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Vol. 14, Issue 7, 2861-2875, July 2003
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INSERM U-559, Unité de Recherche de Physiopathologie des Cellules Epithéliales and Équipe d'Accueil 3289, Université de la Méditerranée, Faculté de Médecine, Marseilles, France
Submitted August 29, 2002; Revised March 13, 2003; Monitoring Editor: Keith Mostov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The
secretion of this digestive enzyme requires the participation of the Grp94
molecular chaperone (Bruneau and Lombardo,
1995; Bruneau et al.,
1998). Once in the lumen of the duodenum, BSDL, in concert with
other pancreatic lipolytic enzymes and preduodenal lipase, acts to complete
the digestion of dietary lipids. Indeed, upon activation by primary bile
salts, BSDL catalyzes the duodenal hydrolysis of cholesterol esters into free
cholesterol and fatty acids before their absorption by intestinal cells
(Howles et al., 1996;
Lombardo et al.,
1980; Lombardo and Guy,
1980; Weng et al.,
1999). Although controversial
(Howles et al.,
1996), BSDL could also participate in the intestinal free
cholesterol absorption (Lopez-Candales
et al., 1993), acting as a cholesterol transfer protein
(Myers-Payne et al.,
1995), or as a cholesterol reesterifying enzyme after cholesterol
absorption by intestinal cells (Bhat and
Brockman, 1982).
A fraction of BSDL, possibly associated with Grp94, reaches the vicinity of
microvilli and upon binding to the surface of intestinal cells is internalized
and transported up to the blood compartment (Bruneau et al.,
1998,
2000a,
2000b,
2003). Consequently, BSDL has
been reported in the plasma of normolipidemic patients
(Lombardo et al.,
1993) where it associates with apolipoprotein B-containing
lipoproteins such as low-density lipoprotein (LDL), chylomicron and
very-low-density lipoprotein (VLDL;
Bruneau et al.,
2003).
The pathway of BSDL throughout intestinal cells has been recently
delineated using Int407 cells (Bruneau
et al., 2001), which do not express BSDL. When added to
the apical reservoir of Transwell-grown Int407 cells, BSDL was shown to first
interact with the apical membrane. Further, BSDL forms clusters that are
internalized via clathrin-coated pits. After endocytosis, BSDL is directed to
a multivesicular compartment, and then the protein transited through the Golgi
apparatus where it colocalized with the KDEL retrieval-receptor. Finally,
enzymatically active intact BSDL that had experienced this transcytotic motion
was released at the basolateral membrane level. The transit of BSDL throughout
the Golgi compartment is compatible with the involvement of the enzyme in the
assembly and secretion of chylomicrons
(Kirby et al., 2002)
and the association of BSDL with these lipoproteins after intestinal
transcytosis (Bruneau et al.,
2003).
Although a receptor for BSDL on intestinal cell membranes has not been
identified, the initial interaction of the protein with CaCo-2 human
intestinal cells is mediated by its binding to putative low-affinity and
high-capacity binding sites (Huang and
Hui, 1990). Structural elements of BSDL potentially implicated in
its interaction with a receptor present at the apical membrane of enterocytes
are multiple. These are as follows: 1) the heparin-binding site present on
BSDL, which could recognize heparin-like molecules lining microvilli membranes
(Bosner et al.,
1988); 2) N-linked glycan structures that can bind to
mannose/fucose lectin receptors (Sallusto
et al., 1995); and 3) finally, O-linked
mucin-type structures of the C-terminal domain of BSDL
(Wang et al., 1995)
susceptible to bind lectin-like receptor or ligands such as selectins. It is
therefore conceivable that receptor(s) present at the apical surface of
intestinal cells could be involved in BSDL uptake by enterocytes (Bruneau
et al., 2001,
2003). The goal of this study
was to characterize (a) receptor(s) involved in the transcytosis mechanism of
BSDL using the human intestinal Int407 cells as model. Data support the view
that the lectin-like oxidized-LDL receptor (LOX-1) is, at least partly,
implicated in the transcytosis of BSDL throughout intestinal cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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7500 Da), dithiobissuccinimidyl
propionate, lectins and ligands used in this study were obtained from Sigma
(St. Louis, MO). Heparin sodium salt from bovine intestine (3000 Da) was
from Fluka (L'Isle-D'Abeau-Chesnes, France). [35S]methionine
(trans-35S label; >1000 Ci/mmol) was from ICN
Biochemicals (Costa Mesa, CA). Affinity-purified rabbit polyclonal antibodies
against human pancreatic BSDL (pAbL64) were homemade
(Abouakil et al.,
1988). An mAb (mAbJ28) to the fucosylated J28 epitope of BSDL
(Mas et al., 1997)
was a gift of Dr. M-J. Escribano (INSERM, Marseilles, France). Mouse
monoclonal antibodies to transferrin receptor (CD71, clone H68.4) were from
Zymed (San Francisco, CA). Rabbit polyclonal antibodies to LDL receptor and to
Grp94 were from Progen (Heidelberg, Germany) and StressGen (Victoria, Canada),
respectively. Goat polyclonal antibodies to the N-terminal (N-14) and to the
C-terminal (E-19) domain of the lectin-like oxidized-LDL receptor (LOX-1) were
from Santa Cruz Biotechnology (Santa Cruz, CA). A functional blocking antibody
JTX92 was provided by Dr. T. Sawamura (Osaka, Japan). Human native LDLs were
isolated from pooled fresh sera by sequential ultracentrifugation, dialyzed,
sterilized by filtration, and stored under nitrogen
(Augé et al.,
1999). LDLs were acetylated (Ac-LDL) according to standard
procedures (Goldstein et al.,
1980). Mildly oxidized (Ox)-LDLs were prepared by UV-C irradiation
(Augé et al.,
1999). Human BSDL was isolated from human pancreatic juice
(Lombardo et al.,
1978). Porcine pancreatic Grp94 was a gift from Dr. C. Nicchitta
(Durham, NC).
