From the Division of Hematology and Oncology, Department of Medicine and Barnes-Jewish Hospital, Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Enteropeptidase is a heterodimeric type II
membrane protein of the brush border of duodenal enterocytes. In this
location, enteropeptidase cleaves and activates trypsinogen, thereby
initiating the activation of other intestinal digestive enzymes.
Recombinant bovine enteropeptidase was sorted directly to the apical
surface of polarized Madin-Darby canine kidney cells. Replacement of
the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal
anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in
secretion both apically and basolaterally. Further deletion of the
noncatalytic heavy chain (L-BEK) resulted in apical
secretion. Thus enteropeptidase appears to have at least three distinct
sorting signals as follows: the light chain (L-BEK) directs
apical sorting, addition of most of the heavy chain (HL-BEK) inhibits
apical sorting, and addition of the mucin-like domain (dSA-BEK)
restores apical sorting. Inhibition of N-linked
glycosylation with tunicamycin or disruption of microtubules with
colchicine caused L-BEK to be secreted equally into apical
and basolateral compartments, whereas brefeldin A caused basolateral
secretion of L-BEK. Full-length BEK was not found in
detergent-resistant raft domains of Madin-Darby canine kidney cells or
baby hamster kidney cells. These results suggest apical sorting of
enteropeptidase depends on N-linked glycosylation of
the serine protease domain and an amino-terminal segment that
includes an O-glycosylated mucin-like domain and three
potential N-glycosylation sites. In contrast to many
apically targeted proteins, enteropeptidase does not form
detergent-resistant associations with sphingolipid-cholesterol rafts.
Enteropeptidase (enterokinase) is a protease of the duodenal brush
border that cleaves and activates trypsinogen. The resultant trypsin
then activates other pancreatic digestive zymogens within the lumen of
the gut. Deficiency of enteropeptidase causes intestinal malabsorption
(1, 2), and intrusion of enteropeptidase into the pancreas may
contribute to hemorrhagic pancreatitis (3, 4). Therefore, the
localization of enteropeptidase is important to normal digestive physiology.
Enteropeptidase consists of a disulfide-linked heterodimer with a heavy
chain of 82-140 kDa and a light chain of 35-62 kDa. Both chains of
mammalian enteropeptidases contain 30-50% carbohydrate, and this
extensive glycosylation may contribute to the apparent variation in
polypeptide masses (reviewed in Ref. 5). Amino acid sequences deduced
by cDNA cloning of bovine (6, 7), porcine (8), human (9), mouse
(10), and rat enteropeptidase (11) indicate that active two-chain
enteropeptidase is derived from a single-chain precursor. The
amino-terminal heavy chain contains domains that are homologous to
sequences of O-glycosylated epithelial mucins, the low
density lipoprotein receptor, complement components C1r and C1s, the
macrophage scavenger receptor, and a recently described MAM motif. The
carboxyl-terminal light chain is homologous to the trypsin-like serine
proteases (reviewed in Ref. 5). Studies of recombinant bovine
enteropeptidase demonstrate that membrane association is mediated by a
signal anchor sequence near the amino terminus (12).
The structures on enteropeptidase that direct it to apical membranes
have not been characterized. Basolateral targeting generally is
mediated by discrete amino acid sequence motifs in the cytoplasmic domains of transmembrane proteins, whereas the nature of apical targeting signals remains controversial. Apical targeting appears to
depend on distributed features of protein ectodomains or transmembrane domains, and there may be several apical targeting mechanisms (13). For
some proteins, apical targeting requires N-linked oligosaccharides (14, 15), or juxtamembrane segments with clustered
O-linked oligosaccharides (16), or interactions of transmembrane domains (17) or glycosylphosphatidylinositol anchors (18,
19) with the lipid bilayer. Apical sorting determinants may function,
in part, by promoting association with sphingolipid-cholesterol rafts
that deliver proteins to the apical cell surface (20). However, some
apical proteins appear not to associate with rafts and lack any of the
currently recognized apical sorting signals (e.g. Ref. 21),
and proteins may be sorted differently in different cell types (13).
Therefore, the mechanism of apical protein targeting remains poorly understood.
We have employed Madin-Darby canine kidney
(MDCK)1 cells, a well
characterized system for the study of protein sorting (13, 22), to
investigate the targeting of enteropeptidase. The results indicate that
signals involved in apical delivery reside in the catalytic domain and
in an amino-terminal segment that includes the mucin-like domain.
Apical delivery of this type II transmembrane protein requires intact
post-Golgi transport vesicles and depends on N-linked
glycosylation. Unlike many other apically targeted proteins, delivery
of enteropeptidase appears not to involve detergent-resistant association with sphingolipid-cholesterol rafts.
Construction of Plasmids--
Plasmid pBEK, containing the
full-length cDNA sequence of bovine enteropeptidase, was assembled
in vector pBluescript II KS+ (Stratagene) from cDNA clones isolated
previously (7). The cDNA insert of pBEK was cloned into the
SmaI site of plasmid pNUT (23) to produce expression plasmid
pNUTBEK. Plasmids pNUTHL, pNUTL, pBlue-HL, and pBlue-L were described
previously (12). In pNUTHL and pBlue-HL, the sequence encoding amino
acids 1-197 was replaced by a signal peptide cassette consisting of
the cleavable signal peptide of prothrombin, a His6 tag,
thrombin cleavage site, and T7 epitope tag. In plasmid pNUTL and
pBlue-L, the same signal peptide cassette replaced the sequence
encoding amino acids 1-783. Additional plasmids derived from pBEK were
constructed using oligonucleotide site-directed mutagenesis as
described previously (12). In plasmid pdSA, the sequence encoding the
signal-anchor domain (residues 1-49) was replaced by the prothrombin
signal peptide cassette from plasmid pNUTHL.
To add the epitope tag DYKDDDDK to the carboxyl terminus of
full-length recombinant enteropeptidase, the DNA sequence 5'-gac tac
aag gac gac gat gac aag tag-3' was inserted before the stop codon of
plasmid pBEK, generating the plasmid pBEKflag. Plasmid pdSALflag was
derived from pdSA by inserting the same oligonucleotide sequence before
the codon for residue 787, thereby deleting the light chain and
appending the DYKDDDDK tag. The cDNA inserts of pBEK, pBEKflag,
pdSA, pdSALflag pBlue-HL, and pBlue-L were excised from the vectors
with NotI and ApaI or NotI and
HindIII and cloned into vectors pcDNA3, pcDNA3.1, or
pcDNAI (Invitrogen) to yield plasmids pcDNA3-BEK,
pcDNA3-BEKflag, pcDNA3.1-dSA, pcDNA3.1-dSAdLflag, pcDNAI-HL, and pcDNAI-L, respectively.
