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,