOH-terminal
segments are fused in frame with the honeybee melittin signal peptide (shaded box).
An 18-kb NotI-SmaI DNA fragment encoding the human apoB100 minigene was excised from pB100LII and inserted into a NotI-SmaI-digested baculovirus transfer vector pVL1392 (Invitrogen) to yield pAcB100.
pB48LII was generated by engineering a translational stop signal at codon 2153 (as described(25) ) of apoB53 in pB53L-L. A 7.2-kb NotI-SmaI fragment encoding human apoB48 was removed from pB48LII and inserted into NotI-SmaI digested pVL1392 to form pAcB48.
The baculovirus transfer vector pVTBac (26) (a gift from T. Vernet) containing the honeybee melittin signal peptide was used for the production of internal and COOH-terminal apoB constructs. Following cloning, all junctions were in frame with the honeybee melittin signal sequence as confirmed by sequencing at the Columbia University Cancer Center (CUCC) Sequencing Facility. Initially, an XbaI oligonucleotide linker with translational stop sequences in all three reading frames (CTAGTCTAGACTAG) was inserted into the SmaI site of pVTBac, yielding pVTBac*. A 6.5-kb BamHI fragment spanning sequences from apoB33 to apoB80 was removed from pB100LII and inserted into the BamHI site of pVTBac* yielding pAcB33-80. A 1.8-kb BamHI-BclI fragment encoding apoB33-46 was inserted into the BamHI site of pVTBac* to produce pAcB33-46. A 4.4-kb BclI-BamHI fragment encoding apoB48-80 was inserted into the BamHI site of pVTBac* to produce pAcB48-80.
To produce apoB69-79, pVTBac was first modified by cutting with KpnI and filling in with the large fragment of DNA polymerase (Klenow) to generate blunt ends. A 63-bp synthetic DNA fragment (CGA ATC GAA GGT CGT AAA GAA ACC GCT GCT GCT AAA TTC GAA CGC CAG CAC ATG AAC AGC TAA) encoding the S peptide of RNase A (as in (27) ), a factor Xa cleavage site, and a translational stop codon were ligated to the blunt-ended pVTBac to generate pVTBac-S. pVTBac-S was digested with PstI and filled in with the Klenow to generate blunt ends. pAcB33-80 was digested with AccI and MscI to generate a 1.4-kb fragment encoding apoB69-79. This fragment was treated with Klenow to generate blunt ends and was subsequently ligated to the linearized blunt-ended pVTBac-S to generate pAcB69-79-S. This construct thus encoded apoB69-79 fused in frame with the S peptide sequence as confirmed by sequencing at the CUCC Sequencing Facility.
To generate apoB78-100, pB100LII was digested with BsiHKAI and was treated with T4 DNA polymerase to remove 3`-protruding sequences. Following digestion with MunI, a 4.5-kb fragment encoding apoB78-100 was isolated. pVTBac was digested with EcoRI and SmaI, and the 4.5-kb apoB fragment was inserted to generate pAcB78-100.
To produce apoB88-100, two oligonucleotides were used to amplify the apoB exon 29 coding sequence from human genomic DNA via the polymerase chain reaction. The 5` primer contained 42 bases (5`-cgg gat cca cAG TCC TCT CCA GAT AAA AAA CTC ACC ATA TTC-3`), with the uppercase letters representing apoB sequence. The non-apoB sequence contains a BamHI site for cloning purposes, and the apoB sequence begins with the final two amino acids of exon 28. The 3` primer contained 27 bases (5`-tcc ccg GGC TGG CTC ACT GTA TGG TTT-3`), with the uppercase letters representing 3` apoB untranslated sequence.The lowercase (non-apoB) sequence contains a XmaI site for cloning purposes. Following amplification, the resulting 1.7-kb fragment was digested with BamHI and XmaI and inserted into pBluescript II SK+/- (Stratagene), which had also been digested with BamHI and XmaI. Following nucleotide sequencing, the same fragment was excised and inserted into pVTBac, which was previously digested with BamHI and XmaI. The construct was in frame with the honeybee melittin signal peptide.
