Biosynthesis and secretion of the mannose 6-phosphate receptor
and its ligands in polarized Caco-2 cells
Debra A.
Wick1,
Bellur
Seetharam1,2, and
Nancy M.
Dahms1
1 Department of Biochemistry,
and 2 Department of Medicine,
Division of Gastroenterology, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
 |
ABSTRACT |
We have analyzed the transport of newly
synthesized mannose 6-phosphate
(Man-6-P)-bearing proteins (i.e.,
lysosomal enzymes) in the polarized human colon adenocarcinoma cell
line, Caco-2, by subjecting filter-grown cells to a pulse-chase
labeling protocol using
[35S]methionine, and
the resulting cell lysate, apical medium, and basolateral medium were
immunoprecipitated with insulin-like growth factor
II/Man-6-P receptor
(IGF-II/MPR)-specific antisera. The results showed that the majority of
secreted lysosomal enzymes accumulated in the apical medium at >2 h
of chase and that this polarized distribution was facilitated by the
IGF-II/MPR selectively endocytosing lysosomal enzymes from the
basolateral surface. Treatment with various agents known to affect
vesicular transport events demonstrated that incubations at 16°C or
incubations with brefeldin A inhibited the secretion of lysosomal
enzymes from both the apical and basolateral surface, whereas treatment
with nocodazole selectively blocked apical secretion. In contrast,
incubation with NH4Cl or nocodazole had a stimulatory effect on basolateral secretion. Taken
together, these results demonstrate that the sorting of Man-6-P-containing proteins into the
apical and basolateral secretory pathways is regulated by distinct
components of the intracellular trafficking machinery.
lysosomal enzymes; intracellular trafficking; insulin-like growth
factor II receptor
 |
INTRODUCTION |
THE SELECTIVE DELIVERY OF newly synthesized lysosomal
enzymes to the lysosome is an essential process for the functional
maturation of this organelle that involves a number of specific
recognition and segregation events. In higher eukaryotic cells, newly
synthesized soluble acid hydrolases acquire mannose 6-phosphate
(Man-6-P) residues on their N-linked
oligosaccharides by the action of
UDP-N-acetylglucosamine:lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase.
The ability of this phosphotransferase to recognize a protein
determinant that is common to lysosomal enzymes provides the
specificity required for the subsequent segregation of lysosomal
enzymes from secretory proteins (5, 27). In the Golgi, phosphomannosyl
residues serve as high-affinity ligands for binding to two distinct
Man-6-P receptors (MPRs), the 300-kDa
insulin-like growth factor II/MPR (IGF-II/MPR) and the 46-kDa
cation-dependent MPR. The removal of acid hydrolases from the secretory
pathway occurs when the receptor-lysosomal enzyme complex enters into
clathrin-coated pits and vesicles for delivery from the
trans-Golgi network (TGN) to an
acidified late endosomal compartment. The acidic pH of this compartment
induces the complex to dissociate. The released lysosomal enzymes are
then delivered to lysosomes, whereas the receptors either return to the
Golgi to repeat the process or move to the plasma membrane where, at
least for the IGF-II/MPR, they function to internalize extracellular
ligands via a recapture pathway (6, 26, 35, 48).
Most cells secrete a small percentage of their newly synthesized
lysosomal enzymes. However, under certain physiological conditions, some acid hydrolases are overproduced and the majority of these enzymes
are secreted instead of being delivered to the lysosome (12, 20). A
number of mechanisms have been proposed to explain how acid hydrolases
may be selectively targeted to escape transport to the lysosome,
resulting in their secretion: 1)
decreased binding to MPRs due to an altered
Man-6-P content on the lysosomal
enzyme (38), 2) decreased binding to
MPRs due to noncarbohydrate effects on the acid hydrolase (28),
3) altered availability of the MPRs to bind ligand due to saturation, downregulation, or redistribution of
the receptors to the plasma membrane (1, 39), and
4) involvement of MPRs in the
delivery of lysosomal enzymes to the cell surface (2). However, the
exact mechanisms by which these processes are regulated to mediate the
delivery of newly synthesized lysosomal enzymes to the cell surface for
secretion, rather than to the lysosome, are not known.
Polarized epithelial cells present a more complicated problem when it
comes to the delivery of proteins to the cell surface in that their
plasma membrane is divided into two morphologically, functionally, and
biochemically distinct cell surface domains: an apical domain that
faces the exterior of the organism and a basolateral domain that faces
the internal environment. Polarized epithelial cells are able to
selectively direct newly synthesized membrane or secretory proteins to
either of these domains (44). To begin to evaluate how the secretion of
lysosomal enzymes may be regulated in a polarized cell, we have
analyzed the biosynthesis and transport of
Man-6-P-containing ligands
(i.e., lysosomal enzymes) in the polarized human intestinal
epithelial cell line, Caco-2. Our results indicate that the
majority of newly synthesized lysosomal enzymes accumulate in the
apical medium. The enrichment of distinct Man-6-P-containing ligands in the
apical and basolateral medium plus the differential effects displayed
by various agents known to alter vesicular transport events indicate
that entry into and passage through the apical and basolateral pathways
are differentially regulated.
 |
MATERIALS AND METHODS |
Materials.
The following reagents were obtained commercially as indicated:
EXPRE35S35S35S
protein labeling mix (1,200 Ci/mmol, NEN Life Science Products); fetal
bovine serum (FBS; HyClone Laboratories); DMEM and trypsin-EDTA (GIBCO
BRL Life Technologies); protein A-Sepharose, brefeldin A (BFA), and
Man-6-P (Sigma); nocodazole (Aldrich);
and endo-
-N-acetylglucosaminidase H
(endo H) (Boehringer Mannheim). Caco-2 cells were kindly provided by
Dr. Ward Olsen of the Veterans Affairs Hospital (Madison, WI).
