A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland

S. G. Venkatesh1 and S.-U. Gorr1,2

1 Department of Periodontics, Endodontics, and Dental Hygiene and 2 Department of Biochemistry and Molecular Biology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

amylase; isoproterenol; xyloside; parotid secretory protein; regulated secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SIGNIFICANT COMPLEMENTS of endocrine and exocrine secretory proteins are stored at high concentration in dense-core secretory granules of endocrine and exocrine cells, respectively (8, 9, 26, 32). These regulated secretory proteins, including peptide hormones, processing enzymes, and digestive enzymes, are released by exocytosis in response to extracellular stimulation of the cell by secretagogues. In addition to this regulated secretory pathway, endocrine and exocrine cells secrete proteins by the constitutive secretory pathway, which is common to all eukaryotic cells (26). Both secretory pathways originate in the trans-Golgi network, where proteins are included in constitutive transport vesicles and immature secretory granules, respectively. Thus sorting steps are involved in targeting secretory proteins to the correct transport vesicle (sorting-for-entry; see Ref. 5).

In many specialized secretory cells including exocrine acinar cells, sorting in the trans-Golgi network is not highly selective. Rather, secretory proteins and lysosomal proteins may enter immature secretory granules but are not stored in the mature secretory granule. These proteins are removed from the granules during maturation by the constitutive-like secretory pathway (1, 8). Two sorting steps, sorting-by-retention (5) and sorting-for-exit (21), refine and concentrate the final protein content of the mature secretory granule.

The mechanisms involved in the sorting of secretory proteins in the trans-Golgi network and immature secretory granules remain poorly understood. Indeed, it appears that different sorting signals and mechanisms function in different proteins and cell types (19, 37). In endocrine cells, receptor-mediated sorting (11) and selective aggregation of regulated secretory proteins (12, 20) have been proposed to mediate sorting to the regulated secretory pathway. Most endocrine-regulated secretory proteins exhibit calcium- and low-pH-induced aggregation (5), which may enhance sorting to the regulated secretory pathway (25). Exocrine pancreatic secretory proteins also exhibit calcium-induced aggregation (15, 28), although it is not clear whether this aggregation plays a role in the sorting and storage of secretory proteins in zymogen granules. In fact, calcium is not necessary for storage of salivary protein in parotid secretory granules, suggesting that these granules do not contain a calcium-dependent storage complex (13). Consistent with this finding, we recently noted that parotid secretory proteins (PSPs) exhibit poor calcium- and low-pH-induced aggregation in vitro (S. G. Venkatesh, D. J. Cowley, and S.-U.Gorr; unpublished observations).

As an alternative to calcium-induced aggregation, it has been proposed that regulated secretory proteins form a storage complex with secretory proteoglycans (33, 34). This hypothesis was supported by in vitro data that showed the interaction of exocrine pancreatic secretory proteins and sulfated proteoglycan (33). In addition, the expression and storage of sulfated proteoglycans and basic proline-rich protein are coregulated by isoproterenol in parotid acinar cells (4). Direct evidence for the role of proteoglycans in protein storage in secretory granules was obtained in transgenic mouse models. Thus disruption of the NDST-2 gene in mice resulted in animals that did not express the fully sulfated, negatively charged glycosaminoglycan, heparin. As a consequence, a severe reduction in the secretory granule contents of the connective tissue-type mast cells was observed, suggesting that storage of the secretory proteins was controlled by heparin through a posttranslational mechanism (14, 23, 24). Despite the data that suggest a role for proteoglycans in the sorting of secretory proteins, no such role was found when the hypothesis was directly tested in endocrine cells (6, 7, 17). In the present report, we used inhibitors of proteoglycan synthesis to show that a sulfated proteoglycan, which is stored in secretory granules, is necessary for efficient storage of secretory proteins in parotid secretory granules. Preliminary evidence suggests that the proteoglycan acts as a buffering agent of the secretory granule to increase retention of secretory proteins.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rat Parotid Tissue Culture and Secretion Experiments

