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Frank Thevenod - One of the best experts on this subject based on the ideXlab platform.
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channels and transporters in Zymogen Granule membranes and their role in Granule function recent progress and a critical assessment
Pancreapedia: The Exocrine Pancreas Knowledge Base, 2015Co-Authors: Frank ThevenodAbstract:Secretory Granules are located at the apex of pancreatic acinar cells. Secretagogues bind to their receptors at the basolateral membrane of acinar cells and trigger the activation of intracellular signaling pathways to elicit fusion of secretory Granules with the apical plasma membrane that is followed by exocytosis of digestive pro-enzymes (the “Zymogens”) into the lumen. This regulated discharge of stored macromolecules is accompanied by the secretion of solutes and water to the cell exterior to hydrate these protein-rich secretory products. Previous functional and pharmacological studies in pancreatic acinar cells and Zymogen Granules (ZG) had suggested that ion channels and transporters are expressed in the membrane of ZG where they contribute to maturation, fusion, exocytosis and/or fluidization of Zymogens. This chapter reviews studies that have been largely published in the postgenomic era and combined biochemical, immunological, electrophysiological, pharmacological, and/or occasionally knockout methodologies to identify cloned transporters and ion channels in the membrane of ZG. Available experimental evidence indicates the presence of several ion channel and transporter proteins in ZG membranes (aquaporins, vacuolar-type HATPase, zinc influx transporter SLC30A2). The evidence for the K channels Kv7.1 and Kir6.1, for ClC Cl channels and the vesicular nucleotide transporter SLC17A9 in ZG is less strong. To better understand the function of these proteins in the secretory pathway further studies are needed. 1. Introductory Remarks A review on the topic of pancreatic Zymogen Granule (ZG) channels and transporters and their function is timely as the last exhaustive review appeared in 2002 (107) and is manageable because of the limited number of publications that had been published in the 12 years since then. These circumstances have allowed me to carry out an in-depth and critical analysis of the published data. The advent of the post-genomic era had raised hopes that – similar to other areas of cell biology – a large number of ZG transport proteins would be identified and their role in pancreatic secretion would be elucidated. Indeed, recent studies have combined functional and molecular approaches to characterize ZG channels and transporters and their role in pancreas physiology. Yet, the fact that only a very limited number of studies have been published in this area of research is surprising as there have been tremendous developments of knowledge and methodologies available to investigate the molecular and cellular biology and physiology of
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cyclosporin a but not fk506 induces osmotic lysis of pancreas Zymogen Granules intra acinar enzyme release and lysosome instability by activating k channel
Pancreas, 2012Co-Authors: Matthias Braun, Christian Langeluddecke, Frank ThevenodAbstract:ObjectivesThe immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared with cyclosporin A (CsA), possibly because of reduced acute pancreatitis following ischemia-reperfusion injury. Ion permeabilities in Zymogen Granule (ZG) membranes, including a KCNQ1 K+ channel, pro
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evidence for kcnq1 k channel expression in rat Zymogen Granule membranes and involvement in cholecystokinin induced pancreatic acinar secretion
American Journal of Physiology-cell Physiology, 2008Co-Authors: Wingkee Lee, Blazej Torchalski, Eleni Roussa, Frank ThevenodAbstract:Secretion of enzymes and fluid induced by Ca2+ in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in Zymogen Granule (ZG) membranes. We hypothesi...
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expression of nhe1 and nhe4 in rat pancreatic Zymogen Granule membranes
Biochemical and Biophysical Research Communications, 1998Co-Authors: Ines Anderie, Winfried Haase, Robert Blum, Sergio Grinstein, Frank ThevenodAbstract:Abstract We previously characterized a Na + /H + exchange activity in rat pancreatic Zymogen Granules [Anderie, I., and Thevenod, F. (1996) J. Membrane Biol. 152, 195-205]. Here we have identified the Na + /H + exchanger (NHE) isoforms present in Zymogen Granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited Zymogen Granule Na + /H + exchange in a concentration dependent manner, maximally to 53 ± 5% of controls at 100nM. The remaining Na + /H + exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC 50 ∼ 25μM) or benzamil (EC 50 ∼ 100μM). Amiloride inhibited weakly suggesting that “amiloride-resistant” and “amiloride-sensitive” NHE are expressed in Zymogen Granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in Zymogen Granule membranes was confirmed by immunoblots of Zymogen Granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic Zymogen Granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in Zymogen Granule membranes of rat exocrine pancreas.