Cell culture.
Caco-2 cells (passages 76-96)
were grown in DMEM (25 mM glucose) supplemented with 20%
heat-inactivated FBS, 4 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5%
CO2. For polarity experiments,
cells were grown as epithelial layers by high-density seeding (1.5 × 106 cells/filter) onto
nitrocellulose membrane filter inserts (Millicell-HA, 30 mm diameter,
0.45 µm pore size, Millipore). The formation and integrity of
monolayers were assessed by the development of a significant
transepithelial electrical resistance of 250-300
/cm2 over the resistance of the
filter alone. Resistance readings were measured with a Millicell-ERS
voltohmeter (Millipore). All polarity studies were performed 14 days
after plating.
Metabolic labeling.
Cells grown on filter inserts were starved for 15 min in DMEM lacking
methionine and cysteine (GIBCO BRL Life Technologies) and containing
10% heat-inactivated FBS (DMEM-FBS). The cells were then incubated in
DMEM-FBS containing
EXPRE35S35S35S
protein labeling mix (0.25-0.5 mCi/ml) (pulse medium) for 1 h
followed by incubation for the indicated times with DMEM containing 20% FBS, 1 mM methionine, and 1 mM cysteine (chase medium). In some
experiments, Man-6-P was added to the
pulse and chase medium to a final concentration of 10 mM. Incubations
at 16°C were carried out in chase medium supplemented with 20 mM
HEPES, pH 7.2. In some experiments, BFA (10 µg/ml) or
NH4Cl (10 mM) was added to the
chase medium. Treatment with nocodazole was carried out by preincubating the cells for 2 h and 45 min in DMEM-20% FBS containing nocodazole (10 µg/ml) followed by the above labeling protocol with
nocodazole (10 µg/ml) present in the starvation, pulse, and chase
media. The apical and basolateral media were harvested. Unless
otherwise indicated, the cells were solubilized for 1 h on ice in
buffer containing 0.1 M Tris, pH 8.0, 0.1 M NaCl, 10 mM EDTA, Triton
X-100 (1% vol/vol), sodium deoxycholate (0.1% wt/vol), aprotinin (1%
vol/vol), antipain (4 µg/ml), benzamidine (20 µg/ml), and 2 µg/ml
each of leupeptin, chymostatin, and pepstatin. The total amount of
protein in the resulting cell lysate was determined using the Bradford
protein assay as recommended by the manufacturer (Bio-Rad). In some
experiments, the cells were labeled under serum-free conditions in DMEM
lacking methionine and cysteine and supplemented with human serum
albumin (0.05% vol/vol), insulin (5 µg/ml), transferrin (5 µg/ml),
and selenium (5 ng/ml).
Immunoprecipitations.
Cell lysates or medium were incubated at 4°C for 16-24 h with
protein A-Sepharose plus anti-MPR polyclonal antibodies. In some
samples, Man-6-P (10 mM) or purified
IGF-II/MPR (0.27 nM), which had been isolated from bovine liver by
pentamannosyl phosphate-agarose affinity chromatography (7, 18), was
added. After recovery by centrifugation, the protein A-Sepharose beads
were washed four times with buffer containing 0.1 M Tris (pH 8.0), 0.1 M NaCl, 10 mM EDTA, and 1% Triton X-100 and once in buffer containing 20 mM Tris (pH 8.0) and 20 mM NaCl. Bound proteins were eluted by the
addition of Laemmli sample buffer and analyzed on 7.5% or 9% SDS
polyacrylamide gels under reducing conditions. The radiolabeled bands
were visualized by fluorography and quantified by using an Ambis
radioanalytical imaging system or a PhosphorImager (Molecular Dynamics
Storm 860) with ImageQuant (version 4.1) software.
Endo H digestion.
Immunoprecipitated samples were eluted from the protein A-Sepharose
beads by incubation with buffer containing 1% SDS and 10 mM
Tris · HCl (pH 7.4) for 5 min at 95°C. The
eluates were precipitated with acetone. Endo H digestion was carried
out in a buffer containing 0.1 M citrate (pH 6.0), 0.075% SDS, 0.2%
2-mercaptoethanol, and 1 mU endo H for 16 h at 37°C. The samples
were analyzed by SDS-PAGE as described above.
IGF-II/MPR-agarose affinity chromatography.
A fraction of the dialyzed medium sample was passed over bovine liver
IGF-II/MPR affinity columns (21). After washing, the columns were
eluted sequentially with 5 mM glucose 6-phosphate in column buffer
followed by 5 mM Man-6-P in column
buffer. The eluates were precipitated with TCA, subjected to SDS-PAGE,
and visualized by fluorography.
 |
RESULTS |
Expression of IGF-II/MPR in Caco-2 cells.
To analyze the biosynthesis of the IGF-II/MPR in polarized Caco-2
cells, cells grown on filter inserts were metabolically labeled and the
cell lysate, apical medium, and basolateral medium were incubated with
IGF-II/MPR-specific antisera. The results of the immunoprecipitation
show the expected band of ~250 kDa, corresponding to the receptor, in
the cell lysate plus multiple lower-molecular-weight species (Fig.