Transfections--
Baby hamster kidney (BHK) cells were grown in
six-well plates and transfected with 5 µg of plasmid pNUT-BEK,
pNUT-HL, and pNUT-L, and 30 µg of Lipofectin (Life Technologies,
Inc.) in serum-free Dulbecco's modified Eagle's medium (DMEM). After
5 h fetal bovine serum was added to 10%. After an additional
18 h, cultures were split 1:10 for selection in 0.5 mg/ml
methotrexate for 10 days. Madin-Darby canine kidney (MDCK-II) cells
(ATCC) were transfected with 5 µg of plasmid pcDNA3-BEK,
pcDNA3-BEKflag, pcDNA3.1-dSA, pcDNA3.1-dSAdLflag,
pcDNAI-HL, or pcDNAI-L and PerFect lipids (pfx-2) (Invitrogen)
according to the manufacturer's recommendations, and clones were
selected with 0.5 mg/ml geneticin (G418) (Life Technologies,
Inc.).
Polyclonal Antibodies--
The cDNA sequence encoding the
light chain of bovine enteropeptidase (amino acids 784-1035) was
cloned into the XhoI site of plasmid pET-28a(+) (Novagen) to
yield plasmid pET-L. Epicurean coli BL21 (DE3) (Stratagene, La Jolla,
CA) transformed with plasmid pET-L were grown in LB broth containing
ampicillin, and protein expression was induced with 1 mM
isopropyl-1-thio-
Antibodies against native enteropeptidase light chain were prepared
similarly. The cDNA insert encoding amino acids 785-1035 of
pBlue-L was cloned into baculovirus expression vector pVL1392 (PharMingen, San Diego, CA). Plasmid pVL1392-L was co-transfected into
Sf9 cells (Life Technologies, Inc.) with BaculoGold DNA
(PharMingen, San Diego, CA) to generate recombinant virus.
Enteropeptidase light chain was expressed by infection of High Five
cells (Life Technologies, Inc.) and purified by affinity chromatography
on soybean trypsin inhibitor-agarose
(Sigma).2 Polyclonal antibody
against the purified active L-BEK was raised in rabbits
(TANA Laboratories, Houston, TX), and the immune serum was designated
anti-Lv.
Preparation of Cell Lysate and Immunoblotting--
Stably
transfected BHK or MDCK II cell lines were maintained in DMEM
containing 10% fetal bovine serum. Cells were lysed directly in the
plate at room temperature with sodium phosphate, pH 7.4, 150 mM NaCl (PBS) containing 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 3 µM aprotinin (1 ml/107 cells). After
centrifugation at 15,000 rpm for 15 min in a microcentrifuge, the
supernatants were stored at Glycosidase Digestions--
To analyze the
N-glycosylation of proenteropeptidase, 10 µl of BHK cell
lysate or conditioned medium was incubated at 100 °C for 5 min in 30 µl total volume of 20 mM sodium phosphate, pH 7.5, 50 mM EDTA-Na, 0.4% SDS, 4%
For digestion with neuraminidase and O-glycanase, BHK cells
expressing BEK were pulse-labeled with 200 µCi/ml
Tran35S-label (>1000 Ci/mmol, containing 75%
[35S]methionine, 15% [35S]cysteine, ICN
Pharmaceuticals) at 37 °C for 30 min and chased for 60 min with
complete DMEM containing an excess of unlabeled methionine (15 µg/ml)
and cysteine (50 µg/ml). Cell lysates were prepared as described
above, pre-cleared for 60 min with protein A-Sepharose 4B (50 µl/ml
sample), and incubated with anti-Lv (5-10 µl/ml lysate) and 5 mg/ml
bovine serum albumin at 4 °C overnight. After addition of protein
A-Sepharose 4B (50 µl), samples were incubated for 60 min at room
temperature. The protein A-Sepharose beads were washed four times with
20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1%
Triton X-100, 0.1% SDS, and once with 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. Antigen-antibody complexes were eluted by boiling at 100 °C for 10 min with 100 µl of 20 mM
Tris-HCl, pH 7.4, 0.5% SDS. Samples of eluate (20 µl) or 20 ng of
purified HL-BEK (12) were digested without or with 0.5 units of PNGase F, 20 milliunits of neuraminidase (Boehringer Mannheim) or 20 milliunits of neuraminidase plus 2 milliunits of O-glycanase
(Genzyme Diagnostic, Cambridge, MA) in 20 mM sodium
cacodylate, pH 6.5, 10 mM calcium acetate, 0.2% SDS, and
2% Nonidet P-40 at 37 °C for 20 h. The products were separated
electrophoretically by 4-20% gradient SDS-PAGE; radiolabeled BEK and
unlabeled HL-BEK were detected by autoradiography and silver staining, respectively.
Cell-surface Biotinylation of BEK and HL-BEK Expressed in BHK
Cells--
BHK cell lines were washed three times with ice-cold PBS,
incubated with 1.5 mg/ml NHS-SS-biotin (Pierce) at 4 °C for 30 min, and reaction was stopped with 50 mM glycine in PBS. Cells
were lysed in 1 ml of PBS containing 1% Triton X-100, 0.5 mM PMSF, and 3 µM aprotinin. Surface
biotin-labeled proteins were precipitated with streptavidin-agarose at
room temperature for 2 h. After washing four times with PBS
containing 0.1% Triton X-100 and 0.1% SDS, the
biotin-streptavidin-agarose complexes were eluted from beads by heating
at 100 °C for 5 min with 30 µl of Laemmli sample buffer (Bio-Rad)
containing 1% Trypsin Accessibility of BEK and HL-BEK Expressed in BHK
Cells--
Transfected BHK cell lines in six-well plates were washed
with PBS and treated without (control) or with 5 µg/ml
trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone
(Worthington) in PBS at room temperature for 10 min. Residual trypsin
was inactivated by addition of a 2-fold excess of soybean trypsin
inhibitor. Cells were pelleted, washed with PBS, and lysed with PBS
containing 0.5% Triton X-100, 0.1% SDS, 100 µg/ml PMSF, 24 µg/ml
aprotinin, 1.5 µg/ml soybean trypsin inhibitor. Cell lysate was
centrifuged at 15,000 rpm in a microcentrifuge, and the supernatant was
collected for SDS-PAGE and Western blotting.
