Journal of Cell Science 113, 2233-2242 (2000) 2233 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1273

A submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells

Katja Schmidt1, Heidrun Dartsch1, Dietmar Linder2, Horst-Franz Kern1,* and Ralf Kleene1 1Institut für Zytobiologie und Zytopathologie, Philipps Universität, Robert-Koch-Str. 5, D-35033 Marburg, Germany 2Institut für Biochemie, Justus Liebig Universität, Gießen, Germany *Author for correspondence (e-mail: [email protected])

Accepted 27 March; published on WWW 25 May 2000

SUMMARY

The secretory lectin ZG16p mediated the binding of antibody directed against chondroitin sulfate, indicating that aggregated zymogens to the granule membrane in the proteins represented proteoglycans. A staining pattern pancreatic acinar cells. Using a recently established in similar to the glycan reaction was observed in immunoblots vitro condensation-sorting assay, we now show that using a polyclonal antibody directed against the whole pretreatment of zymogen granule membranes (ZGM) with bicarbonate extract. Immunogold electron microscopy either sodium bicarbonate at pH 10 or with phosphatidyl revealed that this antibody reacted with components in the inositol-specific phospholipase C (PI-PLC) reduced the periphery of zymogen granules and strongly stained ZGM binding efficiency of zymogens to the same extent, as distinct in the pellet fraction of a standard in vitro condensation- components were liberated from ZGM. Analysis of the sorting assay. The amino acid composition of isolated composition of the bicarbonate extract revealed the presence components of both the acidic and basic group showed of the secretory lectin ZG16p, the serpin ZG46p and the similarities to aggrecan, a cartilage-specific proteoglycan, GPI-linked glycoprotein GP-2, together with several and to glycine-rich glycoproteins, respectively. We therefore unknown proteins, and small amounts of lipase and conclude that a submembranous matrix on the ZGM carboxylester lipase. The unknown proteins detected in 2-D composed of proteoglycans and glycoproteins is involved in gels represented a group of acidic and basic protein spots, granule formation in pancreatic acinar cells. which were positive in a glycan staining reaction and were soluble in methanol. One protein spot of the acidic group and Key words: Rat, Pancreas, Pancreatic zymogen, Submembranous several of the basic group reacted with a monoclonal network, Condensation-sorting, Proteoglycan, Glycoprotein

INTRODUCTION is also present in other members of the granin family and in some secretory proteins within neuro-endocrine cells (Tam et Granule formation in both exocrine and endocrine secretory al., 1993; Krömer et al., 1998). Whether the granin-loop cells involves two major steps through which the regulated interacts directly with the lipid leaflet of the granule membrane secretory proteins are separated from constitutively released or is bound via interaction with as-yet-unidentified membrane products and are packaged in concentrated form within proteins is so far not known. secretion granules. The initial step is a pH-dependent, selective While the selective aggregation of the mixture of 15-20 aggregation of regulated secretory proteins within the acidic enzyme and isoenzyme proteins in pancreatic acinar cells and environment of the trans-Golgi-network (TGN) leading to the thus the formation of the granule core is well documented dense granule core (for review, see Tooze, 1998). In some cells (Leblond et al., 1993; Freedman and Scheele, 1993b; Dartsch it also involves millimolar concentrations of calcium ions and et al., 1998), their binding to the zymogen granule membrane represents a sorting event, since constitutively released (ZGM) is poorly understood. It has long been hypothesized products are excluded from the aggregate (Yoo, 1993; Colomer that the most abundant membrane protein in ZGM, a et al., 1996). The second step in granule formation is the glycoprotein of about 75 kDa referred to as GP-2, might act as binding of the dense core aggregate to specific membrane a sortase for aggregated secretory proteins (Jacob et al., 1992). domains of the TGN and their pinching off as immature GP-2 is attached to the luminal leaflet of ZGM via a glycosyl- secretion granules. In neuro-endocrine cells evidence has been phosphatidyl inositol (GPI) moiety and is liberated from ZGM presented that individual proteins within the aggregate, such as during exocytosis, possibly by an endogenous phosphatidyl chromogranin B, also occur in a tightly membrane-associated inositol-specific phospholipase C (PI-PLC) (Freedman et al., form (Pimplikar and Huttner, 1992). This membrane- 1998a,b). However, it has also been shown that zymogen association seems to be mediated by a sequence at the N- granules can form in the complete absence of GP-2, which is terminus of the protein forming a disulfide-bonded loop, which the case during embryonic development (Dittie and Kern, 2234 K. Schmidt and others