1A,
lane 1). These
lower-molecular-weight species were also detected in both the apical
and basolateral media (Fig. 1A,
lanes 3 and
5) and in immunoprecipitations using monoclonal or affinity-purified antibodies specific for the IGF-II/MPR (data not shown). To determine whether these low-molecular-weight species represented newly synthesized lysosomal enzymes that were bound
to the IGF-II/MPR en route to the prelysosomal compartment, immunoprecipitations were carried out in the presence of
Man-6-P. Figure
1A (lanes
2, 4, and
6) shows that the
lower-molecular-weight species (i.e., major species of 124, 90, doublet
at 65, doublet at 53, and 46 kDa molecular mass) were completely
eliminated when the solubilized cell lysate or medium was incubated
with IGF-II/MPR-specific antiserum in the presence of 10 mM
Man-6-P. In contrast, the ~250-kDa species was efficiently immunoprecipitated from the cell lysate in the
presence or absence of Man-6-P. The
identity of the ~250-kDa species as the IGF-II/MPR was confirmed by
pentamannosyl phosphate-agarose affinity chromatography (data not
shown). Thus these results demonstrate that the multiple
low-molecular-weight species are
Man-6-P-containing ligands (i.e.,
lysosomal enzymes) that remain bound to the receptor and are
coimmunoprecipitated along with the receptor. The observed relative
molecular weight of these species indicates the size of the
Man-6-P-containing proteins rather
than the size of the receptor-ligand complex because of the
dissociation of the complex during the preparation of the samples for
SDS-PAGE. The results also show that no detectable receptor is secreted
into the medium (see also Fig.
2A).
Although a 250-kDa species is detected in the basolateral medium (Fig.
1A, lanes
5 and 6), this band,
which is highly abundant in the basolateral medium as determined by precipitating an aliquot of the medium with TCA (data not shown), is
nonspecific, since it is precipitated with preimmune serum (Fig.
1B, lanes
1 and 6, and Fig.
2A).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Immunoprecipitation of insulin-like growth factor II (IGF-II)/mannose
6-phosphate receptor (MPR) and its ligands.
A: Caco-2 cells grown on filter
inserts were labeled with 35S
protein-labeling mix (250 µCi/ml) for 1 h followed by a 2-h
incubation in medium containing 1 mM unlabeled methionine and cysteine.
Equal aliquots of the cell lysate (Cells), apical medium (Ap), and
basolateral medium (Bl) were immunoprecipitated with polyclonal
antiserum specific for the IGF-II/MPR in the absence ( ) or
presence (+) of 10 mM mannose 6-phosphate (M6P). Samples were analyzed
on a 7.5% SDS-polyacrylamide gel under reducing conditions. Size of
each of the major lower-molecular-weight species [relative
molecular weight
(Mr) × 10 3] is indicated on
right.
B: Caco-2 cells in 60-mm dishes were
labeled with 35S protein-labeling
mix (50 µCi/ml) for 20 h in the absence ( ), lanes
1-5, or presence (+), lanes 6-8, of fetal bovine
serum (FBS). Medium was harvested and immunoprecipitated with preimmune
serum (Pre) or with polyclonal antiserum specific for the IGF-II/MPR
(B2.5) in the absence ( ) or presence (+) of 10 mM mannose
6-phosphate. To the supernatants from the immunoprecipitations in
lanes 2 and
3, purified bovine liver IGF-II/MPR
(0.08 µg) was added along with the B2.5 antibody, and the samples
were analyzed in lanes 4 and
5, respectively. In a separate
experiment, medium from Caco-2 cells labeled with
35S protein-labeling mix (50 µCi/ml) for 20 h in the absence of FBS was chromatographed on an
IGF-II/MPR-Affi-Gel-10 affinity column (MPR column) that was eluted
with glucose 6-phosphate (G, nonspecific ligand, lane
9) followed by mannose 6-phosphate (M, specific
ligand, lane 10). Glucose
6-phosphate and mannose 6-phosphate eluates were precipitated with TCA.
Samples were analyzed on a 9% SDS-polyacrylamide gel under reducing
conditions.
|
|


View larger version (52K):
[in this window]
[in a new window]
|
Fig. 2.
Biosynthesis of the IGF-II/MPR and phosphorylated ligands.
A: Caco-2 cells grown as a polarized
monolayer on filter inserts were labeled with
35S protein-labeling mix (250 µCi/ml) for 1 h followed by incubation for the indicated times in
chase medium containing 1 mM unlabeled methionine and cysteine. Cell
lysates, apical medium, and basolateral medium were immunoprecipitated
with either polyclonal antiserum specific for the IGF-II/MPR or
preimmune serum (Pre). Samples were then incubated in the absence
( ) or presence (+) of endo H before SDS-PAGE on 7.5% resolving
gels. Closed and open arrows indicate the migration of the endo
H-resistant and endo H-sensitive IGF-II/MPR, respectively. *90-kDa
species. , Doublet at 65 kDa.
B: radioactivity present in the
low-molecular-weight species (124, 90, 65, 53, and 46 kDa) shown in
A was quantified, results were
normalized to the total amount of protein in the cell sample, and
percent total (i.e., cells + apical medium + basolateral medium) was
plotted. Values represent means ± SE of 3 experiments performed in
duplicate. , Cells; , apical medium; , apical medium + mannose
6-phosphate; , basolateral medium; , basolateral medium + mannose
6-phosphate.
|
|
Although our results demonstrate that Caco-2 cells do not release
significant amounts of their endogenous IGF-II/MPR into the medium as
has been reported in other cell lines (3, 43), Man-6-P-containing ligands were
coimmunoprecipitated from both the apical and basolateral media using
IGF-II/MPR-specific antisera. This is likely due to the presence of
exogenous soluble IGF-II/MPRs in the chase medium that contains 20%
FBS (50): using quantitative Western analysis, we have found that
typical lots of FBS contain ~1 nM soluble IGF-II/MPR (Y. Zhang and N. M. Dahms, unpublished observations), which would provide
the source of the receptor in the medium to interact with the secreted
ligands. To confirm this hypothesis, medium from Caco-2 cells labeled
in the absence or presence of FBS was immunoprecipitated with
IGF-II/MPR-specific antisera. The results show that, in the absence of
FBS, no low-molecular-weight species were immunoprecipitated (Fig.