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evidence for a 65 kda sulfonylurea receptor in rat pancreatic Zymogen Granule membranes
FEBS Letters, 1997Co-Authors: Matthias Braun, Ines Anderie, Frank ThevenodAbstract:In rat pancreatic Zymogen Granules (ZG), a K+ selective conductance which can be blocked by ATP has been characterized. Here we show that this pathway can be specifically blocked by glibenclamide. Using a rapid filtration assay, we also found specific binding of [3H]glibenclamide to a low-affinity site (Kd 5.6 +/- 1.1 microM) in rat pancreatic Zymogen Granule membranes (ZGM). In photoaffinity labeling experiments with [3H]glibenclamide, a 65 +/- 1.5 kDa polypeptide was specifically labeled. Previously, a approximately 65 kDa mdr1 gene product has been demonstrated to be involved in the regulation of the K+ selective conductance of ZG. We conclude that this protein may be a subunit of, or associated with, a ZG K(ATP) channel.
J M Edwardson - One of the best experts on this subject based on the ideXlab platform.
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cholesterol dependent interaction of syncollin with the membrane of the pancreatic Zymogen Granule
Biochemical Journal, 2001Co-Authors: A Hodel, N J Hansen, J Lawrence, Barbara Wasle, Michael Schrader, J M EdwardsonAbstract:Syncollin is a protein of the pancreatic Zymogen Granule that was isolated through its ability to bind to syntaxin. Despite this in vitro interaction, it is now clear that syncollin is present on the luminal side of the Zymogen Granule membrane. Here we show that there are two pools of syncollin within the Zymogen Granule: one free in the lumen and the other tightly associated with the Granule membrane. When unheated or cross-linked samples of membrane-derived syncollin are analysed by SDS/PAGE, higher-order forms are seen in addition to the monomer, which has an apparent molecular mass of 16 kDa. Extraction of cholesterol from the Granule membrane by treatment with methyl-beta-cyclodextrin causes the detachment of syncollin, and this effect is enhanced at a high salt concentration. Purified syncollin is able to bind to brain liposomes at pH 5.0, but not at pH 11.0, a condition that also causes its extraction from Granule membranes. Syncollin binds only poorly to dioleoyl phosphatidylcholine liposomes, but binding is dramatically enhanced by the inclusion of cholesterol. Finally, cholesterol can be co-immunoprecipitated with syncollin. We conclude that syncollin is able to interact directly with membrane lipids, and to insert into the Granule membrane in a cholesterol-dependent manner. Membrane-associated syncollin apparently exists as a homo-oligomer, possibly consisting of six subunits, and its association with the membrane may be stabilized by electrostatic interactions with either other proteins or phospholipids.
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targeting of the Zymogen Granule protein syncollin in ar42j and att 20 cells
Biochemical Journal, 2000Co-Authors: A Hodel, J M EdwardsonAbstract:Syncollin is a 13-kDa protein associated with the membranes of pancreatic Zymogen Granules. Here we determine the in situ localization of syncollin in pancreatic acinar cells from adult and neonatal rats, and study the targeting of green fluorescent protein-(GFP-) and His(6)-tagged syncollin chimaeras in model exocrine and endocrine secretory cells. Immunocytochemical analysis of the distribution of syncollin in fully differentiated and neonatal acinar cells revealed a granular pattern that corresponded with that of the Zymogen-Granule markers synaptobrevin 2 and amylase. In fully differentiated acinar cells syncollin-positive vesicles were detected in the apical region of the cells, whereas in neonatal acinar cells they were found clustered near the cell nucleus. Both GFP- and His(6)-tagged syncollin entered the secretory pathway when transiently expressed in AR42J or AtT-20 cells. Syncollin-GFP was found predominantly in amylase-positive Granules in AR42J cells and in adrenocorticotrophic hormone- (ACTH-) positive Granules in AtT-20 cells. Syncollin-GFP was also present in the Golgi complex in AR42J cells. Syncollin-His(6) became localized in ACTH-containing Granules in the neuritic processes of AtT-20 cells. In AR42J cells syncollin-His(6) did not co-localize with amylase, but was detected in acidic vesicles. These results show that the exocrine protein syncollin contains intrinsic cell-type-independent targeting information that is retained in both exocrine and endocrine cells after fusion to the GFP tag. In contrast, His(6)-tagged syncollin is efficiently targeted to secretory Granules only in AtT-20 cells and not in AR42J cells.