Animal use was approved by the Institutional Animal Care and Use Committee at the University of Louisville. Male Sprague-Dawley rats (250-300 g) were fasted overnight, with water ad libitum. The rats were then euthanized by carbon dioxide asphyxiation, and their parotid glands were surgically removed, cleaned of fat and connective tissue, and placed in ice-cold Krebs-Ringer HEPES buffer (KRH; 10 mM HEPES, 129 mM NaCl, 5 mM Na2CO3, 4.8 mM KCl, 1.2 mM K2HPO4, 1.2 mM MgCl2, 1 mM CaCl2, and 2.8 mM glucose, pH 7.4). The tissue was sliced into 0.5-mm pieces with a Stadie-Riggs tissue slicer. The weight of the pooled tissue slices was noted, and 0.1-0.2 g of tissue was placed in vials containing 1 ml of KRH buffer. The samples were rinsed for 30 min at 37°C in a shaking water bath. The tissue slices were then preincubated for 30 min in 1 ml of fresh buffer containing 10 mM proteoglycan synthesis inhibitor, either methyl umbelliferyl xyloside (MUX) or paranitrophenyl xyloside (pNPX) in 10 µl of dimethyl sulfoxide. Control samples were incubated in buffer containing an equal volume of dimethyl sulfoxide. The buffer was changed every 10 min during the preincubation period. Samples were gassed with 100% O2 every 15 min during the experiments. In the pilot experiment shown in Fig. 1, oxygenation was omitted.


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Fig. 1.   A: proteoglycan secretion and effect of methyl umbelliferyl xyloside (MUX). Parotid tissue was labeled with Na235SO4 in the presence (+) or absence (-) of MUX. Separate samples were then chase-incubated in the presence or absence of isoproterenol (Isoprot). The labeling and chase media were analyzed by SDS-PAGE and fluorography. Lanes 1 and 2, labeling medium; lanes 3-6, chase medium. Data are from a single experiment. Similar results were obtained in separate experiments with oxygenation. The positions of molecular mass markers are indicated. B: longer exposure (5-fold) of the bottom part of the fluorogram shown in A.

For secretion experiments, the preincubated tissue was incubated in KRH for 2 h in the presence or absence of xyloside (MUX or pNPX) at 37°C. At the end of the incubation period, the tissue was incubated in Dulbecco's modified Eagle's medium (DMEM), pH 7.3, for 15 min. Incubation was continued in fresh DMEM containing either xyloside or dimethyl sulfoxide alone for 1 h (basal secretion medium). This was followed by incubation in fresh DMEM containing 30 µM isoproterenol (stimulated secretion medium). The secretion media were collected, centrifuged (16,000 g for 10 min), and stored at -20°C until use. The tissue slices were homogenized in 1 ml of KRH, and the homogenate was clarified by centrifugation (16,000 g for 15 min). The supernatant (tissue extract) was stored at -20°C until use.

Radiolabeling Experiments

35SO4 labeling of chondroitin sulfate proteoglycans. For radiolabeling, the tissue was preincubated, as described above, and then each tissue sample was pulse-labeled for 2 h in 1 ml of KRH containing 200 µCi Na235SO4. The pulse-labeling medium was replaced with DMEM chase medium, and the tissue was incubated for 1 h. In some samples, the proteoglycan synthesis inhibitor, either MUX or pNPX, was included in the preincubation and pulse-labeling media, as described above. To inhibit only sulfation of the proteoglycan, some samples were incubated with 10 mM of the sulfation inhibitor sodium chlorate (3) for 2 h during the preincubation and labeling periods. Stimulated secretion was achieved by adding 30 µM isoproterenol to the DMEM chase medium. The secretion media and tissue homogenates were centrifuged at 16,000 g for 15 min, and the supernatants were analyzed by SDS-PAGE (27), followed by fluorography (10). In control experiments, secretory granules were isolated from the radiolabeled tissue following the chase, as described below. The secretory granules were lysed, and the contents were analyzed for the presence of labeled proteoglycans by SDS-PAGE.

