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J Biol Chem, Vol. 275, Issue 7, 4759-4765, February 18, 2000
From the Departments of Medicine and Biochemistry, Rush Medical
College, Chicago, Illinois 60612
We have previously established the presence of a
pool of apoE sequestered on the macrophage cell surface by
demonstrating its displacement from a cell monolayer at 4 °C. In
this series of experiments, we use a cell surface biotinylation
protocol to directly quantitate apoE on the macrophage cell surface and
evaluate its transport to and from this cell surface pool. In human
monocyte-derived macrophages labeled to equilibrium and in a mouse
macrophage cell line transfected to constitutively express human apoE3,
approximately 8% of total cellular apoE was present on the surface,
but only a portion of this surface pool served as a direct precursor to secreted apoE. The half-life of apoE on the macrophage cell surface was
calculated to be approximately 12 min. On SDS-polyacrylamide gel
electrophoresis, the apoE isolated from the surface fraction of cells
labeled to equilibrium migrated in an isoform pattern distinct from
that observed from the intracellular fraction, with the surface
fraction migrating predominantly in a higher molecular weight isoform.
Pulse labeling experiments demonstrated that newly synthesized apoE
reached the cell surface by 10 min but was predominantly in a low
molecular weight isoform. There was also a lag between appearance of
apoE on the cell surface and its appearance in the medium. Biotinylated
apoE, which accumulated in the medium, even from pulse labeled cells,
was predominantly in the high molecular weight isoform. Additional
experiments demonstrated that low molecular weight apoE present on the
cell surface was modified to higher molecular weight apoE by the
addition of sialic acid residues prior to secretion and that this
conversion was inhibited by brefeldin A. These results demonstrate an
unexpected complexity in the transport and cellular processing of
macrophage cell surface apoE. Factors that modulate the size and
turnover of the cell surface pool of apoE in the macrophage
remain to be identified and investigated.
The apoprotein E produced by macrophages in the vessel wall has
been reported to have a profound modulatory affect on the atherogenic
process (1-5). ApoE produced locally by macrophages in the vessel wall
may impact on many aspects of vessel wall homeostasis. Based on
in vitro observations, in vivo regulatory effects
of apoE could be predicted for arterial smooth muscle cell growth and
phenotype, cell matrix interactions, modulation of lymphocyte growth
and lymphokine production, platelet aggregability, and retention of
lipoprotein particles by the subendothelial cell matrix (1). The
production of apoE by macrophages appears to be regulated at
transcriptional and post-translational loci (6-9). In addition to its
well established fate as a secretory product of macrophages, it has
also been noted that a substantial portion of newly synthesized apoE is
degraded prior to secretion by macrophages (8, 9). In addition, we have
previously reported that macrophages retain a fraction of apoE on their
cell surface (10). Such plasma membrane-associated surface pools of
apoE have also been reported in other cells types, including
hepatocytes and steroidogenic cells (11-13). In macrophages, it
appears that cell surface proteoglycans, as well as the
LDL1 receptor, are involved
in maintenance in the cell surface fraction of apoE (10, 14).
Several potential fates for cell surface apoE in the macrophage can be
considered. The first would be that this pool serves as the immediate
precursor for secreted apoE. Alternatively, the cell surface pool could
be static and sequestered separate from the secretory pathway.
Additionally, cell surface apoE could be reinternalized prior to its
release from the cell layer. Once internalized, this apoE could be
degraded (thereby contributing to the large fraction of newly
synthesized apoE, which is degraded) or could be recycled to the cell
membrane surface pool or to the secretory pathway. In this series of
studies, we investigated aspects of the distribution, transport, and
metabolism of endogenously synthesized apoE on the macrophage plasma
membrane surface.
Materials--
Recombinant protein G-agarose (r-protein
G-agarose) and Dulbecco's modified Eagles's medium (DMEM) were
obtained from Life Technologies, Inc. Other cell culture materials were
obtained from Falcon. Sulfosuccinimidyl
2-(biotinamido)ethyl-1,3'-dithiopropionate and ImmunoPure immobilized
streptavidin-agarose were purchased from Pierce. BSA,
DL-dithiothreitol, iodoacetamide, BFA, Triton X-100, and
deoxylcholate were from Sigma. Rabbit apoE anti-serum was from
International Immunology Corp. (Murrieta, CA).
[35S]Methionine was purchased from Amersham Pharmacia
Biotech. Monoclonal antibody to tubulin was obtained from Oncogene
Research Products (Cambridge, MA). Neuraminidase from Clostridium
perfringens was purchased from Roche Molecular Biochemicals.