1992), and in partially differentiated acinar carcinoma cell lines 1/10 of methionine and cysteine was applied, and MgSO4 in the Eagle’s (Hansen, 1983). Freedman and Scheele (1993a) have salt solution was replaced by MgCl2. The medium was supplemented demonstrated that globular GP-2 detached from the membrane with 4.5 g glucose/l and was buffered with 10 mM Hepes, pH 7.2. 2 35 at slightly acidic pH forms tetrameric complexes, which might mCi carrier-free Na[ S]O4 (ICN, Eschwege, Germany) were added and be associated with sulfated proteoglycans that were previously incubation continued at 37°C for 120 minutes, with agitation. 100% O2 identified as constituents of pancreatic zymogen granules was supplied every 15 minutes. Subsequently, the lobules were washed in PBS and zymogen granules were prepared as described above. (Tartakoff et al., 1975; Reggio and Palade, 1978). Scheele et For the labeling of glycans the DIG Glycan Labeling Kit al. (1994) have put forward the hypothesis that GP-2, together (Boehringer Mannheim, Germany) was used. Labeling with with the attached proteoglycans, forms a submembranous digoxigenin was performed using method A according to the matrix at the luminal side of ZGM, which might function in manufacturer’s instructions. membrane sorting during granule formation, and also in the stability of granules during storage. They have presented Determination of sulfated glycosaminoglycans and of further evidence (Freedman et al., 1998b) that the protein-bound radioactivity submembranous matrix is released from ZGM during granule To determine the amount of sulfated proteoglycans in fractions of exocytosis, mainly due to the basic pH in the acinar lumen but zymogen granules the Blysan Proteoglycan and Glycosaminoglycan also through the activation of PI-PLC (Freedman et al., 1998a). Assay was performed according to the manufacturer’s protocol (Biocolor, Belfast, Northern Ireland). Briefly, a precipitable complex The release of the matrix seems to be a prerequisite for the was formed by binding of the Blyscan dye to free or proteoglycan- uptake of ZGM by endocytosis following exocytosis. A bound glycosaminoglycans (GAGs). After dissociation of the possible role of the submembranous matrix in the binding of precipitated complex the samples were spectrometrically quantified. the dense granule core to ZGM has been assumed from in vitro For determination of protein-bound radioactivity, samples were studies showing an association of the secretory lectin ZG16p applied to 3MM filters (Whatman, Maidstone, UK). The filters were (Cronshagen et al., 1994) with both the dense granule core and swirled in 10% TCA on ice for 30 minutes, washed twice with ice- the ZGM at acidic pH, and an impairment of in vitro cold 5% TCA for 5 minutes, and once with ethanol. After drying the condensation-sorting after removal of both ZG16p and the protein-bound radioactivity precipitated on the filters was determined proteoglycans from ZGM (Kleene et al., 1999). Since the by liquid scintillation counting using a scintillation counter (Raytest, composition of the submembranous matrix of ZGM is largely Straubenhardt, Germany). unknown, we have attempted to further characterize it and to Methanol precipitation analyze its involvement in granule formation. Since it is known that certain proteoglycans and glycoproteins are methanol-soluble, we subjected the bicarbonate extract of ZGM to methanol precipitation in order to specifically isolate glycosylated components. Methanol precipitation was performed according to MATERIALS AND METHODS Wessel (1984). Briefly, 100 µl of bicarbonate extract corresponding to 100 µg of protein were mixed in consecutive steps with 400 µl of Isolation and subfractionation of zymogen granules methanol, 100 µl of chloroform and 300 µl of dH2O. After Zymogen granules were prepared as described recently (Dartsch et al., centrifugation at 13000 g for 3 minutes the upper phase was collected, 1998). Briefly, the pancreas was removed from fasted male Wistar rats concentrated and subjected to 2-D gel electrophoresis. The interphase (200-230 g body mass) and was homogenized in the following buffer: and lower phase were mixed with 300 µl of methanol and centrifuged 0.25 M sucrose, 5 mM MES, pH 6.25, 0.1 mM MgSO4, 1 mM DTT, at 13000 g for 3 minutes to precipitate methanol-insoluble proteins, 10 µM FOY-305 (Sanol Schwarz, Monheim, Germany), 2.5 mM which were also subjected to 2-D gel electrophoresis. Trasylol (Bayer, Leverkusen, Germany), 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 500 g and 4°C Protein purification for 10 minutes, and the resulting postnuclear supernatant was To isolate the methanol-soluble, glycosylated acidic and basic centrifuged at 2000 g and 4°C for 10 minutes to sediment zymogen components of the bicarbonate extract, samples were subjected to granules. After removal of the brownish layer of mitochondria on top preparative isoelectric focusing using the Rotofor System (Bio-Rad of the pellet, zymogen granules were lysed in 50 mM Hepes, pH 8.0, Laboratories, Munich, Germany). The samples were desalted using and stored at −20°C. After thawing the soluble zymogen granule content PD-10 desalting columns (Amersham Pharmacia Biotech, Uppsala, proteins (ZGC) were separated from the zymogen granule membranes Sweden) and mixed with 3M urea, 2% ampholytes (Servalyte pH 3.0- (ZGM) by centrifugation at 100000 g and 4°C for 30 minutes. 10.0, Serva, Heidelberg, Germany), 10 µM FOY-305 (Sanol Schwarz, Monheim, Germany), 2.5 mM Trasylol (Bayer, Leverkusen, Isolation of ZGM matrix components Germany) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) in a 500 µl of ZGM corresponding to 200 µg of protein were resuspended volume of 20 ml. Isoelectric focusing was performed according to the in 50 mM Hepes, pH 8.0, mixed with 500 µl of 0.3 M NaHCO3, pH manufacturer’s protocol (Bio-Rad Laboratories, Munich, Germany). 11.5, and incubated on ice for 2 hours. To release GPI-anchored After the initial purification step, samples were separated by SDS- proteins (e.g. GP-2), ZGM corresponding to 800 µg of protein were PAGE and recovered by electro-elution using the Whole Gel Eluter incubated in a final volume of 500 µl of 50 mM Hepes, pH 8.0 system (Bio-Rad Laboratories, Munich, Germany). containing 500 mU of PI-PLC (EC 3.1.4.10, Boehringer Mannheim, Germany) at 37°C for 1 hour. After each treatment membranes were Antibody production recovered by centrifugation at 100000 g for 30 minutes. To generate antibodies directed against the bicarbonate extract, immunization of rabbits was performed at Eurogentec (Seraing, Sulfate labeling and detection of glycans Belgium) using a high-speed supernatant of bicarbonate-treated Pancreatic lobules were prepared as described by Scheele and Palade membranes (bicarbonate extract) as antigen in a standard immunization (1975) and were preincubated in 5 ml sulfate-free medium per one protocol (four cycles of injection of 100 µg protein). The antibodies pancreas. The medium was prepared using the SelectAmin Kit (Life directed against the recombinant ZG16p (Cronshagen et al., 1994) and Technologies, Eggenstein, Germany) with the following modifications: the recombinant GP-2 (Dittie and Kern, 1992) were raised in rabbits. The submembranous network of zymogen granules 2235