The apoB17 fusion proteins were generated using pAcB17. Initially, pAcB17 was partially digested with BamHI, and the upstream BamHI site was destroyed by filling it in with Klenow to generate blunt ends. This yielded pAcB17-Bam. A 1.8-kb BamHI-BclI fragment (from pB100LII) encoding apoB33-46 was inserted into the remaining BamHI site of pAcB17-Bam. The BamHI junction was then opened, filled in using Klenow, and religated. This placed the apoB33-46 sequence in frame with the apoB17 sequence and formed pAcB17-(33-46). A 4.4-kb BclI-BamHI fragment encoding apoB48-80 was removed from pB100 LII and was filled in to generate blunt ends using Klenow. This fragment was inserted into the BamHI site of pAcB17-Bam after this site had also been filled in. This placed the apoB48-80 sequence in frame with the apoB17 sequence and resulted in pAcB17-(B48-80).
To generate the apoB17-(69-79) fusion, pAcB17-Bam was digested with BamHI and SnaBI. A 1.5-kb BamHI-SnaBI fragment encoding apoB69-79 was removed from pAcB69-79-S and was inserted into pAcB17-Bam. This construct was linearized with BamHI and was filled in with the Klenow fragment to place apoB69-79 in frame with apoB17. The resulting product was pAcB17-(69-79-S). All fusion junctions were confirmed by sequencing at the CUCC Sequencing Facility.
The resulting transfer vectors encoding regions of apoB were then used to produce recombinant baculoviruses using linearized viral DNA (Invitrogen) according to the manufacturer's suggestions. Recombinant viruses were identified, plaque-purified, amplified, and titered as described(28, 29) .
The BSA-oleate complexes were
made as a 10 stock in PBS, pH 6.2, as follows. 10% (w/v) fatty
acid-free BSA (Sigma) was dissolved in 10 ml of PBS, pH 6.2. Sixty-one
mg of sodium oleate was dissolved in two ml of absolute ethanol plus 40
µl of 4 N NaOH with gentle heating. The ethanol was
evaporated with heating under a stream of nitrogen. In cases where an
oleic acid tracer was used, 1.25 mCi of [
H]oleic
acid was added and then dried under nitrogen. The dried oleate was
redissolved in 10 ml of PBS, pH 6.2, with heating. The oleate/PBS
solution was equilibrated to room temperature while mixing with a stir
bar. The BSA solution was then added, and mixing continued for 10 min.
This 10
solution was filter-sterilized and stored at -20
°C.
Figure 1: Changes in intracellular lipid levels in response to oleic acid treatment. Wild-type virus-infected Sf-21 cells were treated with BSA or BSA complexed with oleic acid. Following treatment, the cellular lipids were extracted and separated via thin layer chromatography. Mass measurements of lipids known to be released from vertebrate and invertebrate cells were made via charring with concentrated sulfuric acid. (PL, phospholipid, DG, diacylglycerol, TG, triacylglycerol, CE, cholesteryl ester) The results are means of three determinations ± S.D. ND, none detected.
Introduction of a
[H] oleic acid tracer, along with the 1 mM oleic acid, resulted in effective incorporation of the tracer in
all lipid pools analyzed except cholesteryl ester (Fig. 2). The
lack of detectable cholesterol ester is consistent with the observation
that Sf-21 cells lack the enzyme required for its
formation(43) . The triacylglycerol pool contained two-thirds
of the intracellular tracer, supporting the mass analysis and
indicating that synthesis of triacylglycerol was a major element of the
lipogenic response. Infections with recombinant viruses did not
significantly alter the intracellular lipid mass levels or tracer
distributions when compared with wild-type virus infection (not shown).
Figure 2:
Distribution of a
[H]oleic acid tracer within intracellular lipids
from cells infected with wild-type virus. The data show the
distribution of labeled oleic acid within lipid classes that are
released by vertebrate and invertebrate cells. The values are expressed
as percentage of total intracellular lipid counts. Each value is the
mean of three determinations ± S.D. ND, none detected; PL, phospholipid, DG, diacylglycerol, TG,
triacylglycerol, CE, cholesteryl
ester.