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analysis of the association of syncollin with the membrane of the pancreatic Zymogen Granule
Journal of Biological Chemistry, 2000Co-Authors: N J Hansen, A Hodel, Reinhard Jahn, J M EdwardsonAbstract:Syncollin is a pancreatic Zymogen Granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from Granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when Granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the Granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When Granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of Granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.
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Association of nucleoside diphosphate kinase with pancreatic Zymogen Granules: effects of local GTP generation on Granule membrane characteristics.
Biochemical Journal, 1996Co-Authors: Stefan J. Marciniak, J M EdwardsonAbstract:It is well established that both GTP-binding proteins and phosphoproteins are involved in the control of exocytosis in the exocrine pancreas. Exocytotic membrane fusion is stimulated by guanosine 5'-[gamma-thio]triphosphate, and the phosphorylation states of several proteins, including at least one on the Zymogen Granule membrane, are known to change during exocytosis. We show here that a nucleoside diphosphate kinase is associated with the cytoplasmic face of pancreatic Zymogen Granules. This enzyme behaves as a phosphoprotein of apparent molecular mass 21 kDa on SDS/polyacrylamide gels, and is able to produce GTP by using ATP to phosphorylate endogenous GDP. GTP production by nucleoside diphosphate kinase is stimulated by the wasp venom peptide mastoparan, both through a direct action on the enzyme and through its ability to increase the availability of endogenous GDP. Two effects of the GTP produced by nucleoside diphosphate kinase are demonstrated: phosphorylation of a 37 kDa Zymogen Granule protein on histidine residues, and stimulation of the fusion of Zymogen Granules with pancreatic plasma membranes in vitro. These results suggest that Granule-associated nucleoside diphosphate kinase is able to maintain local GTP concentrations, and raise the possibility that it might be involved in the control of exocytosis in the pancreatic acinar cell.
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involvement of a phosphoprotein on the Zymogen Granule membrane in the control of regulated exocytosis in the exocrine pancreas
Journal of Cell Science, 1993Co-Authors: C M Maclean, Stefan J. Marciniak, D V Hall, J M EdwardsonAbstract:The pancreatic acinar cell is one of a number of cell types in which phosphoproteins are believed to be involved in the control of regulated exocytosis. We have examined the effects of three agents that affect secretion in the acinar cell on the phosphorylation states of proteins on the Zymogen Granule membrane. We show that Ca2+ and GTP gamma S, which stimulate secretion, also stimulate the phosphorylation of a protein of M(r) 45,000 (p45) on isolated Zymogen Granules. On the other hand, the protein kinase inhibitor genistein inhibits both secretion and phosphorylation of p45. For all three agents, p45 phosphorylation is affected over concentration ranges identical to those that affect secretion. The stimulatory effect of GTP gamma S and the inhibitory effect of genistein are also seen when the phosphorylation state of p45 on Granules within permeabilized cells is examined. Ca2+, however, over the same concentration range, now causes dephosphorylation of p45. Furthermore, the time-course of this effect is similar to that of Ca(2+)-triggered secretion. Phosphorylation of p45 is exclusively on serine, with no detectable phosphorylation on either threonine or tyrosine. We propose that exocytosis in pancreatic acini is controlled at least in part through the phosphorylation/dephosphorylation of p45, with dephosphorylation acting as a trigger for exocytosis.
John A Williams - One of the best experts on this subject based on the ideXlab platform.