Gel electrophoresis and immunoblotting 100 GP-2 Protein samples were separated under denaturing conditions by SDS- ZG16p PAGE (Laemmli, 1970) or by 2-D gel electrophoresis using 8 M urea 80 proteoglycans in the isoelectric focusing (Scheele, 1975). Silver staining was 60 performed according to Hempelmann and Kaminsky (1986). The identification of the individual enzyme spots in 2-D gels was 40 performed according to Schick et al. (1984). For immunoblotting

proteins were transferred to nitrocellulose membranes (Schleicher and soluble [% of total] 20 Schüll, Dassel, Germany) by semi-dry blotting. Membranes were blocked with 1% BSA in TBS for at least 1 hour, subsequently incubated for 2 hours with diluted anti-membrane matrix antibody Hepes pH 8.0 bicarbonate PI-PLC (1:500) or monoclonal anti-chondroitin sulfate antibody (1:50) (Clone Fig. 1. Solubilisation of matrix components from zymogen granule CS-56, Sigma, Deisenhofen, Germany), washed in TBS and finally membranes (ZGM). ZGM (800 µg of protein) were incubated in incubated in diluted peroxidase-conjugated goat anti-rabbit or goat 50 mM Hepes buffer, pH 8.0 (left) or in the presence of 500 mU of anti-mouse antibody (BioRad, Richmond, CA, USA) for 1 hour. PI-PLC (right) for 1 hour at 37°C. Similarly, 200 µg in protein of Immunoblots were processed using diaminobenzidine (DAB) reagent ZGM were incubated in 150 mM NaHCO , pH 10 for 2 hours at 4°C (0.05% DAB, 0.04% CoCl , 0.05% H O in 10 mM Tris-HCl, pH 7.5). 3 3 2 2 (middle). After high speed centrifugation (100000 g, 30 minutes) the In vitro condensation-sorting assay supernatant and pellet fractions were separated by SDS-PAGE and the amounts of GP-2 and ZG16p were determined by densitometric To test the functional significance of the submembranous matrix in quantitation of immunoblots. The amounts of sulfated proteoglycans condensation-sorting a previously established in vitro assay was used were measured using the Blyscan dye, which specifically forms (Dartsch et al., 1998). Briefly, 100 µg of protein extract of precipitable complexes with sulfated glycosaminoglycans on bicarbonate- or PI-PLC-treated membranes were adjusted to pH 8.0 proteoglycans. After dissociation of the precipitated complexes the with 0.5 M MES, pH 4.5, and were added to 1 mg of ZGC. The amount of dye was measured spectrometrically. The values are mixture was adjusted to pH 7.5 or pH 5.9 by adding the same volume expressed as percentage of total supernatant + pellet fractions. (200 µl) of 100 mM MES, pH 4.5 or pH 6.25, respectively. After incubation under rotation at room temperature for 120 minutes the reaction mixture was centrifuged at 13000 g for 30 minutes. The mechanisms by which the granule core is bound to ZGM might resulting pellets were resuspended in 50 mM Hepes, pH 8.0 and be by linkage through the secretory lectin ZG 16p, which was further analyzed. For quantitation of membrane-bound aggregates [35S]methionine/cysteine-labeled ZGC was added to the samples as a found to be associated with proteoglycans and seems to be part tracer (Dartsch et al., 1998). of a matrix on the luminal surface of ZGM (Kleene et al., 1999). To investigate this in more detail, we attempted to remove the Electron microscopy submembranous matrix from the ZGM to characterize its Biopsies from the pancreas of fasted rats, isolated ZGM samples and composition and to analyze the ‘stripped’ ZGM in a standard supernatant and pellet fractions of a standard condensation-sorting condensation-sorting assay (Dartsch et al., 1998). Incubation of assay were fixed in 2% paraformaldehyde/0.1% glutaraldehyde. In the ZGM in Hepes buffer either at 30°C for 1 hour or at 4°C for case of tissue biopsies the fixatives were dissolved in 0.1% cacodylate 2 hours removed about 30-40% each of the GPI-anchored buffer, pH 7.3, while the samples from isolated fractions were fixed in glycoprotein GP-2, the lectin ZG16p and proteoglycans from the their original buffer by adding concentrated fixatives to a final granule membrane (Fig. 1, left). However, incubation in concentration of 1%. The samples were dehydrated in a graded series of alcohol and either embedded in Epon according to standard bicarbonate buffer liberated about 50% of GP-2, 60% of ZG16p procedures or in water-soluble resin Lowicryl K4M (Polysciences Ltd., and 70% of the proteoglycans (Fig. 1, middle). Treatment of Eppenheim, Germany) with polymerisation at −20°C and UV light at ZGM with PI-PLC in Hepes buffer removed only an additional 360 nm wavelength for 48 hours. Thin sections on Formvar-coated 10-15% of the matrix components on top of that removed by grids were stained with uranyl acetate/lead citrate. The matrix Hepes buffer alone (Fig. 1, right). Even after a sequential components were localized in both pancreatic tissue and in isolated treatment (bicarbonate followed by PI-PLC), about 20-30% of fractions using thin sections of K4M-embedded probes, which were the matrix components remained tightly associated with ZGM incubated with a polyclonal antiserum directed against the whole (not shown). The differently treated ZGM and the resulting complement of the isolated ZGM matrix in a dilution of 1:200 to 1:500 extracts were incubated with ZGC proteins at pH 5.9 in a standard and visualized using 10 nm protein A-gold solution (kindly provided in vitro condensation-sorting assay (Fig. 2) (Dartsch et al., 1998). by Dr J. Slot, University of Utrecht, The Netherlands) at a dilution of 1:60 or 1:70, both in 0.5% BSA in PBS. Sections were analyzed using As expected, the binding efficiency of zymogen aggregates to a ZEISS EM 9S electron microscope. To detect proteoglycans in the bicarbonate-treated ZGM was reduced to a lower percentage matrix of ZGM, bicarbonate extract was fixed by adding distilled than to PI-PLC-treated membranes (Fig. 2A), indicating that glutaraldehyde (Serva, Heidelberg, Germany) to a final concentration bicarbonate treatment removed the majority of the matrix of 1% in the presence of 1% Alcian Blue at pH 5.9. The mixture was components required for the binding process. In contrast, addition centrifuged at 13000 g and the precipitate was embedded in Epon and of the liberated matrix components to the assay, which also subjected to electron microscopy of thin sections. contained remnants of ZGM (Dartsch et al., 1998), enhanced the binding capacity (Fig. 2B). This effect was more pronounced with the bicarbonate extract (Fig. 2B). These findings support the RESULTS conclusion that ZGM contain peripheral membrane components which are involved in condensation-sorting. A putative submembranous matrix on ZGM is involved in condensation-sorting Identification of ZGM-associated components We recently obtained evidence that one of the possible To further characterize these components the supernatant 2236 K. Schmidt and others