3H-labeling of rat parotid secretory proteins. Newly synthesized proteins were labeled with 100 µCi/ml 3H-leucine for 2 h, and the tissue was then chase-incubated for 1 h in DMEM supplemented with 1 mM unlabeled leucine. At this point, the tissue was either used to isolate secretory granules containing the labeled proteins or further stimulated with isoproterenol. Stimulated secretion was performed in DMEM with 1 mM cold leucine and 30 µM isoproterenol. The samples were analyzed by SDS-PAGE, and the radioactivity in the protein bands was quantitated by scintillation counting of gel slices, as previously described (18). The degree of stimulation was calculated as the ratio of stimulated secretion to basal secretion.

Isolation of Parotid Secretory Granules

Rat parotid secretory granules were isolated as previously described (2). Briefly, rat parotid gland tissue was weighed and homogenized in 10 volumes of 285 mM sucrose and centrifuged for 10 min at 940 g to pellet cellular debris. The supernatant was centrifuged for 10 min at 1,900 g to obtain the crude granule pellet. The pellet was resuspended in 285 mM sucrose and centrifuged for 10 min at 750 g, followed by centrifugation at 1,900 g for 10 min to obtain the purified granule fraction. To determine the purity of the preparation, the marker enzymes amylase (secretory granules), acid phosphatase (lysosomes), and lactate dehydrogenase (cytosol) were assayed at each stage and in the final pellet. Amylase activity in the samples was quantitated by the Phadebas amylase kit (Pharmacia/Upjohn Diagnostics, Uppsala, Sweden). Acid phosphatase and lactate dehydrogenase activities were determined with their respective assay kits (Sigma Diagnostics, St. Louis, MO). Protein content was quantitated by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Analysis of Rat Parotid Secretory Granule Proteins

Secretory granules were isolated from Na235SO4- or [3H]leucine-labeled tissue slices and lysed by freeze-thawing three times in distilled water. The granule membranes were pelleted by centrifugation at 16,000 g for 10 min. The amylase activity in the granule supernatants was estimated, and samples that contained equal total amylase activities were loaded on SDS-PAGE gels. The protein composition of the soluble granule fraction was analyzed by SDS-PAGE on 15% gels, and the radioactivity in the major bands was quantitated by fluorography or scintillation counting of gel slices.

Treatment of Rat Parotid Tissue With a Weak Acid

Rat parotid tissue was incubated with 10 mM acetic acid either alone or in combination with 10 mM pNPX, and the newly synthesized proteins were labeled with Na235SO4 or 3H-leucine, as described above. The tissue slices were chase-incubated for 2 h in fresh DMEM containing 1 mM cold leucine. The acid was present during the labeling and chase periods. Addition of the acid lowered the pH of the incubation medium from 7.4 to 6.4. Over time, with oxygenation, the pH reverted to near neutral. The control samples were either untreated with any agent or treated with pNPX alone. After the chase-incubation, secretory granules containing labeled secretory proteins were isolated, and their contents were analyzed by SDS-PAGE and fluorography or scintillation counting of gel slices.

Gel Electrophoresis and Immunoblotting

Protein samples were separated by SDS-PAGE on 15% gels (27). The protein bands were visualized by staining with Coomassie brilliant blue for 1 h followed by destaining in methanol-acetic acid-water (10:5:85). For immunoblotting, the unstained protein bands were transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 2% Tween 20 in TBST (Tris-buffered saline, pH 7.4, containing 0.05% Tween-20) for 10 min and subsequently incubated with a rat PSP antibody (a kind gift from Dr. William Ball, Howard University, Washington, DC) (1:10,000) for 1 h. The membrane was then washed with TBST and incubated in diluted (1:5,000) horseradish peroxidase-conjugated goat anti-rabbit IgG (Chemicon) for 1 h. Immunoblots were processed using Supersignal West Pico substrate (Pierce), and the chemiluminescence of the bands was visualized by exposure of the blot to Kodak X-Omat film.

