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Role of sulfated O-linked glycoproteins in zymogen granule formation

Robert C. De Lisle

Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS 66160, USA



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  		Fig. 1. Preparation of Muclin-depleted ZGC and in vitro aggregation of ZGC and purified Muclin. (A) Isolated soluble zymogens were depleted of Muclin on a PNA-agarose column. ZGC, starting material; FT, flow-through fractions; W, wash fractions; E, 0.2 M lactose eluted fractions. FT-4 and W-1 were pooled and used for the in vitro aggregation assay. (B) Muclin-depleted ZGC were incubated at pH 8.0 (Acid -) or acidified to pH 6.3 (Acid +). The aggregates were pelleted, run on 10% SDS-PAGE and stained with Coomassie blue. The control is 50 µg ZGC alone; `Muclin' included 5 µg purified Muclin, which increased ZGC aggregation compared with the control; `BSA' included 10 µg bovine serum albumin, which did not aggregate at acidic pH; `EGTA' included 5 mM EGTA to chelate calcium, which did not affect aggregation; `Ca2+' included 2 mM CaCl2, which did not affect acid-induced aggregation. (C) Quantification of amylase in the pellets by densitometry (arbitrary units) of Coomassie-blue-stained gel. (D) Western blot for Muclin in the pellets. No Muclin was detected in the ZGC-depleted samples, but was detected only when purified Muclin was added (Muclin). The amount of Muclin in the pellet was increased upon acidification (Acid +). (E) By western blot, Muclin in the absence of ZGC does not exhibit acid-mediated aggregation.

 


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  		Fig. 7. In vivo zymogen granule depletion and morphological effects of chlorate and BzlGalNAc after in vitro zymogen granule repletion. Mice were injected with the cholinergic agonist pilocarpine (10 µg/g body weight). After 1 hour, the pancreas was removed, lobules were prepared and incubated in vitro for 4 hours in buffer supplemented with 30 mM NaCl, 30 mM sodium chlorate, 2% DMSO or 32 mM BzlGalNAc. The tissue was then prepared for light level and electron microscopy.

(A) Untreated pancreas; (B) pancreas 1 hour after pilocarpine injection; (C,E) 30 mM NaCl; (D,F) 30 mM sodium chlorate; (G,I) 2% DMSO; (H,J) 32 mM BzlGalNAc. Note that the cells are generally healthy with intact plasma membranes and normal appearing nuclei under all conditions (C,D,G,H) and that they recover from the slight dilation of the endoplasmic reticulum that occurs after pilocarpine depletion of zymogen granules (B).

 


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  		Fig. 2. Effect of chlorate on incorporation of [35S]sulfate and [3H]leucine into acinar cell proteins. (A) [35S]sulfate labeling of isolated acini in the presence of sodium chlorate. Equal aliquots of acini (0.4 mg protein) were labeled in the presence of the indicated concentrations of chlorate. 10% of the cell pellets were run on the gels. (B) Quantification of sulfate labeling versus [chlorate]. Chlorate dose-dependently inhibits sulfation of all the major sulfated proteins and is >95% effective at 30 mM. (C) Trichloroacetic acid-precipitable [3H]-protein synthesized in the presence of sodium chlorate shows that chlorate does not inhibit protein synthesis. Data are means±s.d. of triplicate samples from representative experiments.

 


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  		Fig. 3. Effect of BzlGalNAc on incorporation of [35S]sulfate, [3H]glucosamine and [3H]leucine into acinar cell proteins. (A) Equal aliquots of acini (0.4 mg protein) were incubated with the indicated concentrations of BzlGalNAc. 10% of the cell pellets were run on 7.5% gels and imaged on a phosphor screen for [35S]sulfate. (B) 12.5% gel using 10% of the same cell pellets shown in A. Note the labeled diffuse area representing sulfated BzlGalNAc derivatives. (C) Quantitation of sulfate labeling versus [BzlGalNAc]. Muclin and p82/p75 were quantified from 7.5% gels, and prolipase and proelastase IV were quantified from 12.5% gels. (D) [3H]glucosamine labeling of equal aliquots (1 mg protein) of isolated acini in the presence of BzlGalNAc; 10% of the cell pellets were separated by SDS-PAGE followed by autoradiography. (E) [3H]leucine labeling of isolated acini in the presence of BzlGalNAc. Data are means±s.d. of triplicate samples from representative experiments.