Cell Culture and Biosynthetic Labeling--
Mouse J774 cells
stably transfected to express a human apoE-3 cDNA (J774-E cells)
were used in this study. Detailed information for this cell line has
been previously provided (6-10). Briefly, under standard growth
conditions, these cells produce 900 ng of apoE/mg of cell protein over
24 h, which is similar to the amount produced by mature
cholesterol-loaded human monocyte-derived macrophages in culture. These
cells were maintained in DMEM with 10% fetal bovine serum and 500 µg/ml geneticin. One week prior to initiation of experiments, this
selection medium was replaced by DMEM containing only 10% fetal bovine serum.
Freshly isolated human monocytes used in this study were purified by
elutriation (15). Greater than 95% of the elutriated cells used for
experiments were monocytes, as determined by differential counts of
Wright-stained smears. Human monocytes were cultured and allowed to
differentiate into macrophages in medium containing 20% fetal bovine
serum and 10% pooled human serum.
J774-E cells were seeded in 6-well plates at a density of 2 million
cells/well and incubated at 37 °C in DMEM containing 10% fetal calf
serum for 48 h, when cells reached 95% confluence. After washing
twice with PBS, the cells were labeled with
[35S]methionine. For equilibrium labeling, the cells were
incubated at 37 °C for 16 h with methionine-free DMEM
containing 100 µCi/ml [35S]methionine and 10 µM cold methionine. For short term labeling, the cells
were first preincubated for 30 min at 37 °C with methionine-free medium and then incubated in the same medium containing varying amounts
of [35S]methionine and cold methionine for the times
indicated in each experiment.
Human monocytes were seeded at a density of 6 million/well and cultured
at 37 °C for 5 days in DMEM containing 10% pooled human serum and
20% fetal bovine serum. The cells were labeled with
[35S]methionine for a short term or to equilibrium, as
described above.
Biotinylation of Cell Surface Proteins and Preparation of Cell
Lysate--
Cell surface proteins were biotinylated using the water
soluble biotinylation reagent, ss-biotin, following a previously
published procedure (16) with minor modifications. Briefly, cells were rapidly cooled on ice for 5 min and then washed with PBS at 4 °C.
The cells were then incubated with freshly prepared
sulfo-NHS-ss-biotin/PBS solution (1.0 mg/ml) at 4 °C for 45 min.
After washing twice with PBS at 4 °C, the cells were immediately
harvested for analysis or were incubated in chase medium. As a control
for the biotinylation procedure done in this way, a separate set of
cells were labeled to equilibrium, and the degree of biotinylation of
Cell lysate was prepared by extracting cells with 100 µl of 2% SDS
and heating at 95 °C for 5 min. The extract was then diluted with
0.9 ml of lysis buffer containing 10 mM
Na2HPO4, 15 mM NaCl, 10 mM methionine, 1% Triton X-100, and 1% deoxylcholate. The
cell lysate was sheared three times through a 25-gauge needle and then transferred into a microcentrifuge tube. After centrifugation at 14,000 rpm for 10 min at 4 °C, the supernatant was collected for immunoprecipitation.
ApoE Immunoprecipitation and Isolation of Biotinylated
apoE--
ApoE in cell lysates and in media (selected experiments) was
isolated by immunoprecipitation with goat apo E anti-serum. The samples
were first cleared by incubating for 2 h at 4 °C with 25 µl
of nonimmune goat serum and then with 60 µl of r-protein G-agarose
beads for another 2 h. After centrifugation, the supernatant was
incubated with 18 µl of apoE anti-serum and then with 60 µl of
additional protein G-agarose. Iodoacetamide at a final concentration of
10 mM was included in all incubations to prevent
rearrangement of disulfide bonds.