Radiolabeled proteins were separated by SDS-PAGE, and the gels were impregnated with 1 M salicylate for 30 min (10). Incorporation of 35SO4 into the chondroitin sulfate proteoglycans was observed by fluorography. The gels were dried and exposed to X-ray film (Kodak) for 4-10 days at -20°C. Quantitation of radioactivity in individual bands was performed by densitometric scans of the fluorographs. In some cases, following SDS-PAGE, the gels were dried and exposed to a phosphor screen. The screen was analyzed on a STORM 830 optical scanner (Molecular Dynamics, Sunnyvale, CA), and the bands were quantitated by using ImageQuant software.

Data Analysis

The data obtained from amylase assays, gel slices, scanned electrophoresis gels, and immunoblots were analyzed by Student's t-test or pairwise ANOVA with Dunnett multiple comparisons post test. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rat parotid cells secrete a 40- to 60-kDa sulfated proteoglycan that has been proposed to play a role in protein sorting in parotid acinar cells (4). The proteoglycan undergoes stimulated secretion from rat parotid tissue slices treated with the beta -adrenergic agonist isoproterenol (Figs. 1A and 2A). In contrast, a high molecular weight proteoglycan was detected equally in basal and stimulated secretion medium, suggesting that this proteoglycan was not stored in secretory granules. Consistent with this view, the 40-to 60-kDa proteoglycan, but not the high molecular weight proteoglycan, was detected in isolated secretory granules (Fig. 2B). Thus the 40-to 60-kDa proteoglycan is efficiently sorted to the regulated secretory pathway in parotid tissue.


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Fig. 2.   A: proteoglycan secretion and effect of paranitrophenyl xyloside (pNPX). Parotid tissue was labeled with Na235SO4 in the presence or absence of pNPX. Each sample was then chase-incubated in the absence of isoproterenol, followed by a second chase-incubation in the presence of isoproterenol. The chase media were analyzed by SDS-PAGE and fluorography. Molecular mass standards (kDa) are shown on the left. The experiment was repeated with similar results. B: sulfated proteoglycan in secretory granules of rat parotid gland. Parotid tissue was labeled and chase-incubated without stimulation to maximize storage of newly synthesized proteoglycan to secretory granules. Secretory granules were isolated and total amylase activity in each granule fraction was determined. Equal total amylase activities were loaded in each lane of the SDS-PAGE gels. The sulfated radioactive proteins in the secretory granules were visualized by fluorography of the gels. Molecular mass standards (kDa) are shown on the left. Data are from a single experiment.

The xyloside analogs MUX and pNPX were tested for inhibition of proteoglycan synthesis (16, 34, 36). The proteoglycans were absent from the basal secretion medium, stimulated secretion medium, and the secretory granules from xyloside-treated parotid tissue (Figs. 1A and 2). This finding suggested that MUX and pNPX were equally effective in inhibiting proteoglycan synthesis.

To determine the presence of free glycosaminoglycan (GAG) chains (34), the gel was overexposed (Fig. 1B). A diffuse labeling pattern, representing sulfated GAG chains, was observed in the xyloside-treated samples. The GAG chains were equally present in isoproterenol-stimulated and nonstimulated samples, suggesting that they are secreted by the constitutive secretory pathway and not stored in the secretory granules (Fig. 1B, lanes 5 and 6). The xyloside did not affect the secretion of a 25-kDa sulfated protein (Fig. 2), suggesting that the xyloside treatment was specific for inhibition of proteoglycan synthesis. MUX treatment showed a tendency toward increased protein synthesis when the incorporation of [3H]leucine in cellular and secreted proteins was analyzed in untreated and MUX-treated parotid tissue. In contrast, pNPX treatment of the parotid tissue did not significantly affect the total protein levels (Fig. 3). Thus pNPX was used for further experiments.


