Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS 66160, USA
e-mail: rdelisle{at}kumc.edu
Accepted 1 May 2002
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Summary |
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Key words: Pancreas, Muclin, Secretory granule
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Introduction |
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Common constituents of regulated secretory granules are sulfated
macromolecules, and in this study I investigated the roles of sulfated
O-linked glycoproteins in formation of zymogen granules in the exocrine
pancreas. In the pancreas, the acinar cell synthesizes 20-25 different
digestive enzymes and proenzymes (zymogens), which are all stored in zymogen
granules (Scheele, 1975).
Although these proteins differ widely in their molecular weights and
isoelectric points, they coaggregate at slightly acidic pH values known to
occur in the TGN (Leblond et al.,
1993
), the major sorting station in the secretory pathway. Sorting
continues in the condensing vacuole (immature secretory granule) as the
granule contents further condense and non-regulated secretory proteins are
excluded and trafficked to lysosomes and to the plasma membrane via the
constitutive-like secretory pathway (Arvan
and Castle, 1998
; Arvan and
Chang, 1987
). Upon exocytosis of the zymogen granule, the
digestive enzymes are exposed to the alkaline pH of the acinar/ductal lumen,
and the proteins are solubilized for their transit to the duodenum
(Freedman et al., 1998
). These
facts indicate that ionic interactions mediated by acidic pH in the distal
secretory pathway and basic pH in the acinar lumen control protein packaging
and subsequent release.
Recent findings show that specific molecules in the secretory pathway can
have profound effects on formation of regulated secretory granules and that
post-translational modification of these molecules may be key to their
function. For example, in mast cells, the polyanion glycosaminoglycan heparin
sulfate is required for normal granule formation
(Humphries et al., 1999;
Forsberg et al., 1999
). Also,
in neuroendocrine cells, the sulfated glycoprotein chromogranin A is necessary
for secretory granule formation and proper storage of regulated secretory
proteins (Kim et al.,
2001
).
In contrast to mast cells and neuroendocrine cells, there is less known
about the specific molecules involved in forming zymogen granules in exocrine
cells. The molecules in the pancreatic acinar cell that support this function
are not firmly established, but they are likely to be sulfated glycoproteins
and/or proteoglycans. It appears that there are species differences in that
mice have O-linked glycoproteins (De Lisle,
1994), whereas rats and guinea pigs have proteoglycans in their
zymogen granules (Schmidt et al.,
2000
; Reggio and Palade,
1978
). Recent work using rat pancreas has implicated the
lectin-like molecule ZG16p in zymogen granule formation
(Kleene et al., 1999
) in
concert with granule-membrane-associated proteoglycans
(Schmidt et al., 2000
). The
mouse pancreatic acinar cell contains a limited number of sulfated O-linked
glycoproteins, which are more easily studied biochemically than are
proteoglycans. The major mouse sulfated glycoprotein is Muclin, a 300 kDa
structural protein of the zymogen granule that may have a role in protein
sorting in the regulated pathway (De Lisle
and Ziemer, 2000
). The other major sulfated proteins of the mouse
acinar cell are the zymogens prolipase and proelastase IV, and p82/p75, a
marker of the constitutive-like pathway associated with secretory granule
maturation (De Lisle and Bansal,
1996
).
In this report I focus on these sulfated glycoproteins and the role of their sulfates and O-linked carbohydrates in formation of mouse zymogen granules. When sulfation or O-linked glycosylation are perturbed, the secretory function is reduced, and this is accompanied by morphological alterations that provide a way of interpreting how the regulated pathway is affected by altering sulfated glycoprotein processing.
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Materials and Methods |
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Preparation of pancreatic acini and radiolabeling
Pancreata from mice (ND4, Swiss Webster strain, Harlan, Indianapolis, IN)
were digested with purified collagenase, mechanically dispersed, and acini
were purified by filtration followed by centrifugation on a step gradient of
4% bovine serum albumin as described previously
(De Lisle and Bansal, 1996).
