From the
In its fundamental attributes, the secretion pathway of the
pancreatic bile salt-dependent lipase (BSDL) followed that described
for all enzymes involved in regulated secretion. This route was
inhibited by drugs that affect protein synthesis and intracellular
transport. In the presence of monensin, BSDL was solely detected in
microsome membrane fractions. The association of BSDL with
intracellular membranes involved a protein complex, formed by at least
two proteins of 94 and 56 kDa. In cells experiencing the metabolic
stress due to azetidine-2-carboxylic acid, BSDL was additionally
associated with a protein of 46 kDa. Affinity blotting showed that BSDL
bound directly to the 94-kDa protein (p94). It was suggested that p94
could be a molecular chaperone, further identified as related to the
94-kDa glucose regulated protein (Grp 94). The membrane-associated BSDL
(i.e. BSDL bound to the Grp 94-related p94) was O-
and N-glycosylated and consequently appeared released from
membranes in the trans-Golgi compartment. Therefore and for
the first time, it is suggested that a multiprotein complex including
the chaperone Grp 94-related p94 protein may play an essential role in
the folding and transport of BSDL. One hypothesis is that the
association of BSDL with membrane via the Grp 94-related p94 along its
secretion pathway is required for its complete
O-glycosylation, which occurs on the extended mucin-like
structures present on the C-terminal part of the protein.
Regulated cell secretion is the result of consecutive events
comprising the synthesis and transport of proteins through
intracellular compartments from the endoplasmic reticulum to zymogen
granules. In response to the appropriate signal, zymogen granule
membranes fuse with the luminal plasma membrane of the cell. This
fusion allows the release of the soluble enzymes. During the secretory
process, proteins are adequately folded and assembled and may undergo
post-translational modification. Incompletely assembled, misfolded, or
aggregated products are selectively retained in the endoplasmic
reticulum
(1) . In a similar way, transport to and from the Golgi
apparatus occurs only when nascent proteins have been folded and
assembled in the correct way
(2) . A family of proteins known as
molecular chaperones have been recognized participating in the correct
folding of polypeptidic chains and in driving them into oligomeric
structures, if required
(3) . Molecular chaperones have been
shown to be invaluable to cellular processes such as protein synthesis,
transport, signal transduction, morphogenesis, immune and heat shock,
or stress responses. Members of this family have been recently
localized in distinct compartment along the secretory pathway of the
pancreatic acinar cells
(4) . Elements of the ``quality
control'' mechanisms involved in recognizing, retaining, and
degrading misfolded and misassembled proteins are therefore present in
these cells
(1) . The bile salt-dependent lipase
(BSDL)(
For use in ligand blotting, human
BSDL was biotin-labeled as follows; 1 mg of BSDL in 1 ml of 0.1
M sodium bicarbonate, pH 8.0, buffer was biotinylated with
biotinamidocaproate-N-hydroxysuccinimide ester (5 µl of a
40 mg/ml solution in dimethyl sulfoxide) for 1 h at 4 °C. The BSDL
solution was then dialyzed twice for 24 h against water and diluted to
0.5 mg/ml before use. Approximately 1 ng of biotin-labeled BSDL can be
detected using anti-biotin antibodies conjugated to alkalin phosphatase
as detection system.
Proteins that bind to
BSDL were detected by ligand blotting on membranes obtained after
SDS-PAGE and electrotransfer. Membranes were first blocked overnight
with 0.1 M sodium phosphate, pH 6.0, buffer (3% bovine serum
albumin, 50 mM NaCl) and incubated with biotin-labeled BSDL (5
µg/ml, 2 h, 4 °C). After exhaustive washing (six times) with
0.1 M phosphate buffer (0.3% bovine serum albumin, 0.05% Tween
20), membranes were incubated (1 h, ambient temperature) in 0.1
M sodium phosphate, pH 7.4, buffer (3% bovine serum albumin,
150 mM NaCl) with anti-biotin antibodies conjugated to
alkaline phosphatase. Bands were visualized by incubating replicas with
0.5 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.5
mM nitro blue tetrazolium in 0.1 M Tris/HCl, 0.1
M NaCl, 1 mM MgCl
According to the above results, the effects of
monensin, which is used extensively as an inhibitor of the
trans-Golgi apparatus function
(16) , were further
examined. We first studied the effect of monensin on the time-dependent
secretion of BSDL by the AR 4-2J cells. The rate of BSDL secretion was
linear with time, but a dose-dependent decrease of this rate was
observed in the presence of increasing concentrations of monensin. The
ionophore, at a final concentration of 1
The central dogma of the synthesis and secretion of
pancreatic protein established by Palade and co-workers
(23) stipulated that at the end of the translocation process,
the signal peptide is cleaved by endogenous signal-peptidase, thus
releasing proteins into the soluble cisternal content. Comparative
examination of the intracellular transport of many secreted proteins
showed that the transport from the endoplasmic reticulum to the Golgi
apparatus is selective and can be the rate-limiting step in the
secretion process
(34, 35) . This selectivity may be
associated to different rates of transport between the endoplasmic
reticulum and Golgi apparatus for different secreted proteins within
cells
(36) . This led to the hypothesis that secretory proteins
express specific transport signals within their own structure (34, 35).
This transport may also be
receptor-mediated
(34, 35, 37) .
In its
fundamental attributes, the secretory route of BSDL was similar to that
of other pancreatic secretory proteins. As
We have recently demonstrated
that the N-linked glycosylation of BSDL was essential for the
correct folding and secretion of the enzyme, nevertheless the trimming
and maturation of the N-linked glycosyl structure did not
influence the enzyme secretion
(10) . Therefore, possible
alterations of the N-linked glycosylation processes by
monensin that acted late in the processing continuum
(39) cannot
be advocated as a retention mechanism
(10, 12) . The size
of the membrane-associated BSDL correlated with that of the
glycosylated protein. This was confirmed by lectin
reactivity
(8, 40) and by the lack of effect of the Endo
H on the BSDL electrophoretic mobility, which agreed with the presence
of matured N-linked glycans. The incorporation of fucose
residues in BSDL
(10) and the reactivity of the
membrane-associated protein with the peanut agglutinin after sialidase
action agreed with the presence of Gal-GalNAc-O-Thr/Ser
O-linked structures, possibly fucosylated and sialylated. This
suggested that the glycosylation of BSDL was processed while the
protein is associated with membranes. Since terminal
glycosyltransferases such as galactosyltransferases are localized at
the trans-cisternae and trans-Golgi
network
(25, 41) , we believe that BSDL remains
associated with membranes while transported from the endoplasmic
reticulum to the trans-Golgi apparatus. The release of the
enzyme might take place in a cell compartment posterior to the
trans-Golgi or within it. This agrees with the effect of
monensin, which is described as an inhibitor of trans-Golgi
functions and blocked the normal formation of secretory
vesicles
(16) . Impairment of the secretory process in cancer
pancreatic cells may, therefore, be responsible for the association of
the oncofetal form of BSDL with microsome membranes
(14) .