Figure 3:
Analysis of triacylglycerol secreted from
Sf-21 cells. The data represent the release of
[H]oleic acid-labeled triacylglycerol from cells
that are infected with different combinations of viruses. Lipids were
separated by thin layer chromatography and quantitated by scintillation
counting. Values represent the mean of three determinations ±
S.D. (W, wild-type virus, 48, apoB48 virus, P, PDI virus, M, viruses encoding both MTP
subunits.
Infection of Sf-21 cells with wild-type baculovirus did little to affect the media neutral lipid composition (Fig. 3). This indicates that any change, upon infection with recombinant viruses, would likely reflect the effect of heterologous proteins expressed by these viruses.
In contrast, co-expression of apoB48 with MTP resulted in a dramatic increase in triacylglycerol secretion by the invertebrate cells. The total media triacylglycerol level under these conditions was 10-fold higher than in media from uninfected cells. Thus, apoB48 and MTP are sufficient to efficiently partition cellular triacylglycerol into the secretory pathway. No effect on media diacylglycerol levels was observed in the presence of apoB48 and MTP (not shown), demonstrating that the effect is specific for triacylglycerol.
Figure 4: Analysis of secreted neutral lipids that float at a density of 1.20 g/ml. Following ultracentrifugation at d = 1.20 g/ml, lipoprotein-associated neutral lipids were extracted, separated by thin layer chromatography, and quantitated by scintillation counting. Values represent the mean of three determinations ± S.D. ND, none detected; W, wild-type virus; 48, apoB48 virus; M, viruses encoding both MTP subunits; DG, diacylglycerol; TG, triacylglycerol; CE, cholesteryl ester.
In addition to neutral lipid and protein, lipoproteins contain a surface layer of phospholipid. We therefore assessed the phospholipid content of the Sf-21 cell-produced apoB48 lipoproteins. Analysis of the phospholipids that floated with the apoB48 and triacylglycerol indicated that phosphatidylcholine and phosphatidylethanolamine were both present in the lipoprotein particles (data not shown).
Figure 5: Analysis of apoB48 secretion from cells treated with oleic acid. The relative amounts of apoB48 secreted by Sf-21 cells were analyzed by quantitative immunoblotting, utilizing an anti-apoB polyclonal antibody. ApoB48 was quantitated in the media of control cells (incubated with 0.5% BSA and no exogenous oleic acid) and from cells treated with 0.5% BSA and 1 mM oleic acid. ApoB48 levels from cells without oleate (-oleate) were normalized to 1, and levels from cells with oleate (+oleate) were expressed relative to that. Values represent the mean of four determinations, and error bars represent standard deviation. A representative immunoblot is shown. The increase in reactivity was not paralleled by an increase of intracellular reactivity, and no immunoreactivity occurred when cells infected with wild-type (control) virus were tested (not shown).
To investigate whether the
effect of oleic acid treatment was specific for apoB48 secretion or if
it was influencing general protein secretion, analysis of total cell
protein secretion was carried out. Total S-labeled
secreted proteins were analyzed in the presence and absence of
exogenous oleic acid (Fig. 6). In cells expressing apoB48, oleic
acid stimulated the secretion of apoB48, while the levels of other
secreted proteins were unaffected. The secretion of apoB17 from control
cells was unaffected by the presence of oleic acid, further
demonstrating that the longer apoB48 protein was specifically
influenced by the presence of oleic acid.
Figure 6:
Specificity of the stimulation of apoB48
secretion by oleic acid. To assess the specificity of oleic acid
stimulation of apoB48 secretion, cells were treated with
[S]methionine, and total secreted proteins were
analyzed by fluorography. Oleic acid (1 mM) induction of
apoB48 secretion is clearly seen, while no other proteins secreted by
the cells appear to respond to the treatment. Secretion of apoB17 is
also unresponsive to oleic acid.
([
S]methionine-labeled apoB48 from oleic
acid(-) cells is more readily visible upon longer film
exposure.)