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Fig. 3.   Effect of xyloside on protein synthesis. Parotid tissue slices were labeled with [3H]leucine in the presence or absence (control) of MUX (A) or pNPX (B). The tissue was chase-incubated as described for Figs. 1 and 2, respectively. Tissue extracts and chase media were analyzed by SDS-PAGE and scintillation counting of gel slices. Data represent the total cycles/min (cpm) in chase media and tissue extracts. Values represent means ± SE from 2 to 3 separate experiments (n = 8-13). In A, *P < 0.01; for amylase, P < 0.1.

To determine whether the parotid proteoglycan affects the entry of secretory proteins into secretory granules, pulse-chase experiments were performed in the presence or absence of pNPX. In these experiments, no more than 25-30% of the total newly synthesized amylase and PSP was secreted into the medium. Of this, 3-5% was basal secretion and 22-23% was stimulated secretion in control samples. In inhibitor treated samples, 4-10% of the newly synthesized amylase and PSP, respectively, was secreted under unstimulated conditions, whereas 14-20% of the proteins were released upon stimulation. Thus the fraction of the total radiolabeled proteins that is susceptible to changes in protein sorting represents about 25% of the total radiolabeled protein in the cells. This is the difference between basal (no sorting) and stimulated (fully sorted) secretion. To normalize these data from individual samples, the changes were expressed as the ratio of basal to stimulated secretion. Thus the inhibitor caused a significant (50%) reduction in the degree of stimulation (%stimulated secretion/%basal secretion) of the regulated secretory proteins PSP and amylase (Fig. 4). In the data shown in Fig. 4, the basal and stimulated secretions were assayed consecutively in each sample. Similar results were obtained when basal and stimulated secretions were analyzed in parallel in separate samples (not shown). These results suggest that inhibition of proteoglycan synthesis causes rerouting of newly synthesized regulated secretory proteins from the regulated pathway into a constitutive secretory pathway.


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Fig. 4.   Stimulated secretion of newly synthesized amylase and parotid secretory protein (PSP). Parotid tissue was labeled with [3H]leucine and then chase-incubated to obtain basal secretion medium. The tissue was then stimulated with isoproterenol to obtain stimulated secretion medium. Basal and stimulated media were analyzed by SDS-PAGE, followed by scintillation counting of gel slices. The degree of stimulation was calculated as %stimulated secretion/%basal secretion. Data presented are means ± SE from 2 independent experiments performed in triplicate (n = 6). *P < 0.05.

To rule out the possibility that pNPX caused nonspecific effects on protein storage or secretion rather than a direct effect on protein sorting, we tested the effect of the inhibitor on the secretion of premade PSP and amylase. The fresh rat parotid tissue used for these experiments contains premade secretory granules that store secretory granule proteins that were synthesized before the manipulations described above. Because these proteins were sorted to secretory granules before the inhibition of proteoglycan synthesis, they should not be affected by pNPX treatment. To test this, we analyzed the secretion of total PSP and amylase by immunoblotting and enzyme assay, respectively (Fig. 5). Neither the secretion of total PSP nor the secretion of total amylase showed changes of the magnitude (50% of secreted protein) detected for newly synthesized proteins in Fig. 4. This finding suggests that inhibition of proteoglycan synthesis does not affect the secretion of PSP or amylase that was already stored in secretory granules. This is consistent with the view that the acinar cells have numerous mature secretory granules at any given period, whereas the number of newly synthesized granules during the experiment would constitute a very small fraction of the total. Thus immunocytochemical methods (immunoelectron microscopy and immunofluorescence) would detect primarily the large stores of premade proteins but would not reflect the dynamic changes in the smaller fraction of newly synthesized proteins that occur with the use of the proteoglycan synthesis inhibitor. Therefore, we used pulse-chase protocols and subcellular fractionation to analyze protein storage in nascent secretory granules.