We
further showed that p94 was probably related to Grp 94. Grp 94 is a
resident chaperone of the endoplasmic reticulum
(42) , which
associates with unassembled immunoglobulin
(43) , unassembled
histocompatibility complex class II polypeptides
(44) , and
mutant of herpesvirus glycoproteins
(45) . In all these cases,
additional proteins were found, although their nature and role is still
unknown
(36, 43, 45) . Grp 94 is a glycoprotein
that belongs to the Hsp 90 family implicated in the transport of
important biological molecules within the cell
(46) . Grp 94 has
the KDEL C-terminal sequence for retention of proteins in the lumen of
the endoplasmic reticulum
(33) . Given the fact that chaperones
are identified merely by their molecular weight on SDS-PAGE, it is
possible that Grp 94-related p94 may be the endoplasmic resident Grp
94, or an apparently identical protein associated with the Golgi
apparatus
(2) . Interestingly, the size of both p56 and p46
correlated with those of
A model compatible with all the
data presented here could be as follows. The N-terminal sequence of the
protein, where the two disulfide bridges of BSDL
(7) are
located, folds up either with the help of an undetected chaperone or
spontaneously around the N-linked glycosylation as suggested
by recent data: (i) the maturation of the N-linked structure
did not affect the BSDL secretion; (ii) N-glycosylation of
BSDL was essential for the expression of a fully active
enzyme
(10) . Additionally, the functionality of the
heparin-binding site (see Fig. 7A) located between
residues 61-66
(29, 32) argues for the
``nearly correct'' folding of the N-terminal sequence of the
enzyme. This mechanism might be common to many secretory pancreatic
enzymes such as
Therefore and for the first time, it is
suggested that a multimeric complex including the chaperone Grp
94-related p94 protein may play an essential role in the secretory
process of BSDL, first by accompanying BSDL from its site of synthesis
to its site of release, and second by keeping this protein in close
proximity to membrane glycosyltransferases and presenting the right
conformation of the peptide core of BSDL to these transferases.
Finally, the association with p94 may prevent premature aggregation of
BSDL molecules
(31) .
AR 4-2J cells were incubated (3 h) in
the presence of each inhibitor at the appropriate final concentration,
then cell-free medium was withdrawn and used for the determination of
bile salt-dependent lipase (BSDL) and
We are indebted to V. Sbarra for invaluable technical
assistance, to V. Mariottini for careful typing of the manuscript, and
to B. Khalil and C. Crotte for the artwork. We acknowledge Dr. B.
Lerique for autoradiogram quantitation and Dr. F. Clemente (INSERM
Unité 151, Toulouse, France) for providing us with the AR 4-2J
cell line. We thank Drs. P. Lechne de la Porte and S. Mathieu for
performing microscopy experiments and for fruitful discussions. We are
also indebted to Dr. Franck Haigler III for careful reading of the
manuscript and for suggestions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
is an enzyme found in all examined
pancreatic secretions (for a review, see Ref. 5). The size of this
protein varies by species due to the consequence of the number of
C-terminal tandem-repeated sequences
(6, 7) , which are
O-glycosylated
(8) . The human BSDL possesses 16 of
these tandem repeated sequences and represents the largest (
100
kDa). The rat BSDL has only 4 of these repeats and has the smaller size
(
70 kDa). A site for N-linked glycosylation was located
on Asn-187
(7) close to the Ser-194 of the active
site
(9) . This N-linked glycosylation was shown to be
cotranslational and essential for the secretion and expression of a
fully active enzyme by pancreatic cells
(10) . A glyco-isoform of
BSDL expressed by a neoplastic pancreas has been
described
(11, 12) . This glyco-isoform, referred to as
fetoacinar pancreatic protein or FAP, is associated with oncogenesis
and ontogenesis of the human pancreas
(13) . Immunocytological
experiments performed using J28, a monoclonal antibody that recognizes
FAP, suggested that in tumor cells, this oncofetal form of BSDL was
retained within the endoplasmic reticulum (14). Subcellular
fractionation corroborated this localization and established that FAP
might be associated with microsomal membranes (14). Defective
processing of the N-linked glycosylation in the endoplasmic
reticulum resulting from amino acid mutation was hypothesized to
explain the retention of the protein
(14) . However, we have
recently shown that the maturation of the N-linked structure
along the secretory pathway did not affect enzyme secretion (10).
Variations were therefore observed for the N-linked
glycosylation, which appeared of the biantennary complex type in the
normal variant of the human enzyme
(15) and of the high mannose
type in the variant secreted by inflamed pancreas
(12) .
Moreover, in normal human pancreatic cells, BSDL was partly associated
with membranes of microsomes. The association of BSDL with membranes
was optimal between pH 5 and 6, which agrees with the pH of the
endoplasmic reticulum vesicles. The BSDL can be released from microsome
membranes by salt and trypsin treatment or at pH above
8.(
)
Whereas in human, an in-depth study
concerning the association of BSDL with microsome membranes was not
possible due to the lack of a cell model, we turned to the rat AR 4-2J
cell line. Our experiments showed that BSDL was retained without
degradation within the cell upon tunicamycin or brefeldin A treatment
of cells
(10) . All these data suggest that the secretion or the
folding of BSDL could be under the control of chaperones. In this paper
we have further characterized microsomal proteins that bind to BSDL
molecules synthesized in the presence of monensin, a drug that disrupts
communication between trans-Golgi structures and secretory
vesicules
(16) . Results showed that unfolded, although
glycosylated BSDL, formed a complex with proteins of 94 kDa (to which
the enzyme is bound), 56 kDa, and 46 kDa. The 94-kDa protein is
probably related to the 94-kDa glucose-regulated protein or Grp94.
Materials
Biotin-conjugated lectins, alkaline
phosphatase-conjugated antibodies to biotin, alkaline
phosphatase-conjugated antibodies anti-rabbit, goat anti-rat IgG, goat
anti-mouse IgG, carbonyl cyanide chlorophenylhydrazone (CCCP),
monensin, vinblastin sulfate, defatted serum albumin, Tween 20, nitro
blue tetrazolium, 4-nitrophenyl hexanoate, soybean trypsin inhibitor,
CNBr-activated Sepharose,
biotinamidocaproate-N-hydroxysuccinimide ester,
L-azetidine-2-carboxylic acid, and 5-bromo-4-chloro-3-indolyl
phosphate were from Sigma. Nitrocellulose membranes were from
Schleicher & Schüll (Dassel, Germany). -Glucosidase,
endoglycosidase H (Endo H),
4-nitrophenyl-
-D-maltopentaoside, leupeptin, tunicamycin,
and brefeldin A came from Boehringer (Mannheim, Germany).
Polyvinylpyrolidone K40, phenylmethylsulfonyl fluoride, benzamidine,
-phenyl propionate, heparin, and Coomassie Blue R 250 were from
Fluka (Buchs, Switzerland). Cytochalasin B and cycloheximide were
purchased from Serva (Heidelberg, Germany).
[
S]Methionine (Tran
S-label,
>1000 Ci/mmol) was from ICN Biochemicals (Costa Mesa, CA).
Antibodies
Monoclonal antibody specific for Grp 94
(clone 9G10) was from StessGen Biotechnology Corp. (Victoria, Canada).
Polyclonal antibodies raised using purified secretory rat BSDL and
human -amylase were obtained in our laboratory. These antibodies
did not react with the microsomes of the rat liver.(
)
Antibodies were isolated by affinity chromatography on protein
A-Sepharose as described previously
(6) .