After washing the protein G-agarose beads three times with IP buffer
(lysis buffer + 0.2% SDS), 100 µl of HEPES-buffered saline containing 1% SDS and 1 mM phenylmethylsulfonyl fluoride
is added to the tubes and heated for 3 min at 90 °C to release
apoE-IgG complexes from the protein G-agarose beads (17). Thereafter, 900 µl of IP buffer containing 10 mM iodoacetimide is
added to the tubes, which are mixed and centrifuged to obtain the
supernatant. 900 µl of the supernatant is transferred to tubes that
contain 100 µl of streptavidin-agarose beads that have been washed
twice with IP buffer and pelleted to the bottom. The samples are
incubated for 1 h with rotation at 4 °C. After centrifugation,
the supernatant was collected for determination of unbiotinylated
(intracellular fraction) apoE. The streptavidin-agarose beads were then
washed three times with IP buffer, and then a buffer containing 62.5 mM Tris-HCl (pH 6.8), 20% glycerol, 2% SDS and 5%
For analysis of intracellular apoE, 800 µl of supernatant collected
after incubation with streptavidin-agarose beads was mixed with 4 ml (5 volumes) of acetone, incubated for 30 min at Neuraminidase Digestion of apoE--
ApoE from cell fractions or
human VLDL was digested with neuraminidase as follows. Proteinase
inhibitors were added to final concentrations of 10 µg/ml
benzamidine, 5 µg/ml leupeptin, 100 IU/ml appotinin, and 5 µg/ml
pepstatin. Neuraminidase to a final concentration of 1.5 milliunits/ml
was added in a digestion buffer containing 0.15 M NaCl and
4 mM Ca Cl2 (pH 5.8). Control samples received
digestion buffer alone. After incubation for 3 h at 37 °C,
samples were prepared for SDS-PAGE as described above. Visualization of
labeled cell-derived apoE migration pattern was accomplished using a
PhosphorImager. Visualization of VLDL apoE was accomplished by Western
transfer as described previously in detail (9).
Assessment of the Steady State Distribution of apoE on the
Macrophage Cell Surface--
Biotinylation of cell surface proteins
followed by immunoisolation of cellular apoE and separation of
biotinylated from unbiotinylated apoE was conducted in human
monocytes/macrophages and J774-E cells labeled to equilibrium. In Fig.
1, we present results of a representative human macrophage experiment along with a control in which a similar isolation protocol was used in cells that were labeled but not biotinylated. As shown in the first four lanes of Fig. 1,
derived from biotinylated cells, biotinylated apoE isolated from
streptavidin beads (lanes 1 and 2) was easily
detectable and displayed a different banding pattern on SDS-PAGE
compared with unbiotinylated apoE in the streptavidin supernatant
(lanes 3 and 4), with the biotinylated fraction
having a more prominent high molecular weight band. After correction
for dilution factors used during the isolation, we calculated that
8.3 ± 0.5% of total cellular apoE was accessible to
biotinylation and therefore present on the cell surface. Fractionation of a cell lysate derived from unbiotinylated cells (lanes
5-8) demonstrated two additional points of interest. First, there
was no streptavidin-bound fraction detectable (lanes 5 and
6), confirming no background for this method of analysis.
Second, the total cellular fraction (IC + S) present in the
streptavidin supernatant from unbiotinylated cells (lanes 7 and 8), displayed a banding pattern that was similar to a
combination of the isoform pattern from the surface and intracellular
fractions obtained from biotinylated cells, i.e. prominent
high and low molecular weight apoE bands.
Fig. 2A shows the results of a
similar experiment conducted in J774-E cells except that cells were
chased for the indicated times prior to biotinylation and harvest. For
the time 0 chase, comparable with human monocyte macrophages,
approximately 8% of total cellular apoE is present on the cell surface
after equilibrium labeling (Fig. 2B). Also, as previously
seen with human macrophages, the surface fraction displays a different
molecular weight banding pattern with a predominant higher molecular
weight band, as compared with the intracellular fraction. In cells
chased for 30 min prior to biotinylation and harvest, the intracellular
fraction of apoE fell substantially; however, the percentage of apoE on
the cell surface remained stable because of a comparable fall in the
cell surface fraction. As the chase time is prolonged to 60 and 120 min
prior to biotinylation and harvest; however, there is an increase in
the percentage of total cellular labeled apoE present in the cell
surface fractions, rising to 12%. It is also of interest to note that
the lower molecular weight band of apoE disappears from the IC fraction
prior to its disappearance from the surface fraction. Specifically, the
low molecular weight band of apoE is almost undetectable at the 60- and
120-min time points from the IC fraction but remained easily
distinguishable in the surface fraction.
In Fig. 3, we present quantitative data
from an experiment in which we measured the time course for the fall
(during a chase) of cellular labeled biotinylated apoE and the rise of
medium labeled biotinylated apoE, in cells labeled to equilibrium.