In order to assess the effect of MTP on apoB secretion, cells expressing apoB48 were co-infected with either the individual MTP subunits or with both subunits together. Expression of both MTP subunits with apoB48 in the presence of oleic acid dramatically increased apoB48 secretion (Fig. 7). This increase was 8-fold more than that observed in the presence of either individual subunit alone. This pronounced stimulation of apoB48 secretion by MTP is not seen in the absence of exogenous oleic acid (not shown), demonstrating that MTP's activity is dependent on triacylglycerol availability. By quantitative Western blot analysis of oleate-treated cells, we estimate that the mass of apoB accumulating in the tissue culture medium in the presence of MTP after 17 h was 2-10% of the intracellular apoB mass (data not shown).
Figure 7: Analysis of the effect of MTP on apoB48 secretion from oleate-treated cells. Relative media levels of apoB48 were analyzed by quantitative immunoblotting following co-expression with individual MTP subunits and both subunits together. MTP-mediated stimulation of apoB48 secretion required both MTP subunits. ApoB48 levels from PDI-expressing cells were normalized to 1, and levels from other cells were expressed relative to that. Values represent the mean of three determinations, and error bars represent standard deviation. Representative immunoblots of apoB48 (secreted) and the MTP subunits (intracellular) are shown. No increase in intracellular apoB48 content was detected in the presence of MTP, and no apoB48 was detectable using cells lacking apoB48-encoding viruses (not shown).
To assess whether or not the effect of MTP on apoB48 secretion was specific, total protein secretion was analyzed in cells expressing apoB48 and one or both subunits of MTP (Fig. 8). Induction of apoB48 secretion was clearly seen in the presence of both MTP subunits. No other secreted proteins were stimulated by MTP, demonstrating its specificity for apoB48. An additional media protein (97 kDa) is detectable as a result of MTP expression. We have determined immunochemically that this is the large subunit of MTP (some of which is secreted during overexpression) and not an MTP-stimulated protein (not shown).
Figure 8:
Specificity of the stimulation of apoB48
secretion by MTP. To assess the specificity of MTP stimulation of
apoB48 secretion, oleate-treated cells were treated with
[S]methionine, and total secreted proteins were
analyzed by fluorography. MTP induction of apoB48 secretion is clearly
seen, while no other proteins secreted by the cells appear to respond
to MTP expression. An additional media protein (97 kDa) is detected
from cells expressing both MTP subunits. We have determined
immunochemically that this is the large subunit of MTP (some of which
is secreted during overexpression) and not an MTP-stimulated protein
(not shown).
Figure 9:
Pulse-chase analysis of the intracellular
degradation of apoB. Sf-21 cells were infected with recombinant
apoB48-encoding baculovirus along with MTP or the PDI subunit. The
cells were incubated in medium with or without 1 mM oleate for
17 h and then pulsed for 30 min with
[S]methionine and
[
S]cysteine. They were then chased for the
indicated time periods. Cell lysates were immunoprecipitated and
subjected to SDS-polyacrylamide gel electrophoresis. The apoB48 bands
were excised, and radioactivity was quantitated. The data are expressed
as the percentage of radioactivity at the start of the chase period.
, PDI;
, PDI + oleate;
, MTP +
oleate.
Figure 10:
Buoyant density analysis of secreted
apoB48. To determine if oleic acid treatment and MTP expression were
stimulating the production of apoB48-containing lipoproteins, density
gradient analysis was performed. Total
[S]methionine-labeled, secreted proteins were
subjected to gradient ultracentrifugation followed by Cabosil
precipitation, SDS-PAGE, and fluorography. ApoB48 is clearly detectable
in the HDL density range when stimulated by oleic acid or oleic acid
plus MTP.