1B, compare lanes
2 and 7). Addition
of purified bovine liver IGF-II/MPR to serum-free medium resulted in
the immunoprecipitation of the low-molecular-weight species, which were
eliminated in the presence of 10 mM
Man-6-P (Fig.
1B, lanes
4 and 5), similar to
that observed in the presence of FBS (Fig.
1B, lanes
7 and 8). The
identical low-molecular-weight species could also be isolated directly
from the medium of Caco-2 cells by passage over an IGF-II/MPR affinity
column (Fig. 1B, lanes 9 and
10). Taken together, these results
indicate that newly synthesized IGF-II/MPRs in Caco-2 cells do not
undergo significant proteolysis with subsequent release into the medium
and that Man-6-P-containing ligands
are secreted into both the apical and basolateral medium of Caco-2 cells.
Secretion of Man-6-P-containing
proteins.
To further examine the kinetics of association of lysosomal enzymes
with the receptor and their rates of secretion into the apical and
basolateral medium as well as the biosynthesis of the IGF-II/MPR,
filter-grown cells were pulse labeled with
35S protein-labeling mix for 1 h
and incubated in chase medium containing nonradioactive methionine and
cysteine for various times, and the cell lysates and media were
immunoprecipitated with IGF-II/MPR-specific antisera. The newly
synthesized ~250-kDa IGF-II/MPR was completely endo H sensitive at
0 h of chase, indicating the presence of high mannose
oligosaccharides, and gradually acquired complex-type oligosaccharides
that were completely resistant to endo H digestion (half-life = 4 h)
(Fig. 2A). The half-life of the
receptor was 27 h, which is similar to that reported in several other
cell lines (4, 17, 23).
Analysis of the Man-6-P-containing
ligands revealed a transient appearance of these low-molecular-weight
species in the cell, with no detectable intracellular ligands after 8 h
of chase (Fig. 2). These results are consistent with the dissociation
of newly synthesized lysosomal enzymes from the receptor when they
reach the prelysosomal compartment where the acidic environment of this compartment results in dissociation of the receptor from its ligands (26). The detection of
Man-6-P-containing ligands in the
apical and basolateral media indicates that these proteins have entered the secretory pathway, having escaped interaction with the MPRs and the
subsequent delivery to the prelysosomal compartment. The Man-6-P-containing ligands present in
the cells and media were sensitive to endo H digestion (Fig.
2A), which is consistent with the
presence of Man-6-P on high
mannose-type, N-linked oligosaccharides (15, 47). Although each of the
major species of lysosomal enzymes was detected in both the apical and
basolateral media, the apical medium showed an enrichment of the 90-kDa
species, whereas the basolateral medium showed an enrichment of the
65-kDa species at all time points (Fig.
2A), suggesting the existence of a
selective mechanism for the entry of individual lysosomal enzymes into
the apical or basolateral secretory pathways. Quantitation of the total
phosphorylated population of lysosomal enzymes revealed that
significantly more Man-6-P-containing
ligands were observed in the apical medium than in the basolateral
medium at >2 h of chase time, with ~60% and >80% of the total
lysosomal enzymes detected in the apical medium at 6 and 36 h of chase,
respectively (Fig. 2B). However, the
presence of 10 mM Man-6-P in the pulse and chase media resulted in similar amounts of lysosomal enzymes present in the apical and basolateral media, with 42% and 48% of the
total Man-6-P-containing ligands
detected in the apical medium at 6 and 36 h of chase, respectively
(Fig. 2B). Because the addition of
Man-6-P to the medium of cells has
been shown to inhibit the endocytosis of extracellular ligands by the
IGF-II/MPR (22), these results implicate a role for the receptor in
establishing the polarized steady-state levels of secreted lysosomal
enzymes of Caco-2 cells.
Apical and basolateral sorting pathways.
To determine whether the sorting of lysosomal enzymes into the apical
and basolateral secretory pathways may be regulated differently,
filter-grown Caco-2 cells were treated with several agents known to
affect vesicular transport processes. The results are summarized in
Table 1. Incubation of the cells at
16°C, which is known to block vesicular transport (31, 42),
resulted in the nearly complete inhibition of secretion of lysosomal
enzymes from both the apical and basolateral surfaces, indicating a
similar temperature dependence for transport to the apical and
basolateral cell surface. In addition, the low temperature
significantly decreased the amount of newly synthesized lysosomal
enzymes that associated with the IGF-II/MPR in the cell fraction (Fig.
3 and Table 1), suggesting that 16°C
either blocked the phosphorylation of lysosomal enzymes or prevented
the targeting of lysosomal enzymes and/or the IGF-II/MPR to the
compartment where the receptor normally binds its ligand. Similar
results were obtained with BFA, a fungal metabolite that is known to
interfere with the function of a membrane-bound guanine
nucleotide-exchange protein to prevent association of 20-kDa
GTP-binding ADP ribosylation factors (ARFs) with membranes (11, 16). A
significant decrease was seen in the amount of lysosomal enzymes that
associated with the IGF-II/MPR in the cell fraction as well as the
amount secreted at both surfaces, with a slightly greater impact on
apical secretion (Fig. 4 and Table 1).