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global topology analysis of pancreatic Zymogen Granule membrane proteins
Molecular & Cellular Proteomics, 2008Co-Authors: Xuequn Chen, John A Williams, Peter J Ulintz, Eric S Simon, Philip C AndrewsAbstract:The Zymogen Granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory Granule function. Our long term goal is to develop a comprehensive architectural model for Zymogen Granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Chen, X., Walker, A. K., Strahler, J. R., Simon, E. S., Tomanicek-Volk, S. L., Nelson, B. B., Hurley, M. C., Ernst, S. A., Williams, J. A., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic Zymogen Granule membranes. Mol. Cell. Proteomics 5, 306-312). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their isobaric tag for relative and absolute quantification (iTRAQ) ratios for proteinase K-treated versus control Zymogen Granules. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including myosin V, vesicle-associated membrane proteins, syntaxins, and all the Rab proteins. The second cluster having unchanged ratios included predominantly luminal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. To more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified Zymogen Granule membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.
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organellar proteomics analysis of pancreatic Zymogen Granule membranes
Molecular & Cellular Proteomics, 2006Co-Authors: Xuequn Chen, John A Williams, Eric S Simon, Angela K Walker, John R Strahler, Sarah L Tomanicekvolk, Bradley Nelson, Mary C Hurley, Stephen A Ernst, Philip C AndrewsAbstract:The Zymogen Granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory Granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate Zymogen Granule functions through proteomic and subsequent functional analysis of its membrane components.
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identification of rab 5 but not rab 3a in rat pancreatic Zymogen Granule membranes
Biochemical and Biophysical Research Communications, 1994Co-Authors: A C C Wagner, M Z Strowski, John A WilliamsAbstract:Abstract Low molecular weight GTP-binding (LMWG) proteins are known to be present on secretory organelles and a role for Rab 3A in exocytosis of synaptic vesicles has been postulated. Pancreatic Zymogen Granules possess multiple LMWG proteins which were shown to be on the outside of the Granules by pronase susceptability. Rab 3A, however, could not be identified in rat pancreatic acinar cells or on Zymogen Granules by immunoblots or by northern blots or PCR amplification although all three techniques demonstrated its presence in rat brain, RINm5F and AR42J cells. Immunoblots revealed expression of Rab 5, Rab 7, Rab 11 and ARF in acini. Of these, Rab 5 was identified in purified Zymogen Granule membranes where it may be involved in the endocytotic retrieval of Granule membrane following exocytosis.
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gp 3 a newly characterized glycoprotein on the inner surface of the Zymogen Granule membrane undergoes regulated secretion
Journal of Biological Chemistry, 1994Co-Authors: A C C Wagner, Philip C Andrews, Matthew J Wishart, Sabine M Mulders, Paulette M Blevins, Anson W Lowe, John A WilliamsAbstract:Abstract We have recently reported the cloning of the rat Zymogen Granule membrane glycoprotein GP-3 and the related pancreatic secretory lipase (Wishart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C.D., and Williams, J. A. (1993) J. Biol. Chem. 268, 10303-10311). Specific antipeptide antibodies were generated against both GP-3 and secretory lipase and used for the biochemical and physiological characterization of GP-3. Western blotting confirmed that GP-3 was found exclusively in Zymogen Granule membranes and was absent from Zymogen Granule content which contains the majority of secretory lipase. Extraction of Zymogen Granule membranes with Triton X-114 showed GP-3 to be significantly more hydrophobic than lipase. The GP-3 amino acid sequence contains one potential N-linked glycosylation site at Asn-336. The loss of concanavalin A labeling after both chemical deglycosylation with trifluoromethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Digestion of intact and permeabilized Zymogen Granules with the nonspecific protease Pronase localized GP-3 to the inner surface of Zymogen Granule membranes. Since GP-3 is resident on the inner surface of the Zymogen Granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we observed that GP-3 is released into the pancreatic juice and that secretion of GP-3 was greatly enhanced by cholecystokinin.
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pancreatic Zymogen Granule membrane proteins molecular details begin to emerge
Digestion, 1994Co-Authors: A C C Wagner, John A WilliamsAbstract:Regulated secretion and exocytosis require the controlled docking and fusion of secretory storage organelles with the plasma membrane. Proteins resident in secretory organelle membranes are, therefore, likely to have critical functions as a part of these processes. The exocrine pancreas with its secretory storage organelle, the Zymogen Granule, represents a classical model of regulated secretion and exocytosis. Until recently, however, little has been known of Zymogen Granule membrane protein structure and function. With the recent cloning of two Zymogen Granule membrane proteins, GP-2 and GP-3, and with the identification of the presence of low molecular weight guanosine triphosphate binding proteins, it is now possible to approach the role of the Zymogen Granule membrane proteins at the molecular level.