Different from the experiments presented above, all cells were
biotinylated immediately after the labeling period and chased for the
indicated time prior to harvest. In this way we could follow the apoE
present on the cell surface at chase time 0. A substantial portion of the labeled apoE present on the cell surface at time 0, after equilibrium labeling, rapidly disappears from the cell layer (Fig. 3A) and appears in the medium (Fig. 3B). The most
rapid fall of biotinylated cell-associated labeled apoE occurs prior to
30-40 min, approximately the time course for the rapid phase of
accumulation of biotinylated labeled apoE in the medium. From the data
in Fig. 3A the half-life of apoE on the cell surface is
calculated to be approximately 12 min. Interestingly, the rate of fall
of cellular biotinylated apoE slows substantially after 60 min when the
rate of accumulation of biotinylated apoE in the medium also flattens. This suggests that a portion of the apoE present on the cell surface at
time 0 does not serve as an immediate precursor for secreted apoE. It
should be noted that with this sequence of labeling, i.e.
immediate biotinylation and then chase, the subcellular location of the
biotinylated apoE remaining associated with the cell at the chase times
beyond 0 min can no longer be assigned to the cell surface.
Assessment of Cell Surface apoE in Pulse-labeled Cells--
In
this series of experiments we assessed the time course for the movement
of newly synthesized apoE to the cell surface using pulse-labeled
cells. For the experiments shown in Fig.
4, J774-E cells were pulse labeled for 2, 5, 10, 20, 30, 60, or 90 min. A cell surface fraction for apoE could
not be identified in cells pulse-labeled for 2 or 5 min (not shown);
however, as early as 10 min, apoE was detectable at the cell surface,
representing approximately 3% of total cellular apoE. The percentage
of total cell apoE at the cell surface gradually increased with
increased labeling times. In these experiments, even though apoE could
be detected in the surface fraction at 10 min, no apoE was detected in
the medium fraction until 30-60 min (not shown).
In Fig. 5 we show the SDS-PAGE pattern
for apoE isolated from J774-E cells after short term labeling. Cells
were pulse labeled for 30 min, biotinylated, and harvested. Different
from the equilibrium-labeled cells, the lower molecular weight band of
apoE is prominent in the cell surface fraction (lanes 3 and
4), as well as in the intracellular fraction lanes
1 and 2. A similar but somewhat expanded experiment was
conducted in human macrophages to further evaluate the apoE migration
pattern from pulse-labeled cells. For the experiment shown in Fig.
6, human macrophages were pulse labeled
for 30 min, biotinylated, and harvested or pulse-labeled for 30 min and
chased for 30 or 60 min prior to biotinylation and harvesting. For
cells biotinylated and harvested immediately after labeling, IC and surface fraction are presented (Fig. 6, upper panel). For
the cells chased for 30 or 60 min, surface and medium fractions are presented (Fig. 6, lower panel). Immediately after the
30-min pulse (at 0 chase time) a prominent lower molecular weight apoE band is apparent in both cell surface and intracellular apoE. At this
time, after correction for dilution factors, quantitation indicated
that 2.9 ± 0.3% of total cellular apoE was present on the cell
surface, which is similar to that observed in J774-E cells, as shown in
Fig. 4 (at the 30-min pulse time). With increased chase time prior to
biotinylation (Fig. 6, lower panel), the lower molecular
weight band is observed to diminish in intensity in the surface
fraction, and high molecular weight apoE appears in the medium. No
biotinylated low molecular weight apoE is detected in the medium
fraction.
The results of the above experiments suggested to us that a small
portion of newly synthesized apoE escapes immediate post-translational modification to a high molecular weight form and appears on the cell
surface very rapidly after synthesis. Further, it appears that this
unmodified apoE is not released from the cell surface into the medium
fraction but undergoes modification to a high molecular weight form
prior to its release into the medium. This issue was further examined
in the next series of experiments.
Cellular Processing of Cell Surface apoE--
For this series of
experiments cells were biotinylated immediately after the pulse
labeling period, prior to being chased. In this way, we could follow
the processing of apoE present on the cell surface at time 0 of the
chase in pulse-labeled cells. For the experiments shown in Fig.
7, J774-E cells were pulse-labeled for 25 min. They were then biotinylated and chased for the indicated times. At
time 0, a predominantly lower molecular weight band of apoE is observed
on the cell surface. At 15, 30, 45, 60, and 90 min of chase time,
however, it can be observed that the apoE biotinylated at time 0 is now
distributed into higher molecular weight apoE isoforms in the cell
layer. With increasing chase time, the accumulation of biotinylated
high molecular weight apoE can also be observed in the medium. This
result indicates that low molecular weight apoE present at the cell
surface at time 0 is modified to high molecular weight apoE isoforms
prior to its release into the medium.