Human BSDL Immobilization
Human BSDL was immobilized on CNBr-activated Sepharose (1 mg of protein/0.5
g of wet-gel) as described (Bruneau and
Lombardo, 1995). Before use, the gel was alternatively washed with
basic (0.1 M sodium phosphate, pH 8.0) and acidic (0.1 M Mes, pH 5.5)
buffers.
Cell Culture and Metabolic Labeling
Human intestinal Int407 cells (ECACC Nb:85051004) were cultured in Eagle's
minimum essential medium (EMEM, Invitrogen, Cergy Pontoise, France),
supplemented with 2 mM glutamine, 10% (vol/vol) fetal calf serum (FCS), 1%
(vol/vol) essential amino acids. For each experiment, cells were plated at a
density of 2 x 105 cells per well on 12-mm polycarbonate
permeable supports (Transwell filter inserts, 0.4-µm pore sizes, Corning
Costar, Issy-les-Moulineaux, France) for 5 d, to obtain a tight monolayer
(Bruneau et al.,
2001). Metabolic labeling of proteins was performed on cell grown
in methionine-free DMEM for 45 min. Once starved, cells were pulse-labeled
with [35S]methionine (20 µCi/ml) for 6 h and then washed with
complete PBS.
Chemical Cross-linking
Int407 cells grown on Transwell inserts were washed with the FCS-free EMEM
and incubated with BSDL (100 nM) for 15 min at 4°C. Cells were then washed
with complete PBS and subjected to chemical cross-linking (30 min, 4°C) by
the addition of 1 mM dithiobissuccinimidyl propionate (DSP) in PBS. The
cross-linking reaction was terminated by addition of 10 mM glycine (10 min,
4°C), and cells were washed twice with cold PBS. Cells were then prepared
either for immunofluorescence microscopy or for Western blotting. For
immunofluorescence microscopy, after washing cells were fixed and incubated
with PBS, bovine serum albumin 1% (PBS-BSA) for 30 min and further incubated
for 120 min at room temperature with pAbL64 in PBS-BSA. Cells were washed and
incubated with FITC-anti-IgG antibodies (diluted 1/100 in PBS-BSA). Finally,
cells were washed with PBS, and filters were mounted on glass slides in the
Dabco 10%, glycerol 50%, in PBS. Cells were photographed using a fluorescence
microscope (BHR2-RFCA Olympus, Hamburg, Germany). For Western blotting, cells
were harvested and lysed in lysis buffer (10 mM HEPES, pH 7.4, buffer, 200 mM
NaCl, 2 mM EDTA, and 1.5% Triton X-100, 10 µg/ml leupeptin, 2 mM
benzamidine, PMSF, Soya-bean trypsin inhibitor and 
-phenyl propionate [2
mM each]). Cell lysate was cleared by centrifugation (20 min, 5000 x
g, 4°C). The lysate was electrophoresed, and BSDL complexes were
detected by Western blotting using pAbL64 antibodies.
Biotinylation of Apical Cell Surface Proteins
Monolayers of Int407 cells cultured on Transwell inserts, were washed with
PBS and incubated for 1 h at 4°C with 1 mg/ml
N-hydroxysuccinimide-long chain-biotin (NHS-LC-biotin, Pierce,
Rockford, IL) in sodium bicarbonate, pH 8.0 buffer added in the apical
reservoir. At the end of the incubation, the biotinylation agent was removed
and the reaction stopped with 0.1 M glycine (4°C, 15 min), cells were
washed with PBS and lysed in the Sepharose-immobilized-BSDL column loading
buffer (5 mM Mes pH6.0 buffer, 0.5% Nonidet P-40, 0.1 M NaCl and protease
inhibitors) by sonication (15 s, 4 W). The lysate was centrifuged (20 min,
5000 x g, 4°C) and chromatographed on the
Sepharose-immobilized BSDL column.
Effects of Various Ligands on the Transepithelial Movement of
BSD
Int407 cells were cultured on Transwell inserts, washed twice with FCS-free
EMEM, and preincubated with ligands, lectins, or antibodies in FCS-free medium
for 1 h in an apical reservoir. Cells were incubated for another 3 h with
ligands, lectins, or antibodies in the presence of pancreatic BSDL (100 nM) in
the apical reservoir, whereas fresh FCS-free EMEM medium was added in the
basolateral reservoir. Alternatively, a preincubation of BSDL (100 nM) with
ligands, lectins, or antibodies, followed by the incubation of the putative
complexes thus formed, with cells in the apical reservoir was performed. At
the end of the incubation apical and basolateral media were collected and used
for determination of BSDL (activity and Western blotting).
PAGE and Western Blotting
Gel electrophoreses (SDS-PAGE) were performed according to Laemmli
(1970). After SDS-PAGE,
proteins were electrophoretically transferred onto a nitrocellulose membrane
(Burnette, 1981). Western
blottings were performed using pAbL64 antibodies to BSDL. The antigen-antibody
complexes were detected using chemiluminescence (Roche, Mannheim, Germany).
Quantitations of protein were performed by scanning fluororadiogram using NIH
Image program.
Enzyme Assays
The BSDL activity was determined on 4-nitrophenyl hexanoate
(Gjellesvik et al.,
1992).