Proteins and Enzyme Assays
Proteins were routinely
assayed with the bicinchoninic acid method (Micro-BCA kit from Pierce)
using bovine serum albumin as standard. The activity on 4-nitrophenyl
hexanoate was measured at 410 nm and pH 7.4 in a thermostated cell at
30 °C as described elsewhere
(17) . The -amylase
activity was determined at 410 nm on 4-nitrophenyl maltopentaoside in
pH 7.4, 0.1 M sodium phosphate, 50 mM NaCl buffer in
the presence of
-glucosidase (>24 units/ml).
Human BSDL Immobilization and Biotin
Labeling
Human BSDL was isolated from human pancreatic juice as
described previously
(18) . The pure enzyme was immobilized on
CNBr-activated Sepharose (1 mg of protein/0.5 g of wet gel) in 0.1
M sodium borate buffer (0.25 M NaCl, 5 mM
CaCl, pH 8.0). After an overnight incubation at 4 °C
under agitation, the gel was allowed to settle and quenched with 10 gel
volumes of 0.1 M ethanolamine (4 h, 4 °C). Before use, the
gel with immobilized BSDL was extensively washed alternatively with
basic (0.1 M sodium phosphate, pH 8.0) and acidic (0.1
M MES, pH 5.5) buffers.
Polyacrylamide Gel Electrophoresis and Western
Blotting
Gel electrophoresis (SDS-PAGE) were performed on a slab
gel of polyacrylamide (5 or 7.5%) and 0.1% sodium dodecyl sulfate under
reducing conditions according to Laemmli
(19) using a Bio-Rad
Mini-Protean II apparatus. Non-reducing conditions were obtained by
omitting dithiothreitol in the denaturation buffer. Proteins were
electrophoretically transferred to a nitrocellulose membrane
(20) in 0.2 M Tris/HCl, pH 9.2, buffer (10% methanol),
at 150 mA for 3 h in a cold room. Completeness of transfer was verified
by staining polyacrylamide gels with Coomassie Blue R250. Destaining
was performed in ethanol/acetic acid/water (2/3/35 by volume).
Membranes were then treated as described previously
(8) using
the polyclonal antiserums to rat pancreatic BSDL. The BSDL protein mass
was determined by densitometric scannings of Western blotting as
described by Abouakil et al.(10) , corrected for the
volume fraction loaded on SDS-PAGE. Alternatively, gels were stained
using the Bio-Rad silver stain kit following the protocol given by the
supplier.
Lectins and Ligand Blotting
After electrotransfer
to nitrocellulose membranes, replicas were blocked 2 h with 50
mM Tris/HCl, 150 mM NaCl, pH 8.0, buffer containing
3% bovine serum albumin by weight. Glycoproteins were detected by
lectin blotting using biotin-conjugated lectins (10 µg/ml) as
described by Mas et al.(8) .
, pH 9.5, buffer.
Cell Culture
AR 4-2J cells, a rat pancreatic tumor
cell line derived from a transplantable tumor of the acinar pancreas
(CRL.1492, ATCC, Rockville, MD), kindly provided by Dr. F. Clemente
(INSERM, Unité 151, Toulouse, France), were grown in
Dulbecco's modified Eagle's medium (DMEM) as described
previously
(10) . In this cell line, most of the activity on
4-nitrophenyl hexanoate (>85%) can be ascribed to BSDL
(10) .
Isolation of Microsomes
The AR 4-2J cells grown to
confluence were washed with incomplete PBS buffer (10 mM
phosphate buffer, pH 7.0, 0.15 M NaCl without Ca and Mg
), harvested by scraping with a rubber
policeman, and homogenized with a Polytron in a 5 mM Tris/HCl,
pH 7.4, buffer (0.25 M sucrose, 0.1 mM EDTA, 2
mM benzamidine, 2 mM phenylmethylsulfonyl fluoride).
Isolation of microsomes was done by serial centrifugation as follows.
First, cell debris and nuclei were pelleted by centrifugation at 600
g for 10 min. The supernatant was first centrifuged at
3000
g, then at 8000
g, and finally
at 15,000
g for 10 min to precipitate zymogen
granules, mitochondria, and lysosomes, respectively. The microsomes
were then pelleted by centrifugation of the post-lysosomal supernatant
at 100,000
g for 1 h. The pellet was then washed with
the Tris/HCl/sucrose buffer and once again centrifuged under the same
conditions.
Differential Solubilization and Temperature-induced Phase
Separation in Triton X-114
Separation of membrane proteins from
soluble proteins was done by differential solubilization and
temperature-induced phase separation using Triton X-114 according to
the three-phase method described by Hooper and Bashir
(21) .
Inhibitor Treatment
The rat pancreatoma AR 4-2J
cells (80% confluence) were used for inhibition experiments. For this
purpose, the drug was added from a stock solution (monensin, 10
mM in ethanol; brefeldin A, 1 mg/ml in PBS; cytochalasin B, 5
mg/ml in PBS; vinblastin sulfate, 1 mg/ml in PBS; CCCP, 5 mM
in ethanol and cycloheximide, 1 mg/ml in PBS) to dishes at the
appropriate final concentration. At the required time of incubation (3
h), the cell-free medium was withdrawn and used for the analysis of
BSDL and -amylase activities. When variable concentrations of
monensin were used, the BSDL activity was determined in the cell-free
medium and the cell membrane fraction. For this purpose, after
withdrawal of the cell-free medium, the corresponding cell layer was
washed with PBS, scraped with a rubber policeman and lysed by
sonication in a 10 mM Tris/HCl, pH 7.4, buffer (0.1 M
NaCl, 0.1% Triton X-100, 2 mM benzamidine, and 1 mM
EDTA). The homogenate was then centrifuged for 1 h at 17,000
g, the supernatant was discarded, and the membrane pellet was
washed twice with a 10 mM Tris/HCl, pH 7.4, buffer and
resuspended in the lysis buffer. Tunicamycin treatment of cells was
performed as described previously before protein labeling with
[
S]methionine
(10) . Briefly, cells were
starved for 2.5 h in the presence of tunicamycin (8 µg/ml) in DMEM
without methionine. After washing twice with PBS, cells were pulsed for
1.5 h with [
S]methionine (20 µCi/ml) and
then chased for 30 min while still in the presence of tunicamycin.
Cells were washed and lysed as above.
Pulse-chase Protocol
AR 4-2J cells were grown in
methionine-free DMEM to starve the cells of this amino acid. After
45-60 min of starvation, dishes were pulse-labeled with
[S]methionine (40 µCi/ml). The pulse medium
was removed after 5 min followed by two quick washes with the chase
medium, and cells were chased in DMEM with 10% fetal calf serum for
different time intervals. The chase was stopped by aspirating the
medium and adding ice-cold PBS without CaCl
and
MgCl
. Cells were then lysed in 10 mM Hepes, pH
7.4, buffer (200 mM NaCl, 2 mM CaCl
, 2.5
mM MgCl
, 1.5% Triton X-100, 10 µg/ml
leupeptin, 2 mM benzamidine, 2 mM
phenylmethylsulfonyl fluoride, 2 mM soybean trypsin inhibitor,
and 2 mM
-phenyl propionate). The cell lysates were
centrifuged at 2000
g for 15 min at 4 °C to pellet
nuclei and cell debris. The supernatant was used for
immunoprecipitation. When indicated, chase medium was also used for
immunoprecipitation. Some cells were further starved and pulsed in the
presence of 5 mM azetidine-2-carboxylic acid.