Immunofluorescence and Confocal Microscopy--
Stably
transfected MDCK II cells were grown on clear Costar Transwell filters
(pore size 0.4 µm) until a tight monolayer was formed as shown by the
transepithelial resistance. The filters were washed with PBS and fixed
at 4 °C for 15 min in PBS containing 1.5% paraformaldehyde (25) or
ethanol:acetic acid (9:1). Cells were washed sequentially with PBS
followed by PBS containing 0.5% Triton X-100, 1% BSA, and 1% goat
serum and then incubated in PBS containing 20 µg/ml rabbit anti-Lv
serum (1:100) or anti-caveolin IgG (1:500) (Transduction Laboratories,
Lexington, KY), 1% BSA, and 1% goat serum at 4 °C overnight. Cells
were washed three times with PBS containing 0.05% Tween 20 and once
with PBS and then incubated with Cy3-labeled goat anti-rabbit antibody
(15 µg/ml diluted with TBS containing 1.5% non-immune goat serum)
(Jackson ImmunoResearch, West Grove, PA) at room temperature for 60 min. After washing, the filters were cut out and mounted on slides with
glycerol mounting medium (Sigma) and examined with a confocal laser-scanning microscope (Zeiss Axioplan/Bio-Rad MRC1024).
Analysis of Enteropeptidase Polarity on MDCK Cells--
To
detect membrane-bound enteropeptidase, stably transfected MDCK cells
were grown on six-well Costar Transwell filters (pore size 0.4 µm)
until a tight monolayer was formed as shown by the transepithelial
resistance. Cells were pulse-labeled with 150-200 µCi/ml
Tran35S-label at 37 °C for 30 min and chased for 30 min
with complete DMEM containing 150 µg/ml unlabeled methionine. Either
the apical or basolateral cell surface was incubated with 1.5 mg/ml
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-biotin, Pierce) at 4 °C for 30 min, and the reaction was stopped with 50 mM glycine in PBS (10 ml per 100-mm dish),
and cell lysates were prepared as described above. Enteropeptidase was
immunoprecipitated with anti-Lv (10-30 µg/ml sample) and protein A-Sepharose 4B (50 µl). Antigen-antibody complexes were eluted by
boiling at 100 °C for 10 min with 100 µl of 20 mM
Tris-HCl, pH 7.4, containing 0.5% SDS. The eluate was diluted to 500 µl with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl,
and biotin-labeled proteins were precipitated with streptavidin-agarose
as described above. Proteins were eluted from streptavidin-agarose with
Laemmli sample buffer containing 1% Effects of Tunicamycin, Brefeldin A, and Colchicine on Targeting
of Enteropeptidase--
MDCK II cells transfected with pcDNAI-L
were grown on Costar Transwell filters until formation of a tight
monolayer as determined by transepithelial resistance measurements.
Cells were then treated without or with 2 µg/ml tunicamycin or 10 µg/ml brefeldin A in serum-free DMEM for 24 h. The cell
viability and integrity of the monolayer were confirmed after treatment
by measuring the transepithelial resistance. Conditioned media were
collected from either apical or basolateral surfaces and concentrated
10-fold by ultrafiltration (Centriprep-10). Samples (20 µl) of
concentrated media were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting as described above. The amount of
L-BEK secreted from either surface was quantitated with NIH
Image version 1.61 (26).
Triton X-100 Extraction--
Detergent extractability of
enteropeptidase was determined according to Brown and Rose (27).
Confluent cell monolayers in 10-cm dishes were labeled with 200 µCi/ml Tran35S-label in serum-free DMEM without
methionine for 30 min at 37 °C and chased at 37 or 20 °C with
DMEM, F-12 HEPES medium containing an excess of unlabeled methionine
(15 µg/ml) and cysteine (50 µg/ml). The cells were lysed with 1 ml
of extraction buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5 mM PMSF, and 3 µM aprotinin) for 10 min on ice. Lysates were centrifuged for 5 min in a microcentrifuge at 15,000 rpm at 4 °C. The
supernatant (Triton X-100 soluble fraction) was removed. The pellet
(Triton X-100 insoluble fraction) was dissolved in 100 µl of 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 1% SDS;
DNA was sheared by passage through a 22-gauge needle, and the solution
was diluted by addition of 900 µl of extraction buffer.
Enteropeptidase was immunoprecipitated from soluble and insoluble
fractions with anti-Lv (5 µl/ml) for analysis by SDS-PAGE and
autoradiography as described above.
Isolation of Low Density Membrane
Domains--
Detergent-insoluble glycosphingolipid-enriched raft
domains were prepared by a modification of the Triton X-100 procedure of Pike and Casey (28). Confluent cells in 100-mm dishes were rinsed
twice with ice-cold PBS and scraped into 1 ml of lysis buffer
containing 25 mM MES, pH 6.5, 150 mM NaCl, 1%
Triton X-100, 10 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, and 0.3 mM aprotinin.
Lysates were incubated for 20 min on ice with intermittent gentle
agitation and then mixed with an equal volume of 80% sucrose in MBS
(25 mM MES, pH 6.5, 150 mM NaCl). Six ml of
30% sucrose in MBS followed by 4 ml of 5% sucrose in MBS were layered
on top of each sample. The gradients were centrifuged at 4 °C for
23 h at 175,000 × g (39,000 rpm, Beckman SW40
rotor). Fractions of 1.2 ml were collected, and the small insoluble
pellet was resuspended in 600 µl of lysis buffer by homogenization
(25,000 rpm, 1 min) with a Brinkmann homogenizer (Kinematica AG,
Switzerland). Proteins in the fractions were precipitated with 10%
trichloroacetic acid on ice for 30 min, and pellets were resuspended in
100 µl of 0.2 N NaOH. Samples (20 µl) of
trichloroacetic acid-concentrated fractions were analyzed by SDS-PAGE
and immunoblotting with either anti-Lv (1:4,000) or anti-caveolin IgG
(1:10,000) (Transduction Laboratories, Lexington, KY).
Antibody-induced Patching--
BHK cells expressing BEK were
cultured in 150-mm dishes to 90% confluency in DMEM, 10% fetal bovine
serum and washed twice with 10 ml of ice-cold PBS. Cells were incubated
without (control) or with anti-Lv (preabsorbed with non-transfected BHK
cell lysate coupled on Affi-Gel 10) at a dilution of 1:1000 in
serum-free DMEM, 0.1% BSA at 12 °C for 60 min. After washing three
times with 10 ml of ice-cold PBS, cells were further incubated with goat anti-rabbit IgG (1:1000) at 12 °C for 60 min. Cells were scraped into 25 mM MES, pH 6.5, 150 mM NaCl,
1% Triton X-100, 10 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, and 0.3 mM aprotinin.