One-cycle Immunoprecipitation and Recapture of LOX-1 by BSDL
Radiolabeled 35S-labeled proteins eluted from the
Sepharose-immobilized BSDL column were used for immunoprecipitation. For
one-cycle immunoprecipitation the eluted material was incubated overnight
(4°C) in presence 20 µg of antibodies to LOX-1 receptor (antibodies
N-14 and E-19, 1/1 by vol mix). Prewashed protein A- and protein G-Sepharose
(5 mg each) were added to antibody-antigen complexes and incubated under
agitation (4 h, 4°C). The antigen-antibody protein-A/G complexes were then
recovered by centrifugation (10,000 x g, 20 min). The pellet
was washed twice successively with the washing buffer (10 mM Tris/HCl buffer,
pH 7.4, 25 mM EDTA, and 1% Triton X-100), with the washing buffer supplemented
with 1 M NaCl, with the last buffer supplemented with 0.1% SDS and with 10 mM
Tris/HCl buffer, pH 7.4, containing 5 mM EDTA. Recapture of immunoprecipitated
material was performed using E-19 and N-14 antibodies to LOX-1 receptor
coupled with the gel of the Seize primary immunoprecipitation kit (Pierce,
Brebières, France). For this purpose radiolabeled cell proteins were
subjected to immunoprecipitation with E-19–or N-14–coupled gel.
After elution the immunoprecipitated material was recaptured by incubation
with BSDL (100 nM) or without BSDL (control) for 4 h, and additional pAbL64
antibodies to BSDL were added and incubated overnight (4°C). Protein
A-Sepharose (10 mg) prewashed with the binding buffer was incubated with
receptor-ligand antibody complexes for another 4 h. At the end of incubation,
these complexes were recovered by centrifugation (10,000 x g,
15 min, 4°C). The pellet was then washed twice with the washing buffer (10
mM Tris/HCl, pH 7.4, buffer, 25 mM EDTA, and 1% Triton X-100), twice with the
washing buffer supplemented with 1 M NaCl, and twice again with 10 mM
Tris/HCl, pH 7.4, 5 mM EDTA.
Pellets obtained in both experiments were transferred into the Laemmli's
sample buffer (Laemmli, 1970),
boiled for 2 min, centrifuged, and electrophoresed on SDS-PAGE. Gels were
stained with Coomassie blue R250, destained in ethanol/acetic acid water
(2/3/35 by volume), and autoradiographed using BioMax MS FILM (Eastman Kodak,
Rochester, NY).
Reverse Transcription and PCR
Total mRNA was isolated with Trizol (Invitrogen) from Int-407 cells. RNA (1
µg) was reverse-transcribed (RT) with oligo-dT primers using the M-MLV
reverse transcriptase (Promega, Madison, WI), following manufacturer
instructions. RT material (5 µl) was amplified by PCR with Taq DNA
polymerase (Ozyme, St. Quentin/Yvelines, France) using forward primer,
(5'-TCTATCATTCTTAgCTTgAATTTggAAATg-3') and reverse primer,
(5'-TCACTgTgCTCTTAggTTTgCCTT-3') specific to LOX-1 (Genset, Evry,
France). For PCR, 30 cycles were used at 94°C for 40 s, 55°C for 1
min, and 72°C for 1 min. The RT-PCR fragment (0.9 kb) was then cloned
in the pCR-II TOPO plasmid (Invitrogen) and sequenced. The sequence of this
transcript matched totally that of the cDNA encoding human LOX-1 receptor
(Adachi et al., 1997).
This cDNA was subcloned into pBK vector (Stratagene, Amsterdam, The
Netherlands) and named pBK-LOX-1 and alternatively into pEGFP-N1 (Clontech,
Saint Quentin Yvelines, France), without fusion of LOX-1 with the EGFP, to
give pLOX-1.
Expression of LOX-1 Receptor
Stable transfection of Int407 cells was performed using a
lipopolyamine-mediated transfection procedure according to the manufacturer
(Lipofectamine, Invitrogen). The selection of stable clones was performed for
3 weeks in selection EMEM-medium with G418 (500 µg/ml medium). The
surviving cells were trypsinized and cloned by end-limiting dilution method.
Stably transfected clones were maintained under the same selection conditions.
Two positive clones, one referred to as Int-407 clone 14 (cells transfected
with pLOX-1) and another referred to as Int-407 clone 2 (cells transfected
with pBK-LOX-1) were selected for further experiments.
Flow Cytometry
Detection of LOX-1 at the surface of Int407 cells was carried out by
indirect fluorescence. Cells were released from culture plates by treatment
with nonenzymatic cell dissociation solution (Sigma) for 15 min at 37°C.
All subsequent steps were carried out at 4°C. The cells were washed three
times in PBS, fixed with 2% paraformaldehyde in PBS for 10 min, and washed
extensively with PBS-BSA. Int407 cells were incubated with E-19 antibodies to
the extracellular domain of the LOX-1 receptor for 1 h, washed three times
with PBS, and finally incubated for 30 min with FITC-labeled secondary
antibodies. The cells were then washed, resuspended in Isoflow buffer, and
analyzed on an EPICS Profile II flow cytometer (Coulter, Hialeah, FL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The intestinal human cell line Int407 constitute
a potent epithelial intestinal cell model to characterize a putative receptor
involved in the transcytosis of BSDL.
We have first attempted to demonstrate the association of BSDL with
proteins present at the apical membrane of Int407 cells. For this purpose,
cell monolayers were grown on a Transwell insert to obtain a tight epithelium
and incubated with human BSDL for 15 min at 4°C. At the end of the
incubation period, cells were exhaustively washed with complete PBS and
subjected to chemical cross-linking with DSP. In control experiments, DSP was
omitted. Once the cross-linking reaction was terminated, cells were washed
with complete PBS and incubated with pAbL64 antibodies, followed by
FITC-labeled anti-IgG and examined for immunofluorescence. As shown in
Figure 1A, no fluorescence can
be detected on control cell membranes, whereas a strong reactivity was
obtained with cells that have experienced the chemical cross-linking. Thus
BSDL can be coupled to proteins present at the membrane of Int407 cells. These
cells were then harvested and lysed, and proteins were resolved by SDS-PAGE.