The isolated acini were suspended in Hepes-buffered Ringer's solution (HR
buffer) supplemented with 0-30 mM NaClO3 (chlorate) or 0-32 mM
benzyl-N-acetyl-
-galactosaminide (BzlGalNAc) from a 1.6 M stock in
dimethylsulfoxide (DMSO). For experiments with chlorate-treated cells, all
media were supplemented with NaCl to obtain a final 30 mEq extra sodium,
matching the highest concentration of Na added as sodium chlorate. For
experiments with BzlGalNAc-treated cells, the final concentration of DMSO was
2% in all cell aliquots, matching the maximum [DMSO] added with 32 mM
BzlGalNAc. The cells were biosynthetically labeled in the presence of the
indicated chemicals: 60 minutes with 100 µCi/ml [35S]sulfate; 60
minutes with 1 µCi/ml [3H]leucine; or 16 hours with 40 µCi/ml
[3H]glucosamine using media without glucose and supplemented with
22 mM pyruvate to enhance uptake of the labeled sugar. After labeling, cells
were pelleted and aliquots were run on SDS-PAGE. Gels of
[35S]-labeled cell proteins were phosphorimaged (Cyclone, Packard
Instruments, Meriden, CT). Gels of [3H]glucosamine labeled protein
were treated with Enhance (NEN, Boston, MA) followed by fluorography for 20
days on X-ray film. [3H]leucine-labeled protein was precipitated
with 10% trichloroacetic acid followed by liquid scintillation counting.
Pulse-chase analysis
For pulse chase analysis, met/cys-free medium (Sigma) was used during the
cell isolation procedure to deplete the cells of these amino acids. The
isolated acini were then pulse labeled with 0.5 mCi/ml [35S]met/cys
(Tran35S-Label, ICN, Costa Mesa, CA) for 30 minutes in met/cys-free
medium. The cells were washed and resuspended at 0.4-1 mg cell protein per ml
of chase medium (HR with three times the usual amino acid concentration and 50
µg/ml soybean trypsin inhibitor) alone (control) or supplemented with 30 mM
NaCl, 30 mM chlorate, 2% DMSO or 32 mM BzlGalNAc. The cells were aliquoted at
1 ml per well into 24-well plates and incubated at 37°C. As indicated,
cells received a final concentration of 1 µM carbachol and 1 mM 8-Br-cAMP
for the final 30 minutes of incubation to stimulate regulated secretion of
[35S]-labeled newly made protein. At the indicated times, cells
were transferred to siliconized microfuge tubes (Sigma), pelleted and the
media and cell pellets saved. The entire 1 ml of the media samples was
precipitated with 10% trichloroacetic acid, and the pellets were solubilized
in SDS-PAGE sample buffer plus 1-2 µl 1 M Tris-OH to keep the pH alkaline.
Cell pellets (10% of the total) and the entire precipitated media samples were
run on SDS-PAGE followed by phosphorimaging. In separate experiments, media
from unlabeled cells were precipitated, run on 10% SDS-PAGE and Coomassie blue
stained for quantification of released total amylase. Protein bands were
quantified from scanned gels (Hewlett Packard ScanJet IIcx, Palo Alto, CA)
using OptiQuant software (Packard Instruments). Muclin and its precursor
pro-Muclin are of unique Mr in the acinar cell, which has
been verified by immunoprecipitation (De
Lisle and Ziemer, 2000). Therefore, it is straightforward to
examine pro-Muclin and Muclin in whole tissue and cell homogenates by
SDS-PAGE. p80, the C-terminal cleavage product of pro-Muclin, was
immunopreciptated from cell homogenates as described using an antiserum to the
13 C-terminal amino acids (De Lisle and
Ziemer, 2000
). Carbohydrates on Muclin were assessed by lectin
blotting with peanut agglutinin (PNA; specific for Gal ß(1-3)GalNAc) and
Maackia amurensis agglutinin (MAA; specific for NANA
(2-3)Gal) as
described previously (De Lisle et al.,
1998
).
In vivo zymogen granule depletion and in vitro recovery
To deplete stored zymogen granules, mice were injected with a cholinergic
agonist (10 µg pilocarpine per g body weight i.p.). After 1 hour, the
pancreas contained 50% of its initial amylase and Muclin content (not
shown), and the majority of zymogen granules were depleted (c.f.