Figure 12: MTP stimulation of different apoB segments. To determine if specific regions of apoB are required for secretion and MTP responsiveness, nine apoB segments (Fig. 11) were expressed with or without both MTP subunits in oleic acid-treated cells. B-``48'' represents the apoB48-like protein secreted by cells infected with the apoB100-encoding virus. B-100H (B-100 heavy) represents the full-length protein secreted by the same cells. Quantitative immunoblotting was used to assess levels of induction. For each construct, secretion levels in the presence of PDI alone were normalized to a value of 1, and levels in the presence of both subunits are expressed relative to that. Five of the segments were retained intracellularly and were not detected in the media (ND), even in the presence of MTP. Error bars represent the standard deviation from three independent determinations. Independent t-test analysis for apoB17 resulted in p = 0.06005 and for apoB88-100 p = 0.32946. Representative immunoblots are shown for secreted and intracellular apoB. Odd-numbered lanes represent samples from cells expressing the apoB segment and PDI. Even-numbered lanes represent samples expressing apoB and both MTP subunits.
Figure 13:
Analysis of apoB17 fusion proteins. To
assess any requirement for the NH terminus of apoB for
secretion and MTP sensitivity, apoB17 was fused in frame with three
internal segments of the protein. The length of the fusions (relative
to apoB100) is indicated. Quantitative immunoblotting (as for Fig. 12) demonstrated that all three fusions were detectable in
the media of infected cells and that secretion of the longest fusion
was stimulated by MTP. ND indicates no detectable induction.
The induction value for apoB17-(48-80) is a mean of three
determinations ± S.D.
To understand the mechanism of triacylglycerol targeting to the secretory pathway of cells, we introduced mammalian gene products into invertebrate host cells. Invertebrate cells are unable to effectively secrete triacylglycerol, although they actively synthesize triacylglycerol when given media supplemented with free fatty acid.
Individually, expression of the mammalian genes for apoB or MTP did not promote substantial triacylglycerol secretion. However, in combination, apoB and MTP promoted a striking increase in the levels of triacylglycerol secreted from the cells. This observation suggests that the productive partitioning of triacylglycerol into the secretory pathway requires an ER-associated neutral lipid transfer activity (provided by MTP) and a vehicle by which the lipid can be shuttled through the pathway and out of the cell (provided by apoB).
The lipoproteins secreted by Sf-21 cells expressing apoB and MTP contained phospholipid and triacylglycerol. Unlike mammalian cells, cultured Sf-21 insect cells do not synthesize cholesterol esters(43) , thus none were detected in the secreted lipoproteins. This suggests that cholesteryl ester is not a required substrate for the formation of apoB-containing lipoproteins.
Invertebrate fat body tissue produces apolipoproteins that are capable of binding and transporting neutral lipids. However, their neutral lipid transfer activity (lipid transfer particle) is localized extracellularly, in the hemolymph. It is therefore likely that the differences in lipoprotein assembly that have arisen within the animal kingdom (substrate usage and site of assembly) are due to differences in the secretion or retention of the respective lipid transfer activities.
The location of the animal's lipid transfer activity is likely to influence the neutral lipid species (triacylglycerol or diacylglycerol) that is released from the cell. Vertebrates and invertebrates both appear to mobilize cytosolic triacylglycerol stores by first hydrolyzing them into more soluble diacylglycerol(11, 45) . In vertebrates, it appears that the diacylglycerol is re-esterified to triacylglycerol at the cytoplasmic face of the ER(45) . The ER localization of MTP places it in close proximity to the re-esterification reaction, making it accessible to triacylglycerol. In addition MTP transports nonpolar lipids more actively than amphipathic lipids(46) . MTP's subcellular location and substrate specificity are therefore likely to contribute to the partitioning of triacylglycerol rather than diacylglycerol into the secretory pathway where it is complexed with apoB. In contrast, the invertebrate neutral lipid transfer activity (lipid transfer particle) is extracellular. The lack of triacylglycerol re-esterification activity at the plasma membrane would likely limit the availability of this lipid for extracellular assembly. However, diacylglycerol is more soluble and membrane-permeable than triacylglycerol(47, 48) , making it a better candidate for transfer across the plasma membrane for extracellular lipoprotein assembly.