After transfer onto nitrocellulose membranes, blots were probed with pAbL64.
As shown in Figure 1B, BSDL can
be detected in cell lysates independently of the presence of the cross-linker.
However, when DSP was present in the incubation of cells with BSDL, two extra
bands with lower migration than BSDL were detected. These bands correspond to
proteins of 200 and 150 kDa. Although the upper band could correspond to
cross-linked BSDL dimers, that migrating at 150 kDa might be formed of human
BSDL monomer (100 kDa) coupled to a protein of 50 kDa.
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To confirm these results and to further attempt to locate the 50-kDa
protein, Int407 cells were grown in the presence of [35S]methionine
and lysed. The cell lysate was clarified by centrifugation and chromatographed
on an affinity column made of BSDL-immobilized on Sepharose beads to isolate
proteins having an affinity for the enzyme. After elution of unbound material
and washing out of the column with the lysis buffer, bound proteins were
tentatively eluted, first with 0.1 M sodium acetate, pH 4.0, buffer (buffer A,
Figure 2A) and second with the
buffer A supplemented with Nonidet P-40 (1%; buffer B,
Figure 2A).
35S-methionine–labeled material, eluted with buffer A and B,
was pooled to give pool 1 and pool 2, respectively, then concentrated,
analyzed on SDS-PAGE, and autoradiographed. Alternatively, the buffer B
supplemented with 3 mg/ml heparin was used for elution and eluted fractions
were pooled to give pool 3. As shown on
Figure 2B, one band
representing 30% of the material and migrating at 50 kDa was detected in
fractions eluted with buffer B, along with two other bands around 30 kDa (pool
2, lane 2). However only the 50-kDa protein was eluted with buffer B
supplemented with heparin (pool 3, lane 3). No trace of this material was
detected in the material eluted with buffer A (pool 1, lane 1). Therefore,
BSDL seems to form a complex with a 50-kDa protein, and the complex, which is
insensitive to acidic pH, is dissociated in the presence of detergent or
heparin. To further determine whether this 50-kDa protein locates at the
apical membrane of Int407 cells, a biotinylation of cell surface proteins was
performed on cells cultured in Transwell inserts to form a tight epithelium.
After biotinylation, cells were extensively washed and lysed, and the cleared
lysate was chromatographed on the affinity column. The bound material was
eluted with buffer B and analyzed by Western blotting using
peroxidase-conjugated antibodies to biotin. Although many cell surface
proteins were biotinylated (unpublished data), only three proteins interacted
with immobilized BSDL (Figure
2B, lane 4). The two proteins with the lower migration (90
and 150 kDa) could be aggregates of the third one, the migration of which
coincides with the 50-kDa protein. Because only apical proteins can be
biotinylated under conditions used, one has to conclude that the 50-kDa
protein that binds BSDL is a protein located at the apical membrane of Int407
cells. This protein could represent a specific receptor involved in the uptake
of BSDL by intestinal cells (Huang and
Hui, 1990; Bruneau et
al., 2001).
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BSDL Transcytosis and Ligand Competition
We furthered this study by determining ligand specificity of the intestinal
receptor of BSDL. The enzyme has been shown to associate with the molecular
chaperone Grp94 (Bruneau et al.,
1998,
2000b) and with transferrin
(Erlanson-Albertsson et al.,
1985) in pancreatic and duodenal fluids. Therefore, BSDL may be
taken up by receptors that internalize Grp94 or transferrin. To determine
whether these companion proteins are involved in the BSDL uptake by and
transcytosis throughout Int407 cells, we incubated the human enzyme with
either Grp94 (10 µg/ml) or transferrin (10 µg/ml) in FCS-free medium for
30 min at 37°C to restore in vitro eventual complexes. Then the mixtures
were added in the apical reservoir and incubated for 3 h; thereafter the
amount of BSDL that had transcytosed up to the bottom reservoir was determined
by recording the enzyme activity. Any of the ligands used had an effect on the
BSDL activity. As illustrated in Figure
3 and in the conditions used, neither Grp94 nor transferrin
affected the amount of transcytosed BSDL. Also, a 30-min preincubation of
Int407 cells with 10 µg/ml antibodies specific to Grp94 or to transferrin
receptor (which is expressed by Int407 cells; unpublished data) had no
significant effect on BSDL transcytosis. Only specific polyclonal pAbL64 and
monoclonal mAbJ28 antibodies to BSDL significantly decrease the transcytosis
of the enzyme throughout Int407 cells.
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These data indicated that under our experimental conditions, the
transcytosis of BSDL may neither implicate the Grp94 protein and the
Grp94-receptor (Wassenberg et
al., 1999) nor be mediated via the transferrin receptor
(Dautry-Varsat et al.,
1983; Marsh et al.,
1995). Therefore, only antibodies to BSDL impaired the
transcytosis of the protein possibly by binding to structural elements
involved in the interaction of the protein with its intestinal receptor.