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Fig. 5.   Secretion of total PSP and amylase from unlabeled parotid tissue. Secretion medium from untreated (-pNPX) and pNPX-treated (+pNPX) parotid tissue was collected in the presence or absence of isoproterenol. The medium was analyzed by immunoblotting for PSP (A). Medium and tissue extracts were analyzed by amylase assay (B). %secreted = (amylase activity in medium/amylase activity in tissue) × 100%. Data presented are means ± SE from 2 independent experiments performed in duplicate (n = 4).

The sulfated proteoglycan found in parotid secretory granules consists of acidic, proline-rich core protein modified by sulfated chondroitin chains (9). To test whether sulfation alone could explain the rerouting of secretory proteins, parotid tissue was pulse-labeled in the presence of sodium chlorate, an inhibitor of sulfation (3, 18). Chlorate treatment of the rat parotid tissue completely inhibited the sulfation of the proteoglycan (data not shown). However, this did not affect the secretion of amylase or PSP (Fig. 6), suggesting that sulfation of the proteoglycans alone is not sufficient for sorting of PSP and amylase.


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Fig. 6.   Inhibition of proteoglycan sulfation. Parotid tissue slices were labeled with [3H]-leucine in the presence or absence of 10 mM chlorate, followed by a chase-incubation (-Isoprot). The tissue was then stimulated with isoproterenol. Basal and stimulated secretion media and tissue extracts were analyzed by SDS-PAGE and scintillation counting of gel slices. %secreted = (cpm in medium/cpm in tissue extract) × 100%. Data presented are means ± SE from a single experiments (n = 4). Similar results were obtained in a separate experiment. *P < 0.5.

To directly test whether inhibition of proteoglycan synthesis affected the subcellular localization of regulated secretory proteins, secretory granules were isolated from rat parotid tissue. A 10-fold increase in the specific activity of amylase, compared with nongranule fractions, and lack of acid phosphatase and lactate dehydrogenase activity in the final granule pellet, suggested that the preparation was free of lysosomes and other cellular contaminants (Table 1). Analysis of the granular content showed that the amounts of newly synthesized proteins stored in secretory granules that were formed in the presence of the proteoglycan synthesis inhibitor were significantly reduced compared with control granules (Fig. 7). Reduced storage was detected for each of the three major proteins found in [3H]leucine-labeled samples: PSP, amylase, and p32.

                              
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Table 1.   Isolation of parotid secretory granules



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Fig. 7.   Secretory granule proteins in untreated (solid line) and pNPX-treated (broken line) parotid tissue. Parotid tissue was labeled with [3H]leucine in the absence (control) or presence of pNPX (pNPX-treated), followed by chase-incubation. Secretory granules were then isolated from the tissue and lysed in water. Amylase activity was quantitated in each lysate sample, and equal amylase activity was loaded in each lane of SDS-PAGE gels. The radioactivity in protein bands was quantitated by scintillation counting of gel slices.

The acidic, sulfated proteoglycan and basic secretory proteins have been proposed to modify the ionic milieu of parotid secretory granules (4). Indeed, the perturbation of storage of regulated secretory proteins upon inhibition of proteoglycan synthesis is akin to the effect of mild alkalization of secretory granules. Thus von Zastrow et al. (38) observed that addition of 20 mM ammonium chloride increases the output and substantially alters the relative composition of newly synthesized proteins in the granule compartment. This led us to hypothesize that the acidic proteoglycan could be involved in buffering the contents of the secretory granule. A direct test of this model, using cationic dyes, was not feasible because only nascent secretory granules were affected by xyloside treatment (Fig. 5), as discussed above. Instead, we employed a dynamic approach consisting of pulse-chase experiments in the presence of the mildly acidifying agent acetic acid to reverse the proposed alkalization (22).