The ability of Sf-21 cells to secrete apoB48 enabled us to study the effect of MTP on apoB secretion and determine whether specific regions of apoB are important for its secretion and MTP-responsiveness. These experiments demonstrate a clear role for both subunits in apoB secretion. In Sf-21 cells, co-expression of apoB48 with the 97-kDa MTP subunit alone had little or no effect on apoB48 secretion (Fig. 7). This suggests that the 97-kDa subunit alone is incapable of stimulating apoB secretion and that this subunit is also incapable of utilizing endogenous insect PDI as a productive subunit. Co-expression of both subunits of MTP in Sf-21 cells has been shown to result in detectable levels of neutral lipid transfer activity(23) . In the present study, expression of apoB48 in the presence of both MTP subunits increased apoB48 secretion 8-fold over either individual subunit alone. This demonstrates a requirement for both MTP subunits and active lipid transfer for the stimulation of apoB secretion. Analysis of total protein secretion demonstrated specificity of the MTP effect for apoB48 (Fig. 8).
ApoB
produced in hepatoma cells is subject to rapid intracellular
proteolysis under conditions that do not favor apoB secretion. Its
secretion is enhanced by oleate through rescue from
proteolysis(44, 49) . In primary rat hepatocytes, apoB
is rapidly degraded, but its secretion rate is unaffected by
oleate(50) . Pulse-chase experiments demonstrated that apoB is
not rapidly degraded in Sf-21 cells (Fig. 9). Accordingly, the
protease inhibitor, N-acetyl-Leu-Leu-norleucinal, which
inhibits apoB degradation in mammalian cells(51, 52) ,
seems to have little if any effect on Sf-21 apoB levels. ()The fact that we see a basal level of apoB secretion in
the Sf-21cells in the absence of MTP might be attributable to the lack
of an intracellular apoB degradation system. Therefore, the ability of
oleate and MTP to stimulate apoB secretion in this system might not
occur as a result of rescue of apoB from proteolysis. Our data also
suggest that apoB synthesis is not changed by oleate or MTP. Our
results are consistent with intracellular transport rather than
degradation being rate-limiting for apoB secretion, as recently
suggested by Bonnardel and Davis(53) . Therefore, this system
might be useful for identifying the primary impediment to apoB
secretion and the mechanism by which MTP exerts its effect.
Many studies have been carried out to define regions of apoB that may participate in lipoprotein formation. Limited trypsin proteolysis has demonstrated that some regions of apoB100, when trypsinized, readily dissociate from LDL, while other regions remain tightly associated with the lipoprotein particle(54) . Complementary studies where proteolytic fragments of apoB100 were incubated with lipid microemulsions demonstrated that specific regions of apoB100 are more lipophilic than others(55, 56) . Although these studies suggest that certain regions of apoB may readily bind lipid, they provide little information about which regions are essential for lipoprotein assembly.
Sequence based predictions suggest that
apoB100 contains several amphipathic motifs throughout its sequence. A
recent analysis suggests that apoB has a pentapartite structure of
three amphipathic -helical stretches alternating with two
amphipathic
-sheet stretches (57) . The
-sheet
regions are predicted to exist as irreversible lipid-associating
domains that encompass amino acids 827-1961 and 2611-3867.
Systematic COOH-terminal truncation of apoB100 suggests that there
are no specialized regions within the COOH-terminal 70% of the protein
that are essential for lipoprotein
assembly(16, 17, 18) . These studies suggest
that the lipid binding capacity of apoB is a function of its total
length. While these studies thoroughly examined the effect of apoB
COOH-terminal deletions, all constructs retained the NH terminus of apoB.
The present study tested whether apoB
contains specific regions that are required for protein secretion and
MTP responsiveness. Nine apoB segments spanning the entire length of
the protein were expressed with and without MTP. These segments
included NH-terminal, COOH-terminal, and internal apoB
fragments (Fig. 11) and varied in length from 10% of apoB
through the full-length apoB100.
Of the initial constructs tested,
only those containing the NH terminus of apoB were secreted
by the cells and were stimulated by MTP ( Fig. 7and Fig. 12). In contrast, none of the constructs that lacked the
NH
terminus of apoB were stimulated by MTP. The majority of
these apoB segments (5 out of 6) were trapped inside the cells and
remained so, even in the presence of MTP. The trapped segments
consisted mainly of internal regions of apoB. All of the trapped
segments overlapped with apoB sequences predicted to contain
irreversible lipid-associating amphipathic
-sheets(57) .