Fig. 7A and B). The pancreas
was then removed and lobules prepared by injecting HR buffer into the tissue
and dissecting out the distended lobules. The lobules were injected with HR
supplemented with 30 mM NaCl, 30 mM chlorate, 2% DMSO (vehicle) or 32 mM
BzlGalNAc. This allowed the zymogen-granule-depleted tissue to be exposed to
the test chemicals within 15 minutes of sacrifice of the mouse. The lobules
were incubated at 37°C with agitation at 120 rpm in a shaking waterbath
and oxygenation every 30 minutes. In control lobules, the amylase content was
90% of untreated levels after 4 hours of incubation (data not shown), and
the cells were refilled with zymogen granules
(Fig. 7C,G).
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Morphology and immunocytochemistry
For morphology studies, tissues were fixed in 4% paraformaldehyde, 1.6%
glutaraldehyde in phosphate-buffered saline and processed for light and
electron microscopy. Thick plastic sections (1 µm) were stained with
toluidine blue, and images were obtained with differential interference
contrast optics on a Nikon Diaphot microscope (Nikon, New York, NY). Thin
sections were prepared and imaged on a JEOL 100CX-11 electron microscope. For
indirect immunofluorescence, the tissue was fixed for 1 hour in 2%
paraformaldehyde, 0.1% saponin to permeabilize cell membranes and
cryoprotected as described previously
(Barthel and Raymond, 1990).
Cryosections were cut at 5 µm. Immunostaining was performed with rabbit
anti-Muclin (De Lisle and Ziemer,
2000
), sheep anti-amylase (The Binding Site, San Diego, CA), both
diluted 1:500 in 2% normal donkey serum or rat monoclonal antibody to the
lysosomal membrane protein LAMP-1 (clone 1DB4; Development Studies Hybridoma
Bank, University of Iowa), diluted 1:5. The secondary antibodies were donkey
anti-rabbit-FITC, donkey anti-sheep-Texas Red and donkey anti-rat-Texas Red
(Jackson ImmunoResearch, West Grove, PA), and
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to label
the nuclei. Images were obtained with a SPOT II digital camera (Diagnostic
Instruments, Sterling Heights, MI) on a Nikon Diaphot microscope with
appropriate filter sets.
Cell viability was assessed using the LIVE/DEAD kit from Molecular Probes (Eugene, OR). Acini were incubated at 37°C under the indicated conditions for the indicated times. The LIVE/DEAD reagent, which consists of calcein-acetoxymethyl ester and ethidium-homodimer, was added and the incubation continued for 10 minutes at 37°C. The cells were then imaged on the Nikon inverted microscope, and green and red images obtained to show live (calcein-positive) and dead (ethidium-homodimer labeled nuclei) cells. The images were analyzed using Scion Image software (Scion Corp., www.scioncorp.com ) to quantify the area occupied by red-labeled nuclei. Total cell death was achieved by adding Triton X-100 to 1% and was used to calculate the percentage of dead cells under the different incubation conditions.
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Results |
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The specificity of the aggregation was tested by including a constitutively secreted protein, bovine serum albumin. Serum albumin did not exhibit acid-mediated aggregation itself nor did it affect acid-mediated ZGC aggregation (Fig. 1B,C). Also tested was whether aggregation is affected by the presence or absence of Ca2+, which is known to be at millimolar concentrations in the TGN. Chelation of calcium associated with the ZGC by adding 5 mM EGTA had little effect on acid-mediated ZGC aggregation nor did addition of 2 mM exogenous CaCl2 (Fig. 1B,C). Neither EGTA nor Ca2+ had any effect up to 10 mM (not shown).
Inhibition of sulfation with sodium chlorate
Chemical inhibitors of post-translational processing were used with
isolated mouse acinar cells and pancreatic lobules from in vivo
zymogen-granule-depleted tissue to test the role of sulfated O-glycoproteins
in granule formation and maturation. Sodium chlorate was used as a competitive
inhibitor of ATP-sulfurylase, a key enzyme in synthesis of the high energy
sulfate donor 3'-phosphoadenosine-5'-phosphosulfate
(Baeuerle and Huttner, 1986).
Chlorate dose-dependently inhibited incorporation of [35S]sulfate
into pancreatic acinar cell proteins (Fig.