Treatment of Caco-2 cells with nocodazole, which causes
depolymerization of microtubules (10), resulted in about a 50%
reduction in the amount of lysosomal enzymes secreted apically and a
twofold increase both in the amount secreted basolaterally as well as
the amount associated with the IGF-II/MPR in the cell fraction at the
6-h chase time (Fig. 5 and Table 1). These
results demonstrate the requirement for an intact microtubular network
for lysosomal enzymes to efficiently traverse the apical secretory
pathway. Treatment of the cells with the weak base,
NH4Cl, which raises the pH of intracellular organelles (9, 33), resulted in a selective threefold
increase in the secretion of lysosomal enzymes at the basolateral
surface with a slight inhibitory effect on the apical secretory
pathway. In contrast to that observed with nocodazole, the amount of
lysosomal enzymes associated with the cell fraction was unchanged at
the 6-h chase time (Fig. 6 and Table 1).
Taken together, these results demonstrate that the secretion of
lysosomal enzymes via the apical and basolateral secretory pathways is
mediated through distinct machinery.
View this table:
[in this window]
[in a new window]
|
Table 1.
Quantitation of mannose 6-phosphate-containing proteins in Caco-2
cells and media following various treatments
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Secretion of lysosomal enzymes following incubation at 16°C. Caco-2
cells grown as a polarized monolayer on filter inserts were labeled
with 35S protein-labeling mix (250 µCi/ml) for 1 h and then incubated in chase medium for 2 or 6 h at
37°C or 16°C. Cells (C), apical medium (A), and basolateral
medium (B) were immunoprecipitated with polyclonal antisera specific
for the IGF-II/MPR, and samples were subjected to SDS-PAGE on 7.5%
resolving gels.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Secretion of lysosomal enzymes in the presence of brefeldin A (BFA).
Caco-2 cells grown as a polarized monolayer on filter inserts were
labeled with 35S protein-labeling
mix (250 µCi/ml) for 1 h and then incubated in chase medium
containing 0.2% DMSO ( ) or in chase medium containing 0.2%
DMSO plus BFA (+) for 2 or 6 h. Cells (C), apical medium (A), and
basolateral medium (B) were immunoprecipitated with polyclonal antisera
specific for the IGF-II/MPR, and samples were subjected to SDS-PAGE on
7.5% resolving gels.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Secretion of lysosomal enzymes in the presence of nocodazole. Caco-2
cells grown as a polarized monolayer on filter inserts were
preincubated for 2 h and 45 min, labeled with
35S protein-labeling mix (250 µCi/ml) for 1 h, and then incubated in chase medium for 2 or 6 h. All
media contained 0.2% DMSO in the absence ( ) or presence (+) of
nocodazole. Cells (C), apical medium (A), and basolateral medium (B)
were immunoprecipitated with polyclonal antiserum specific for the
IGF-II/MPR, and samples were subjected to SDS-PAGE on 7.5% resolving
gels.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Secretion of lysosomal enzymes in the presence of
NH4Cl. Caco-2 cells grown as a
polarized monolayer on filter inserts were labeled with
35S protein-labeling mix (250 µCi/ml) for 1 h and then incubated in chase medium alone ( ) or
in chase medium containing NH4Cl
(+) for 2 or 6 h. Cells (C), apical medium (A), and basolateral medium
(B) were immunoprecipitated with polyclonal antisera specific for the
IGF-II/MPR, and samples were subjected to SDS-PAGE on 7.5% resolving
gels.
|
|
 |
DISCUSSION |
MPRs mediate the lysosomal targeting of
Man-6-P-containing soluble acid
hydrolases, a heterogeneous population of >40 enzymes that differ in
size, oligomeric state, number of N-linked oligosaccharides, and extent
of phosphorylation of their oligosaccharide chains. Although much
information is available concerning the essential role of this
phosphomannosyl recognition system in the biogenesis of lysosomes in
nonpolarized cells (36), little is known about the trafficking of the
receptor and its ligands in polarized epithelial cells. To analyze the
biosynthesis and transport of newly synthesized lysosomal enzymes in
polarized cells, we have used IGF-II/MPR-specific antisera to detect by
coimmunoprecipitation the total pool of Man-6-P-containing proteins in the
human colon adenocarcinoma cell line, Caco-2.
It has been reported that Caco-2 cells release the majority of their
secretory glycoproteins and lipoproteins (41, 46) as well as some
nonglycoproteins (40) into the basolateral medium, and it was suggested
(41, 46) that the basolateral pathway represents a default pathway for
exocytosis. Subsequent studies have revealed that the acid hydrolase,
-glucosidase, is secreted predominantly from the apical surface in
Caco-2 cells, whereas the other lysosomal enzymes tested, namely,
cathepsin D,
-glucuronidase, and
-hexosaminidase, are secreted
predominantly via the basolateral pathway (24). Our current studies
clearly demonstrate that the majority of secreted phosphorylated
lysosomal enzymes accumulate in the apical medium of Caco-2 cells. The
discrepancy between our results and those of Klumperman et al. (24) may
be due to the difference in the type of assay utilized: Klumperman et
al. measured the activity of selected lysosomal enzymes, whereas in the
current study IGF-II/MPR-specific antiserum was used to detect the
total endogenous pool of phosphorylated lysosomal enzymes, which
coimmunoprecipitate with the receptor. The observation that the
presence of 10 mM Man-6-P in the
medium resulted in an increase in the amount of lysosomal enzymes in
the basolateral, but not the apical, medium (Fig.
2B) is consistent with our previous
findings that, although present on both plasma membrane domains, the
IGF-II/MPR is capable of endocytosing phosphorylated ligands via the
recapture pathway exclusively from the basolateral surface (8).
Therefore, the presence of Man-6-P in
the medium would be expected to inhibit the uptake of lysosomal enzymes
only from the basolateral surface, as observed in the current report.
In addition, the observed decrease in the total amount of
phosphorylated lysosomal enzymes in the apical medium following
incubation with Man-6-P (Fig.