These structural elements can be the N- and O-linked
glycans of BSDL, which bore epitopes recognized by pAbL64 and mAbJ28 and that
carry out oligomannosidic and fucosylated structures
(Sugo et al., 1993;
Mas et al., 1993;
Wang et al., 1995,
1997). The heparin-like binding site of the enzyme
(Wang and Hartsuck, 1993)
could also be implicated. We thus investigated whether these structural
elements are involved and further attempted to characterize ligand specificity
of the BSDL putative intestinal receptor. For this purpose the effects of
various ligands, which have been shown to hinder glycoprotein interaction with
specific receptors or to inhibit oxidized-LDL binding to class A and B
scavenger receptors and to the lectin-like Ox-LDL receptor (LOX-1;
Moriwaki et al.,
1998; Shirai et al.,
1999), were examined. First of all, to rule out the possibility
that BSDL interacted with the cell via the mannose/fucose lectin receptor
(Avraméas et al.,
1996), we performed competition experiments using mannan as a
competitor (Arnold-Schild et al.,
1999). No inhibitory effect was observed using this ligand at a
concentration of 1 mg/ml (Figure
4A), whereas 0.3 mg/ml mannan was shown to inhibit completely the
uptake of FITC-dextran by the mannose/fucose lectin receptor of dendritic
cells (Sallusto et al.,
1995). Heparin (0.1 and 1 mg/ml) partially decreased the amount of
BSDL present in the bottom reservoir, whereas neither heparan sulfate (1
mg/ml) nor chondroitin sulfate (200 µg/ml) had important effect on BSDL
transcytosis (Figure 4A). Other
carbohydrate compounds used at lower concentration (100 µg/ml) such as
kappa-carrageenan (a polysaccharide extracted from the lichen Eucheuma
cottonii and composed predominantly of sulfated galactose residues) quite
totally annihilates the transcytosis of BSDL. Fucoidan (a polysaccharide
extracted from the brown algae Fucus vesiculosus and composed
predominantly of sulfated fucose residues) at comparable concentration is a
little less efficient than carrageenan in inhibiting the enzyme transcytosis.
Polyanionic nucleic acid such as polycytidylic acid had also no effect, albeit
the four-stranded polyinosinic acid had an inhibitory effect at either 100 or
200 µg/ml. This study was completed by determining the effects of various
lectins on BSDL transcytosis. For this purpose monolayers of Int407 cells were
preincubated with lectin (20 µg/ml in the culture medium) applied for 1 h
in the apical reservoir. The culture medium was then removed, and BSDL (100
nM) was added with lectin in the apical reservoir. After 3-h incubation the
amount of transcytosed BSDL was determined in the basolateral reservoir. As
shown on Figure 4B, only
concanavalin A (ConA), which primarily recognizes oligomannosidic structures,
decreased by some 70% the transcytosis of BSDL, whereas peanut agglutinin
(PNA) and Ulex europeaus I lectin (UEA-I) had a poor effect. These two lectins
bound to O-linked glycans and to fucose residues 
(1–2) linked to
galactose, respectively.
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These data suggest that the uptake of BSDL that precedes its transcytosis
throughout Int407 cells did not involve the mannose/fucose lectin receptor,
which is implicated in the macropinocytosis of glycosylated macromolecules
(Arnold-Schild et al.,
1999). Nevertheless, it seems evident that carbohydrate structures
of BSDL are implicated in the interaction of the enzyme with its receptor.
Furthermore, the inhibition of BSDL transcytosis promoted by reagents used
above also suggest that class A or B scavenger receptors or LOX-1 could be, at
least partly, implicated in the BSDL uptake by Int407 cells. Many facts
indicate that these receptors could be actually involved in BSDL interaction
with intestinal cell membranes. First, scavenger receptors, in general, have
been shown to bind polysaccharides
(Hampton et al.,
1991). Second, class B scavenger receptors were located in
intestine (Cai et al.,
2001) where they catalyze cholesterol uptake
(Hauser et al.,
1998). Third, BSDL, which is involved in cholesterol absorption
(Lombardo, 2001), is also
susceptible to associate with lipoprotein structures such as LDL
(Caillol et al.,
1997), receptors of which are also expressed by intestinal cells
(Fong et al.,
1995).
All these results suggest that the putative intestinal receptor of BSDL is
a 50-kDa protein that recognizes glycan structures. This receptor could be a
lectin-like receptor. The 50-kDa receptor LOX-1 has been characterized as such
lectin-like receptor (Metha and Li,
2002); therefore we focused on this receptor. First, we determined
whether the intestinal cell line Int407 synthesized LOX-1 and expressed the
receptor at their cell surface. The cell surface expression of this receptor
was analyzed by flow cytometry using E-19 antibodies to the extracellular
domain of LOX-1 receptor. As shown in
Figure 5A, compared with
control in which these primary antibodies were omitted, a net increase in
fluorescence was observed when Int407 cells were treated with E-19 antibodies.
This result strongly suggests that LOX-1 receptor is expressed at the apical
surface of Int407 grown to form a tight epithelium. Second, the radioactive
material eluted with the buffer B in Figure
2 has been further immunoprecipitated with a vol/vol mixture of
E-19 and N-14 antibodies to LOX-1. The material thus immunoprecipitated was
resolved on SDS-PAGE and autoradiographed. As shown in
Figure 6A, a main band
representing a protein of 50 kDa can be detected, suggesting that this
protein interacting with the Sepharose-immobilized BSDL is actually LOX-1.
Finally, recapture experiment was performed
(Figure 6B). When radiolabeled
cell proteins were immunoprecipitated with antibodies to LOX-1 (E-19 and N-14
coupled to Seize gel), eluted, and analyzed on SDS-PAGE, three main bands were
detected (Figure 6B, lane 1).
The material eluted, using pH 2.8 glycine buffer and neutralized with Tris/HCl
buffer (pH 9.5) to avoid denaturation as specified in the protocol of the
immunoprecipitation kit, was further recaptured by incubation with BSDL and
material susceptible to bind BSDL was then immunoprecipitated with pAbL64.
