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Department of Medical Biochemistry and a The Wallenberg Laboratory, Göteborg University, Göteborg, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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KEY WORDS: • apolipoproteinB-HDL • Brefeldin A • hepatoma cells • microsomal triglyceride transfer protein • secretion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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). Apolipoprotein B occurs in
two forms, apoB100 and apoB48 (Kane et al. 1980
). The larger apoB100
consists of 4536 amino acids, whereas apoB48 corresponds to the
N-terminal 48% of apoB100. Both proteins are coded for by the same
gene (Innerarity et al. 1996
, Scott 1989
, Young 1990
), and the apoB100
mRNA is cotranscriptionally edited by a deaminase that converts codon
2153 into a translational stop codon (Chen et al. 1987
, Powell et al. 1987
). In humans, apoB100 is virtually the only form of apoB that is
expressed in liver, whereas apoB48 is the dominating form of apoB that
is synthesized in the intestine (Glickman et al. 1986
). However, both
apoB100 and apoB48 can assemble VLDL when expressed in the liver of
certain animals such as rats (Innerarity et al. 1996
). In this article,
we present a brief overview of the assembly process of VLDL based on
our studies of cultured human (HepG2) and rat (McA-RH7777) hepatoma
cells. This is a complex process in which apoB interacts with lipids to
form lipoprotein particles.
The secretory pathway of the cell has important roles in the
intracellular sorting of proteins, and the molecular mechanisms for the
segregation of secretory and membrane proteins have been elucidated in
great detail (Pfeffer and Rothman 1987
, Rothman and Orci 1992
).
However, the assembly and secretion of plasma lipoproteins is a complex
process that is still not completely understood. This process involves
the interaction between apoB and the membrane of the endoplasmic
reticulum and, subsequently, with an amphipathic monolayer of a
phospholipid and cholesterol-containing surface that covers a core of
neutral lipids that constitutes a lipoprotein particle. Apolipoprotein
B exists in three forms in the secretory pathway as follows (Fig. 1
): I) a form that is associated with the microsomal membrane
(Borén et al.
and 1993b
), II) a more dense form
(characterized as a lipoprotein particle with high density) and
III) VLDL-sized particles that correspond to the particles
that are secreted from the cells.
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). The deoxycholic acid/carbonate procedure had two major
advantages compared with the standard carbonate method. First, it
improved recovery of VLDL from the secretory pathway. Second, virtually
all apoB was extracted from the microsomal membrane. The standard
carbonate method typically yields a major amount of membrane-associated
apoB, making this latter finding strikingly different and unexpected.
The concentration of deoxycholate used in the extraction method is well
below the critical micellar concentration. Thus, the detergent does not
interfere with the membrane structure, and integral membrane proteins
such as calnexin and ribophorin are left membrane bound (Rustaeus et
al. 1998
). Furthermore, control experiments demonstrated that
deoxycholic acid/carbonate did not interfere with the structure of
VLDL. Characterization of the apoB that was released from the membranes
with the deoxycholic acid/carbonate method showed that it could be
isolated on lipoprotein particles with high density. These results
showed that apoB is not integrated into the microsomal membrane as a
classical membrane protein but instead is associated with the membrane
in a different way (Rustaeus et al. 1998
).
While the dense apoB100-containing particle is retained in the
secretory pathway of the cell, the dense apoB48-containing lipoprotein
particle is secreted (we will refer to these dense particles as
"apoB48 HDL") (Borén et al. 1994
). This difference allowed a
comparison between the membrane-associated apoB48 HDL that were
extracted by the deoxycholate/carbonate method and apoB48 HDL that were
secreted from the cell. The results indicated that the apoB48 HDL
extracted from the membrane with the deoxycholate/carbonate technique
had the same density as apoB48 HDL secreted from the cells (Rustaeus et
al. 1998
). Moreover, our results indicated that both of these forms of
apoB48 were associated with lipids. These data indicate that the
membrane-associated apoB48 is partially lipidated. Kinetic experiments
using pulse-chase methodology indicated that the partially lipidated
membrane-associated form of apoB48 was a precursor to both the dense
apoB48 HDL and apoB48 VLDL (Borén et al. 1994
). These results
indicate that the first event in the formation of VLDL is formation of
a dense, partially lipidated precursor particle that is loosely
associated with the microsomal membrane (Fig. 2
).The molecular mechanism through which this precursor particle is
associated with the membrane is still unclear, but we speculate that
its retention is mediated by chaperones.