A 140 120 100 Hepes treated ZGM 80 PI-PLC treated ZGM 60 NaHCO3 treated ZGM 40

cpm in pellet [% of control] 20

B 140 120

100 extract of Hepes treated ZGM 80 extract of 60 PI-PLC treated ZGM extract of 40 NaHCO3 treated ZGM

cpm in pellet [% of control] 20

Fig. 2. Effect of stripped ZGM and of membrane-matrix components on in vitro condensation-sorting. ZGM (100 µg of protein) incubated in 50 mM Hepes buffer, pH 8.0, in the presence or absence of PI- PLC or incubated in NaHCO3, pH 10 (see Fig. 1), were added to a standard in vitro condensation-sorting assay (Dartsch et al., 1998) containing 35S-labeled ZGC as a tracer (A). Similarly, 100 µg of protein of the high speed extracts containing the membrane matrix components were added to the assay in a separate set of experiments (B). The mixtures were incubated for 2 hours at pH 5.9, and after centrifugation at 13000 g the amount of protein-bound radioactivity in the pellet fractions representing aggregated zymogens bound to the membranes was determined. The data are normalized to Hepes- treated ZGM (A) or to the respective supernatant (B) and are representative of three independent experiments.

fractions of PI-PLC- and bicarbonate-treated membranes were separated by 2-D gel electrophoresis and after silver staining the protein patterns were compared to those of separated ZGC proteins (Fig. 3A). The PI-PLC extract contained the GPI- anchored glycoprotein GP2, the membrane-associated serpin ZG46p (Chen et al., 1997), the secretory lectin ZG16p, lipase and, in addition, 6-7 spots in the acidic range and 4 in the neutral Fig. 3. Comparison of membrane-matrix components with zymogen or basic range (Fig. 3B). These protein spots did not cross-react granule content proteins (ZGC). The whole complement of ZGC (A) with antibodies directed to chymotrypsinogen, trypsinogen, and the supernatant fractions of ZGM incubated in Hepes buffer plus procarboxypeptidase or amylase (data not shown), and were not PI-PLC (B) or in bicarbonate buffer (C) were subjected to 2-D gel identical with major zymogen proteins. The bicarbonate extract electrophoresis followed by silver staining. In (A) the known spectrum showed a similar pattern, except that it contained carboxylester of pancreatic enzymes is shown: A, amylase; CEL, carboxyl ester lipase (CEL) and additional protein spots in both the acidic and lipase; C1, C2, isoforms of chymotrypsinogen; L, lipase; PCP, group of procarboxypeptidases; PE1, PE2, PE4, isoforms of proelastase; basic range (Fig. 3C). We then used the whole complement of R, ribonuclease; T1, T2, T4 isoforms of trypsinogen; ZG46p, matrix components released from ZGM by bicarbonate pancreatic serpin; STI, soybean trypsin inhibitor. (B) The PI-PLC treatment to raise a polyclonal antiserum in rabbits. This extract contains the glycoprotein GP-2, the serpin ZG46p, carboxyl antibody was used in immunoblots on ZGC (Fig. 4A), the PI- ester lipase, lipase and a number of additional spots which do not PLC extract (Fig. 4B) and the bicarbonate extract (Fig. 4C). In cross-react with antibodies directed against major ZGC proteins. ZGC the antiserum recognized carboxylester lipase, lipase, the (C) In addition to the protein spots shown in B the bicarbonate extract serpin ZG46p and in trace amounts the secretory lectin ZG16p, contains the secretory lectin ZG16p and several protein spots which do which were known to be associated with the membrane matrix not cross-react with antibodies directed against the major zymogens. (Fig. 4A). In addition, several proteins in the acidic and basic range of the 2-D gel showed a reaction with the antibody, but PLC extract showed a strong reaction of the antibody with GP- the exact nature of these protein spots is still unknown. The PI- 2, ZG46p and weak reactions with both ZG16p and a major spot The submembranous network of zymogen granules 2237