Results of this recapture cycle showed that only one protein of 50 kDa
can be specifically isolated (Figure
6B, lane 2, arrowhead). These data indicated that the 50-kDa
protein immunoprecipitated with antibodies to LOX-1 recognized BSDL. Control
omitting BSDL in recapture experiment did not allow isolating the 50-kDa
protein (Figure 6B, lane
3).
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To define the involvement of LOX-1 in the BSDL transcytosis, monolayers of Int407 cells were incubated for 1 h with polyclonal antibodies (N-14 or E-19) directed against LOX-1 or with mAb JTX92 (described as a blocking antibody for the LOX-1 receptor function) or with antibodies to the LDL-receptor applied in the apical reservoir. Cells were then washed and BSDL added in fresh medium containing each antibodies. After 3-h incubation under standard conditions, the amount of transcytosed BSDL was determined by recording the enzyme activity in the basolateral reservoir. As shown on Figure 3, although antibodies to the LDL receptor or to LOX-1 (N-14 or E-19) were ineffective in inhibiting the transcytosis of the enzyme, mAb JTX92 decreased the transport of BSDL throughout Int407 cells.
These data demonstrated that the LOX-1 receptor is actually expressed at the apical surface of Int407 cells and that LOX-1 may interact with BSDL and could be involved in the transcytosis of BSDL.
LOX-1 Is Implicated in BSDL Transcytosis throughout Int407 Cells
Because the expression of LOX-1 may be upregulated by Ox-LDL and by
atherogenic components of oxidized lipoproteins (Aoyama et al.,
1999,
2000), we examined the effects
of incubation of Int407 cells with native and modified LDL (40 µg/ml), on
the amount of transcytosed BSDL. For this purpose, Int407 cells were incubated
for 24 h with LDL (native or modified), and then the apical reservoir medium
containing LDL was withdrawn, and fresh medium containing BSDL (100 nM) was
added and incubated for another 3 h before recording BSDL in the bottom
reservoir. As illustrated in Figure
7, Ox-LDL does increase the amount of BSDL detected in the bottom
reservoir, whereas neither native LDL nor Ac-LDL promote such effect. This
result suggested that Ox-LDL increases the rate of the BSDL transcytosis
possibly by an enhancement of the expression of the LOX-1 receptor.
|
We next examined the effect of Ox-LDL on the expression of lox-1
RNA transcripts in Int-407 cells. For this goal, intestinal cells were grown
for 24 h in the absence or in the presence of Ox-LDL (40 µg/ml); at the end
of the incubation period, cells were washed and harvested, and RNA was
extracted. RNA was then used as matrix for RT-PCR using a pair of
oligonucleotide primers specific for human LOX-1. Products were further
analyzed on agarose gel (Figure
8). Under conditions used and even though at least three attempts
were done, it was quite difficult to detect RT-PCR product from RNA of Int407
cells grown in absence of Ox-LDL, whereas a 0.9-kb transcript was clearly
obtained after the RT-PCR using RNA extracted from cells incubated with
Ox-LDL. This transcript shown an open reading frame of 933 base pairs, the
sequence of which (unpublished data) matched totally that of human LOX-1
(Adachi et al., 1997;
Sawamura et al.,
1997). This cDNA was then subcloned into two eukaryotic expression
vectors, pEGFP-N1 and pBK-CMV under the control of SV40 and CMV promoter to
give pLOX-1 and pBK-LOX-1 vectors, respectively. Int407 cells were transfected
with these expression vectors and then selected by end-dilution, and two cell
clones, one referred to as Int-407 clone 14 (cells transfected with pLOX-1)
and another referred to as Int-407 clone 2 (cells transfected with pBK-LOX-1),
were also used. These two clones expressed different amount of LOX-1
transcripts as assessed by RT-PCR (Figure
9A). Flow cytometry experiments demonstrated (see
Figure 5B) that the cell
surface expression of LOX-1 is higher in transfected Int407 cells than in the
wild-type cell. Furthermore, clone 2 fluorescence is a little higher than that
of clone 14, suggesting that the amount of LOX-1 receptor expressed at the
surface is higher in clone 2 than in clone 14. Therefore, these two selected
clones were grown on Transwell inserts until a tight epithelium was reached
and assayed for BSDL transcytosis under standard conditions. As shown in
Figure 9B, compared with wild
Int407 control cells, transfected cells allow a significant increase in BSDL
transcytosis, by 300 and 500% by Int407 clone 14 and clone 2, respectively.
Therefore, amount of BSDL that had transcytosed is directly proportional to
the amount of RT-PCR transcripts (compare
Figure 9, A and B) and to the
amount of LOX-1 receptors expressed at the cell surface (see
Figure 5B). These data
confirmed that LOX-1 receptor participates in the BSDL transcytosis. We
further examined the BSDL transcytosis under conditions in which the apical
reservoir was buffered between pH 4 and pH 8. No modification in BSDL
transcytosis occurs under these conditions (our unpublished results).
|
|
Bidirectional Transport of BSDL
We next wondered whether the transport of BSDL may be bidirectional. For
this purpose BSDL (100 nM) was applied to apical or basolateral surface of
Int407 cells. As assessed by Western blotting and activity, after 3 h at
37°C intact BSDL applied to apical or basolateral cell surfaces was
transported in both luminal and abluminal directions across Int407 cell
monolayers. However, when monolayers were incubated at 4°C, the transport
of BSDL was largely reduced (Figure
10). The same relative decrease in BSDL transcytosis rate at 4 vs.
37°C was also observed with LOX-1–transfected Int407 cells
(unpublished data). Thus, transepithelial flux of BSDL did not occur by
passive diffusion through intercellular tight junctions or monolayer leaks.