2B) suggests that inhibition of the
IGF-II/MPR recapture pathway at the basolateral surface by
Man-6-P partially inhibits the
delivery of lysosomal enzymes to the apical surface. Thus the
IGF-II/MPR plays a role in mediating the delivery of at least a portion
of lysosomal enzymes to the apical surface via an indirect secretory
pathway (Fig. 7). Taken together, these
results indicate that the IGF-II/MPR recapture pathway plays a critical
role in establishing the steady-state polarized distribution (i.e.,
apical enrichment) of phosphorylated lysosomal enzymes in intestinal
epithelial cells.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 7.
Proposed secretory pathway of lysosomal enzymes in Caco-2 cells.
Majority of newly synthesized soluble mannose 6-phosphate-containing
lysosomal enzymes ( ) accumulate in the apical medium, at least a
portion of which arrive by an indirect pathway that is IGF-II/MPR
dependent and involves the recapture pathway (RECAP) at the
basolateral surface. Inhibition (//) of selected pathways by the
indicated treatments (in italics) is shown. Enrichment of the MPRs
(y-shaped receptor symbol) on the basolateral cell surface is also
indicated (8). TGN, trans-Golgi
network.
|
|
The fungal metabolite BFA has been shown to interfere with various
steps of intracellular vesicular transport. With respect to endoplasmic
reticulum (ER)-to-Golgi transport, BFA treatment leads to an inhibition
of anterograde vesicular transport, whereas the retrograde pathway
seems to remain unaffected. At the morphological level, this results in
an almost complete disappearance of the Golgi apparatus and relocation
of Golgi constituents to the ER. These effects are caused by the
inability of cytosolic coat components to bind to organellar surfaces:
BFA inhibits GDP-GTP exchange on ARF proteins, which are key components
for the recruitment of coat proteins (30, 34, 37). Our results show
that BFA, as well as incubation at 16°C, inhibits both apical and
basolateral secretion to a similar extent. In addition, little
association of the enzymes with the IGF-II/MPR was detected in the
cellular fraction (see Table 1 and Figs. 3 and 4). These results are
consistent with BFA and low temperature blocking anterograde movement
through the Golgi, thus either preventing the newly synthesized
lysosomal enzymes from entering into compartments containing the
phosphotransferase and/or preventing the phosphorylated enzymes from
interacting with the MPRs in the TGN (Fig. 7).
The transport of brush-border enzymes to the apical membrane has been
shown to occur in Caco-2 cells via either a direct pathway or an
indirect pathway in which the proteins are first delivered to the
basolateral membrane and then undergo transcytosis for delivery to the
apical membrane (29, 32). Studies using nocodazole, a
microtubule-depolymerizing agent, demonstrated that the delivery of
membrane and secretory proteins to the apical surface was inhibited in
the presence of the drug, whereas the delivery of basolateral proteins
was unaffected (13). Consistent with these studies are our results that
show that treatment with nocodazole resulted in an overall decrease in
the amount of lysosomal enzymes secreted apically (see Table 1 and Fig.
5). The overall increase in the amount of lysosomal enzymes secreted at
the basolateral surface and contained within the cell fraction in the
presence of nocodazole (see Table 1 and Fig. 5) is likely due to an
increase in the overall flux of the enzymes through the basolateral
secretory pathway caused by an accumulation of the acid hydrolases in
the TGN and/or endosomal compartments that results from the blockage of
the apical secretory pathway (Fig. 7).
The targeting of newly synthesized acid hydrolases involves both the
binding and release of ligand. The receptor-enzyme complex forms in the
Golgi and is transported to a prelysosomal compartment. The low pH of
this late endosomal compartment induces dissociation of the complex by
causing a change in the conformation of the receptor (49). A change in
the ability to dissociate ligand can have an overall impact in the
targeting pathway. This is demonstrated in the treatment of cells with
lysosomotropic amines, such as NH4Cl, which accumulate in acidic
intracellular compartments, causing an increase in the
intracompartmental pH and an impairment of receptor-enzyme
dissociation. The inability to dissociate ligand results in constantly
occupied MPRs, which stimulate the secretion of newly synthesized acid
hydrolases (14, 45). Our results show that treatment of Caco-2 cells
with NH4Cl had a slight inhibitory effect on the secretion of lysosomal enzymes at the apical surface but
significantly increased (3-fold) the amount of secretion at the
basolateral surface (see Table 1 and Fig. 6). A similar result was
observed when cells were incubated in the presence of 10 mM Man-6-P: the amount of lysosomal
enzymes in the cells, apical medium, and basolateral medium was 96%,
67%, and 348% of control, respectively, at >6 h of chase. These
results indicate the involvement of an acidified compartment(s) in
regulating the entry of lysosomal enzymes into the apical and
basolateral secretory pathway, with the loss of acidified compartments
having a selective inhibitory effect on apical secretion (Fig. 7).
In summary, we propose that targeting of lysosomal enzymes to the
apical surface occurs in part via an indirect pathway: the IGF-II/MPR
recaptures secreted lysosomal enzymes at the basolateral surface and
the enzymes are subsequently delivered to an apical endosomal
compartment (Fig. 7), a site where the endocytic pathways from the
apical and basolateral surfaces meet (19, 25). The ability of the
IGF-II/MPR to internalize secreted lysosomal enzymes solely from the
basolateral surface (8) and the observed decrease in the accumulation
of lysosomal enzymes in the apical medium in cells treated with
NH4Cl or
Man-6-P, two treatments that result in
the unavailability of the receptor, support the role of IGF-II/MPR in
generating the steady-state polarized distribution (i.e., apical enrichment) of secreted phosphorylated lysosomal enzymes in intestinal epithelial cells. Further studies are required to address the issue of
how lysosomal enzymes are selectively segregated into the apical
secretory pathway and whether the number and/or extent of
phosphorylation of N-linked oligosaccharides present on an acid
hydrolase plays any role in this process.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-44200. This work was done during the
tenure of an Established Investigatorship from the American Heart
Association to N. M. Dahms.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. M. Dahms,
Medical College of Wisconsin, Dept. of Biochemistry, 8701 Watertown
Plank Road, Milwaukee, WI 53226 (E-mail:
ndahms{at}mcw.edu).