Secretory granules were isolated from the pulse-chased tissue and analyzed for protein storage. Figure 8A shows that acid treatment of the tissue did not abrogate the effect of pNPX, although acid by itself slightly increased the synthesis of the proteoglycan. In contrast, Fig. 8B shows that the acid treatment did not significantly change the storage of newly synthesized amylase or PSP in the granules. pNPX treatment, on the other hand, significantly reduced the granule contents of both PSP and amylase, consistent with the data in Figs. 4 and 7. The effect of pNPX was completely reversed by the addition of weak acid, suggesting that inhibition of proteoglycan synthesis indeed affects the pH of the secretory granules.


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Fig. 8.   Effect of weak acid on the storage of PSP and amylase in pNPX-treated and untreated tissue. Rat parotid tissue was labeled with Na235SO4 (A) or [3H]leucine (B) in the presence or absence of pNPX and/or acetic acid, as indicated. After a chase period, secretory granules were isolated and the contents analyzed by SDS-PAGE. A: sulfated proteoglycans in secretory granule lysates. The sulfate-labeled proteins were separated by SDS-PAGE and analyzed by phosphorimaging. The leftmost lane represents radiolabeled molecular mass markers. The position of the 45-kDa marker band is indicated. B: [3H]leucine-labeled proteins were analyzed by SDS-PAGE and scintillation counting of gel slices. Data presented are means ± SE from 5 independent experiments (n = 6-9). *P < 0.05, different from untreated control (-acid, -pNPX).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sulfated proteoglycans have long been thought to play a role in the sorting and storage of secretory proteins in endocrine and exocrine secretory granules (26, 32). Despite indirect evidence in support of this hypothesis, a direct role of proteoglycans in the sorting of endocrine secretory proteins could not be demonstrated (6, 17). On the other hand, recent findings show that transgenic mice, in which the NDST-2 gene has been disrupted, fail to synthesize heparin, which leads to a severe reduction of protein storage in the secretory granules of the connective tissue-type mast cells (14, 23, 24). We now provide evidence that a sulfated proteoglycan, which is stored in parotid secretory granules, is necessary for efficient storage of other exocrine-regulated secretory proteins. Thus it appears that this sorting mechanism functions in some mast cells and exocrine cells but not in endocrine cells. In fact, it is increasingly recognized that sorting of secretory proteins can be cell-type and protein specific (19, 37). The mechanism of action of the proteoglycan could be through direct interaction with other granule components or through an effect on the granule milieu.

Possible direct interactions of proteoglycans and other secretory proteins include a "docking" function, wherein these molecules could be attached to the membranes and bind other molecules in the granule (34). Schmidt and coworkers (35) have proposed that a submembranous matrix of proteoglycans on zymogen granule membranes aids in granule formation via a secretory lectin in pancreatic acinar cells. Also, complexes between proteoglycan and basic secretory protein have been demonstrated in vitro (33). However, the parotid proteoglycan did not coimmunoprecipitate with other granule components (unpublished observation), prompting us to consider alternate mechanisms of action.

The parotid proteoglycan is costored with basic secretory proteins including basic proline-rich protein (4). Thus the mature proteoglycan (acidic) and basic granule proteins exhibit opposing charges. In fact, the inhibition of protein sorting in the absence of the proteoglycan was reminiscent of the reported effects of weak bases on the sorting of regulated secretory proteins. Several studies have shown that the sorting of regulated proteins into secretory granules is perturbed by mild alkalinization (29, 38). Thus an increase in the pH of nascent parotid secretory granules results in a preferential increase in the basal secretion of PSP and p32 protein (38). Together, these studies show that the pH of immature secretory granules is vital in ensuring the proper sorting of secretory proteins.

