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Protein Composition and Biogenesis of the Pancreatic Zymogen Granules

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Protein Composition and Biogenesis of the Pancreatic Zymogen Granules Protein Composition and Biogenesis of the Pancreatic Zymogen Granules Xuequn Chen Department of Physiology, Wayne State University School of Medicine. Detroit, MI e-mail: [email protected] Version 2.0, January 27th, 2021 [DOI: 10.3998/panc.2021.03] I. INTRODUCTION Physiological stimulation of acinar cells by secretagogues triggers local apical Ca2+ spiking, The exocrine pancreas produces and secretes fusion of ZG membrane with the apical membrane, multiple digestive enzymes and has been the and exocytosis (53, 60, 61, 85). In contrast to the model in which the structure and functional physiological condition, supramaximal CCK organization of the mammalian secretory pathway stimulation elicits sustained elevation of cytosolic was originally discovered and intensively studied [Ca2+] and leads to mistrafficking of digestive and (59, 83) The pancreatic acinar cells exhibit one of lysosomal enzymes, inhibition of apical secretion the highest protein synthesis rates among and abnormal exocytosis redirected to basolateral mammalian cells. More than 90% of the newly plasma membrane all of which are believed to synthesized proteins are targeted to the secretory contribute to the pathogenesis of acute pancreatitis pathway (71) and packaged into large secretory (23, 68). The ZG content contains the digestive granules, called zymogen granules (ZG). In enzymes and associated proteins which are the contrast to the smaller neuroendocrine and major protein components of the pancreatic juice endocrine granules, ZGs have an averaged secreted into the duodenum. The ZG membrane diameter of around 1µm. They are responsible for carries at least part of the molecular machinery transport, storage and secretion of digestive responsible for digestive enzyme sorting, granule enzymes and have long been a model for studying trafficking and exocytosis. For example, digestive the general mechanisms of secretory granule enzyme sorting and packaging will, at least to biogenesis and regulated exocytosis. Stimulation some extent, depend on interactions between ZG of the acinar cells by secretagogues such as content and ZG membrane components exposed acetylcholine and cholecystokinin triggers fusion of to the lumen of ZGs. The cytoplasmic surface of ZG membrane with the apical plasma membrane ZG membrane must contain vesicular trafficking and the release of digestive enzymes into the proteins including Rabs, SNARE proteins as well pancreatic ductal system. In the duodenum, as molecular motors to interact with cytoskeleton. trypsinogen is converted to trypsin by proteolytic Defective ZG biogenesis and trafficking can result cleavage via enterokinase and activated trypsin in various pancreatic diseases such as acute and then proteolytically activates the other zymogen chronic pancreatitis (23, 31, 68). A comprehensive enzymes (9, 58). understanding of the protein composition of the ZG content and membrane is necessary to provide 1 critical insights in the biogenesis and regulated It is believed that the integral and peripheral ZG secretion of pancreatic ZGs. membrane proteins serve critical functions for zymogen sorting/packaging, vesicular trafficking II. ZG PROTEIN COMPOSITION and regulated exocytosis. Therefore, a comprehensive identification of ZG membrane A. Zymogens and digestive enzymes proteins is expected to shed new lights on our understanding of ZG biogenesis and secretion. In The major secretory products of the acinar cells, early studies (20, 37, 52), characterization of rat namely the content of ZGs, are digestive enzymes ZG membranes by SDS-PAGE indicated a which belong to five functional groups of hydrolytic relatively simple protein composition of about 10 enzymes including endo- and exo-proteases, components with GP2 (glycoprotein 2) accounting lipases, glycosidases, and nucleases. In contrast for 40% of the proteins. In the past decade, studies to endocrine cells which often produces a have been carried out to characterize ZG predominant peptide or protein product such as membrane proteins using two-dimensional gel insulin, acinar cells synthesize, package and electrophoresis. These efforts led to the secrete a mixture of nearly 20 different enzymes identifications of two additional major ZG and isoenzymes including amylase, trypsinogens, membrane components, GP3 (glycoprotein 3) (79) chymotrypsinogens, carboxypeptidases, and membrane dipeptidase (35), by N-terminal esterases, lipases and ribonucleases. Most amino acid sequence analysis. However, due to pancreatic proteases are synthesized as inactive the lack of sensitive tools for protein identification, precursors or zymogens which only become the identities of many spots resolved on