2A,B), which, in addition to Muclin, are p82/75, a marker of the
constitutive-like secretory pathway (but unrelated to the
80 kDa
proteolytic fragment of pro-Muclin called p80) and the pancreatic digestive
enzymes prolipase and proelastase IV (De
Lisle and Bansal, 1996
). The maximal effect on sulfation was
between 10-30 mM chlorate, with >95% inhibition of sulfation for all
proteins (Fig. 2B). By
contrast, chlorate had no effect on protein synthesis as measured by
incorporation of [3H]leucine into trichloroacetic acid precipitable
protein (Fig. 2C).
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Inhibition of sulfation and O-glycosylation with BzlGalNAc
Benzyl-N-acetyl--galactosaminide (BzlGalNAc), which acts as an
acceptor for O-linked oligosaccharides, was used to block elongation of these
sugars on glycoproteins (Delannoy et al.,
1996
). In the acinar cell, the majority of sulfate is on O-linked
sugars (De Lisle and Bansal,
1996
), so it was expected that blockage of O-linked
oligosaccharide elongation would reduce sulfation as the normal sites would
not be synthesized. BzlGalNAc dose-dependently inhibited sulfation of acinar
cell proteins (Fig. 3A). It was
noticed that a broad area of radioactivity appeared on the phosphorimages of
BzlGalNAc-treated cells, that this labeled material migrated to the same
region of the gels largely independently of the percentage acrylamide used and
was best separated from the labeled proteins on 12.5% acrylamide
(Fig. 3B). This labeled
material probably represents sulfated BzlGalNAc metabolites
(Delannoy et al., 1996
;
Huang et al., 1992
). Although
the incorporation of [35S]sulfate into BzlGalNac derivatives was
diffuse, the maximum amount of radioactivity in this material was equivalent
to 169% and 49% of that incorporated into prolipase and proelastase,
respectively, under control conditions. To avoid these areas, samples were run
on both 7.5% and 12.5% acrylamide gels
(Fig. 3A,B) for quantification
of the specific protein bands. The maximum effect of BzlGalNAc on glycoprotein
sulfation was at 16-32 mM BzlGalNAc, and inhibition ranged from 54% to 75%,
showing some differences among the different proteins
(Fig. 3C).
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The effect of BzlGalNAc on total protein oligosaccharide synthesis was
estimated by labeling with [3H]glucosamine, which can be used by
cells as a precursor for GlcNAc, GalNAc and sialic acid and thus will be
incorporated into most N- and O-linked oligosaccharides
(Beeley, 1985). Only Muclin was
appreciably labeled with [3H]glucosamine, and BzlGalNAc
dose-dependently inhibited this labeling, with almost complete inhibition at
16-32 mM (Fig. 3D). BzlGalNAc
had only minor effects on total protein synthesis and caused
20%
inhibition of [3H]leucine incorporation into TCA-precipitable
protein at 32 mM (Fig. 3E).
Effects of chlorate and BzlGalNAc on post-translational processing of
pro-Muclin
I next examined the effects of chlorate and BzlGalNAc on the biosynthetic
maturation of Muclin. Muclin is synthesized as pro-Muclin, a type I membrane
protein that undergoes extensive N- and O-linked glycosylation and sulfation
of its O-linked sugars in the TGN. In a post-Golgi compartment pro-Muclin is
proteolytically cleaved to mature sulfated Muclin, which is luminal and
remains in the zymogen granule, and an 80 kDa non-sulfated glycoprotein (p80),
which is trafficked to the apical plasma membrane of the acinar cell
(De Lisle and Ziemer, 2000).
As shown in Fig. 4, chlorate
had little effect on processing of pro-Muclin to Muclin and p80. By contrast,
BzlGalNac slowed the maturation process, resulting in slower disappearance of
pro-Muclin (Fig. 4B) and
reduced production of Muclin (Fig.
4C) and p80 (Fig.
4D). There was a slight effect of the vehicle (DMSO) on the
accumulation of mature Muclin, but it was nowhere near as great as the
BzlGalNAc effect. In addition, the Mr of Muclin in the
presence of BzlGalNAc was less than normal, reflecting the inhibition of
O-linked oligosaccharide elongation (Fig.
4A, asterisk).
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Previous work using BzlGalNAc in various cultured cell lines has reported
alterations in terminal sugar composition of glycoproteins, specifically
increased T antigen and decreased sialic acid
(Delannoy et al., 1996).