The nature of the cellular processing of the surface fraction (Fig. 7)
of apoE, which resulted in its maturation from a low molecular weight
to a high molecular weight form, was further investigated. These
investigations utilized two complementary approaches. Based on previous
reports (18-20), we hypothesized that the modification that accounted
for the increased apparent molecular weight for the apoE during the
chase incubation in Fig. 7 was due to the addition of sialic acid
residues. As a first approach, we utilized BFA. This agent has been
shown to disrupt Golgi function and inhibit Golgi-mediated
post-translational glycosylation of proteins (21, 22). For the
experiments shown in Fig. 8, cells were
pulse-labeled for 25 min and immediately biotinylated, as was done for
the experiment shown in Fig. 7. Cells were then chased for 30 or 60 min
prior to harvest; BFA was included in half of the cultures. Cell
surface apoE (Fig. 8, upper panel) at time 0 is, again,
predominantly in lower molecular weight apoE. With increasing times of
chase in the absence of BFA there is an increase in high molecular
weight isoforms of apoE relative to low molecular weight form; and an
overall loss of apoE signal intensity with the accumulation of
biotinylated apoE in the medium (Fig. 8, lower panel). In
the presence of BFA, however, there is no release of biotinylated apoE
into the medium, and further, the apoE present at the cell surface at
time 0 remains in the low molecular weight unmodified isoform.
As a second approach we specifically evaluated the nature of the
modification accounting for the change in molecular weight of cell
surface apoE, and the results of these experiments are shown in Figs.
9 and 10.
In Fig. 9, we first document the effect of neuraminidase digestion on
the mobility of apoE from J774-E cells. As shown, digestion of total
cell-derived apoE with neuraminidase resulted in the appearance of a
single isoform with mobility identical to that of similarly digested
apoE isolated from human VLDL. Thus, the presence of sialic acid
residues accounted for the high molecular weight isoforms present in
cell-derived apoE. For the experiment shown in Fig. 10, we used
neuraminidase digestion to evaluate the modification of cell surface
apoE, which occurred during the chase incubations for the experiment
shown in Fig. 7. Cells were pulse-labeled and immediately biotinylated
and then harvested immediately or chased for 20 min prior to harvest,
exactly as described in Fig. 7. For the harvest, total cellular
biotinylated apoE was isolated for the 0- and 20-min chase times, and
medium biotinylated apoE was isolated from the 20-min chase time. Half
of the isolates were then subjected to neuraminidase digestion. As
shown, the neuraminidase digestion resulted in the disappearance of
high molecular weight apoE isoforms from the cell, as well as from the
medium fractions; indicating that the maturation from the low to high
molecular weight biotinylated isoform was due to the addition of sialic
acid during the chase period. This provides additional evidence for the
role of the Golgi in this maturation in that addition of sialic acid
residues to endogenously synthesized protein is a marker for
movement of proteins through the trans-Golgi network (16).
The results of our studies indicate an unexpected complexity in
the transport of the cell surface fraction of apoE in the macrophage.
Under equilibrium conditions, approximately 8% of total cellular apoE
is present in the cell surface fraction. Cell surface apoE, in
equilibrium-labeled cells, is distributed in high molecular weight and
low molecular weight isoforms, with the high molecular weight form
predominating (Figs. 1 and 2). In cells labeled to equilibrium, as the
apoE is allowed to mature during a chase prior to cell surface
biotinylation, the amount of apoE remaining in the intracellular
fraction falls. The amount of labeled apoE on the cell surface also
falls during the chase period but more slowly so that the percentage of
total labeled apoE in the surface fraction increases (Fig. 2). The fall
in cell surface apoE is mirrored by the accumulation of biotinylated
apoE in the medium of equilibrium-labeled cells, and the half-life of
apoE on the macrophage cell surface is 12 min (Fig. 3). These results
suggest a model in which intracellular apoE is the precursor for cell
surface apoE, and cell surface apoE is the precursor for secreted apoE.
Further analysis of our experimental results, however, requires
modification of this relatively straightforward model for apoE
transport from the intracellular compartment to the medium. A small
portion of newly synthesized apoE reaches the plasma membrane very
rapidly, and there is a lag between the time when newly synthesized apoE can be identified in the cell surface fraction, and when it can be
detected in the medium. These studies also demonstrate that the apoE
isoform, which accumulates in the surface fraction in cells that are
pulse-labeled, is mostly in the low molecular weight isoform, not the
high molecular weight isoform that is detected on the surface in
equilibrium-labeled cells. Further, the apoE that accumulates in the
medium is predominantly the high molecular weight isoform (Fig. 6).