Sucrose gradient centrifugation and Western blotting were performed as
described above. For immunofluorescence, cells grown on slide chambers
were either fixed with acetic acid:ethanol (1:9) on ice for 10 min and
incubated with anti-Lv (1:1000) and Cy3-conjugated anti-rabbit IgG
(1:50), or first cross-linked with primary and secondary antibodies at
12 °C for 60 min before fixing with acetic acid ethanol (1:9) on ice
for 10 min. After washing with PBS, cells were mounted with 30%
glycerol in 20 mM Tris-HCl, pH 7.4, and photographed with a
Zeiss Axiophot microscope.
Enteropeptidase Is Expressed as a Single-chain Zymogen with
Extensive N-Linked and O-Linked Glycosylation--
BHK cells expressed
two major species of cell-associated full-length proenteropeptidase
(BEK) with molecular masses of 180 and 150 kDa (Fig.
1A, lane 1). These apparent
sizes are similar to those for secreted and intracellular forms,
respectively, of proenteropeptidase lacking the transmembrane and
mucin-like domains, HL-BEK (Fig. 1A, lanes 4 and
7), as reported previously (12). Digestion of BEK (Fig.
1A, lane 3) or HL-BEK (Fig. 1A, lanes 6 and
9) with protein N-glycosidase F (PNGase) reduced
the apparent mass of all species to
Digestion of proenteropeptidase (BEK) with neuraminidase and
O-glycanase also decreased its apparent mass, in this case
by
None of these variants of enteropeptidase had detectable enzymatic
activity prior to cleavage and activation by trypsin, as reported
previously (12), indicating that all are synthesized predominantly as
single chain zymogens.
Enteropeptidase Is Localized to the Apical Surface of Polarized
Cells--
Biotin surface labeling of transfected BHK cell lines
showed that virtually all of the 180-kDa form of BEK was exposed on the
plasma membrane, whereas the 150-kDa species of both BEK and HL-BEK
were mainly intracellular (Fig.
2A). Surface localization of
180-kDa BEK also was demonstrated by digestion with trypsin (Fig.
2B). The 180-kDa form of BEK was cleaved into catalytically active two-chain enteropeptidase, whereas the intracellular 150-kDa forms of BEK and HL-BEK were inaccessible to trypsin. When expressed in
polarized MDCK cells, BEK was localized to the apical surface as shown
by immunofluorescence staining and confocal laser-scanning microscopy
(Fig. 3), and by selective biotinylation
of apical and basolateral membrane proteins (Fig.
4). As observed for several other apical
membrane proteins in MDCK cells (22, 29), pulse labeling of BEK showed
that it was delivered directly to the apical surface within 30 min of
chase (data not shown).
Multiple Signals Contribute to Apical Sorting of
Enteropeptidase--
Targeting signals in enteropeptidase were
localized further by deletion mutagenesis (Fig. 4). Constructs were
prepared that lacked the transmembrane domain (dSA-BEK), the mucin-like
domain (HL-BEK), the entire heavy chain (L-BEK), or the
light chain (dSAL-BEK). Because the available antibodies were specific
for the enteropeptidase light chain, a short peptide epitope (DYKDDDDK)
was attached to the carboxyl terminus of construct dSAL-BEK to
facilitate immunoprecipitation and Western blotting. Adding this
epitope to full-length BEK in construct BEKflag did not impair
expression or apical targeting (Fig. 4), suggesting that the tag would
not perturb biosynthesis of other constructs. Cells were grown as tight
monolayers on porous filters in Transwell units. The targeting of
membrane-associated BEK and BEKflag was assessed by surface
biotinylation. The targeting of secreted proteins dSA-BEK, dSAL-BEK,
HL-BEK, and L-BEK was assessed by analysis of culture
medium exposed selectively to apical and basolateral surfaces.
Replacement of the signal anchor domain with a cleavable signal peptide
resulted in the secretion of construct dSA-BEK from the apical cell
surface, whereas amino-terminal deletion including the mucin-like
domain resulted in both apical and basolateral secretion of construct
HL-BEK (Fig. 4). Therefore, an apical targeting determinant appears to
reside near the amino terminus of enteropeptidase in a segment
(residues 48-197) that contains a mucin-like domain and is
O-glycosylated (Fig. 1B). Deletion of the entire
heavy chain again resulted in apical secretion of construct
L-BEK (Fig. 4), suggesting that the heavy chain can inhibit
the function of a second apical targeting determinant that resides in
the enteropeptidase light chain. This light chain sorting determinant
also is not required for correct targeting because deletion of the
light chain did not impair apical localization of construct dSAL-BEK
(Fig. 4). Thus, enteropeptidase appears to have at least three distinct targeting signals as follows: the enteropeptidase light chain (L-BEK) directs apical secretion, addition of the heavy
chain (HL-BEK) inhibits apical sorting and causes delivery to both
apical and basolateral surfaces, and addition of an amino-terminal
segment containing the mucin-like domain (dSA-BEK) restores apical sorting.
Effect of Tunicamycin, Colchicine, and Brefeldin A on L-BEK
Secretion in MDCK Cells--
Some secretory proteins (14) and
transmembrane proteins (15) appear to employ N-glycans as
apical sorting signals, and inhibition of N-glycosylation
often causes delivery to both apical and basolateral surfaces. The
serine protease domain of bovine enteropeptidase contains three
potential N-linked glycosylation sites (7), and to assess
the role of N-glycans in the sorting of L-BEK,
transfected MDCK cells were treated with tunicamycin (Fig.
5). Glycosylation was inhibited
effectively as shown by the
Apical targeting of some secreted and membrane proteins in MDCK cells
is selectively impaired by agents that depolymerize microtubules (30,
31) or by treatment with brefeldin A (32, 33). The behavior of
L-BEK is consistent with these findings. Treatment of MDCK
cells with colchicine resulted in the delivery of L-BEK
randomly to apical and basolateral surfaces (Fig. 5). Treatment with
brefeldin A reversed the polarity of L-BEK secretion so
that nearly 75% was recovered from the basolateral compartment (Fig.
5). Transmembrane conductivity measurements confirmed that neither
colchicine nor brefeldin A altered the integrity of the polarized cell
monolayer (Fig. 5C). Apical secretion of L-BEK therefore depends on an intact microtubular network and on brefeldin A-sensitive vesicular transport, probably at a site between the trans-Golgi network and the apical plasma membrane (33).
Enteropeptidase Is Not Associated with Detergent-resistant
Glycosphingolipid-Cholesterol Rafts--
Many apically directed
membrane proteins can be recovered in specialized membrane domains that
are rich in glycosphingolipids and cholesterol and that are relatively
insoluble in Triton X-100, termed "rafts" (20). However,
full-length enteropeptidase expressed in either BHK or MDCK cells was
completely solubilized by brief treatment of the cells with Triton
X-100 at 4 °C (Fig. 6A).