in the acidic range of 30-31 kDa (rectangle, Fig. 4B). However, several protein spots of PI-PLC extracts visible after silver staining did not react with the antibody (compare Figs 3B and 4B). In the bicarbonate extract of ZGM carboxylester lipase, 8- 10 spots in the acidic (arrows) and 6-8 spots in the basic range (arrowhead) were detectable in addition to the proteins shown in Fig. 4B. These spots were not visible in silver-stained gels (compare Figs 3C and 4C). Immunolocalization of matrix components We then used the antiserum directed against the components of the bicarbonate extract to stain various tissues from the rat. At the light microscope level the strongest reaction was found in pancreatic acinar cells but not in duct cells or the islets of Langerhans. In the parotid gland the reaction was restricted to certain portions of the duct system, and a strong reaction was found in the apical cytoplasm of the cells of the prostate (data not shown). Using immunogold techniques at the electron microscope level (Fig. 5) the anti-ZGM matrix antibody reacted with the Golgi complex, with zymogen granules and with the content released into the acinar lumen (Fig. 5A). Inside zymogen granules a concentration of gold particles in the periphery of the condensed content was observed (Fig. 5B). During exocytosis only few gold particles remained associated with the ZGM, which is inserted into the apical plasma membrane (Fig. 5C, arrows). The content proteins released into the acinar lumen formed a fibrillar network with immunogold particles often arranged in line. This observation supports the existence of a network-like structure (Fig. 5D). The matrix components are methanol-soluble proteoglycans and glycoproteins Certain proteoglycans are known to be methanol-soluble (Faltynek et al., 1982). It was therefore likely that the acidic and basic proteins of the bicarbonate extract, which were only visible after immunoblotting but not in silver-stained gels, represented methanol-soluble proteoglycans. To test this assumption we subjected the high-speed supernatant of bicarbonate-treated ZGM to a standard methanol precipitation according to Wessel (1984). After centrifugation, both the supernatant and the pellet fractions were separated on 2-D gels and analyzed by immunoblotting using the anti-matrix antibody. The pellet representing the methanol-insoluble fraction contained predominantly carboxylester lipase, ZG46p, lipase, ZG16p and a few minor, as yet unidentified protein spots (Fig. 6A). The methanol-soluble fraction revealed a similar staining pattern to the whole bicarbonate extract shown in Fig. 4C, consisting of a prominent group of acidic and basic Fig. 4. Immunoblot analysis using an anti-membrane matrix proteins (Fig. 6B). To further verify their proteoglycan nature antibody. 50 µg in protein of ZGC (A), 150 µg in protein of the PI- we used both a monoclonal antibody directed against PLC extract (B), and 50 µg in protein of the bicarbonate extract (C) chondroitin sulfate A and C (clone CS-56, Sigma), and a were separated on 2-D gels and immunoblots were performed with commercial glycan labeling kit. The anti-chondroitin sulfate an antibody directed against the whole bicarbonate extract antibody selectively reacted with one protein spot of the acidic representing ZGM matrix components. (A) In ZGC the antibody group (Fig. 7A, rectangle), which was prominent in the PI-PLC reacts with carboxylester lipase, lipase, ZG46p and ZG16p and in and in the bicarbonate extract (compare Figs 4B,C and 7A), addition with several spots in the acidic and basic range of the gel. and also with four spots of the basic group (arrowheads). We (B) In the PI-PLC extract the antibody strongly reacts with GP-2, conclude that these protein spots represent chondroitin sulfate- ZG46p, ZG16p, CEL, lipase, and with a prominent protein spot of 31 kDa and a pI of 5.9 (rectangle in B and C). (C) The bicarbonate containing proteoglycans. The glycan detection reaction extract also contains GP-2, ZG46p, ZG16p, carboxylester lipase revealed strong labeling of the methanol-soluble acidic group (CEL), lipase and in addition a characteristic group of spots in the of matrix components supporting their glycoconjugate nature acidic (arrows) and basic (arrowheads) range of the gel. (arrows, Fig. 7B). 2238 K. Schmidt and others