Anson W Lowe - One of the best experts on this subject based on the ideXlab platform.
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the pancreatic Zymogen Granule membrane protein gp2 binds escherichia coli type 1 fimbriae
BMC Gastroenterology, 2009Co-Authors: Anson W LoweAbstract:GP2 is the major membrane protein present in the pancreatic Zymogen Granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.
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Escherichia coli type 1 Fimbriae
2009Co-Authors: Anson W LoweAbstract:The pancreatic Zymogen Granule membrane protein, GP2, bind
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absence of the major Zymogen Granule membrane protein gp2 does not affect pancreatic morphology or secretion
Journal of Biological Chemistry, 2004Co-Authors: Sara A Michie, Anson W LoweAbstract:The majority of digestive enzymes in humans are produced in the pancreas where they are stored in Zymogen Granules before secretion into the intestine. GP2 is the major membrane protein present in Zymogen Granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the Zymogen Granule. Other studies have suggested that GP2 is important in the formation of Zymogen Granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malabsorption such as weight loss, growth retardation, or diarrhea. Zymogen Granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or Zymogen Granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.
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processing of the major pancreatic Zymogen Granule membrane protein gp2
Pancreas, 2002Co-Authors: Benjamin A Fritz, Clinton S Poppel, Matthew W Fei, Anson W LoweAbstract:Introduction The pancreatic exocrine secretory Granule, the Zymogen Granule, releases digestive enzymes into the intestine. GP2 is the most abundant Zymogen Granule membrane protein. Coincident with exocrine secretion, GP2 is released from the membrane and secreted into the pancreatic duct. Aim To characterize changes in the structure of GP2 as it progresses through the secretory pathway. Methodology Polarized MDCK cells that express the rat GP2 gene were used to examine the sequential processing of the polypeptide backbone. Results Within the cell, GP2 is initially proteolytically processed from a 55- to a 53-kd form at or before the trans-Golgi network. The protein is then processed to a 51-kd form, which is found on the apical plasma membrane and in secretions. Similar processing was also observed in primary rat pancreatic cultures and in MDCK cells that express human GP2. The amino-terminal sequence of human GP2 derived from pancreatic secretions was determined for two human patients and began at Gly39, revealing a potential processing site. Conclusions In contrast to other digestive enzymes secreted by the pancreas that are activated by proteolysis in the intestine, GP2 undergoes sequential intracellular cleavage. Alterations in GP2 structure by proteolysis may regulate GP2 function at specific sites within the pancreatic cell.
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gp 3 a newly characterized glycoprotein on the inner surface of the Zymogen Granule membrane undergoes regulated secretion
Journal of Biological Chemistry, 1994Co-Authors: A C C Wagner, Philip C Andrews, Matthew J Wishart, Sabine M Mulders, Paulette M Blevins, Anson W Lowe, John A WilliamsAbstract:Abstract We have recently reported the cloning of the rat Zymogen Granule membrane glycoprotein GP-3 and the related pancreatic secretory lipase (Wishart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C.D., and Williams, J. A. (1993) J. Biol. Chem. 268, 10303-10311). Specific antipeptide antibodies were generated against both GP-3 and secretory lipase and used for the biochemical and physiological characterization of GP-3. Western blotting confirmed that GP-3 was found exclusively in Zymogen Granule membranes and was absent from Zymogen Granule content which contains the majority of secretory lipase. Extraction of Zymogen Granule membranes with Triton X-114 showed GP-3 to be significantly more hydrophobic than lipase. The GP-3 amino acid sequence contains one potential N-linked glycosylation site at Asn-336. The loss of concanavalin A labeling after both chemical deglycosylation with trifluoromethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Digestion of intact and permeabilized Zymogen Granules with the nonspecific protease Pronase localized GP-3 to the inner surface of Zymogen Granule membranes. Since GP-3 is resident on the inner surface of the Zymogen Granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we observed that GP-3 is released into the pancreatic juice and that secretion of GP-3 was greatly enhanced by cholecystokinin.
Michael J Edwardson - One of the best experts on this subject based on the ideXlab platform.