Also, when rafts were prepared by sucrose density gradient
centrifugation of Triton X-100 extracts, no enteropeptidase (BEK) was
recovered in the low density fractions 4 and 5 that contain caveolin, a marker for rafts; instead BEK was found in fractions that contain soluble proteins (Fig. 7).
Some apical transmembrane proteins (34) and
glycosylphosphatidylinositol-anchored proteins (27) that are
incorporated into rafts during biosynthesis appear to acquire
resistance to Triton X-100 extraction after departure from the
endoplasmic reticulum. Certain intestinal brush border enzymes begin to
associate with detergent-resistant rafts while still in their transient
high mannose-glycosylated forms (35). To evaluate the possibility that
enteropeptidase could associate with rafts transiently en route to the
cell surface, pulse-chase analyses were performed with BHK cells
expressing BEK. When cells labeled with radioactive methionine and
cysteine were chased at 37 °C (Fig. 6B), enteropeptidase with endo H-sensitive oligosaccharides was detected in the
detergent-soluble fractions from the earliest time point and decreased
during the following 2 h. Endo H-resistant enteropeptidase
appeared after 1 h of chase, consistent with oligosaccharide
processing in the Golgi. At no time of chase was enteropeptidase found
to be resistant to detergent extraction.
Growth of cells at 20 °C blocks the intracellular transport of
certain proteins, often resulting in their accumulation within the
trans-Golgi of MDCK cells (36) or BHK cells (37). To determine whether
this block would cause the intracellular accumulation of a transient
raft-associated enteropeptidase species, BHK cells expressing BEK were
pulse-labeled with radioactive methionine and cysteine and chased at
20 °C (Fig. 6, B and C). Under these conditions enteropeptidase contained endo H-sensitive oligosaccharides at all times of chase through 5 h, indicating that transport was blocked at or before the mid-Golgi, possibly in the endoplasmic reticulum. No detergent-insoluble enteropeptidase was detected. Pulse-labeled MDCK cells expressing BEK also did not contain
detergent-insoluble enteropeptidase when chased at 20 °C for 1-5 h
(data not shown). The results of these pulse-chase experiments suggest
that intracellular association of enteropeptidase with rafts either
does not occur or is extremely transient.
Association of certain membrane proteins with rafts may depend on
clustering, and patching with antibodies is reported to allow increased
recovery of placental alkaline phosphatase in detergent-resistant
membrane domains (38). To determine whether patching could force
enteropeptidase to associate with rafts, transfected BHK cells were
incubated with anti-Lv and anti-rabbit immunoglobulin either before or
after fixation for immunofluorescence microscopy (Fig.
8). When cells were fixed prior to the
addition of antibodies, enteropeptidase was diffusely distributed on
the cell surface with a fine punctate pattern (Fig. 8A).
Fixation with acetic acid:ethanol permeabilizes the cells, so that
intracellular enteropeptidase in the endoplasmic reticulum was also
seen. This method was selected because a weak immunofluorescence signal
was obtained with anti-Lv after fixation with 4% paraformaldehyde, which does not permeabilize cells. However, cell-surface staining is
clearly distinguished by focusing through the cell, and the typical
surface immunofluorescence pattern can be seen over the nucleus where
the endoplasmic reticulum does not interfere. A similar fine punctate
surface pattern was seen for caveolin (Fig. 8C).
Preincuation of cells with anti-caveolin and anti-rabbit immunoglobulin
gave no signal, confirming the integrity of the cells prior to
permeabilization and the low background immunofluorescence of the
method (Fig. 8D). Preincubation of cells with anti-Lv and anti-rabbit immunoglobulin resulted in redistribution of
enteropeptidase into large surface patches (Fig. 8B).
Despite patching with anti-Lv, enteropeptidase still was not recovered
in detergent-resistant low density membrane fractions after treatment
of cells with Triton X-100 at 4 °C (Fig.
9).
Enteropeptidase is a type II integral membrane protein that is
found in the apical microvilli of enterocytes in the duodenum and
proximal jejunum (5). As shown by immunofluorescence microscopy (Fig.
3) and by selective cell-surface biotinylation (Fig. 4), the signals
that specify its apical localization in enterocytes are functional in
MDCK cells, a well characterized model system for the study of protein
sorting in which most apical proteins are delivered directly to the
apical cell surface (22, 29). The correct localization of
enteropeptidase and other apical membrane proteins may be critical to
their biological function, but the mechanisms of apical protein
targeting are not well characterized. Whereas basolateral targeting
often depends on compact structural motifs exposed in the cytoplasmic
domains of transmembrane proteins, apical targeting instead appears to
depend on any of several distributed features of other domains. These
may include characteristics of the transmembrane domain (17), the
presence of a glycosylphosphatidylinositol membrane anchor (18, 19),
extracellular N-glycans (14, 15), or an
O-glycosylated juxtamembrane segment (16).
In enteropeptidase at least two such apical sorting determinants appear
to coexist, N-glycans and an O-glycosylated
segment near the amino-terminal transmembrane domain. Bovine
enteropeptidase has 19 potential N-linked glycosylation
sites, three of which are in the carboxyl-terminal light chain (7).
Many of these sites are utilized, as indicated by the large shift in
apparent mass upon digestion with PNGase (Fig. 1). Unfortunately,
inhibition of N-glycosylation with tunicamycin caused the
intracellular degradation of full-length enteropeptidase (BEK) so that
the role of N-glycans in targeting of the native protein
could not be evaluated. However, the enteropeptidase light chain
(L-BEK) was secreted apically and inhibition of
N-glycosylation randomized its sorting (Fig. 5). Thus,
N-glycans are required for apical sorting of the
enteropeptidase light chain, as observed for several other secreted and
membrane-bound glycoproteins (13).
A second sorting determinant is present near the amino terminus of the
enteropeptidase heavy chain, and like the N-glycosylated light chain, it can direct the apical targeting of secreted variants of
enteropeptidase. Replacement of the endogenous transmembrane domain
with a cleavable signal peptide (dSA-BEK) and additional deletion of
the light chain (dSAL-BEK) did not prevent apical targeting, but
removal of residues 48-197 (HL-BEK) caused secretion from both apical
and basolateral surfaces (Fig. 4). This segment is
O-glycosylated (Fig. 1B), probably within a
mucin-like domain between residues 166 and 192, and it also contains
three potential N-glycosylation sites. Whether these
oligosaccharides contribute to the apical targeting function of this
segment has not been determined.