Secretion-negative apoB segments obtained from tunicamycin-treated
cells showed greater electrophoretic mobility than those from untreated
cells, indicating that the trapped internal segments were at least
targeted to the ER. (
)This suggests that the heterologous
signal sequence functioned properly.
The one segment that lacked the
NH terminus of apoB but was secreted was apoB88-100.
This segment corresponds almost exactly with the final exon of apoB
(exon 29). The apoB88-100 region does not overlap with a
predicted irreversible lipid-associating region of apoB, but it
contains sequences predicted to form reversible lipid-binding
-helixes much like non-apoB apolipoproteins. Although
apoB88-100 was capable of being secreted, it was not responsive
to MTP. This is also the case for non-apoB apolipoproteins.
Analysis
of the nine apoB segments tested suggested that the NH terminus may be important for secretion of internal regions of
apoB and for MTP responsiveness. To further explore this idea, three
fusion proteins were generated. These contained three internal
fragments that were secretion-defective and nonresponsive to MTP. Each
was fused in frame with the NH
-terminal 17% of apoB
(apoB17). Following fusion with apoB17, all of the internal fragments
could be detected in the media of expressing cells (Fig. 13),
suggesting that apoB17 had conferred secretion competence on them. Only
the longest of the three was responsive to MTP. This construct,
apoB17-(48-80), represents 49% of the total apoB sequence, and
its secretion was stimulated 3.6-fold by MTP.
The reason for the
importance of the NH terminus of apoB in directing apoB
secretion and enabling MTP responsiveness is currently unclear. This
region may interact directly with MTP and may mediate MTP sensitivity
by providing a nucleation point for lipid acquisition by apoB. Previous
work suggests this region must be completely translated before nascent
apoB can be lipidated(58) . Other studies have suggested that
the NH
terminus of apoB undergoes novel translocational
pausing, resulting in transient transmembrane intermediates (19, 20) and may integrate into the inner leaflet of
the membrane(59) . (An alternative model is that apoB pauses
during translation rather than translocation(59) .) Additional
studies suggest that the NH
terminus eventually
translocates into the ER lumen(21) . It has been suggested that
translocation then pauses so that apoB exists as a transient
transmembrane protein with the NH
terminus extending into
the lumen of the ER, while the remainder of the protein remains
cytosolic. Without the impetus to resume translocation (i.e. binding of lipid presented by MTP within the ER lumen), the
arrested apoB is susceptible to cytoplasmic degradation and subsequent
secretion of the already lumenal NH
terminus(21) .
The NH terminus of apoB is very rich in cysteine
residues. Twelve of the protein's 25 cysteine residues are found
in the NH
-terminal 11% of the
protein(60, 61) . All 12 of these cysteines are
involved in disulfide bond formation, while only 4 of the remaining 13
cysteines are found in disulfide bonds(62) . Without ER lumenal
proteins to hold them in place and promote their forward translocation,
secretory proteins are free to retrotranslocate and free themselves
from the ER membrane(63) . A protein such as apoB that is
capable of periods of translocation arrest may require additional
mechanisms to prevent retrotranslocation. A compact, lumenal NH
terminus (held together with disulfide bonds) may form a
disulfide knot, thus preventing retrotranslocation and keeping apoB
accessible to lipids presented by MTP. Subsequent lipid acquisition is
likely to stimulate the forward translocation of the rest of the
protein, ultimately leading to lipoprotein maturation and the
subsequent secretion of the lipid and apoB complex.
In summary, we
have created a system with which to study the process of apoB and
MTP-mediated lipoprotein assembly. Our ability to promote lipoprotein
formation in insect cells (utilizing several gene products encoded by
separate viruses) has allowed us to probe the importance of each
individual protein. A detailed analysis of apoB suggests that the
NH terminus of this protein is essential but not sufficient
for MTP-mediated lipoprotein formation. Future studies should allow a
better understanding of the mechanism by which the NH
terminus has its effect and of which NH
-terminal
elements are essential. Similar studies may help define the functional
elements of MTP and should aid in the analysis of other gene products
that might participate in lipoprotein formation.