Because low molecular weight isoform apoE does not accumulate in the
medium, its disappearance from the cell surface must be due to
degradation or its conversion to the high molecular weight isoform.
Fig. 7 clearly indicates that some portion of the low molecular weight
isoform on the cell surface does, in fact, serve as precursor to high
molecular weight isoform cellular apoE and secreted apoE. The results
of experiments utilizing neuraminidase digestion and BFA indicate that
cell transport through the Golgi is required for the conversion of low
molecular weight apoE on the cell surface to a high molecular weight isoform.
It also is of interest that cell surface apoE, after falling rapidly in
the first 30-40 min, falls more slowly (Fig. 3) and that there is a
persistent low molecular weight isoform on the surface at least 60 min
after the lower molecular weight isoform has virtually disappeared from
the intracellular compartment (Fig. 2). The results in Fig. 7 also
demonstrate the persistence of a low molecular weight isoform of apoE,
biotinylated at time 0, for at least 90 min. These observations support
the notion that there is a fraction of cell surface apoE that has a
longer residence time on the cell surface or that undergoes repeated
recycling. This issue, along with a number of others raised by our
observations will require additional investigation. For example, does
only low molecular weight isoform apoE get recycled, or can high
molecular weight isoform also be recycled from the cell surface for
eventual secretion? What proportion of apoE reinternalized from the
cell surface is degraded instead of recycled? The mechanism by which a
portion of newly synthesized apoE reaches the macrophage cell surface
before complete sialation is not clear. Completion of post-translational glycosylation likely requires sequential and ordered
movement through the Golgi compartments (23). The affinity of apoE for
lipid membranes or for plasma membrane receptor proteins that are
transported to the cell surface (for example, proteoglycans or LDL
receptor) could be involved in interrupting such orderly movement. The
biologic importance of the sialation of apoE is also not clearly
established. It has been shown in Chinese hamster ovary cells that are
defective in post-translational glycosylation, for example, that such
modification is not required for efficient apoE secretion (24).
Interestingly, Fazio et al. have recently reported that apoE
present on the surface of lipoproteins and injected into intact mice is
spared degradation after uptake by hepatocytes and is recycled through
the Golgi for resecretion (25). Whether factors involved in recycling
of apoE taken up in a lipoprotein particle by hepatocytes and those
factors involved in recycling of newly synthesized apoE in the
macrophage are the same or different will require additional
investigation. They may, in fact, be different given the different role
of apoE synthesized by each of these cell types, the unique complement
of cell surface proteoglycans and cell surface receptors for each cell
type, and the divergent function of these cell types. Recycling of
other endogenously synthesized cell surface proteins through the Golgi
has been demonstrated (16).
In summary, our data indicate a dynamic role for the cell surface apoE
fraction in the macrophage. The major implications of our findings are
as follows. First, there appears to be segregation of cell surface
apoE, such that predominantly modified forms are released even when
unmodified forms are predominant at the cell surface. There also
appears to be a difference in cell surface retention times between
asialo and sialated forms of apoE. Second, the observation that
unmodified apoE appears at the cell surface provided a tool with which
we were able to establish that recycling of this asialo form of cell
surface apoE occurs, and the addition of sialic acid indicates that
recycling occurs through the Golgi. Third, our observations raise the
possibility that alterations in cell surface binding sites (for example
differential expression of receptors or amount or type of proteoglycan)
can alter the characteristics of the cell surface pool with respect to
recycling. Based on our observations we formulate the following
model/hypothesis for the distribution and transport of macrophage cell
surface apoE. After synthesis, a portion of apoE can be rapidly
transported to the cell surface where it appears as a low molecular
weight unglycosylated isoform. At this time the majority of this apoE is reinternalized, and at least a portion is recycled through the Golgi
to be converted to a high molecular weight glycosylated form that is
eventually secreted into the medium. A portion of cell surface apoE is
also reinternalized for degradation (10). Based on the persistence of
an unglycosylated form of apoE associated with the cell in both
equilibrium-labeled and pulse-labeled cells, it appears that there may
be a subfraction of apoE that either has a prolonged cell surface
residence time or undergoes repeated recycling. At least a portion of
the high molecular weight form of apoE can be secreted, but a fraction
of the high molecular weight apoE at the cell surface has a prolonged
residence time or undergoes repeated recycling prior to release or
degradation. Factors that modulate the size and movement of the cell
surface apoE fraction in macrophages remain to be identified and investigated.