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). Incubating the cells with oleic acid could influence this
relationship. Thus, the secretion of apoB48 VLDL was highly dependent
upon the availability of fatty acids. We believe that the results could
be explained in the following way (Fig. 3
):the apoB48 VLDL assembly consists of two discrete steps, the first
being the formation of partially lipidated apoB48 HDL. In the second
step, bona fide apoB48 VLDL are formed from these pre-VLDL particles by
the addition of the bulk of the lipid. Thus, apoB48 HDL can be
converted into apoB48 VLDL in the presence of oleic acid, but these
pre-apoB48 VLDL (apoB48 HDL) particles can also be released into the
secretory pathway and secreted when the second step is not active,
i.e., in the absence of oleic acid. The conclusion that apoB48 VLDL is
assembled in two steps fits well with the classical
immunoelectronmicroscopy study by Alexander et al. (1976)
. In that
study, it appeared that apoB translated in the rough endoplasmic
reticulum fused with triacylglycerol-rich particles further down the
secretory pathway.
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and 1998
).
The assembly of VLDL is highly dependent upon microsomal triglyceride
transfer protein (MTP) (Gordon 1997
). Using a photoactivatable
inhibitor of MTP, we demonstrated that inhibition of MTP during the
translation of apoB48 inhibited the assembly of apoB48 VLDL. In
contrast, inhibition of MTP after formation of apoB48 HDL (during the
second step) did not inhibit the apoB48 VLDL assembly process (Gordon et al. 1996
). This indicated that the main effect of MTP was on the
first step, i.e., during and/or immediately after translation of the
protein. To define the exact localization of the MTP dependence in the
assembly process, we turned to studies of the role of MTP in the
assembly of apoB100 VLDL (Rustaeus et al. 1998
). As in the case of
apoB48, MTP was essential during translation. In a series of
experiments, we used BFA to block the VLDL assembly process and to
accumulate membrane-associated full-length apoB100. We then removed the
BFA and followed the effect on the assembly and secretion of apoB100
VLDL during the subsequent chase period in the absence of the drug. At
various times during this chase, we inhibited MTP and followed the
effect on the assembly and secretion of apoB100 VLDL, always comparing
the MTP-inhibited cells with control cells treated in the same way
(Rustaeus et al. 1998
).
The results of these pulse-chase studies indicated that there was a post-translational step during which the assembly process was MTP dependent. This step occurred before the major amount of lipid was added to the particle. We propose that the role of MTP is to cotranslationally assemble an "apoB100 precursor" and to post-translationally adjust the structure of this particle so that it can interact with the second step. Thus, a functional definition of the first step would be the step that is dependent on MTP and during which a precursor particle that can interact with the second step assembly process(es) is assembled (Fig. 3) .
The second step has the potential to be important for regulating the
assembly process because membrane-associated apoB100 that fails to
assemble VLDL is degraded. Inhibition of the second step with a low
concentration of BFA resulted in the accumulation of membrane-bound
apoB100 precursor particles, and kinetic studies showed that
membrane-associated apoB100 was sorted to post-translational
degradation when the VLDL assembly was blocked with BFA (Rustaeus
1998
). We have previously demonstrated that the calpain inhibitor
N-acetyl-Leu-Leu-norleucinal (ALLN) (Adeli et al. 1997
) did
not inhibit the degradation of the standard carbonate-unextractable
form of apoB100 (i.e., the apoB100 precursor form). This means that the
precursor is degraded via an ALLN-insensitive protease system. This is
interesting because it suggests the presence of two sites for
intracellular degradation of apoB100 in the secretory pathway, i.e., an
early ALLN-sensitive pathway, most likely via proteasomes, and a late
ALLN-insensitive degradation of apoB100 precursor. There may be a
difference in the mechanisms regulating apoB100 and apoB48 degradation
because the apoB48 HDL particles can be secreted, in contrast to the
dense apoB100 precursor (see above).