Immunoprecipitation
Radiolabeled cells were lysed
in 10 mM Hepes, pH 7.4, buffer in the presence of salts,
detergent, and protease inhibitors as described above. Lysates were
then clarified by centrifugation (2000 g, 15 min, 4
°C). One ml of either lysate or isolated microsomes was incubated
overnight at 4 °C with 25 µg of antibodies to rat BSDL. Protein
A-Sepharose (10 mg) pre-adsorbed with non-radioactive proteins from
either lysate or microsome was added to antibody-antigen complexes and
incubated for 4 h at 4 °C under agitation. At the end of the
incubation, the antigen-antibody-protein A complexes were recovered by
centrifugation (10,000
g, 15 min, 4 °C). When
required, a two-cycle immunoprecipitation procedure slightly modified
from Doolittle et al.(22) was used. The final 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 buffer. The
pellet was then transferred into the SDS-PAGE Laemmli's sample
buffer, warmed for 2 min at 95 °C, centrifuged, and electrophoresed
on SDS-PAGE. Gels were stained with Coomassie Blue R 250 and destained
as above, immersed (30-60 min) in Amplify (Amersham), and
autoradiographed using Hyperfilm MP (Amersham).
Endoglycosidase H Treatment
After metabolic
labeling with [S]methionine, cells were lysed,
clarified, and immunoprecipitated with antibodies to rat BSDL. The
pellet was dissolved in 100 µl of Tris/HCl buffer (0.1 M
Tris/HCl, pH 6.8, 2% SDS, and 10% glycerol), boiled for 2 min and
centrifuged (10,000
g, 5 min). The supernatant was
10-fold diluted in 50 mM sodium phosphate, pH 5.6, buffer, 3%
Triton X-100. Half of the sample was then treated by Endo H (40
milliunits/ml) for 10 h at 37 °C. The remaining was incubated in
the absence of Endo H and used as control. Both parts were
immunoprecipitated with antibodies to rat BSDL and analyzed on
SDS-PAGE.
Effect of Monensin on Bile Salt-dependent Lipase
Secretion by AR 4-2J Cell Line
We have shown recently that the
rat pancreatic cell line AR 4-2J is able to synthesize and secrete the
74-kDa N-glycosylated BSDL
(10) . The effect of various
drugs affecting protein synthesis or transport mechanisms were
therefore examined. As listed in , drugs that affect the
integrity of the cytoskeleton such as cytochalasin B or vinblastin
sulfate caused an approximately 40% decrease of the BSDL and
-amylase activities, both detected in the cell free medium. CCCP,
which is an uncoupler of the oxidative phosphorylation, had the same
effect as the protein synthesis inhibitor cycloheximide. The most
effective inhibitors of the secretion were the carboxyl ionophore
monensin and the fungal metabolite brefeldin A, which impairs the
trans-Golgi to secretory vesicles traffic and the endoplasmic
reticulum to Golgi traffic, respectively. These two agents inhibited
the secretion of BSDL by 80-90%; consequently, it appeared that
the secretion (and transport) of the glycosylated BSDL was sensitive to
energy depletion or to sodium/potassium equilibration in cells, as is
the transport of
-amylase. All these data suggested that the
secretion pathway of the BSDL paralleled that of the
-amylase and
was therefore similar to that of all other secretory pancreatic
enzymes
(23) .
10
M, totally inhibited the BSDL and the
-amylase
secretion in less than 3 h. Therefore, AR 4-2J cells were incubated for
the appropriate time in the presence of increasing concentrations of
monensin. As depicted in Fig. 1, increasing concentrations of
monensin from 10
to 10
M
inhibited the BSDL secretion; meanwhile, the esterolytic activity on
4-nitrophenyl hexanoate increased in the cell membrane fraction. As
expected, monensin induced the retention of BSDL within the cell
membrane compartment. We were interested in whether the retention of
BSDL was specific to a cell compartment. Electron microscopy indicated
that upon menensin treatment of AR 4-2J cells (10
M, 3 h), colloidal gold particles following the
formation of the BSDL-antibody complexes accumulated in vacuoles likely
originating from the Golgi apparatus. As these vacuoles were dispersed
throughout the cytoplasm, microsomes of AR 4-2J cells were isolated
from control cells and from cells after monensin treatment. Microsomes
were then subjected to the temperature-induced phase separation with
Triton X-114. Each phase was analyzed by SDS-PAGE and Western blot
using polyclonal antibodies to rat BSDL (Fig. 2). In the absence
of monensin, as judged from activity and Western blot scanning, BSDL
was mainly detected in the aqueous phase. Following the monensin
treatment of AR 4-2J cells, nearly 80% of the total BSDL protein mass
was detected in the fractions containing membrane proteins (Triton
X-114-rich phase and insoluble-detergent phase; Fig. 2). As
expected, independent of monensin treatment, 80-90% of the
-amylase determined from either its activity or by quantitation of
Western blot was found in the aqueous phase of pancreatic microsomes.
Hence, the association of BSDL with membrane microsomes was favored by
monensin.
Figure 1:
Effect of monensin on the secretion of
the BSDL of rat pancreatic AR 4-2 J cells. Confluent AR 4-2 J cells
were grown for 3 h with increasing concentrations of monensin. The BSDL
activity was then determined either in cell-free medium () or
in cell membrane fraction obtained after cell lysis (
). Control
activity was taken as the activity in cell-free medium or in
cell-membrane fraction of AR 4-2J cells grown in the absence of
monensin. Each value represents means ± S.E. from at least three
independent experiments.
Figure 2:
Triton X-114 phase separation of
microsomes from AR 4-2 J cells. Microsomes of AR 4-2 J cells were
isolated by centrifugation and then subjected to the
temperature-induced phase separation induced by Triton X-114. Cells
were previously treated for 3 h with (+ monensin) or without
(- monensin) 10M monensin. 7 µg
of protein from the aqueous phase (lanea), the
Triton X-114-rich phase (laneb), and the
detergent-insoluble phase (lanec) were separated by
SDS-PAGE and electrotransferred on nitrocellulose membranes.
Immunodetection was performed using polyclonal antibodies to rat BSDL.
The total BSDL protein mass was determined by densitometric scanning,
taking into account the volume fraction of each phase loaded onto
SDS-PAGE. The BSDL activity was measured on 4-nitrophenyl hexanoate.
ND, not detected. mU stands for 10
units.
The rate of synthesis and secretion of this protein was
very fast as suggested by pulse-chase experiments, immunoprecipitation,
and quantitation of band intensities on SDS-PAGE. After starvation (45
min) and a 5-min pulse with [S]methionine, the
maximal synthesis of BSDL occurred after only a 10 min chase, while the
enzyme could be immunoprecipitated from the extracellular medium after
a chase of approximately 20 min (Fig. 3). The fast rate of
secretion of BSDL by AR 4-2J cells gives a good explanation to the fact
that in cells which were not treated with monensin, the
membrane-associated BSDL cannot be detected. Because storage granules
are poorly represented in AR 4-2J cells, we suggest that the rate of
secretion of BSDL, which is about 30 min, corresponds to that of its
synthesis, maturation, and transport. Consequently, BSDL is transiently
associated with intracellular membranes and its release occurs within
(or consecutive to) a cellular compartment sensitive to monensin.
Because monensin affects regions of the trans-Golgi apparatus
primarily associated with the final stages of secretory vesicle
maturation
(16) , it is suggested that BSDL reached those regions
in a membrane-associated state. For the purpose of this study, AR 4-2J
cells were (unless otherwise noted) grown for 3 h in the presence of
10
M monensin before use. They will be
referred to as monensin-treated AR 4-2J cells.