We thank Stephanie Thompson for assistance
with manuscript preparation.
*
This work was supported by National Institutes of Health
Grants HL 39653 and HL 57489.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
LDL, low density
lipoprotein;
VLDL, very low density lipoprotein;
BFA, brefeldin A;
J774-E, apoE expressing J774 macrophages;
ss-biotin, sulfosuccinimidyl
2-(biotinamido)ethyl-1,3'-dithioproprionate;
IC, intracellular
fraction;
S, surface fraction;
M, medium fraction;
C, total cell
fraction;
PAGE, polyacrylamide gel electrophoresis;
DMEM, Dulbecco's
modified Eagle's medium;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline.
Transport and Processing of Endogenously Synthesized ApoE on
the Macrophage Cell Surface*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin (an intracellular cytoskeletal protein) was measured as
<0.5% of total cellular -tubulin.
-mercaptethanol, 50 mM dithiothreitol was used to
release biotinylated (surface fraction) apoE from the beads.
20 °C (dry ice in
ethanol), and then centrifuged at 28,000 rpm for 30 min. The pellets
were resuspended in 0.1 ml of buffer containing 10 mM
Tris-HCl (pH 6.8), 100 mM NaCl, 1% SDS, 1 mM
EDTA, and 1 mM EGTA. 15 µl of the resuspended sample was
mixed with 15 µl of sample buffer, boiled at 95 °C for 5 min, and
then applied to 10% SDS-PAGE. For analysis of cell surface apoE,
biotinylated apoE released from streptavidin-agarose beads was boiled
at 95 °C for 5 min and then mixed with a density buffer containing
40% glycerol and 5% bromphenol blue. The samples were applied to 10% SDS-PAGE. ApoE signals on SDS-PAGE were detected using a Molecular Dynamics PhosphorImager and quantitated using the ImageQuant software program. Results are expressed in scanning cpm. Titration experiments utilizing a range of antiserum concentrations and a range of labeled apoE indicated that the polyclonal antiserum used for these experiments immunoprecipitated asialo and sialated apoE with equal efficacy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cell surface apoE fraction in human
macrophages labeled to equilibrium. Human monocytes were isolated,
grown, and labeled to equilibrium as described under "Experimental
Procedures." Cell surface proteins were biotinylated by incubating
with freshly prepared ss-biotin/PBS solution (1.0 mg/ml) at 4 °C for
45 min. Cells that were incubated with PBS alone (without biotin) were
used as a nonbiotinylation control. After washing twice with PBS, the
cells were lysed, and apoE in both unbiotinylated and biotinylated
fractions was determined, as described under "Experimental
Procedures." Lanes 1, 2, 5, and
6 represent the apoE recovered from immobilized streptavidin
in lysates from biotinylated (lanes 1 and 2) or
unbiotinylated (lanes 5 and 6) cells. Lanes
3, 4, 7, and 8 represent the apoE
in the streptavidin supernatant from biotinylated (lanes 3 and 4) and unbiotinylated (lanes 7 and
8) cells. Note the different mobility patterns of apoE in
lanes 1 and 2 versus lanes
3 and 4.
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Fig. 2.
Cell surface apoE fraction in apoE J774-E
cells labeled to equilibrium. J774-E cells were cultured and
labeled to equilibrium as detailed under "Experimental Procedures."
After washing the cells twice with PBS, labeled cells were chased at
37 °C for 0, 30, 60, or 120 min (as indicated) with DMEM containing
0.1% BSA and 500 µM unlabeled methionine. After the
indicated chase period, cell surface proteins were biotinylated. After
washing twice with PBS, the cells were lysed, and apoE in both IC and
cell surface (S) fractions was determined. A is
the scanning image of an SDS-PAGE. B presents the
quantitation of intracellular apoE (in scanning cpm on the right
axis) and the surface/intracellular apoE ratio (expressed as a
percentage on the left axis). Results shown are the means ± S.D.
of triplicate samples from a representative experiment.
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Fig. 3.
Disappearance of cell surface apoE in
equilibrium-labeled cells. J774-E cells were incubated and labeled
to equilibrium as described under "Experimental Procedures."