Other O-glycosylated proteins may be targeted apically
independent of N-glycosylation. For example, the apical
sorting of a neurotrophin receptor (p75NTR), a type I
membrane protein, depends on an O-glycosylated juxtamembrane segment that remains functional in the absence of
N-glycosylation and does not require membrane association
for targeting (16). The O-glycans of p75NTR have
not been proved to play a direct role in sorting, and it remains
possible that other structural features constitute the apical sorting
determinants. However, these results suggest that O-glycans
as well as N-glycans may support apical targeting. Cells that are unable to complete complex-type oligosaccharides appear to
target apical glycoproteins correctly (39, 40), suggesting that the
mannose-rich core regions of N-glycans may comprise a sorting signal. Because mannose does not commonly occur in
O-glycans, functional targeting motifs within
N-linked and O-linked oligosaccharides could
differ structurally and be recognized by distinct lectin-like components of the sorting pathway, as suggested by Scheiffele and
colleagues (14). For example, VIP36 is an
N-acetylgalactosamine-binding lectin of exocytic vesicles
that might mediate targeting by O-glycans (41), and similar
mannose-binding lectins might mediate targeting by
N-glycans.
The heavy chain of enteropeptidase appears to contain a third
functionally defined signal that can inhibit apical targeting in
certain contexts. Addition of most of the heavy chain to the light
chain caused the delivery of construct HL-BEK to both apical and
basolateral surfaces, suggesting that the heavy chain inhibits apical
targeting directed by the light chain (Fig. 4). In contrast, construct
dSAL-BEK was secreted apically, indicating that the targeting function
of the juxtamembrane region was not inhibited by inclusion of the
remainder of the heavy chain. Both chains are extensively
N-glycosylated, and the mechanism by which a portion of the
glycosylated heavy chain could inhibit the
N-glycan-dependent targeting of the light chain
is unknown. Additional targeting determinants may be present in
enteropeptidase. In particular, our studies do not exclude an
independent or supplementary targeting function of the enteropeptidase
transmembrane domain.
Although they may employ distinct targeting signals, many apically
sorted membrane proteins associate with glycosphingolipid and
cholesterol-rich rafts (20). Rafts are resistant to dissolution by
Triton X-100 at 4 °C, and raft proteins are operationally defined by
their recovery in low density Triton X-100-insoluble membrane domains
that are isolated by differential centrifugation. For example, apical
targeting can be mediated by the transmembrane domain of influenza
virus hemagglutinin or the glycosylphosphatidylinositol anchor of
alkaline phosphatase, and these proteins are recovered in
detergent-resistant rafts (17, 42, 43). Several apical N-glycosylated intestinal hydrolases with type I or type II
transmembrane topology also are found in rafts (35).
Enteropeptidase does not fit this pattern because full-length
membrane-bound BEK was solubilized completely by extraction of cells
with Triton X-100 (Fig. 6); transient intracellular association with
detergent-resistant rafts was not evident, with or without blockage of
delivery to the plasma membrane by incubation of cells at 20 °C
(Fig. 6, B and C). Enteropeptidase also was not
recovered in low density membrane fractions upon sucrose density
gradient centrifugation (Fig. 7). The association of
glycosylphosphatidylinositol-linked placental alkaline phosphatase with
rafts reportedly was enhanced by patching with antibody prior to
detergent extraction so that raft association was stable even at
30 °C, a temperature at which alkaline phosphatase otherwise is
completely soluble in Triton X-100 (38). In the case of enteropeptidase
this maneuver caused it to cluster into large membrane aggregates (Fig.
8) but did not prevent solubilization by Triton X-100 at 4 °C (Fig.
9). Thus, apical targeting of enteropeptidase does not appear to depend on detergent-resistant association with rafts.
Certain other apical proteins also do not appear to associate tightly
with rafts. Intestinal maltase-glucoamylase shares with enteropeptidase
the structural features of type II transmembrane topology, extensive
N-linked glycosylation, and a juxtamembrane mucin-like
domain that may be O-glycosylated (44); both proteins are
sorted apically and are solubilized by Triton X-100 (35). Lactase-phlorizin hydrolase is an extensively N-glycosylated
type I transmembrane protein that also is sorted to the apical surface of enterocytes and is solubilized by Triton X-100 (35). Based on these
examples, efficient apical targeting of N-glycosylated transmembrane proteins can occur with or without detergent-resistant raft association.
Secreted glycoproteins also are not incorporated directly into rafts
but nevertheless can be targeted apically, again raising the question
of whether some apical targeting pathways may be independent of rafts.
However, transient raft association within the cell could be sufficient
for delivery to the apical plasma membrane, after which secreted or
transmembrane proteins could dissociate and diffuse away from the rafts
that brought them there. Apical secretory proteins could bind to rafts
indirectly, perhaps through interaction of their N-glycans
with lectin-like sorting receptors (14). Similarly, apical
transmembrane glycoproteins could interact with rafts directly through
transmembrane domain-lipid interactions or indirectly through
protein-protein interactions that involve intrinsic raft proteins, and
these interactions may not be stable after disruption of the supporting
lipid bilayer with detergents (38, 45). If such a model were correct,
then the ability to detect raft association could be sensitive to the assay conditions. For example, the glycoprotein clusterin is secreted apically from MDCK cells and could be solubilized completely by extraction of cells with Triton X-100 at pH 7.5 (46); however, extraction at pH 6.2 was reported to allow detection of CHAPS-insoluble clusterin, suggesting a weak association with rafts (45). Depletion of
cellular cholesterol also caused mis-sorting of clusterin, and this
result indirectly supports a function of cholesterol-rich rafts in its
apical targeting (45). At the present time we have not recovered
enteropeptidase in low density raft membranes at pH 6.5, even after
patching enteropeptidase on the cell surface with antibodies.
Therefore, if rafts participate in targeting enteropeptidase, the mode
of involvement remains to be defined.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-D-galactopyranoside, and cells were
lysed with 40 mM MOPS, pH 7.5, 100 mM NaCl, 1% Triton X-100, 6 M urea, 300 mM imidazole, 10 mM dithiothreitol, and 100 µg/ml soybean trypsin
inhibitor. After centrifugation, the supernatant solution was dialyzed
against binding buffer (20 mM MOPS, pH 7.9, 50 mM NaCl, 0.5% Triton X-100) containing decreasing concentrations of urea (4 M to zero). Recombinant
enteropeptidase light chain was purified by chromatography on
Ni2+-nitrilotriacetic acid-agarose (12). Polyclonal
antibodies to the purified light chain were prepared in rabbits by
standard methods (24). Specific immune IgG against denatured light
chain (anti-L) was purified by sequential affinity chromatography on protein A-agarose and on enteropeptidase light chain immobilized on
Affi-Gel 10 (Bio-Rad).