Figure 3:
Time-dependent secretion of BSDL. The
AR4-2J cells were grown in the absence of methionine (45 min), washed,
pulsed for 5 min with [S]methionine, and chased
for the time indicated. BSDL was then immunoprecipitated from the
cell-free medium using antibodies to rat BSDL and analyzed on
SDS-PAGE.
Glycosylation of the Membrane-associated Form of Bile
Salt-dependent Lipase
The BSDL detected in the lysates of
isolated microsomes from rat pancreatic monensin-treated AR 4-2J cells
had a molecular mass of approximately 74 kDa (Fig. 2), compatible
with that of the N-glycosylated BSDL of AR 4-2J
cells
(10) . Among proteins present in the Triton X-114-rich
phase (Fig. 4, laneT), only one protein
reacted with antibodies to rat BSDL (Fig. 4, lanea). In spite of the presence of many proteins in this
phase only a small number of them reacted with lectins. Among these
proteins, the membrane-associated BSDL from microsomes of
monensin-treated AR 4-2J cells reacted with concanavalin A
(Fig. 4, laneb, Con A) and
Galanthusnivalis agglutinin (Fig. 4, lanec, GNA). These lectins recognized biantennary
hybrid and high mannose type N-linked glycans, respectively,
demonstrating that the membrane-associated BSDL was
N-glycosylated. Nevertheless, incorporation of
[H]fucose in BSDL of AR 4-2J
(10) suggested that the rat enzyme, as its human
homologue
(8) , could also be O-glycosylated as
indicated by the presence of four putative sites for
O-glycosylation
(24) . The presence of O-linked
glycan structures was detected by means of lectin blotting using the
peanut agglutinin (PNA) on the secretory rat BSDL (Fig. 4,
laned) but also on the membrane-associated BSDL
(Fig. 4, lanee). Prior to incubation with PNA,
replicas were treated by sialidase as described by Mas et
al.(8) .
Figure 4:
Glycosylation of the membrane-associated
rat BSDL. Rat pancreatic juice proteins were separated by SDS-PAGE and
electrotransferred onto nitrocellulose membrane. Rat BSDL was localized
on the replicate by immunodetection using antibodies to rat BSDL
(lanea, 3 µg of loaded proteins). Proteins of
the Triton X-114-rich phase from microsomes of monensin-treated (3 h,
10M) AR 4-2 J cells were separated on
SDS-PAGE and transferred onto nitrocellulose, N-linked sugars
were detected by lectin blotting with concanavalin A (ConA)
and G. nivalis agglutinin (GNA) (lanes b and
c, 7 µg of loaded proteins), and O-linked sugars
were detected by lectin blotting with the PNA (lanee, 7 µg of loaded proteins). The presence of
O-linked sugars on secretory rat BSDL was detected by lectin
blotting as above (laned, 7 µg of loaded
proteins). Silver staining of proteins of the Triton X-114-rich phase
from microsomes of monensin-treated cells after SDS-PAGE was also shown
(laneT, 5 µg of loaded
proteins).
All these data clearly indicated that
N-linked and O-linked oligosaccharide structures were
present on the membrane-associated BSDL. The reactivity of PNA with the
membrane-associated BSDL demonstrated the presence of
Gal-GalNAc-O-Thr/Ser structures. The addition of galactose
residues to oligosaccharide structures was a late event occurring in
the trans-Golgi compartment
(25) . The high reactivity
of BSDL with lectins can be explained, first by the presence of both
N- and O-linked carbohydrate structures on BSDL (8),
and second because this protein was not degraded upon impairment of
secretory processes
(10) . Nevertheless one can note that Con A
also strongly reacted with proteins of approximately 90-100 kDa
while PNA recognized a 69-kDa protein. Other reactive proteins appeared
after longer development times. As shown in Fig. 3, BSDL appeared
outside the cell within 20 min of chase; hence, it is suggested that at
this time the enzyme had passed through the Golgi apparatus where its
N-linked glycosylated structure matured. Even though this fact
has already been suggested by lectin blotting (Fig. 4), the
sensitivity of BSDL to Endo H treatment was further determined. For
this purpose BSDL was immunoprecipitated from the radiolabeled cell
lysate following a 20-min chase (see Fig. 8for conditions) and
treated with Endo H. As unglycosylated BSDL was still reactive with its
specific antibodies
(10) , the reaction product was again
immunoprecipitated and analyzed by SDS-PAGE. As shown on Fig. 5,
the electrophoretic mobility of BSDL was not modified following Endo H
treatment. In comparison, intracellular BSDL from tunicamycin-treated
cells presented a higher mobility. This indicates that within 20 min,
BSDL may reach the trans-Golgi compartment, where its
N-linked sugar matured, while still associated with membranes.
Figure 8:
Association of BSDL to intracellular
proteins in azetidine-2-carboxylic acid treated AR 4-2J cells. AR 4-2J
cells grown in the absence of monensin were starved with methionine for
45 min in the presence (+) or absence (-) of 5 mM
azetidine-2-carboxylic acid (Azetidine). Cells were then
pulsed for 5 min with [S]methionine in the
presence or absence of 5 mM azetidine-2-carboxylic acid and
chased for the required time with complete DMEM. Cells were then lysed,
and clarified lysates were immunoprecipitated with specific polyclonal
antibodies to rat BSDL before electrophoresis on SDS-PAGE and
autoradiography.
Figure 5:
Endoglycosidase treatment of BSDL. AR4-2J
cells were radiolabeled with [S]methionine,
chased for 20 min in the absence of the radioactive amino acid, and
lysed. The lysate was clarified and BSDL immunoprecipitated. The
precipitated material was either treated by Endo H (40 milliunits/ml)
for 10 h (+) or incubated without the glycosidase (-) and
again immunoprecipitated with antibodies to rat BSDL before SDS-PAGE.
For comparison, cells were preincubated in the presence of 8 µg/ml
tunicamycin (Tm) in the absence of tunicamycin and
radiolabeled with [
S]methionine (30 min) while
still in the presence (+) or absence (-) of tunicamycin.
Cells were then washed twice and lysed. Cell lysates were then
immunoprecipitated with antibodies to rat BSDL and submitted to
SDS-PAGE.
Activity of the Membrane-associated Form of the Bile
Salt-dependent Lipase
As shown on Fig. 1, the impairment
of cell transport by monensin favored the association of BSDL with
membranes. However, an increase in membrane-associated BSDL activity
was not directly correlated with a decrease in secreted activity. These
data suggested that membrane-associated BSDL was, at least, poorly
active. We, therefore, attempted to determine whether
membrane-associated BSDL was enzymatically active. For this purpose
microsomes of monensin-treated AR 4-2J cells were subjected to
temperature-induced phase separation with Triton X-114. Enzyme activity
present in each phase was determined and corrected for their respective
volume (see Fig. 2). Approximately 90% of the activity on
4-nitrophenyl hexanoate was detected in the soluble phase, in spite of
the fact that only a small amount of BSDL protein (<5%) was
associated with this phase. In contrast, the largest amount of BSDL
protein was found in the Triton X-114-rich phase, where a very weak
enzymatic activity on 4-nitrophenyl hexanoate was detected. In
microsomes isolated from control cells, BSDL activity was exclusively
detected in the soluble phase, in which we also detected all the BSDL
protein. Furthermore, we observed that Triton X-114 did not affect BSDL
activity, indicating that membrane-associated BSDL was poorly active.