the 2D activated by a cascade of limited proteolysis within gels remained unknown. In another study, fourteen the intestinal lumen. Because of the importance of spots corresponding to small GTP binding proteins ZG to digestive enzyme storage and regulated were resolved on a 2D gel of ZG membrane secretion and as a general model for secretory proteins by [35S]GTPγS blotting analysis (28). vesicles, the identification and characterization of However, the identities of the spots were not both the soluble and membrane proteins of ZGs determined. Different from the above abundant ZG have been of great interest in the field. In an early membrane proteins, a number of low abundance pioneering study, the secreted ZG contents from regulatory proteins have been identified on ZG the guinea pig exocrine pancreas were analyzed membrane by immunoblotting and by two-dimensional gel electrophoresis which immunocytochemistry. Examples of these proteins resolved 19 distinct high molecular weight proteins. included the small GTPase, Rab3D (55, 77) and Thirteen of the 19 proteins were identified by actual the SNARE proteins, VAMP2 (vesicle associated or potential enzymatic activity (69). In more recent membrane protein 2) (25) and syntaxin 3 (22, 34). studies using mass spectrometry-based More recently, VAMP 8 was found on ZG proteomics analyses, the identities of these membrane and to play a major physiological role in enzymes have been confirmed and additional regulated exocytosis (81). Despite the significant isoforms were found (12, 63). The ZG contents amount of knowledge of ZG membrane proteins make up the major protein components of the accumulated in the past decades on an individual pancreatic juice secreted into the duodenum. basis, a comprehensive characterization of the Therefore, the identification of ZG content proteins membrane protein components of this organelle also has a significant impact on biomarker studies was not achieved until the application of modern in the pancreatic juice (15). mass spectrometry revealed a much more B. Components and topology of the ZG complex makeup of the ZG membrane (11, 12, 63). membrane proteins 2 Organellar proteomics represents an analytical in two consecutive low speed steps to generate a crude strategy that combines biochemical fractionation particulate fraction (P2) enriched in ZGs. The particulate was resuspended, mixed with equal volume of Percoll, and comprehensive protein identification. Initial and ultracentrifuged. The dense white ZG band was purification of organelles leads to reduced sample then collected and washed. To purify ZG membrane, the complexity and links proteomics data to functional isolated ZGs were lysed with nigericin and analysis (7, 80, 90). In the past decade, organellar ultracentrifuged to separate contents and membranes. The membrane pellet was then washed sequentially proteomic analysis has been carried out for with 250 mM KBr and 0.1 M Na2CO3 (pH 11.0). Right: virtually every subcellular compartment in the Top shows a cartoon to illustrate the Percoll gradient mammalian secretory pathway including Golgi, ER ultracentrifugation; at the bottom are Nomarski and and secretory granules (80). The first fluorescent images of purified ZGs to demonstrate the purity of ZGs and the positive staining of a ZG marker, comprehensive analysis of rat ZG membrane were Rab3D. (Reproduced from reference (12)). carried out by combining modern mass spectrometry-based proteomics technologies and These results indicated a much more complex a well-established protocol of ZG purification (12). protein composition of the ZG membrane. By Using this protocol (outlined in Figure 1, left), a combining multiple separation strategies including crude granule pellet (P2) was prepared by two one-, two-dimensional gel electrophoresis and two- consecutive low speed centrifugations and then dimensional HPLC with tandem mass further purified by an ultracentrifugation in a self- spectrometry, over 100 proteins were identified forming Percoll gradient. A heavy white band, from purified ZG membrane (12). Most of the containing highly purified ZGs, was observed and known ZG membrane proteins were identified, collected just above the bottom of the tube. The including high abundance matrix proteins such as ZG membrane and content proteins were then GP2, GP3, ZG16 and syncollin which are likely separated by osmotic lysis of ZGs with the involved in ZG sorting and packaging, and low ionophore, nigericin, followed by abundance proteins such as dynactin2 (48) and ultracentrifugation. The membrane pellet was VAMP2 which are involved in ZG trafficking and washed first with 0.25M KBr and then with 0.1M exocytosis. A large number of novel ZG membrane Na2CO3 (pH 11.0) to remove soluble content proteins were also identified, including the SNARE proteins and loosely associated proteins. The protein,
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