These data indicate that BSDL can enter both apically and basolaterally
directed transcytosis pathway in Int407cells. Measurements of band intensities
and of activities suggest that the apically directed transport pathway was as
efficient than the basolaterally directed transport. Finally, we sought to
determine whether the transport of BSDL from the basolateral to the apical
compartment also depended upon LOX-1. First, at 37°C this abluminal
transport was increased in the same proportion as the luminal transport in
Int407 cells overexpressing LOX-1 compared with wild-type cells (unpublished
data). This motion was also reduced when recorded at 4°C. Second, we
furthered this study using LOX-1 inhibitors as described in
Figure 4. Data indicated that,
as the apical-to-basolateral transport of BSDL, the basolateral-to-apical
transport of the enzyme is also inhibited by carrageenan (0.1 mg/ml, 95
± 1% inhibition), fucoidan (0.1 mg/ml, 82 ± 5% inhibition), and
Con A (20 µg/ml, 90.3 ± 15.5% inhibition), whereas heparin still
remained a poor inhibitor (1 mg/ml, 38.3 ± 15.6% inhibition). These
data strongly support that LOX-1 mediates BSDL transport in both
apical-to-basolateral and basolateral-to-apical directions.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), a result that was confirmed by us in human
(Lechêne de la Porte et
al., 1987) and rat
(Bruneau et al.,
1998). We have further shown that BSDL is present on microvilli,
in the endosomal compartment, and in interdigitations of the basolateral
membrane of enterocytes (Bruneau et
al., 1998). Recently, using Int407 human intestinal cells, we
have shown that pancreatic BSDL formed clusters, in association with
clathrin-coated pits at the apical surface, which were then internalized.
Subsequent to internalization, coated vesicle-containing BSDL fuse with a
nocodazole-sensitive multivesicular compartment. The protein then transited
through the Golgi apparatus and the intact enzymatically active protein is
further released at the basolateral level
(Bruneau et al.,
2001).
We have further demonstrated that the circulating BSDL originated from the
pancreatic secretion via the intestinal transcytosis
(Bruneau et al.,
2003). Very few macromolecules traverse the epithelial barrier
intact by passive diffusion (Harada et al.,
1994a,
1994b). However some
macromolecules are efficiently transported across epithelial cells. This
transcellular pathway of macromolecular transport termed transcytosis is most
often receptor-mediated, as exemplified by the mechanism for the mucosal
secretion of polymeric IgA by the polymeric immunoglobulin receptor (pIgA-R,
Mostov and Blobel, 1983) and
IgG by the MHC-class-I-related Fc receptor (FcRn; Dickinson et al.,
1999). Macromolecular transcellular transport can occur by fluid-phase or
non–receptor-mediated endocytosis, but this pathway is inefficient, and
most of endocytosed cargo is delivered to lysosomes for degradation
(Futter et al.,
1996). Therefore, we can suggest that BSDL interacted with an
apical receptor likely located in clathrin-coated pits, before its motion
through intestinal cells. Although a receptor for BSDL in intestinal cells has
not been identified, binding studies using a heterologous system (i.e., human
intestinal CaCo-2 cells and porcine BSDL) have shown that the initial
interaction of the enzyme with these cells is mediated by the binding of BSDL
to putative low-affinity and high-capacity binding sites at the cell surface
(Huang and Hui, 1990). In the
present study we have attempted to characterize this receptor.
It has been hypothesized that the heparin-binding site present on BSDL
(Fält et al.,
2001) could be involved in such binding
(Bosner et al.,
1988). As shown here, heparin at concentration up to 1 mg/ml only
partially impaired the uptake of BSDL by the cells (as assessed by the amount
of enzyme activity recorded in the basal reservoir of Transwell inserts). This
suggests that the enzyme could interact, albeit with a poor avidity, with
brush-border membrane-associated, heparin-like molecules before
internalization. These heparin-like molecules could be part of proteoglycans.
However, the lack of heparan sulfate effect indicated that these proteoglycans
are not related to the syndecan family, which is composed of transmembrane
heparan sulfate proteoglycans (Mali et
al., 1990) described as alternative receptors or coreceptors
for the uptake of lipoprotein lipase-bridged atherogenic lipoproteins
(Williams et al.,
1992; Fuki et al.,
1997). Other sulfated glycans such as chondroitin sulfate are also
poorly efficient in inhibiting the transcytosis of BSDL throughout Int407
cells. Also internalization of the enzyme via the transferrin receptor
(present at the cell surface of Int407 cells), which is clathrin dependent
(Hansen et al.,
1993), may not represent a way for BSDL to associate with
clathrin-coated pits of the apical membrane of enterocytes because neither
transferrin nor antibodies to transferrin-receptor modulate the BSDL motion
through intestinal cells.
Another possibility was the involvement of the N-linked glycan
structure of BSDL (Mas et al.,
1993). This structure, which is highly variable
(Sugo et al., 1993)
may help BSDL to interact with mannose/fucose lectin receptor
(Avraméas et al.,
1996). The molecular chaperone Grp94, once associated with BSDL
(Bruneau et al.,
1998) can also be internalized via this receptor and
macropinocytosis (Arnold-Schild et
al., 1999; Wassenberg
et al., 1999). Mannose/fucose lectin receptor seems to be
not involved in BSDL transcytosis because mannan, a specific competitor of
this receptor (Arnold-Schild et
al., 1999), does not impair BSDL transcytosis. However, the
competitive effect of Con A, a lectin that recognizes the oligomannosidic core
of N-linked glycans, suggests that the N-glycosylation of
BSDL could be involved.