This could be the result of a tertiary structure, different from that
of the soluble protein which displays full activity. In all
probability, the impairment of the trans-Golgi apparatus
function by monensin
(16) interrupted the folding process of
BSDL. Consequently, only incompletely folded forms of BSDL might be
associated with intracellular membranes. We therefore attempted to gain
information about the folding state of the membrane-associated BSDL by
pulse-chase experiments. AR 4-2J cells were pulsed for 1-5 min
and then chased for 2 min. Cell lysates were immunoprecipitated and
analyzed on SDS-PAGE under reducing and non-reducing conditions. The
electrophoretic mobility of the membrane-associated BSDL was not
modified on SDS-PAGE under reducing or non-reducing running conditions.
This means that the two disulfide bonds present on BSDL formed loops
that were too small
(7) to influence protein mobility once
reduced. This point was supported by the fact that the electrophoretic
mobility of the soluble form of BSDL was also not influenced by the
SDS-PAGE running conditions.
Identification of Proteins That Bind to BSDL
Since
the association of BSDL with microsome membranes involved neither a
glycosyl phosphatidylinositol tail
(54) nor a Golgi or ER
retention signal
(26, 27) , but appeared sensitive to KBr
treatment, we sought to determine whether BSDL was associated with
microsome membranes by means of membrane proteins. For this purpose,
monensin-treated AR 4-2J cells were grown in the presence of
[S]methionine and lysed in 5 mM MES, pH
6.0, buffer containing 0.5% Nonidet P-40, 0.1 M NaCl, and 1
mM EDTA. The cell lysates were clarified by centrifugation at
2000
g for 10 min and then chromatographed on an
affinity column made of BSDL immobilized on Sepharose gel to isolate
proteins that bind to BSDL. After elution of unbound fraction with the
lysis buffer and washing of the column with the same buffer at pH 5.0,
bound proteins were eluted at pH 8.0 (0.1 M sodium phosphate,
0.15 M NaCl, and 0.5% Nonidet P-40). The eluted
[
S]methionine-labeled proteins were
electrophoresed on SDS-PAGE and autoradiographed. As shown in
Fig. 6
(lanea), two bands at approximately
90-120 kDa and 74 kDa were detected. A third band at 56 kDa,
although less intense, was also present. Control experiments performed
on a column of Sepharose without immobilized BSDL indicated that no
protein was nonspecifically absorbed by the gel. When microsomes,
isolated from monensin-treated AR 4-2J cells, were chromatographed on
the immobilized BSDL column two signal bands were detected on SDS-PAGE
(Fig. 6, laneb). They corresponded to proteins
of approximately 94 kDa and 70-74 kDa. Diffuse bands were also
detected at 56 kDa and around 40-50 kDa. Because antibodies to
rat BSDL reacted with the 70-74-kDa band in Western blotting
experiment (Fig. 6, lanec), other bands
detected on the autoradiogram were thought to be representative of
proteins that specifically bound to BSDL. Therefore, BSDL present in
microsomes was associated with at least two proteins of 94 kDa (p94)
and 56 kDa (p56), respectively. A third protein at <50 kDa may also
be suspected to bind to BSDL. To determine whether these proteins were
all together associated with membranes or to the lumen content of
microsomes, the soluble phase obtained after temperature-induced phase
separation with Triton X-114 was chromatographed on the immobilized
BSDL column as described above. No radioactive material was eluted from
this column. Nevertheless, fractions eluted at pH 8.0 were collected,
pooled, and concentrated before electrophoresis on SDS-PAGE and
autoradiography; as expected, no signal was detected on the film. To
confirm this result, radioactive material present in the Triton
X-114-rich phase obtained after temperature-induced phase separation
with Triton X-114 was immunoprecipitated with antibodies to rat BSDL
and electrophoresed on SDS-PAGE. As shown on Fig. 6(laned), p94 and p56 as well as BSDL were found in this
phase-containing membrane proteins
(21) . Taken together, these
data suggested that the multimeric complex might be effectively
associated with membranes of microsomes. The band intensities, assuming
the same methionine/cysteine contents for each protein, suggested that
p94 and BSDL associate stoichiometrically while 1 or 2 p56 molecules
could be involved in a putative complex.
Figure 6:
Identification of proteins that bind to
BSDL. [S]Methionine-labeled AR 4-2 J cell lysate
or microsomes from monensin-treated AR 4-2J cells were chromatographed
on an immobilized BSDL column. The column was washed with 5 mM
MES, pH 5.0, buffer with 0.5% Nonidet P-40, 0.1 M NaCl, and 1
mM EDTA to elute unbound material. Bound proteins were then
eluted with 0.1 M sodium phosphate, pH 8.0, buffer (0.15 NaCl
and 0.5% Nonidet P-40). Eluted proteins were concentrated and
electrophoresed on SDS-PAGE before autoradiography. Figure shows an
autoradiogram of the material bound to the immobilized BSDL column
starting from AR 4-2J cell lysate (lanea) or from AR
4-2J cell microsomes (laneb). Material eluted from
the immobilized BSDL column starting from microsomes was
electrophoresed on SDS-PAGE, transferred to a nitrocellulose membrane,
and probed with antibodies to rat BSDL (lanec). The
Triton X-114-rich phase obtained after temperature-induced phase
separation with Triton X-114 of
[
S]methionine-labeled microsomes was
immunoprecipitated with antibodies to rat BSDL. The precipitated
material was then electrophoresed on SDS-PAGE and autoradiographed
(lane d).
Since the co-isolation of
p94 and p56 with BSDL implies their physical association as a complex,
we sought to confirm this by taking advantage of the affinity of BSDL
for heparin
(28, 29) . For this purpose, radioactive
material eluted from the immobilized BSDL column was loaded onto a
heparin-Sepharose column equilibrated in a 10 mM Tris/HCl, pH
7.4, buffer. As shown on Fig. 7A, two radioactive peaks
were eluted with the Tris/HCl buffer containing 0.4 M NaCl and
1 NaCl, respectively. Most of the radioactive material (70%)
applied to the column was eluted with 0.4 M NaCl. Nearly 80%
of the activity on 4-nitrophenyl hexanoate of the starting material was
also detected in this fraction. The remaining radioactive material and
BSDL activity was associated with the material eluted with 1 M
NaCl and in the washing buffer. Radioactive material eluted in each
fraction was electrophoresed on SDS-PAGE and autoradiographed. As shown
on Fig. 7B (laneb), material eluted
at 0.4 M NaCl included p94, BSDL, and p56, while the material
with the lower mass (<50 kDa) became almost undetectable. No signal
was obtained after analysis of the material eluted at 1 M NaCl
(Fig. 7B, lane c) or eluted in the washing
fractions (Fig. 7B, lanea). These
data suggested that in microsomes at least two proteins, p94 and p56,
formed a membrane multimeric complex that associates with BSDL.
Figure 7:
Analysis of proteins that bind to BSDL on
heparin-Sepharose column. A, radioactive material eluted at
basic pH from the immobilized BSDL column was chromatographed on a
heparin-Sepharose column (1 ml) eluted with 10 mM Tris/HCl
buffer, pH 7.4. Bound material was eluted with the same buffer
containing 0.4 M NaCl and 1 M NaCl. Radioactivity
() and activity on 4-nitrophenyl hexanoate (
) were
determined in each eluted fraction. B, SDS-PAGE and
autoradiography of fractions eluted from the heparin-Sepharose column.