Therefore, I examined binding of PNA (T antigen) and MAA (sialic acid) to
acinar cell proteins synthesized in the presence of chlorate or BzlGalNAc for
4 hours. It was shown previously that Muclin is the major PNA- and MAA-binding
glycoprotein in the mouse acinar cell (De
Lisle, 1994
), and this is apparent on the lectin blots of whole
pancreatic tissue shown in Fig.
4E. BzlGalNAc caused an increase in PNA binding and a reduction in
MAA binding to Muclin (Fig.
4E). In addition, the reactive band in both lectin blots was of
lower Mr in BzlGalNAc-treated cells, and the shift was
about the same magnitude as seen in [35S]met/cys pulse-labeled and
chased protein (Fig. 4A).
Effects of chlorate and BzlGalNAc on basal and stimulated protein
secretion
I next examined the effects of these chemicals on basal and regulated
protein secretion by [35S]met/cys pulse-chase analysis using
freshly isolated pancreatic acini. Cells were labeled in control medium and
chased in the presence or absence of the inhibitory chemicals. As shown in
Fig. 5A and quantified in
Fig. 5B,C, untreated acini have
a basal release of newly made protein of 1.7% per hour (control). In the
presence of chlorate during the chase, there was a modest inhibitory effect on
basal secretion by 6 hours of chase to 65% of control
(Fig. 5A,B). When stimulated
with a cholinergic agonist (1 µM carbamylcholine chloride) and a
membrane-permeant cAMP analog (1 mM 8-Br-cAMP) for the last 30 minutes of
chase, the control cells responded with an increase in the rate of protein
release of 6.8-fold compared with the basal level. Treatment with chlorate had
no effect on the stimulated rate of protein secretion (7.1-fold increase
compared with basal), but the cumulative amount of newly synthesized protein
released was reduced, largely reflecting the decrease in basal release
(Fig. 5B). Also shown in
Fig. 5 is the control using 30
mM NaCl to match the additional salt added as sodium chlorate in these
experiments; NaCl had no effect on basal or stimulated secretion.
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Isolated acini treated with BzlGalNAc exhibited decreased basal release of
newly synthesized protein to 50% of control by 6 hours
(Fig. 5A,C). More dramatically,
stimulated secretion was almost totally inhibited by BzlGalNAc
(Fig. 5A,C). Also shown in
Fig. 5 is the effect of
dimethylsulfoxide (DMSO) used as a vehicle for BzlGalNAc. DMSO had a slight
effect on basal secretion (82% of control) but none on the increased rate upon
stimulation of the cells.
To see if BzlGalNAc also blocked release of amylase from prestored zymogen granules, Coomassie-blue-stained gels of secreted proteins from separate experiments using unlabeled cells under the same conditions were examined. As shown in Fig. 5D, BzlGalNAc slowed basal release of prestored amylase compared with the other conditions used. However, when secretion was stimulated, there was a similar increase in the rate of amylase release from BzlGalNAc-treated cells as from the other conditions used. Thus, BzlGalNAc inhibits traffic through the secretory pathway but does not affect stimulated release of amylase that had already reached zymogen granules before addition of BzlGalNAc.
Since secretion of amylase was reduced by these treatments, it was of interest to determine whether newly made amylase was accumulating or being degraded in the treated cells. The [35S]-labeled amylase remaining in the cells after 6 hours of chase was found to be similar under all conditions tested but was slightly elevated in the presence of BzlGalNAc (Fig. 5E). This result indicates that blockage of amylase release in the presence of BzlGalNAc causes some accumulation in the cell.
Morphological examination of treated cells (below) indicated that these chemical treatments were not toxic to the cells. To measure this more directly, a combination of calcein-acetoxymethyl ester and ethidium homodimer was used to label healthy and dead cells, respectively (see Materials and Methods). As shown in Fig. 6, freshly isolated acini had a small number of dead cells but most were healthy (Fig. 1A,E). Use of Triton X-100 to kill the cells shows the opposite labeling pattern with no calcein staining and all nuclei labeled with ethidium homodimer (Fig. 6B,F). Under control conditions, after 6 hours of incubation, there was a small increase in dead cells, and there was a similar increase in the presence of NaCl or chlorate (Fig. 6I). By contrast, there was a greater amount of dead cells after 6 hours of incubation in the presence of BzlGalNAc (Fig. 6D,H,I). However, DMSO alone showed a similar increase in cell death (Fig. 6D) but, importantly, DMSO did not exhibit the blockage of stimulated release of newly synthesized amylase that BzlGalNAc did (Fig. 5C).