ZGM matrix components are enriched after methanol-soluble acidic and basic proteins were separated condensation-sorting preparatively by vertical SDS-PAGE. The proteins were In Fig. 2 it has been demonstrated that solubilized recovered from the gel by electro-elution. The fractions submembranous matrix components enhance the binding containing the methanol-soluble proteins were again identified efficiency of aggregated zymogens to ZGM. To further by immunoblotting using the anti-ZGM matrix antibody. The identify which components of the matrix were involved in amino acid composition of these proteins was determined. It condensation-sorting, the bicarbonate extract was incubated revealed a similarity of one protein of the acidic group (fraction with ZGC either at pH 7.4 or at pH 5.9. After cenrifugation no. 13) with the proteoglycan aggrecan of the rat, a major the pellet fractions were subjected to 2-D gel electrophoresis constituent of the extracellular matrix. The amino acid and immunoblotting using the anti-matrix antibody. A composition of the other proteins of the acidic group (fraction significant enrichment of both the acidic and the basic group numbers 12, 14, 15) were similar and revealed a high degree of glycoproteins was observed in the pellet fractions at pH of glycine residues, characteristic of glycine-rich proteins 5.9, indicating their association with either aggregated found in the cell wall of plants (Table 1). The amino acid zymogens or with ZGM present in the assay (Fig. 8B). This composition of the basic group of proteins showed similarities was further examined by immunogold electron microscopy to the putative polysaccharide binding protein precursor from (Fig. 9). The antibody stained isolated ZGM (Fig. 9A) and porphyra purpurea and to a 27 kDa primary mesenchyme- also the matrix components after their detachment by specific spicule protein precursor of the purple sea urchin bicarbonate treatment (Fig. 9C). When the matrix was fixed (Table 2). N-terminal sequencing of the acidic glycine-rich in the presence of Alcian Blue, a cationic dye which binds to protein no. 15 revealed that the protein starts with six acidic groups on mucosubstances and proteoglycans, a fine consecutive glycines, while N-terminal sequencing of the fibrillar network was observed (Fig. 9B). Addition of the matrix components to a standard condensation-sorting assay revealed a significant enrichment of immunogold particles along the membranes and in the periphery of aggregated zymogens (Fig. 9D- F). Characterization of individual matrix components To isolate the methanol-soluble glycosylated acidic and basic components the bicarbonate extract was subjected to a preparative isoelectric focusing in solution. The fractions obtained after focusing were analyzed by immunoblotting using the anti-ZGM matrix antibody to identify the methanol-soluble proteins. In a second purification step the samples containing the

Fig. 5. Fine structural localization of membrane-matrix components. The polyclonal antibody directed against the complete bicarbonate extract labels the cisternae of the Golgi complex (G in A) and zymogen granules of rat pancreas (A; arrows, B). During exocytosis of granules immunogold particles are seen along the inserted ZGM and in the content (arrows, C). Fibrillar material within the acinar lumen is also labeled (D). Bars, 1 µm (A), 0.1 µm (B-D). The submembranous network of zymogen granules 2239

Fig. 6. Identification of methanol- soluble proteins of the bicarbonate extract. The supernatant of bicarbonate-treated membranes obtained after high speed centrifugation was subjected to methanol precipitation. After centrifugation the pellet fraction representing methanol-insoluble proteins (A) and the supernatant fraction representing methanol- soluble proteins (B) were separated by 2-D gel electrophoresis followed by immunoblotting using the anti- membrane matrix antibody. Note that the unidentified proteins of the basic group (arrowheads) and especially those of the acidic group (arrows) were exclusively detectable in the methanol-soluble fraction. CEL, carboxyl ester lipase; L, lipase; ZG16, secretory lectin; ZG46, secretory serpin. Box in B, see Fig. 4.

Fig. 7. Detection of chondroitin sulfate-containing components and glycoproteins in the bicarbonate extract. 2.5 mg of bicarbonate extract were desalted using a PD 10 desalting column and subjected to 2-D gel electrophoresis followed by immunoblotting using an anti- chondroitin sulfate antibody (A) or subjected to glycan detection (B). Note that the basic group of proteins (arrowheads, A) and only one protein of the acidic group are recognized by the antibody against chondroitin sulfate, while all other proteins of the acidic group are glycosylated proteins (arrows, B). CEL, carboxyl ester lipase.