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identification of snares that mediate Zymogen Granule exocytosis
Biochemical and Biophysical Research Communications, 2007Co-Authors: James A Pickett, Manuel Campostoimil, Paul Thomas, Michael J EdwardsonAbstract:A secretagogue-stimulated pancreatic acinar cell releases digestive enzymes from its apical pole. We attempted to identify the SNAREs involved in Zymogen Granule exocytosis. Antibodies against syntaxins 2 and 3, SNAP-23 and VAMP 8, and the corresponding recombinant SNAREs, inhibited amylase secretion from streptolysin O-permeabilised acini; other anti-SNARE antibodies and SNAREs had no effect. Botulinum neurotoxin C, which cleaved syntaxin 2 and (to a lesser extent) syntaxin 3, but not syntaxins 4, 7 or 8, also inhibited exocytosis. We propose that syntaxin 2, SNAP-23 and VAMP 8 mediate primary Granule-plasma membrane fusion. Syntaxin 3 may be involved in secondary Granule-Granule fusion.
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syncollin is required for efficient Zymogen Granule exocytosis
Biochemical Journal, 2005Co-Authors: Barbara Wasle, Matthew R Turvey, Olga Larina, Peter Thorn, Jeremy N Skepper, Jennifer A Morton, Michael J EdwardsonAbstract:Syncollin is a 13 kDa protein that is present in the exocrine pancreas, where the majority of the protein is tightly attached to the luminal surface of the Zymogen Granule membrane. We have addressed the physiological role of syncollin by studying the phenotype of syncollin KO (knockout) mice. These mice show pancreatic hypertrophy and elevated pancreatic amylase levels. Further, secretagogue-stimulated amylase release from pancreatic lobules of syncollin KO mice was found to be reduced by about 45% compared with wild-type lobules, and the delivery of newly synthesized protein to Zymogen Granules was delayed, indicating that the mice have a pancreatic secretory defect. As determined by two-photon imaging, the number of secretagogue-stimulated exocytotic events in acini from syncollin KO mice was reduced by 50%. This reduction was accounted for predominantly by a loss of later, 'secondary' fusion events between Zymogen Granules and other Granules that had already fused with the plasma membrane. We conclude that syncollin is required for efficient exocytosis in the pancreatic acinar cell, and that it plays a particularly important role in compound exocytosis.
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the plasma membrane q snare syntaxin 2 enters the Zymogen Granule membrane during exocytosis in the pancreatic acinar cell
Journal of Biological Chemistry, 2005Co-Authors: James A Pickett, Peter Thorn, Michael J EdwardsonAbstract:Abstract During exocytosis in the pancreatic acinar cell, Zymogen Granules fuse directly with the apical plasma membrane and also with Granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused Granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused Granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused Granules, as judged by its location around dye-filled structures of 1-μm diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled Granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled Granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused Zymogen Granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion.
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acetylcholine induced Zymogen Granule exocytosis comparison between acini and single pancreatic acinar cells
Pancreas, 2002Co-Authors: Manuel Campostoimil, Michael J Edwardson, Paul ThomasAbstract:INTRODUCTION Numerous studies have been carried out on the agonist-evoked calcium responses of single pancreatic acinar cells; however, several reports have shown that dissociation of the exocrine pancreas into predominantly single cells has an adverse effect on agonist-evoked amylase secretion. AIMS AND METHODOLOGY To determine whether single acinar cells behave in an anomalous manner compared with cells within an intact acinus, we measured exocytosis in both single acinar cells and acini (2-5 cells) present in the same preparation. Exocytosis of individual Zymogen Granules was quantified in real-time by using the technique of continuous time-differential analysis of brightfield digital images. RESULTS Basal rates of exocytosis were low in both single cells and intact acini. Application of 10 microM acetylcholine for 6 minutes stimulated a biphasic secretory response in acinar cells. Additionally, we found that exocytotic events occur repetitively in specific locations within the apical domain; i.e., there are exocytotic "hot spots." There were no statistically significant differences between the exocytotic rates, nor were there any differences in the characteristics of the exocytotic hot spots of single cells compared with those of acini. CONCLUSION We conclude that time-differential analysis of brightfield images appears to be a useful tool for the investigation of the role of gap junctions in Zymogen Granule exocytosis and that single acinar cells provide a reasonable model for studies of acinar cell signaling and secretion.