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Morphological effects on acinar cell secretory organelles of chlorate
and BzlGalNAc
I next examined the morphological effects of these chemicals on the
secretory pathway of the acinar cell. To help visualize changes in newly
forming secretory granules, zymogen granules were first depleted in vivo and
then allowed to reform during in vitro incubations (see Materials and
Methods). As shown by comparing uninjected control
(Fig. 7A) with 1 hour
post-pilocarpine (Fig. 7B),
there was an almost total loss of zymogen granules after in vivo stimulation.
The pilocarpine ZG-depleted tissue was healthy, and during a 4 hour in vitro
incubation under control conditions the cells refilled with zymogen granules
(Fig. 7C,E,G,I) and the amylase
content recovered to about 90% of initial levels observed in unstimulated
control tissue (data not shown).
When zymogen-granule-depleted lobules were incubated in vitro with chlorate, they synthesized some new zymogen granules, but the cells also had an accumulation of low density vacuoles in the perinuclear region of the cell (Fig. 7D), whereas control cells had a normal morphology (Fig. 7C). At the ultrastructural level, an accumulation of electron-lucent vacuoles was observed near the nucleus as well as normal appearing zymogen granules (Fig. 7F). The vacuoles contained varying densities of loose protein aggregates. By contrast, the control cells had normal zymogen granules, including protein aggregates in the Golgi indicating ongoing granule formation (Fig. 7E).
When zymogen-granule-depleted lobules were incubated in vitro with BzlGalNAc, they made very few normal appearing granules and had an extensive accumulation of low density vacuoles reaching from the perinuclear region toward the apical surface of the cells (Fig. 7H). At the ultrastructural level, the BzlGalNAc-treated cells contained an accumulation of electron-lucent vacuoles with some poorly condensed protein in their lumina (Fig. 7J). The control cells, treated with DMSO, were normal in appearance and had normal electron-dense zymogen granules (Fig. 7G,I).
Localization of Muclin and amylase in chlorate and BzlGalNAc-treated
pancreatic cells
To examine the subcellular localizations of the major regulated secretory
protein, amylase, and the major granule structural component, Muclin, frozen
sections of lobules were examined following a 4 hour in vitro recovery
incubation in the presence of chlorate or BzlGalNAc. As shown in
Fig. 8, amylase and Muclin are
largely colocalized in control cells (A: NaCl and C: DMSO) after a 4 hour
recovery incubation, as they are in untreated pancreas (not shown). The
cytoplasm from the perinuclear region up to the apical plasma membrane is
replete with zymogen granules. Treatment with chlorate during the recovery
period caused the appearance of some larger labeled structures for both
proteins (Fig. 8B; arrows).
However, amylase and Muclin were still largely colocalized after chlorate
treatment. By contrast, after treatment with BzlGalNAc during the recovery
incubation, there are noticeable changes in amylase and Muclin distributions
(Fig. 8D). Amylase labeling is
in large clusters in the perinuclear region of the cells
(Fig. 8D; arrowheads),
corresponding to tthe vacuoles in Fig.
7J. On the other hand, Muclin is somewhat more diffusely localized
in the cytoplasm apical to that of amylase
(Fig. 8D; arrows). After
BzlGalNAc treatment, amylase and Muclin are in separate compartments of the
cells, with amylase remaining in the perinuclear vacuoles and Muclin
progressing further towards that apical pole of the cell.
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It was of interest to see if protein sorting from the TGN in general was perturbed by BzlGalNAc treatment. Therefore, the localization of the lysosomal marker protein LAMP-1 was compared with Muclin and amylase in treated tissue. Normal acinar cells have relatively few lysosomes that are located peripherally to the ZG-rich apical cytoplasm (Fig. 9A). Treatment with chlorate had no discernable alteration in LAMP-1 distribution (not shown). By contrast, BzlGalNAc-treated cells appeared to have more LAMP-1-positive structures that were somewhat larger (Fig. 9B,C). However, LAMP-1 was not seen to overlap with either Muclin (Fig. 9B) or amylase (Fig. 9C) in BzlGalNAc-treated cells. This is consistent with continued proper sorting of lysosomal proteins from the TGN in the presence of BzlGalNAc.