The acidic PRP core protein is not stored efficiently in secretory granules in the absence of attached glycosaminoglycan chains (7). Therefore, we considered that inhibition of proteoglycan synthesis would lead to a relative loss of negative charges or a net gain in positive charges due to the basic proline-rich proteins (7). This would be equivalent to the gain in positive charges mediated by weak bases that enter the secretory granule and become protonated. The parotid secretory granule is a well-buffered organelle (2), and a complete removal of sulfated, acidic sugars was required for an effect on sorting. Removal of only the sulfate groups had no effect on sorting of PSP and amylase. Presumably, the acidic PRP is still stored in secretory granules after inhibition of sulfation alone.

To test the hypothesis that the acidic sulfated proteoglycan acts as a buffering agent in the sorting of PSPs, the tissue was subjected to acidification by weak acid. This treatment reversed the effect of the proteoglycan synthesis inhibitor on protein storage. Importantly, the acid did not prevent the inhibition of proteoglycan synthesis. Instead, the acid treatment could directly affect granule pH, supporting the idea that the proteoglycan affects the internal pH of secretory granules. Alternatively, acid treatment could exert an independent effect (e.g., on the pH of the cytosol) that increases protein storage and thereby counters the effect of the proteoglycan inhibitor. Indeed, cytosolic acidification could inhibit the formation of clathrin-coated vesicles (22) and thus inhibit the removal of secretory proteins from immature secretory granules (1). In this case, acid treatment alone should increase protein retention in secretory granules. Because this was not observed, we propose that the primary effect of acid treatment is to directly reverse the effect of the proteoglycan inhibitor in the secretory pathway. We cannot yet exclude the possibility that the pNPX and acid treatments affected secretory granule mechanisms other than granule pH.

In contrast to the effect of the proteoglycan inhibitor, an increase in proteoglycan synthesis did not result in enhanced storage of other granule components. This finding suggests that, whereas the pH of the granule is critical for efficient sorting of secretory proteins, there is also a limit to the amount of protein that can be stored in the granule. In general, an acidic pH is prevalent in the immature granules of both exocrine and endocrine cells. However, in endocrine cells, the pH of the granule continues to decrease during maturation, becoming more acidic, whereas in exocrine cells, it returns to a nearly neutral pH (31). Why, then, are sulfated proteoglycans not necessary for the sorting of regulated secretory proteins in endocrine cells (6, 7, 17)? A potentially significant difference between endocrine and exocrine cells is the ubiquitous expression of chromogranins in endocrine and neuroendocrine cells, whereas chromogranins are absent from exocrine cells (30). Chromogranins are acidic sulfated glycoproteins (pI around 5) that may act as buffering agents in endocrine secretory granules. Hence, the internal pH of these granules is not sufficiently affected by proteoglycan synthesis inhibitors to affect protein sorting. The finding that the sorting of basic proline-rich protein is not affected by the expression of the parotid proteoglycan in endocrine cells, which express chromogranin A, is consistent with this model (7). Indeed, endocrine secretory granules show increased resistance to alkalinization when chromogranin A is overexpressed in the cells (S.-U. Gorr, B. H. Fasciotto, and R. Jain, unpublished observation). Together, these findings suggest that acidic secretory proteins are buffering agents that play a role in protein sorting and storage in secretory cells. This role would be maintained by chromogranins in endocrine and neuroendocrine cells, heparin proteoglycan in mast cells, and chondroitin sulfate proteoglycan in parotid acinar cells.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Dental Research Grant R01-DE12205.


    FOOTNOTES

Address for reprint requests and other correspondence: S.-U. Gorr, Department of Periodontics, Orthodontics and Dental Hygiene, University of Louisville Health Sciences Center, Louisville, Kentucky 40292 (E-mail: sven.gorr{at}louisville.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 18, 2002;10.1152/ajpcell.00552.2001

Received 18 November 2001; accepted in final form 10 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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