 


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  		Fig. 4. Effects of chlorate and BzlGalNAc on post-translational maturation of pro-Muclin. (A) Pancreatic lobules were prepared from zymogen-granule-depleted mouse pancreas, pulse-labeled for 30 minutes in the presence of 30 mM NaCl, 30 mM chlorate, 2% DMSO or 32 mM BzlGalNAc. The samples were washed and chased in the continued presence of the chemicals for the indicated times. Equal amounts of tissue were run on SDS-PAGE (DNA equivalent to 3x105 cells) and phosphorimaged. p80 was immunoprecipitated from equal aliquots (DNA equivalent to 2.7x106 cells). The asterisk indicates the lower Mr form of Muclin produced in BzlGalNAc-treated cells. (B) Quantification of labeled pro-Muclin by phosphorimaging as a function of chase time expressed relative to the time zero incorporation of the appropriate control (NaCl and DMSO, respectively). (C) Quantification of labeled Muclin by phosphorimaging as a function of chase time expressed relative to the time zero incorporation into pro-Muclin of the appropriate control. (D) Quantification of p80 by phosphorimaging as a function of chase time expressed relative to the level at 4 hours of chase of the appropriate control. Data in B to D are from triplicate samples of a representative experiment and are means±s.d. (E) Lectin binding to pancreatic proteins. Pancreatic lobules were prepared from zymogen-granule-depleted mouse pancreas and incubated with 2% DMSO (vehicle) or 32 mM BzlGalNAc for 4 hours. Equal amounts of tissue were separated by SDS-PAGE followed by western blotting and probing with PNA (T antigen) and MAA (sialic acid).

 


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  		Fig. 5. Effects of chlorate and BzlGalNAc on basal and stimulated secretion of newly made proteins. (A) Phosphorimage of secreted [35S]met/cys-labeled proteins. Pulse-labeled acini were washed and chased at 1 mg cell protein per ml in the indicated buffers. During the final 30 minutes of chase, 1 µM carbachol and 1 mM 8-Br-cAMP were added to stimulate regulated secretion (S). The lane marked `0' is 10% of a time zero pellet (0 hour chase) used to quantify the percentage release values. (B) The effect of 30 mM chlorate on basal and stimulated secretion (arrow indicates addition of stimulus for final 30 minutes of chase) of [35S]met/cys-labeled proteins. Amylase release was quantified relative to cell content just after the pulse labeling. Data are means±s.e., n=4 independent experiments. (C) The effect of 32 mM BzlGalNAc on basal and stimulated secretion (arrow indicates addition of stimulus for final 30 minutes of chase) of [35S]met/cys-labeled proteins. For comparison, the control data from panel B are reproduced here. Data are means±s.e.m., n=4 independent experiments. *P=0.005 compared with the DMSO control after stimulation. (D) Quantification of basal and stimulated secretion (arrow indicates addition of stimulus for final 30 minutes of incubation) of prestored protein by Coomassie blue staining of secreted media from a representative independent experiment. (E) Quantitation of [35S]labeled amylase 6 hours after chase under the different conditions. Data are expressed as a percentage of initial level of labeled amylase; means±range of values from two independent experiments.

 


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  		Fig. 6. Assessment of cell viability after incubation with NaCl, chlorate, DMSO or BzlGalNAc. Acini were incubated for the indicated times and then labeled for 10 minutes with calcein-acetoxymethyl ester and ethidium homodimer to label live and dead cells, respectively. (A-H) The cells were imaged by fluorescence microscopy and representative images are shown. Live cells accumulate calcein and fluoresce green (E,G,H). Dead cells are stained by ethidium homodimer and fluoresce red (A-D). Triton X-100 (1%) was used to kill the cells which cannot accumulate calcein (F) but admit ethidium homodimer, which fluorescently stains nuclei red (B). (I) Scion Image analysis software was used to quantify the amount of red staining ethidium homodimer under the different conditions. The amount of dead cells in each sample was calculated as the percentage of ethidium homodimer labeled nuclei relative to Triton X-100 killed cells.

 


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  		Fig. 8. Effects of chlorate and BzlGalNAc on immunolocalization of Muclin and amylase after in vitro zymogen granule repletion. Pancreatic lobules were prepared from zymogen-granule-depleted mice and incubated in vitro for 4 hours in buffer supplemented with (A) 30 mM NaCl, (B) 30 mM chlorate, (C) 2% DMSO or (D) 32 mM BzlGalNAc. Cryosections were prepared and dual immunostained for Muclin (green) and amylase (red) and for DNA with DAPI (blue). Note larger Muclin- and amylase-positive structures in chlorate-treated tissue (arrows in B) compared with controls; and dramatic dissociation of Muclin (arrows in D) and amylase (arrowheads in D) in BzlGalNAc-treated tissue.

 


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  		Fig. 9. Effect of BzlGalNAc on immunolocalization of LAMP-1, Muclin and amylase after in vitro zymogen granule repletion. Cryosections were prepared the same as in Fig. 8 and immunolabeled for the lysosomal membrane protein LAMP-1, Muclin and amylase. (A) In control tissue, there are sparse lysosomes (arrow) that are negative for Muclin. (B) BzlGalNAc-treated tissue has larger LAMP-1-positive structures (arrow) that are negative for Muclin. (C) BzlGalNAc-treated tissue has larger LAMP-1-positive structures (arrow) that are negative for amylase.

 

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© The Company of Biologists Ltd 2002