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Discussion |
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Understanding of the second process, association of the aggregates with TGN
membranes, is less well established, but recent data show that there are cargo
receptors that mediate association of the aggregating proteins with the TGN
membrane. A strong case has been made that carboxypeptidease E is such a cargo
receptor. In mice with defective carboxypeptidase E
(Cpefat/Cpefat), redirection from the regulated pathway
to the constitutive pathway occurs for several hormones and neuropeptides
(Cool et al., 1997). However,
whether this effect is due to loss of carboxypeptidase E itself or is
secondary is not clear. These mice also have decreased expression of
prohormone convertase enzymes, which are required for proteolytic cleavage of
hormone precursors (Berman et al.,
2001
), and this processing affects hormone storage in regulated
granules (Irminger et al.,
1997
).
The data presented in the current paper have significance for both of these processes and demonstrate that correct sulfation and O-linked oligosaccharide addition to glycoproteins in the pancreatic acinar cell are required for normal zymogen granule formation and regulated protein secretion. Inhibition of sulfation has a relatively mild effect on transit through the regulated secretory pathway in the acinar cell. The main effect is to slow delivery of newly made proteins to a regulated secretory compartment without blocking their subsequent stimulated release. Nevertheless, inhibition of sulfation causes the accumulation of vacuoles in the Golgi region of the cell, although normal zymogen granules still form. These observations indicate that when sulfation is blocked, the kinetics of granule formation are slowed but not totally blocked and that protein sorting to the regulated secretory pathway still occurs. Along with pro-Muclin, also participating in the condensation process are two sulfated digestive enzyme precursors, prolipase and proelastase.
In contrast to inhibition of sulfation by chlorate, BzlGalNAc, which blocks O-linked oligosaccharide elongation and only partially blocks sulfation, has more profound effects on the regulated secretory pathway. Not only do immature granules accumulate to a greater extent, but both unstimulated and regulated secretion of newly made protein is blocked. These effects are not caused by toxicity of BzlGalNAc for the following reasons: (i) protein synthesis is only mildly affected by BzlGalNAc; (ii) other than accumulation of immature secretory vacuoles, cellular morphology is healthy looking, with intact plasma membranes and normal appearing nuclei; (iii) although there is a small increase in cell death, this is due to the vehicle (DMSO), and the small increase in cell damage is not sufficient to account for the profound effect on regulated secretion; and (iv) the stimulated secretion of prestored amylase, which is present in mature zymogen granules, from BzlGalNAc-treated cells is similar to controls. The effects of BzlGalNAc appear to be specific for protein transport in the regulated pathway, as lysosomal protein sorting is not perturbed by either chlorate or BzlGalNAc, as shown by immunolabeling for LAMP-1. In BzlGalNAc-treated tissue, there appeared to be larger organelles labeled for LAMP-1, but there was no colocalization of LAMP-1 with either Muclin or amylase.
At this time it is not possible to directly identify the key glycoprotein
affected by BzlGalNAc, but we propose that these effects are caused by its
perturbation of processing of pro-Muclin, which we have put forth as a
putative cargo receptor in the acinar cell
(De Lisle and Ziemer, 2000).
Pro-Muclin is a type I membrane protein that is cleaved in a post-Golgi
compartment, presumably the immature granule, to yield Muclin and the membrane
protein p80 (De Lisle and Ziemer,
2000
). Pro-Muclin acquires fixed negative charges via sulfation of
its O-linked sugars in the TGN. Also in the TGN, the zymogens aggregate at the
mildly acidic pH of this organelle and are likely to interact ionically with
pro-Muclin's sulfates, as shown by the in vitro interaction of purified Muclin
with isolated zymogens in a pH-dependent manner. This association is expected
to facilitate packaging into immature secretory granules.