The importance of the second step in the regulation of lipoprotein assembly is also indicated by recent studies of the liver fatty acid binding protein (L-FABP) on the assembly and secretion of apoB100 VLDL (K. Lindberg, this group, unpublished observations). Liver fatty acid binding protein inhibited the VLDL secretion without interfering with the biosynthesis of apoB100. The effect of L-FABP appeared to be an inhibition of the second step, thereby promoting a "late" intracellular degradation of apoB100. This effect of L-FABP could be explained by a diversion of fatty acids from the second step. It should be pointed out that this effect of L-FABP was not due to changes in the biosynthesis of triglycerides in the cell but appeared to be a direct effect on the second step.
There appears to be a structural requirement for apoB to undergo the
second step in the assembly process. Experiments with cycloheximide to
produce truncated, nascent apoB polypeptides released by puromycin
demonstrated that it was necessary for apoB to reach a length of at
least apoB40 (i.e., 200 kDa) in order to go through the second step,
whereas a size of 80–120 kDa was sufficient to form a dense "apoB
HDL" particle (Borén et al. 1992
, P. Stillemark, this group,
unpublished observations).
Recently, apoB was demonstrated to associate with calnexin, a chaperone
present in the secretory pathway (Chen et al. 1998
). Most intracellular
apoB100 occurs in the membrane-associated precursor form in cultured
hepatocytes and hepatoma cells. The observation that this is the first
synthesized form of full-length apoB100 and that it requires MTP to
fold correctly indicates that this form undergoes an active folding
that requires chaperones. However, chaperones may also be required for
the "second step." To investigate this hypothesis, we isolated
lipoprotein particles with a density of d < 1.010 g/mL from
rat liver microsomes and characterized the proteins that were
associated with these lipoproteins. The proteins were purified by
polyacrylamide gel electrophoresis under denaturing conditions and
identified by mass spectrometry after trypsin treatment (Larsson et al. 1997
). The results indicated that lipoprotein particles with d< 1.010 g/mL were also associated with chaperones, a finding that
suggests that the second step is a highly active process supported by
chaperones.
In summary, the second step in the VLDL assembly process has a key role in the regulation of apoB secretion and is a very active process that can be inhibited by BFA.
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FOOTNOTES |
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1 Presented at the symposium "Assembly and
Physiology of Apolipoprotein B-Containing Lipoproteins It's Not Just
for Heart Disease Anymore!" as part of Experimental Biology 98, April
18–22, 1998, San Francisco, CA. The symposium was sponsored by the
Energy and Macronutrient Research Interest Section of the American
Society for Nutritional Sciences, the Egg Nutrition Center, the
American Heart Association-Western States Affiliate, Merck Research
Laboratories, Bristol-Meyers Squibb Pharmaceutical Research Institute
and Parke-Davis Pharmaceutical Research. Published as a supplement to
The Journal of Nutrition. Guest editors for this supplement
were Rosemary L. Walzem, University of California, Davis, and Robert L.
Hamilton, University of California, San Francisco, CA.
2 Supported by grant #7142 from the Swedish
Medical Research Council, the Swedish Heart and Lung Foundation, the
Swedish Oleo-Margarine Foundation of Nutritional Research and the Novo
Nordisk Insulin Foundation.
3 Abbreviations used: ALLN,
N-acetyl-Leu-Leu-norleucinal; apo, apolipoprotein; BFA,
Brefeldin A; L-FABP, liver fatty acid-binding protein; MTP, microsomal
triglyceride transfer protein.
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