Received 7 December 1998; accepted in final form 28 May 1999.
 |
REFERENCES |
1.
Achkar, C.,
Q. M. Gong,
A. Frankfater,
and
A. S. Bajkowski.
Differences in targeting and secretion of cathepsins B and L by BALB/3T3 fibroblasts and Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts.
J. Biol. Chem.
265:
13650-13654,
1990[Abstract/Free Full Text].
2.
Chao, H. H.,
A. Waheed,
R. Pohlmann,
A. Hille,
and
K. von Figura.
Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.
EMBO J.
9:
3507-3513,
1990[Abstract].
3.
Clairmont, K. B.,
and
M. P. Czech.
Extracellular release as the major degradative pathway of the insulin- like growth factor II/mannose 6-phosphate receptor.
J. Biol. Chem.
266:
12131-12134,
1991[Abstract/Free Full Text].
4.
Creek, K. E.,
and
W. S. Sly.
Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.
Biochem. J.
214:
353-360,
1983[Medline].
5.
Cuozzo, J. W.,
K. Tao,
Q. L. Wu,
W. Young,
and
G. G. Sahagian.
Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation.
J. Biol. Chem.
270:
15611-15619,
1995[Abstract/Free Full Text].
6.
Dahms, N. M.,
P. Lobel,
and
S. Kornfeld.
Mannose 6-phosphate receptors and lysosomal enzyme targeting.
J. Biol. Chem.
264:
12115-12118,
1989[Free Full Text].
7.
Dahms, N. M.,
P. A. Rose,
J. D. Molkentin,
Y. Zhang,
and
M. A. Brzycki.
The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding.
J. Biol. Chem.
268:
5457-5463,
1993[Abstract/Free Full Text].
8.
Dahms, N. M.,
B. Seetharam,
and
D. A. Wick.
Expression of insulin-like growth factor (IGF)-I receptors, IGF- II/cation-independent mannose 6-phosphate receptors (CI-MPRs), and cation-dependent MPRs in polarized human intestinal Caco-2 cells.
Biochim. Biophys. Acta
1279:
84-92,
1996[Medline].
9.
Dean, R. T.,
W. Jessup,
and
C. R. Roberts.
Effects of exogenous amines on mammalian cells, with particular reference to membrane flow.
Biochem. J.
217:
27-40,
1984[Medline].
10.
De Brabander, M. J.,
R. M. Van de Veire,
F. E. Aerts,
M. Borgers,
and
P. A. Janssen.
The effects of methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl]carbamate (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro.
Cancer Res.
36:
905-916,
1976[Abstract].
11.
Donaldson, J. G.,
D. Finazzi,
and
R. D. Klausner.
Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.
Nature
360:
350-352,
1992[Medline].
12.
Dong, J. M.,
E. M. Prence,
and
G. G. Sahagian.
Mechanism for selective secretion of a lysosomal protease by transformed mouse fibroblasts.
J. Biol. Chem.
264:
7377-7383,
1989[Abstract/Free Full Text].
13.
Eilers, U.,
J. Klumperman,
and
H. P. Hauri.
Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2).
J. Cell Biol.
108:
13-22,
1989[Abstract].
14.
Gonzalez-Noriega, A.,
J. H. Grubb,
V. Talkad,
and
W. S. Sly.
Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling.
J. Cell Biol.
85:
839-852,
1980[Abstract].
15.
Hasilik, A.,
U. Klein,
A. Waheed,
G. Strecker,
and
K. von Figura.
Phosphorylated oligosaccharides in lysosomal enzymes: identification of
-N-acetylglucosamine(1)phospho(6)mannose diester groups.
Proc. Natl. Acad. Sci. USA
77:
7074-7078,
1980[Abstract].
16.
Helms, J. B.,
and
J. E. Rothman.
Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF.
Nature
360:
352-354,
1992[Medline].
17.
Hemer, F.,
C. Korner,
and
T. Braulke.
Phosphorylation of the human 46-kDa mannose 6-phosphate receptor in the cytoplasmic domain at serine 56.
J. Biol. Chem.
268:
17108-17113,
1993[Abstract/Free Full Text].
18.
Hoflack, B.,
and
S. Kornfeld.
Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver.
J. Biol. Chem.
260:
12008-12014,
1985[Abstract/Free Full Text].
19.
Hughson, E. J.,
and
C. R. Hopkins.
Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces.
J. Cell Biol.
110:
337-348,
1990[Abstract].
20.
Isidoro, C.,
M. Horst,
F. M. Baccino,
and
A. Hasilik.
Differential segregation of human and hamster cathepsin D in transfected baby-hamster kidney cells.
Biochem. J.
273:
363-367,
1991[Medline].
21.
Jadot, M.,
W. M. Canfield,
W. Gregory,
and
S. Kornfeld.
Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor.
J. Biol. Chem.
267:
11069-11077,
1992[Abstract/Free Full Text].
22.
Kasper, D.,
F. Dittmer,
K. von Figura,
and
R. Pohlmann.
Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes.
J. Cell Biol.
134:
615-623,
1996[Abstract].
23.
Kiess, W.,
L. A. Greenstein,
L. Lee,
C. Thomas,
and
S. P. Nissley.
Biosynthesis of the insulin-like growth factor-II (IGF-II)/mannose-6- phosphate receptor in rat C6 glial cells: the role of N-linked glycosylation in binding of IGF-II to the receptor.