Lanea, unbound fraction; laneb,
fraction eluted with 0.4 M NaCl; lanec,
fraction eluted with 1 M NaCl.
Preventing Folding of BSDL Induced the Formation of a
Multimeric Complex
We tested whether the formation of the
membrane multimeric complex involving BSDL, p94, and p56 or possibly
lower molecular mass proteins was related to the folding state of BSDL,
as suspected from the activity deficiency of the membrane-associated
enzyme. For this experiment, monensin treatment was omitted and AR 4-2J
cells were grown in methionine-free DMEM in the presence of the proline
analogue, azetidine-2-carboxylic acid, which prevents folding of
nascent proteins
(30) . Under these conditions, misfolding of
BSDL should be drastic, as proline represents about 7% of the amino
acid residues of the protein (40/592). In addition, at the level of the
O-glycosylated C-terminal tandem repeated sequences, 1 amino
acid residue out of 3 is a proline
(24, 29) . After
starvation (45 min) in methionine-free DMEM in the presence of
azetidine-2-carboxylic acid (5 mM), cells were pulsed for 5
min with [S]methionine while still in the
presence of azetidine-2-carboxylic acid and chased with DMEM in the
absence of the proline analogue. Cell lysates were subjected to
immunoprecipitation with antibodies to rat BSDL before analysis on
SDS-PAGE. As shown on Fig. 8, in cells experiencing the metabolic
stress of azetidine-2-carboxylic acid, proteins of 94, 56, and 46 kDa
were co-precipitated with BSDL. As expected, neither BSDL activity nor
BSDL immunoreactive protein could be detected in the cell-free medium
of AR 4-2J cells incubated in the presence of the proline analogue.
However, when cells were grown in the absence of the proline analogue,
only traces of p94 and p46 were co-precipitated with BSDL, while the
enzyme was normally secreted. Therefore, misfolded BSDL interacts with
proteins p94, p56, and p46 involved in the multiprotein complex, which
appears to function as a folding complex. It is likely that this
folding complex formed within the ER and continued up to the Golgi
apparatus.
Ligand Blotting of Proteins in AR 4-2J Cells
We
next attempted to determine whether BSDL could bind directly to 94-,
56-, or 46-kDa proteins to form a well ordered complex. For this
purpose the biotin-labeled BSDL was used in ligand blotting to detect
proteins able to bind BSDL. The rat monensin-treated AR 4-2J cells were
lysed as described previously. After centrifugation (2000
g, 15 min), proteins were separated on SDS-PAGE and
electrotransferred onto a nitrocellulose membrane. The electrophoretic
migration of the BSDL was first observed by Western blotting using the
polyclonal antibodies to the rat enzyme (Fig. 9, lanea). The enzyme electrophoresed as a doublet at 70 and 74
kDa, probably representing unglycosylated and glycosylated BSDL,
respectively. When nitrocellulose membranes were incubated with
biotin-labeled BSDL, two bands were immunodetected by anti-biotin
antibodies conjugated to alkaline phosphatase (Fig. 9, laneb). The first one at
70-74 kDa corresponded to
the self-aggregation of BSDL molecules
(32) , and the second one,
at approximately 95 kDa correlated well with the previously detected
p94 protein that binds to the immobilized BSDL. Similar data were
obtained when isolated microsomes were analyzed (Fig. 9, lanee). These two bands were not detected in the control
experiment in which biotin-labeled BSDL was omitted (Fig. 9,
laned). A heparin-like binding site was detected
within the sequence of BSDL
(24, 29) . This site,
probably involved in the binding of the enzyme to the intestinal
brush-border
(32) , may be essential to the association of BSDL
with microsome membranes. To test this possibility, we performed a
ligand blotting in the presence of saturating amount of heparin (2
mg/ml). As shown on Fig. 9(lanec), heparin
did not hinder the self-association of BSDL and its binding to the
95-kDa protein. This last feature means that the interaction between
the 95-kDa protein and BSDL does not involve the heparin-binding site
present on the enzyme.
Figure 9:
Western blotting and ligand blotting.
Clarified lysates of monensin-treated AR 4-2J cell were electrophoresed
on SDS-PAGE and electrotransferred to nitrocellulose membrane (40
µg of cell proteins). Lanea, the membrane was
incubated with antibodies to rat BSDL and the immunodetection performed
with anti-IgG antibodies labeled with alkalin phosphatase;
lanes b-d, the membrane was incubated with
biotin-labeled BSDL and the binding reaction revealed with anti-biotin
antibodies labeled with alkaline phosphatase (laneb). Incubation was performed in the presence of heparin
(2 mg/ml, lanec), or the biotin-labeled BSDL was
omitted (laned). Lanee, proteins
from AR 4-2 J cells microsomes (10 µg) were separated on SDS-PAGE
before transfer and affinity blotting performed as in lane
b.
Characterization of the p94 as a Grp 94-related
Protein
The presence of chaperone proteins along the secretion
pathway of acinar pancreatic cells has been described
recently
(4) . Because of the size homology between the p94 and
the 94-kDa glucose-regulated protein (Grp 94) and because the Grp 94
was frequently associated with proteins around 40-50 kDa
(2) as found here, we investigated the possible participation of
this former chaperone protein in the secretory process of BSDL. The Grp
94 chaperone was detected in monensin-treated AR 4-2J cell lysates by
Western blot using a specific monoclonal antibody (clone 9G10). Since
this chaperone protein may be involved in the multimeric complex and
might assist the folding of BSDL, we first attempted to detect Grp 94
in the material isolated from the immobilized BSDL column. As shown
above, when microsomes isolated from monensin-treated AR 4-2J cells
were chromatographed on the immobilized BSDL column, a multimeric
complex was isolated (see Fig. 6, laneb or
Fig. 10A, lanea). In this complex, we
demonstrated that the 70-74-kDa protein was the BSDL as this
material reacted with antibodies to rat BSDL (see Fig. 6,
lanec). We then, used monoclonal antibody to Grp 94
to determine whether this chaperone protein could be detected in the
multimeric complex involving BSDL. As shown in Fig. 10A (laneb), the antibody specific for Grp 94
actually recognized the 94-kDa band. The antibody to Grp 94 was
therefore used to immunoprecipitate the radioactive material eluted
from the column of immobilized BSDL and concentrated by lyophilization.
After SDS-PAGE and autoradiography, immunoprecipitated proteins
resolved into proteins at 94 and 74 kDa (Fig. 10A,
lanec). The two other bands detected at 56 and 46
kDa were also co-precipitated
(Fig. 10A, lanea).