Fig. 8. Distribution of the group of acidic and basic proteins in a standard in vitro condensation- sorting assay at pH 5.9. ZGC proteins were incubated at pH 5.9 in the absence (A) or in the presence (B) of an extract obtained from bicarbonate-treated ZGM. After centrifugation at 13000 g the pellets were analyzed by 2-D gel electrophoresis followed by immunoblotting using the anti-membrane matrix antibody. Note that the group of acidic (arrows) and basic proteins (arrowheads) were only present in the pellet fraction when bicarbonate extract was added. CEL, carboxyl ester lipase; L, lipase; GP2, glycoprotein 2; ZG16, secretory lectin. chondroitinase-treated basic proteins revealed the sequence DISCUSSION (D,S,GorT)(Q,VorL)DFP(EorR)TGA(AorV), which does not match to any known sequence. Sequencing of tryptic peptides Proteoglycans are macromolecules composed of was not possible, because all proteins of the acidic and basic glycosaminoglycan (GAG) chains covalently bound to a protein group were protease-resistant, supporting their high degree of core. The GAGs are arranged in an alternating unbranched glycosylation. sequence and carry sulfate substituents in various positions Although the exact identification of individual matrix (Kjellen and Lindahl, 1991). Within the organism proteoglycans proteins is so far not possible, we conclude that proteoglycans occur in three major locations: as components of the and glycine-rich glycoproteins are components of the extracellular matrix (ECM; for review, see Iozzo, 1998), as part submembranous matrix. of the glycocalyx on the cell surface (reviewed in Bernfield et 2240 K. Schmidt and others al., 1999) and as a component of intracellular compartments, incorporation in vitro into pancreatic lobules of the guinea pig, predominantly in secretion granules (for review see Kolset and sulfated proteoglycans have been identified within zymogen Gallagher, 1990). Proteoglycans in both the ECM and on the granules of acinar cells and in the secretion of the duct system cell surface are involved in a variety of biological processes, (Tartakoff et al., 1975; Reggio and Palade, 1978). Scheele et al. such as growth and organization of cells and tissues, acting as (1994) reported that more than 90% of sulfate-labeled filters or modulating growth factor activities. These complex proteoglycans in pancreatic acinar cells of the rat were regulatory processes rely on interactions of both the GAG associated with zymogen granule membranes (ZGM). 35 chains and domains in the protein core with various partners in Furthermore, they mentioned that 70% of the SO4-labeled their surrounding. Intracellular proteoglycans have been molecules were released from ZGM by treatment with sodium observed in various cellular compartments, such as in the carbonate at pH 11.2, while the glycoprotein GP-2 remained endosomal compartment, in lysosomes, in the nucleus and most bound to ZGM. Since PI-PLC treatment in this study liberated predominantly in secretion granules of the various cell types of about 50% of GP-2 together with 27% of radiolabeled the haemopoetic system including mast cells in the gut and in proteoglycans, the authors postulated a submembranous matrix connective tissues (for review, see Kolset and Gallagher, 1990). at the luminal surface of ZGM, which at least in part is Secretory granule proteoglycans contain heparan sulfate and composed of the GPI-anchored glycoprotein GP-2 with sulfated chondroitin sulfate chains attached to a unique core protein, proteoglycans attached to it. The present study corroborates and which is composed of repetitive units of serine and glycine, and therefore is referred to as serglycin (Ruoslahti, 1988). Within the secretion granules of haemopoetic cells and mast cells the proteoglycans are complexed to basic proteins, such as proteases and cytolytic agents, and therefore these proteins have been considered as matrix structures involved in the efficient concentration of secretory products (Matsumoto et al., 1995). Sulfation of the GAG chains on granule proteoglycans seems to be important for an efficient storage of secretory products, as indicated by recent studies in transgenic animals in which a key enzyme for sulfation has been knocked out (Forsberg et al., 1999; Humphries et al., 1999a,b). Using sulfate

Fig. 9. Immunolocalisation of membrane-matrix components on ZGM and in the in vitro condensation-sorting assay. The anti-membrane-matrix antibody shows a strong reaction with isolated ZGM (A, enlarged 180000× in inset) and also with the fixed bicarbonate extract (C). When the fixation of bicarbonate extract is performed in the presence of Alcian Blue a fibrillar network is observed (B, arrows). In a standard in vitro condensation-sorting assay to which matrix components have been added, immunogold particles are observed in the periphery of aggregated zymogens (D) and along the membranes which have aggregated zymogens attached (arrows, E,F). Bars, 0.1 µm. The submembranous network of zymogen granules 2241

Table 1. Amino acid composition of the acidic methanol- Table 2. Amino acid composition of the basic methanol- soluble proteins of the membrane matrix soluble proteins of the membrane matrix Acidic protein Acidic proteins Glycine-rich Basic proteins PSBP* PM27‡ no. 13 CSPCP* nos 12, 14, 15 CWSP2P‡ (23-35 kDa, pI 8-9) (22 kDa) (27 kDa) Ala 5.9 6.3 2.0 3.7 Ala 3.0 10.0 6.0 Arg 5.3 3.6 0.8 6.1 Arg 1.6 1.5 3.7 Asx 5.6 6.8 2.1 4.7 Asx 3.8 2.6 8.1 Glx 11.7 12.6 2.6 7.9 Glx 5.7 10.0 11.0 Gly 12.7 12.1 45.0 44.9 Gly 22.1 16.7 18.3 Ile 2.6 3.5 0.6 0.9 Ile 1.2 1.1 3.7 Leu 5.4 6.8 1.2 0.9 Leu 3.8 5.3 8.9 Lys 1.8 1.4 0.3 2.8 Lys 0.8 7.4 3.2 Met 0.0 0.8 0.5 0.5 Met 15.5 9.5 8.5 Phe 1.8 2.8 1.0 4.7 Phe 1.8 3.6 2.8 Pro 8.0 8.0 1.0 0.9 Pro 1.4 7.8 10.5 Ser 12.8 13.2 7.2 7.0 Ser 9.2 7.8 4.4 Thr 6.4 9.6 1.1 2.8 Thr 2.1 4.2 3.7 Tyr 1.2 2.3 0.3 4.2 Tyr 0.6 5.7 2.1 Val 6.1 6.5 3.3 3.3 Val 3.6 5.3 4.9