The effects of BzlGalNAc on post-translational processing of pro-Muclin are several and support the idea that pro-Muclin is a cargo receptor. First, BzlGalNAc inhibits sulfation by about 60%, and this may interfere with content aggregation and association of pro-Muclin with the content, similar to the chlorate effect. Second, cleavage of pro-Muclin is retarded by BzlGalNAc, and pro-Muclin persists longer than in control cells. Third, the eventual production of Muclin and p80 are both greatly reduced, indicating that incorrectly O-glycosylated pro-Muclin is degraded in the cell. Accompanying these biochemical changes, there is a dramatic dissociation of immunoreactive pro-Muclin/Muclin from amylase, which under normal conditions almost totally colocalize in the acinar cell. In addition, newly synthesized proteins fail to reach a stimulus-responsive compartment. Thus, incorrect post-translational processing of pro-Muclin is associated with a blockage of transport of proteins in the regulated pathway.
The mechanism of the BzlGalNAc blockage of the regulated pathway may be
directly caused by loss of normally processed pro-Muclin. Others have provided
evidence that lectin-like interactions of granule contents with
carbohydrate-binding proteins may be important in granule formation and
regulated secretion. In the mucin-secreting cell line HT29 MTX, treatment with
BzlGalNAc interferes with mucin granule formation and also inhibits basal and
stimulated secretion of mucins
(Hennebicq-Reig et al., 1998).
In addition, the cells accumulate numerous small vesicles containing
brush-border-associated glycoproteins, which normally are delivered to the
apical membrane (Huet et al.,
1998
). Because BzlGalNAc reduces glycoprotein sialylation (MAA
binding) and increases terminal galactose (PNA binding), the authors of these
studies suggest that sialic acid may play a key role in recognition of
secretory proteins and their sorting to the apical membrane. These changes in
PNA and MAA binding are also seen on Muclin in BzlGalNAc-treated acinar cells,
consistent with the idea that terminal sugars on glycoproteins are important
for protein sorting and granule formation in the secretory pathway. A similar
proposal has been made for the lectin-like protein ZG16p, which associates
with cholesterol-rich lipid rafts, the glycosylphosphatidyl-inositol-linked
membrane protein GP-2 and sulfated proteoglycans in the rat acinar cell
(Schmidt et al., 2001
). The
basic model put forth in both cases is that carbohydrates mediate association
of secretory proteins with lectin-like membrane proteins of the TGN/immature
granule, fostering granule formation and maturation. This model does not
directly take into account the effect of the acidic pH of these organelles,
but the lectin activity could easily be pH dependent, involving binding of
protonated secretory proteins to the negatively charged sialic acid residues
on the glycoproteins, which in turn are bound by the lectin-like membrane
protein.
Additional effects of BzlGalNAc may be caused by accumulation of its
metabolites in the secretory pathway. It has been shown that BzlGalNAc is
modified by glycosyltransferases and complex structures such as NANA
(2-3)Gal ß(1-3)BzlGalNAc are formed in BzlGalNAc-treated cells
(Delannoy et al., 1996
;
Huang et al., 1992
). We
observed [35S]sulfate-labeled BzlGalNAc derivatives in treated
acinar cells, which may accumulate to a significant degree in the TGN and
distal secretory pathway. Such molecules are expected to have an osmotic
effect, contributing to the dilute, watery state of vacuoles in the treated
cells. In addition, the monovalent, sulfated BzlGalNAc derivatives may
associate ionically with the aggregating mixed-charge secretory proteins.
Normally, the secretory proteins are expected to interact with the polyvalent
anionic sulfated glycoprotein pro-Muclin. Because the sulfated BzlGalNAc
derivatives are monovalent they cannot assist in condensation. The possibility
that BzlGalNAc might interfere with stimulus-secretion coupling in the acinar
cell is disproved by the fact that BzlGalNAc does not affect stimulated
release of prestored zymogens. Thus, its effects on secretion of newly
synthesized proteins are likely to be specific to its inhibition of
O-glycosylation and the possible osmotic effects on the TGN and immature
granules.
In summary, there is convincing evidence that protein packaging in the regulated secretory pathway depends on electrostatic interactions driven by the charged substituents of macromolecules in the pathway and mediated by the acidic luminal pH of the TGN and post-Golgi compartments. There is also accumulating evidence that these processes involve lectin-like interactions with accessory proteins' terminal carbohydrate structures, possibly sialic acid.
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References |
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