Presumably, these two bands corresponded to those already detected (see
Fig. 8
). These results suggested that the Grp 94 was retained by
the immobilized BSDL column and was actually involved in the multimeric
complex that associates p56, p46, and BSDL. In another set of
experiments, monensin-treated AR 4-2J cells were labeled with
[S]methionine and lysed. The cell lysate was
clarified by centrifugation and immunoprecipitated with antibody to Grp
94. Analysis of the immunoprecipitated material on SDS-PAGE indicated
that essentially three proteins of 120, 70/74, and 58 kDa
co-precipitated with the Grp 94 (Fig. 10B, lanea, asterisk), one of which was suspected to be
BSDL (arrow). A second immunoprecipitation was therefore
performed on this material after dissociation of complexes
(22) using either monoclonal antibody specific for Grp 94 or
polyclonal antibodies to rat BSDL. As shown on Fig. 10B,
the first antibody immunoprecipitated two proteins of 95 and 87 kDa
(laneb), while antibodies to rat BSDL precipitated
only one protein, the size of which correlated with BSDL (lanec). The 87-kDa protein could be the unprocessed Grp
94
(33) . When the two-cycle immunoprecipitation procedure was
performed as above, first with antibodies to rat BSDL and subsequently
with either antibodies to rat BSDL or Grp 94, the same pattern was
obtained. As shown on Fig. 10C, the first
immunoprecipitation (lanea) indicates that BSDL was
effectively precipitated (arrow) and that a protein at 94 kDa
(asterisk) is co-precipitated along with proteins at 50/56 and
43 kDa. Subsequent precipitation with either antibodies to rat BSDL
(laneb) or Grp 94 (lanec)
detected the respective protein. Therefore, we can conclude that the
p94 might be related to the Grp 94. The association between the Grp
94-related protein and BSDL was indicated first by the co-precipitation
of the two proteins by specific antibodies directed against each
individual species, and second by their co-isolation by chromatography
on immobilized BSDL column (see Fig. 6, lanec and Fig. 10A, laneb).
Figure 10:
Characterization of p94 as a Grp
94-related protein. A, detection of the Grp 94 chaperone
protein in the multimeric complex that binds to the immobilized BSDL
column. [S]Methionine-labeled proteins from
microsomes of monensin-treated AR 4-2J cells were chromatographed on
the immobilized BSDL column. Material eluted at basic pH was
concentrated and submitted to SDS-PAGE (lanea) or
electrotransferred to nitrocellulose membrane and probed with the
monoclonal antibody to Grp 94 (laneb). The material
eluted at basic pH was also concentrated and immunoprecipitated using
the monoclonal antibody to Grp 94 (lanec). B and C, two-cycle immunoprecipitation. Two-cycle
immunoprecipitation was performed on cell lysate of monensin-treated AR
4-2J cells after metabolic labeling with
[
S]methionine. The first cycle was performed
using either monoclonal antibody to Grp 94 (B, lanea) or antibodies to rat BSDL (C, lane
a), followed by a second immunoprecipitation with monoclonal
antibody to Grp 94 (B, laneb; C,
lanec) or polyclonal antibodies to rat BSDL
(B, lanec; C, laneb).
-amylase, the secretion
of BSDL was sensitive to the impairment of the Golgi apparatus by
monensin and brefeldin A. Cytoskeleton-disrupting agents (cytochalasin
B and vinblastin sulfate) had a poor effect on the secretion (at least
at the concentration used here). CCCP, a respiratory inhibitor, blocked
the secretion of both
-amylase and BSDL, probably by inhibiting
the fusion of secretory vesicules with target membranes, an event that
is energy-dependent
(38) . As shown by temperature-induced phase
separation with Triton X-114, the impairment of the secretion pathway
by the ionophore monensin induced the association of BSDL to microsome
membranes. This was in contrast to the behavior of
-amylase which,
even after cell treatment by monensin, remained associated with the
soluble fraction of microsomes. The association of the former enzyme
did not involve signal sequence (KDEL sequence or other) or anchoring
by means of lipids (acylation or glycosyl phosphatidylinositol tail)
but by a membrane multiprotein complex. This multiprotein complex
implicated at least three proteins, p94, p56, and p46, of which p94
binds directly to BSDL. The association of BSDL with microsome
membranes upon monensin treatment might therefore be due to a direct
interaction of the enzyme with the 94-kDa protein. The
membrane-associated BSDL was enzymatically inactive or poorly active in
contrast to its soluble form. This provides evidence that the
membrane-associated BSDL might not be correctly folded, either because
monensin interrupted the secretory process and consequently stopped the
folding process or because this drug acted directly on the folding
process by affecting, for example, the maturation of the BSDL
glycosylation
(39) . In the absence of monensin, the multiprotein
complex cannot be co-immunoprecipitated with BSDL which therefore
appeared transiently associated to p94. In conditions of metabolic
stress, such as protein synthesis in the presence of the proline
analogue azetidine-2-carboxylic acid, the association appeared
permanent. A model consistent with all these findings suggests that the
multimeric complex, more precisely the association with p94, may
facilitate the proper folding of BSDL.
-galactoside
1,2-fucosyltransferase
and
-galactoside
2,6-sialyltransferase, respectively, two
transferases most probably involved in the terminal glycosylation of
BSDL
(15) . The protein disulfide isomerase (molecular mass
56 kDa), the expression of which is induced by the calcium
ionophore A 23187 (47), may also be involved in the folding complex of
BSDL. Unfortunately, precipitating antibodies to these proteins are not
available to test this hypothesis.
-amylase. Therefore, the particular secretion
pathway of BSDL, involving the association with the Grp 94-related p94,
may be due to specific structural properties of this protein. Although
multiple reasons may be advocated, the most probable one would lie in
the fact that BSDL is the unique secretory pancreatic protein with
mucin-like tandem-repeated sequences on which O-linked
oligosaccharides are clustered. This cluster encoded by exon 11
(48) is located on the extended C-terminal domains rich in
serine, threonine, and proline residues
(7) . When this domain
was deleted by recombinant DNA techniques, the truncated BSDL was
normally expressed and displayed full activity
(49, 50) .
In contrast, transfection of BSDL cDNA with 88% of the exon 11 residues
deleted from the sequence resulted in a protein that was not
secreted
(49) . These data suggested that a domain encoded by
exon 11 of BSDL is required for intracellular processing and secretion
of the normal enzyme
(49) . This domain, between residues
490-534
(49) , may be responsible for the interaction
between p94 and BSDL. We can therefore suggest that the association of
BSDL with the Grp 94-related p94 protein is essential for the
processing (i.e. glycosylation) of the C-terminal
tandem-repeated sequences. Proteins that are rapidly degraded within
eukaryotic cells contain regions rich in proline, glutamic acid,
serine, and threonine (PEST region) associated with abundant doublets
of basic amino acids (51). Many of these doublets are present within
the sequence of BSDL, and the PEST regions coexist with the cluster of
O-linked oligosaccharides
(24, 52) . A survey of
the literature demonstrated that these sequence features are absent in
sequences of other enzymes of the pancreatic secretion. Of significant
importance is that no intracellular degradation of BSDL occurred in
pancreatic cells upon treatment with drugs that inhibits the enzyme
secretion
(10) . Hence, association with membranes by means of
the Grp 94-related p94 chaperone protein and consequently glycosylation
of PEST sequences may contribute to take the pancreatic BSDL away from
a possible degradation route and to its secretion by acinar pancreatic
cells. Once fully glycosylated, the enzyme is released from the Grp
94-related p94 either in or after the trans-Golgi compartment
and aggregated in the trans-Golgi network
(31) . The
releasing mechanism is still unknown, but proteoglycans involved in the
condensing process of secretory proteins may play a role
(53) .
Whenever the PEST sequences and the domain 490-534 are deleted or
absent, BSDL enters the pathway shared by all pancreatic secreted
proteins
(23) .
Table:
Effect of drugs on the secretion of BSDL and
-amylase by AR 4-2J cells
-amylase activity. Values
are means ± S.E. of at least six independent experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.