The bicarbonate extract of ZGM was subjected to preparative isoelectric The methanol-soluble proteins of the basic group were isolated as focusing in solution. The fractions obtained after focusing containing the described in Table 1 and subjected to amino acid analysis. unidentified glycoconjugates were separated preparatively by vertical SDS- *PSBP, putative polysaccharide binding protein precursor, porphyra PAGE and recovered from the gel by electro-elution using the Whole Gel purpurea, 22 kDa, pI 8.5; ‡PM 27, 27 kDa primary mesenchyme-specific Eluter. The methanol-soluble proteins of the acidic group were subjected to spicule protein precursor, purple sea urchin, 29 kDa, pI 8.1. amino acid analysis. *CSPCP, aggrecan core protein precursor (cartilage-specific proteoglycan core protein), rat, 221 kDa, pI 4.2; ‡CWSP2P, cell wall structural protein 2 bicarbonate-soluble membrane matrix components (Fig. 4C). In precursor, wood tobacco, 20 kDa, pI 5.6. immunogold electron microscopy this antibody reacts with No. 13 (36 kDa, pI 5.5); no. 12 (42 kDa, pI 4.2); no. 14 (40 kDa, pI 5.0); components in the periphery of zymogen granules (Fig. 5B,C), no. 15 (34 kDa, pI 5.0). but most importantly it intensely stains ZGM in a standard condensation-sorting assay to which the bicarbonate-soluble extends the previous findings by Scheele et al. (1994) regarding membrane matrix components have been added (Fig. 9D-F). the submembranous matrix on ZGM. Using antibodies directed The equivalent to this staining reaction is the enrichment of the against GP-2 and the lectin ZG16p in immunoblots and a same components in the pellet fraction of the assay at pH 5.9 precipitation assay for proteoglycans we could show that 60- (Fig. 8B). The exact biochemical nature of these membrane- 70% of proteoglycans and about 50-60% of the lectin ZG16p matrix components has not yet been elucidated. are removed from ZGM by bicarbonate treatment at pH 10. At Microsequencing of individual components separated by variance with the previous study the bicarbonate treatment also isolelectric focusing in solution followed by preparative vertical liberates GP-2 to about 50% (Fig. 1). With PI-PLC treatment SDS-gels has so far failed, because most of the components are we could release about 50% of the three components, the protease-resistent. The methanol-soluble acidic protein no. 13 majority of which, however, is liberated by incubation in the (Table 1) shows a similar amino acid composition to the respective buffer alone. This indicates a limited efficiency of PI- cartilage-specific proteoglycan of the rat, although aggrecan has PLC in removing the GPI-anchor of GP-2, which might be due a high molecular weight core protein with a mass of 221 kDa. to the fact that GP2 is masked by the attachment of With the exception of protein no. 13, the other proteins of the proteoglycans at the luminal side of ZGM. However, a acidic group seem to be glycoproteins. They could be consecutive treatment of ZGM with first bicarbonate and then deglycosylated using trifluoro methan sulfonic (TFMS) and PI-PLC also still leaves about 30% of GP-2 in tight association their amino acid composition suggests that they represent with ZGM. Using these pretreated membranes in a standard in glycine-rich glycoproteins. A number of different studies vitro condensation-sorting assay, an inhibition of the attachment indicate that glycine-rich glycoproteins can function as of aggregated zymogens to ZGM was observed to the same adhesion molecules (Schirmer et al., 1994) and that they act as extent as components of the submembranous matrix had scaffold structures in plants (Wojtaszek and Bolwell, 1995). We previously been removed (Fig. 2A). Addition of the extract to therefore assume that the acidic glycine-rich proteins as the assay could compensate for the inhibitory effect (Fig. 2B). components of the ZGM matrix could function as a The 2-D gel analysis has revealed that the membrane matrix submembranous scaffold involved in binding of the granule released by bicarbonate, besides known membrane-associated core and in supporting granule shape and stability. The proteins such as GP-2, carboxylester lipase, lipase, ZG16p and chondroitin sulfate-containing basic proteins (Fig. 7A) have a ZG46p, contains a group of acidic and basic protein spots, the similar amino acid composition, pI and molecular mass to the majority of which behave like proteoglycans or glycoproteins. 27 kDa primary mesenchyme-specific spicule protein precursor They are soluble in methanol (Fig. 6B), they react in a glycan of the sea urchin (Table 2), which represents a skeletal protein staining assay (Fig. 7B), and at least one spot in the acidic group containing a lectin domain (Harkey et al., 1995). and several in the basic group react with a monoclonal antibody Future work is required to further identify the exact nature directed against chondroitin sulfate (Fig. 7A). The same pattern of these membrane-matrix components. It is, however, already of protein spots appears after immunoblotting using a possible to conclude that the binding of aggregated zymogens polyclonal antibody directed against the whole mixture of to the granule membrane is ensured by a macromolecular 2242 K. Schmidt and others matrix consisting of several glycoconjugate components, Humphries, D. E., Wong, G. W., Friend, D. S., Gurish, M. F. and Stevens, which are reversible bound to ZGM during granule formation R. L. (1999b). 14 Heparin-null Transgenic Mice are Unable to Store Certain in the TGN. They are released from ZGM during exocytosis, Granule Proteases in Their Mast Cells. J. Histochem. Cytochem. 47, 16450- 11646. predominantly by bicarbonate ions, which in turn are secreted Iozzo, R. V. (1998). Matrix proteoglycans: from molecular design to cellular into the acinar lumen by the initial duct system. function. Annu. Rev. Biochem. 67, 609-652. Jacob, M., Laine, J. and LeBel, D. (1992). 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