70 °C. For Western blotting, proteins
were fractionated in 4-15% gradient polyacrylamide electrophoresis gels (Bio-Rad) and transferred by electroblotting onto supported nitrocellulose membranes (pore size 0.4 µm,, Bio-Rad). Membranes were
blocked with 3% non-fat milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS) containing 0.05% Tween 20 (TBST) at room
temperature for 30 min. The blocked membrane was incubated with 1.5 µg/ml affinity purified anti-L-IgG or anti-Lv-antiserum
(1:4000) diluted in TBST containing 1.5% non-fat milk at 4 °C
overnight. Membranes were washed four times with TBST, once with TBS,
and incubated with peroxidase-conjugated affinity purified swine
anti-rabbit IgG (Dako Corp., Carpinteria, CA) 0.12 µg/ml in TBST
containing 1.5% milk at room temperature for 1-2 h. Bound second
antibody was detected with the chemiluminescent ECL detection system
(Amersham Pharmacia Biotech).
-mercaptoethanol, and 0.02% sodium azide. Denatured and reduced proteins (30 µl) were then digested without (control) or with 0.5 units of
peptide-N-glycosidase F (Oxford Glycosciences, Bedford, MA)
or 1 milliunit of endoglycosidase H (Boehringer Mannheim) in a total
volume of 30 µl at 37 °C for 16 h. Samples (10 µl) of these
digested materials were analyzed by Western blotting.
-mercaptoethanol. The cell lysate, flow-through, and
eluate fractions from streptavidin-agarose were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting.
-mercaptoethanol, separated on
4-15% gradient mini-ready gels (Bio-Rad), fixed with 40% methanol,
10% acetic acid, treated with Amplify fluorographic reagent (Amersham
Pharmacia Biotech) for 30 min, dried, and exposed to XAR5 film (Eastman Kodak Co.) at
70 °C. Alternatively, to detect secreted forms of
enteropeptidase, conditioned medium from either apical or basolateral surfaces was collected, immunoprecipitated with anti-Ly, and
fractionated by SDS-PAGE. The enteropeptidase then was visualized by
either autoradiography (if labeled with Tran35S-label) or
Western blotting.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
120 kDa, indicating that much of
the difference between the observed sizes and the predicted polypeptide
mass of 110 kDa is due to N-linked glycosylation. The
150-kDa species were sensitive to both endoglycosidase H (endo H) and
PNGase, indicating that the N-linked oligosaccharides
consist of high mannose structures. The 180-kDa species were resistant
to endo H (Fig. 1A, lane 2), indicating the presence of
complex-type oligosaccharides. Therefore, the 150-kDa species appears
to be localized in the endoplasmic reticulum and are converted to
180-kDa species by oligosaccharide processing in the Golgi. Deletion of
the remainder of the heavy chain yielded a secreted 45-50-kDa protein,
L-BEK, consisting of the enteropeptidase catalytic domain.
As expected for a secreted glycoprotein, the N-linked
oligosaccharides of L-BEK were resistant to endo H (Fig.
1A, lane 11) but sensitive to PNGase (Fig. 1A, lane
12).
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Fig. 1.
Glycosidase digestion of wild-type and mutant
recombinant enteropeptidases expressed in BHK cells. A,
to evaluate N-linked glycosylation patterns, cell lysates or
conditioned media were prepared from stably transfected BHK cells
expressing distinct forms of enteropeptidase. Samples (10 µl) were
digested without ( ) or with (+) peptide N-glycosidase F
(PNGase) or endoglycosidase H (Endo H) and
analyzed by gel electrophoresis and Western blotting as described under
"Experimental Procedures." B, to evaluate
O-linked glycosylation patterns, BHK cells expressing
full-length enteropeptidase (BEK) were pulse-labeled with
Tran35S-label, and cell lysates or conditioned media were
prepared and immunoprecipitated with anti-enteropeptidase antibody.
Samples of immunoprecipitates or purified HL-BEK were digested without
(
) or with (+) PNGase F, neuraminidase (Neur), or
neuraminidase plus O-glycanase. After gel electrophoresis as
described under "Experimental Procedures," BEK was visualized by
autoradiography and HL-BEK was visualized by silver staining.
14 kDa (Fig. 1B). Deletion of the amino-terminal 197 residues in HL-BEK (Fig. 1B) prevented the change in
mobility associated with O-glycanase digestion, suggesting a
major O-glycosylated region lies between residues 48 and 197 in the exoplasmic part of the heavy chain. A possible site for this
O-linked glycosylation is the mucin-like domain between
residues 166 and 192.
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Fig. 2.
Cell-surface exposure of BEK and HL-BEK in
BHK cell lines. A, accessibility to surface
biotinylation. BHK cell lines expressing BEK or HL-BEK were labeled
with 1.5 mg/ml NHS-SS-biotin, and cell lysates were subjected to
precipitation with streptavidin-agarose. Samples of total cell lysate
(T), streptavidin-agarose flow-through (F), and
eluate (S) were analyzed by gel electrophoresis and Western
blotting as described under "Experimental Procedures."
B, accessibility to extracellular trypsin. BHK cell lines
expressing BEK or HL-BEK were treated without ( ) or with (+) trypsin,
and cell lysates were prepared, and samples were analyzed by gel
electrophoresis and Western blotting as described under "Experimental
Procedures."
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Fig. 3.
Immunofluorescence and confocal microscopy of
proenteropeptidase (BEK) in transfected MDCK II cells. Transfected
MDCK II cells expressing full-length enteropeptidase (BEK) were grown
on Costar clear Transwell filters to form a tight monolayer, fixed in
1.5% paraformaldehyde (A and a) or
ethanol:acetic acid (9:1) (B and b), and
incubated with either anti-Lv (A) or anti-caveolin IgG
(B) and Cy3-conjugated goat anti-rabbit IgG, and examined by
confocal laser-scanning microscopy as described under "Experimental
Procedures." A and B show x-y scans
parallel to the plane of the filter near the apex of the cells.
Enteropeptidase exhibits an apical staining pattern (A),
whereas caveolin is localized laterally (B). Some cells in
this mixed cell line do not express enteropeptidase, and this accounts
for the patchy staining in A. The insets (a
and b) show scans along the x-z dimension and
demonstrate the apical localization of enteropeptidase (a)
and basolateral localization of caveolin (b).
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Fig. 4.