Mol. Endocrinol.
5:
281-291,
1991[Abstract].
24.
Klumperman, J.,
J. A. Fransen,
T. C. Boekestijn,
R. P. Oude Elferink,
K. Matter,
H. P. Hauri,
J. M. Tager,
and
L. A. Ginsel.
Biosynthesis and transport of lysosomal
-glucosidase in the human colon carcinoma cell line Caco-2: secretion from the apical surface.
J. Cell Sci.
100:
339-347,
1991[Abstract].
25.
Knight, A.,
E. Hughson,
C. R. Hopkins,
and
D. F. Cutler.
Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells.
Mol. Biol. Cell
6:
597-610,
1995[Abstract].
26.
Kornfeld, S.
Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors.
Annu. Rev. Biochem.
61:
307-330,
1992[Medline].
27.
Lang, L.,
M. Reitman,
J. Tang,
R. M. Roberts,
and
S. Kornfeld.
Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes.
J. Biol. Chem.
259:
14663-14671,
1984[Abstract/Free Full Text].
28.
Lazzarino, D.,
and
C. A. Gabel.
Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6- phosphate receptor.
J. Biol. Chem.
265:
11864-11871,
1990[Abstract/Free Full Text].
29.
Le Bivic, A.,
A. Quaroni,
B. Nichols,
and
E. Rodriguez-Boulan.
Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line.
J. Cell Biol.
111:
1351-1361,
1990[Abstract].
30.
Lippincott-Schwartz, J.,
L. C. Yuan,
J. S. Bonifacino,
and
R. D. Klausner.
Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER.
Cell
56:
801-813,
1989[Medline].
31.
Matlin, K. S.,
and
K. Simons.
Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation.
Cell
34:
233-243,
1983[Medline].
32.
Matter, K.,
M. Brauchbar,
K. Bucher,
and
H. P. Hauri.
Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2).
Cell
60:
429-437,
1990[Medline].
33.
Maxfield, F. R.
Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts.
J. Cell Biol.
95:
676-681,
1982[Abstract].
34.
Misumi, Y.,
K. Miki,
A. Takatsuki,
G. Tamura,
and
Y. Ikehara.
Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes.
J. Biol. Chem.
261:
11398-11403,
1986[Abstract/Free Full Text].
35.
Munier-Lehmann, H.,
F. Mauxion,
and
B. Hoflack.
Function of the two mannose 6-phosphate receptors in lysosomal enzyme transport.
Biochem. Soc. Trans.
24:
133-136,
1996[Medline].
36.
Neufeld, E. F.
Lysosomal storage diseases.
Annu. Rev. Biochem.
60:
257-280,
1991[Medline].
37.
Pelham, H. R.
Multiple targets for brefeldin A.
Cell
67:
449-451,
1991[Medline].
38.
Pohlmann, R.,
M. W. Boeker,
and
K. von Figura.
The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins.
J. Biol. Chem.
270:
27311-27318,
1995[Abstract/Free Full Text].
39.
Prence, E. M.,
J. M. Dong,
and
G. G. Sahagian.
Modulation of the transport of a lysosomal enzyme by PDGF.
J. Cell Biol.
110:
319-326,
1990[Abstract].
40.
Ramanujam, K. S.,
S. Seetharam,
M. Ramasamy,
and
B. Seetharam.
Expression of cobalamin transport proteins and cobalamin transcytosis by colon adenocarcinoma cells.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G416-G422,
1991[Abstract/Free Full Text].
41.
Rindler, M. J.,
and
M. G. Traber.
A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.
J. Cell Biol.
107:
471-479,
1988[Abstract].
42.
Saraste, J.,
G. E. Palade,
and
M. G. Farquhar.
Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells.
Proc. Natl. Acad. Sci. USA
83:
6425-6429,
1986[Abstract].
43.
Scott, C. D.,
and
R. C. Baxter.
Regulation of soluble insulin-like growth factor-II/mannose 6-phosphate receptor in hepatocytes from intact and regenerating rat liver.
Endocrinology
137:
3864-3870,
1996[Abstract].
44.
Simons, K.,
and
S. D. Fuller.
Cell surface polarity in epithelia.
Annu. Rev. Cell Biol.
1:
243-288,
1985.
45.
Stein, M.,
T. Braulke,
K. von Figura,
and
A. Hasilik.
Effects of differentiation-inducing agents on synthesis, maturation and secretion of cathepsin D in U937 and HL-60 cells.
Biol Chem Hoppe-Seyler
368:
413-418,
1987[Medline].
46.
Traber, M. G.,
H. J. Kayden,
and
M. J. Rindler.
Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line.
J. Lipid Res.
28:
1350-1363,
1987[Abstract].
47.
Varki, A.,
and
S. Kornfeld.
Structural studies of phosphorylated high mannose-type oligosaccharides.
J. Biol. Chem.
255:
10847-10858,
1980[Abstract/Free Full Text].
48.
Von Figura, K.,
and
A. Hasilik.
Lysosomal enzymes and their receptors.
Annu. Rev. Biochem.
55:
167-193,
1986[Medline].
49.
Westcott, K. R.,
R. P. Searles,
and
L. H. Rome.
Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor.
J. Biol. Chem.
262:
6101-6107,
1987[Abstract/Free Full Text].
50.
Yang, Y. W.,
A. R. Robbins,
S. P. Nissley,
and
M. M. Rechler.
The chick embryo fibroblast cation-independent mannose 6-phosphate receptor is functional and immunologically related to the mammalian insulin-like growth factor II (IGF-II)/man 6-P receptor but does not bind IGF-II.
Endocrinology
128:
1177-1189,
1991[Abstract].
Am J Physiol Gastroint Liver Physiol 277(3):G506-G514
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society