Immediately after labeling, cells were biotinylated and then chased for
the indicated time periods. At each chase time the amount of
biotinylated apoE was determined in the cell layer (A) and
in the medium (B). The values shown are the means ± S.D. of triplicate samples. The curves were generated using
SigmaPlot 4.0 (SPSS, Chicago, IL).
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[in a new window]
Fig. 4.
Rate of movement of newly synthesized apoE to
the cell surface. J774-E cells were incubated with methionine-free
DMEM containing 250 µCi/ml [35S]methionine at 37 °C
for 10, 20, 30, 60, or 90 min. After removing the labeling medium, cell
surface proteins were immediately biotinylated, apoE in the cell
surface and intracellular fractions was determined as described under
"Experimental Procedures," and the percentage of total cell apoE in
the surface fraction after each labeling period was calculated. Values
shown are means ± S.D. of triplicate samples.
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[in a new window]
Fig. 5.
Analysis of surface apoE in J774-E cells
after short term labeling. J774-E cells were labeled with
[35S]methionine (250 µCi/ml) for 30 min. Cells were
biotinylated and harvested immediately. Lanes 1 and
2, IC fraction; lanes 3 and 4, surface
fraction.
View larger version (31K):
[in a new window]
Fig. 6.
Pulse/chase analysis of surface apoE in human
macrophages after short term labeling. Human macrophages were
labeled for 30 min with methionine-free DMEM containing 250 µCi/ml
[35S]methionine. Cells were then chased at 37 °C with
DMEM containing 0.1% BSA and 500 µM unlabeled methionine
for 0, 30, or 60 min (as indicated) and then biotinylated and
harvested. ApoE in the IC fraction and surface (S) fraction
was measured for the 0-min chase (upper panel). ApoE in the
medium and in cell surface fractions was determined at the 30- and
60-min chase (lower panel).
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[in a new window]
Fig. 7.
Modification of macrophage surface apoE.
J774-E cells were labeled at 37 °C for 25 min with methionine-free
DMEM containing 250 µCi/ml [35S]methionine. The cell
surface proteins were then immediately biotinylated. After washing with
DMEM + 0.1% BSA at 37 °C, the cells were chased at 37 °C for 0, 15, 30, 45, 60, or 90 min with DMEM containing 0.1% BSA and 500 µM unlabeled methionine. After each chase time, total
biotinylated apoE remaining associated with the cells (C),
and that present in the medium (M) was determined. A
representative gel from two experiments, each done in duplicate, is
shown.
View larger version (25K):
[in a new window]
Fig. 8.
BFA inhibits the modification of macrophage
surface apoE. J774-E cells were labeled with 250 µCi/ml of
[35S]methionine for 25 min and then biotinylated. BFA at
a concentration of 5 µg/ml was included in the biotinylation solution
for cells to be chased in the presence of BFA, whereas 0.5% ethanol
(vehicle control) was included in control cells. The cells were then
chased with DMEM containing 5 µg/ml BFA, or carrier alone, for 30 or
60 min. Biotinylated apoE remaining associated with the cells
(upper panel) and that present in the chase medium
(lower panel) was determined.
View larger version (18K):
[in a new window]
Fig. 9.
Digestion of apoE by neuraminidase.
Freshly prepared human VLDL (isolated by density gradient
ultracentrifugation, d < 1.006 gm/ml) was diluted with
digestion buffer and digested with neuraminidase, as described under
"Experimental Procedures." The VLDL was then subjected to SDS-PAGE
and detected by Western blot transfer (lane 1). J774-E cells
were cultured and labeled to equilibrium, as described under
"Experimental Procedures." Total cellular apoE was isolated by
immunoprecipitation and digested with neuraminidase, as described under
"Experimental Procedures." Undigested (lane 2) and
digested (lane 3) labeled cell-derived apoE were analyzed by
SDS-PAGE. The migration of 35-kDa molecular mass markers are
shown.
View larger version (20K):
[in a new window]
Fig. 10.
Analysis of the modification of biotinylated
labeled cell surface apoE. J774-E cells were pulse-labeled for 25 min and immediately biotinylated exactly as described in the legend to
Fig. 7. Half of the cells were harvested immediately for analysis. The
balance were chased for 20 min. Biotinylated apoE remaining associated
with the cell at time 0 and after the 20-min chase, and biotinylated
apoE in the medium after the 20-min chase was isolated and subjected to
digestion with neuraminidase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Rush Medical College,
1653 W. Congress Pkwy., Chicago, IL 60612. Tel.: 312-942-8231; Fax:
312-942-8233; E-mail: tmazzone@rush.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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