Domains required for apical sorting of
enteropeptidase. Stably transfected MDCK II cell lines expressing
variants of enteropeptidase were grown on Costar Transwell units. For
detection of membrane-associated enteropeptidase, cells were labeled
from either the apical (Ap) or basolateral (Bl)
side with NHS-SS-biotin. Biotinylated proteins in cell lysates were
isolated with streptavidin-agarose for SDS-PAGE and Western blotting
with anti-L antibody (BEK) or anti-flag antibody
(BEK.Flag). For analysis of secreted forms of
enteropeptidase (dSA, dSAdL.Flag, HL, and L),
conditioned media were collected from either the apical or basolateral
side surface of MDCK cells. Cell lines expressing dSA or dSAdL.Flag
were biosynthetically labeled with Tran35S-label, and
conditioned medium was analyzed by immunoprecipitation and
autoradiography. Conditioned medium from cells expressing BEK,
BEK.Flag, HL, or L was analyzed by Western blotting with anti-Lv. The
amount of apically sorted enteropeptidase was quantitated from two
independent experiments and expressed as a percentage of the
total.
15-kDa decrease in the apparent mass of
L-BEK, and secretion was reduced significantly by retention
within the endoplasmic reticulum. However, in contrast to the apical
secretion of glycosylated L-BEK, the under-glycosylated
protein was secreted randomly from both basolateral and apical surfaces
(Fig. 5). A small residual quantity of fully glycosylated
L-BEK was secreted apically, indicating that the sorting
pathway remained capable of correctly delivering apical glycoproteins
after exposure of cells to tunicamycin. Thus, the apical targeting
signal of L-BEK appears to include N-glycans. Similar experiments could not be performed for membrane-bound BEK or
secreted variants containing the heavy chain because inhibition of
glycosylation with tunicamycin caused complete intracellular retention
(data not shown).
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Fig. 5.
Effects of tunicamycin, brefeldin A, and
colchicine on sorting of the secreted enteropeptidase light chain.
Tight monolayers of MDCK II cells expressing enteropeptidase light
chain (L) were grown on Costar Transwell filters and treated
with 2 µg/ml tunicamycin (Tun), 10 µg/ml brefeldin A
(BFA), or 12 µg/ml colchicine (Colch) in
serum-free medium for 18-24 h. Control cells (Con) were
incubated similarly. A, conditioned medium was collected
from the apical (Ap) or basolateral (Bl) surface
and analyzed by SDS-PAGE and Western blotting. gp,
glycosylated; dgp, deglycosylated. B, the amount
of apically secreted L-BEK was quantitated from three
independent experiments and expressed as a percentage of the total.
C, the transepithelial resistance was not significantly
affected by the various treatments.
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Fig. 6.
Detergent solubility of enteropeptidase in
MDCK and BHK cells. A, cells expressing full-length
enteropeptidase (BEK) were labeled with Tran35S-label for
30 min, chased at 37 °C for 30 min, and extracted on ice with buffer
containing 1% Triton X-100. Detergent- soluble (S) and
detergent-insoluble fractions (I) of cell lysates were
separated by centrifugation and analyzed by immunoprecipitation,
SDS-PAGE, and autoradiography as described under "Experimental
Procedures." B, BHK cells expressing BEK were labeled with
Tran35S-label and chased at 37 or 20 °C. At the
indicated times, samples were extracted with Triton X-100,
immunoprecipitated, and incubated with (+) or without ( ) endo H. Reactions were analyzed by SDS-PAGE and autoradiography. The mobilities
of glycosylated (gp) and deglycosylated (dgp)
enteropeptidase are indicated. Conditions in C are the same
as in B, except that the time of chase was extended to 300 min.
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Fig. 7.
Sucrose density gradient analysis of the raft
association of enteropeptidase in MDCK and BHK cells. Cells
expressing BEK were grown in 150-mm dishes and extracted on ice in MES
buffer, pH 6.5, containing 1% Triton X-100. Lysates were subjected to
sucrose density gradient centrifugation, and fractions were analyzed
for enteropeptidase (EK) or caveolin (Cav) by
Western blotting as described under "Experimental
Procedures."
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Fig. 8.
Antibody-induced patching of
enteropeptidase. Cells expressing full-length enteropeptidase
(BEK) were fixed on ice and subsequently incubated with Cy3-conjugated
goat anti-rabbit IgG and rabbit antibody to either enteropeptidase
(A) or caveolin (C). Under these conditions, both
antigens display a diffuse, finely punctate immunofluorescence pattern.
Alternatively, cells were first incubated with Cy3-conjugated goat
anti-rabbit IgG and antibody to either enteropeptidase (B)
or caveolin (D), followed by fixation. Under these
conditions, anti-caveolin does not have access to caveolin on the inner
leaflet of the plasma membrane and there is only background
immunofluorescence (D); anti-enteropeptidase causes the
accumulation of enteropeptidase in large surface patches. Photographs
were taken focusing on the apex of the cells to emphasize the pattern
visible on the plasma membrane.
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Fig. 9.
Effect of antibody-induced cross-linking on
the association of enteropeptidase with detergent-resistant rafts.
BHK cells expressing enteropeptidase (BEK) were incubated at 12 °C
without ( Abs) or with (+Abs) rabbit anti-Lv and
goat anti-rabbit IgG antibodies. Cells were extracted on ice with MES
buffer, pH 6.5, containing 1% Triton X-100 and subjected to sucrose
gradient centrifugation. Fractions were analyzed for enteropeptidase
and caveolin (Cav) by SDS-PAGE and Western blotting as
described under "Experimental Procedures."
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENT |
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We thank Dr. Linda Pike (Washington University) for assistance in the preparation of detergent-insoluble rafts.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK50053.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Howard Hughes Medical
Institute, Washington University School of Medicine, 660 South Euclid
Ave., Box 8022, St. Louis, MO 63110. Tel.: 314-362-9029; Fax:
314-454-3012; E-mail: esadler{at}im.wustl.edu.
The abbreviations used are: MDCK, Madin-Darby canine kidney; BEK, recombinant bovine proenteropeptidase; dSA-BEK, mutant proenteropeptidase lacking the amino-terminal signal anchor domain; dSAdL-BEK, mutant proenteropeptidase lacking the signal anchor, mucin-like, and light chain domains; HL-BEK, mutant proenteropeptidase lacking the amino-terminal signal anchor and mucin-like domains; L-BEK, mutant proenteropeptidase containing the light chain and 17 carboxyl-terminal residues of the heavy chain; NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; BHK, baby hamster kidney; PNGase, peptide N-glycosidase; endo H, endoglycosidase H; MES, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin.
2 D. Lu, X. Zheng, and J. E. Sadler, manuscript in preparation.
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