Finally, the O-glycosylated C-terminal mucin-like tail of BSDL
(Wang and Hartsuck, 1993;
Wang et al., 1995;
Mas et al.,
1997), which differs between species both in the amount of
repeated identical sequences (Sbarra
et al., 1998) and in glycosylation
(Wang and Hartsuck, 1993),
could also be involved in the transcytosis of the enzyme. The use of
heterologous systems, e.g., binding of BSDL to human CaCo-2 cells
(Huang and Hui, 1990) and
transcytosis of rat BSDL through human Int407 cells
(Bruneau et al.,
2001) and the lack of effect of PNA allowed us to rule out in
first instance this assumption. Antibodies to BSDL (pAbL64 and mAbJ28)
impaired transcytosis throughout Int407 cells and steric hindrance may be
advocated to explain this effect.
Many receptors could be involved in BSDL uptake and transcytosis throughout
Int407 cells (Ramprasad et al.,
1995; Adachi et al.,
1997; Dhaliwal and
Steinbrecher, 1999; Shirai
et al., 1999; Cai
et al., 2001; Lobo
et al., 2001; Werder
et al., 2001;
Gillotte-Taylor et al.,
2001). Among them the lectin-like oxidized LDL receptor or LOX-1,
a 50-kDa protein detected in nearly all tissues examined
(Sawamura et al.,
1997) has retained our attention. Although carrageenan and
polyinosinic acid abolished Ox-LDL binding to LOX-1, fucoidan, chondroitin
sulfate and polycytidylic acid are less effective
(Moriwaki et al.,
1998). A unique feature of LOX-1 is the presence of a lectin-like
domain in a conserved C-terminal domain, which is essential for Ox-LDL binding
(Chen et al., 2001b).
As mentioned above, the fact that Con A, which is specific for the
N-linked glycans of BSDL, blocked the transcytosis of this latter
protein indicated that a lectin-type receptor could be involved in the
transcytotic mechanism of BSDL.
However, we cannot rule out the binding of Con A to LOX-1, which is also a
N-glycosylated protein. From current results it seems obvious that
the BSDL receptor of Int407 cells is partially blocked by mAb to LOX-1,
upregulated by Ox-LDL as is LOX-1 (Aoyama
et al., 1999; Metha
and Li, 2002), the mRNA of which can be easily detected upon
stimulation of Int407 cells by Ox-LDL. The implication of LOX-1 in BSDL
transcytosis is confirmed by the overexpression of LOX-1 in Int407 cells
transfected with the cDNA encoding the human LOX-1. This overexpression leads
to a substantial increase in the amount of transcytosed enzyme. Taken as a
whole, data demonstrated that the transcytosis of BSDL throughout intestinal
cells implicates LOX-1. A striking result is the decrease in BSDL transcytosis
upon fucoidan treatment of Int407 cells, because fucoidan does not compete
with Ox-LDL for binding to LOX-1 (Moriwaki
et al., 1998). No clear explanation can be done to this
result; however, it is possible that the interaction of BSDL with LOX-1
involves fucose residues of BSDL, an interaction that should be impaired by
nonsulfated fucose residues of fucoidan, whereas the interaction between LOX-1
and the protein moiety of Ox-LDL (Moriwaki
et al. 1998) could involve ionic and hydrophilic
interactions and not actual LOX-1 lectin-binding properties (Chen et
al., 2001a,
2001b;
Shi et al., 2001).
Clearly, if fucose residues of BSDL are involved in the interaction of the
enzyme with the LOX-1 receptor, they should not be (1–2) linked
to galactose residues as UEA-1 lectin did not impair BSDL transcytosis. A way
to conciliate the binding of BSDL to heparin-like molecules lining the brush
border membrane (see Fält et
al., 2001; Bosner et
al., 1988) and the implication of LOX-1 would be that BSDL
first bind to heparin-like molecules lining intestinal cells and then are
transferred to LOX-1 with which the enzyme associates by mean of glycannic
determinants. Once bound to LOX-1, BSDL could form clusters that are taken up
via clathrin-coated pits of intestinal cells
(Bruneau et al.,
2001).
The mechanism of LOX-1–mediated BSDL transport in Int407 cells
possibly differs from that defined for the pIgA-R (Mostov et al.,
1980,
2000;
Mostov and Blobel, 1983;
Apocada et al., 1994) but instead resembles that of FcRn (Dickinson
et al., 1999). As observed for this latter receptor, the
bidirectional transcytosis of BSDL through Int407 cells suggests that LOX-1
may reenter the cell for return to the opposite membrane. Thus unlike pIgA-R,
which is committed to only one round of dimeric IgA transport, the same LOX-1
molecule could transport BSDL for multiple rounds in both luminal and
abluminal direction. Nevertheless, contrary to FcRn, which first binds its
ligand within the acidic endosome after fluid phase uptake (Dickinson et
al., 1999), as suggested by cross-linking experiments, LOX-1 should
rather bind BSDL at the cell surface as pIgA-R does with IgA
(Mostov and Blobel, 1983;
Apocada et al., 1994). As a consequence, IgG transport across
epithelial cells by FcRn is dependent on the pH and sensitive to bafilomycin
A1, which collapses pH gradient in intracellular vesicles, whereas transport
of BSDL across Int407 cells did not depend upon pH (our unpublished results)
and is insensitive to bafilomycin A1
(Bruneau et al.,
2001). Of course the exact mechanism of BSDL transcytosis and the
pathway of LOX-1 receptor through Int407 cells need further studies. Another
question concerns the release of BSDL from LOX-1 once the complex reached the
basolateral domain of intestinal cell membrane. One possibility is that
intestinally neo-synthesized lipoprotein particles such as chylomicrons
(Hui and Howles, 2002;
Bruneau et al., 2003)
may help the enzyme to dissociate from LOX-1. However, the involvement of
LOX-1 in the BSDL transcytosis does not exclude that of other receptor(s).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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* Corresponding author. E-mail address: nadine.bruneau{at}medecine.univ-mrs.fr.
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