Protein Composition and Biogenesis of the Pancreatic Zymogen Granules Xuequn Chen, Ph.D. Department of Physiology, Wayne State University e-mail: [email protected] Version 1.0 April 19, 2018 DOI: 10.3998/panc.2018.16 1. 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 (50, 57, 58, 81). 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 (56) and intensively [Ca2+] and leads to mistrafficking of digestive and studied thereafter. The acini of the exocrine lysosomal enzymes, inhibition of apical secretion pancreas are the functional units of digestive and abnormal exocytosis redirected to basolateral enzyme synthesis, storage and regulated plasma membrane all of which are believed to secretion (79). The pancreatic acinar cells exhibit contribute to the pathogenesis of experimental one of the highest protein synthesis rates among acute pancreatitis (21, 65). The ZG content mammalian cells. More than 90% of the newly contains primarily the digestive enzymes and synthesized proteins are targeted to the secretory associated proteins which are the major protein pathway (68) and packaged into large secretory components of the pancreatic juice secreted into granules, called zymogen granules (ZG). In the duodenum. The ZG membrane carries at least contrast to the relatively small neuroendocrine part of the molecular machinery responsible for and endocrine granules, ZGs have an averaged digestive enzyme sorting, granule trafficking and diameter of around 1µm. ZGs are responsible for exocytosis. For example, digestive enzyme transport, storage and secretion of digestive sorting and packaging will, at least to some enzymes and has long been a model for studying extent, depend on interactions between ZG the mechanism of secretory granule biogenesis content and ZG membrane components exposed and regulated exocytosis. Stimulation of the to the lumen of ZGs. The cytoplasmic surface of acinar cells by secretagogues such as ZG membrane must contain vesicular trafficking acetylcholine and cholecystokinin triggers fusion proteins including Rabs, SNARE proteins as well of ZG membrane with the apical plasma as molecular motors to interact with cytoskeleton. membrane and the release of digestive enzymes Defective ZG biogenesis and trafficking can result into the pancreatic ductal system. In the in various pancreatic diseases such as acute and duodenum, trypsinogen is converted to trypsin by chronic pancreatitis (21, 29, 65). A proteolytic cleavage via enterokinase and comprehensive understanding of the protein activated trypsin then proteolytically activates the composition of the ZG content and membrane will other zymogen enzymes (8, 55). provide critical insights in the biogenesis and regulated secretion of pancreatic ZGs. 1 It is believed that the integral and peripheral ZG 2. ZG Protein Composition membrane proteins serve critical functions for zymogen sorting/packaging, vesicular trafficking Zymogens and digestive enzymes and regulated exocytosis. Therefore, a The major secretory products of the acinar cells, comprehensive identification of ZG membrane namely the content of ZGs, are digestive enzymes proteins is expected to shed new lights on our which belong to five functional groups of understanding of ZG biogenesis and secretion. In hydrolytic enzymes including endo- and exo- early studies (20, 35, 49), characterization of rat proteases, lipases, glycosidases, and nucleases. ZG membranes by SDS-PAGE indicated a In contrast to endocrine cells which often relatively simple protein composition of about 10 produces a predominant peptide or protein components with the glycoprotein GP2 accounting product such as insulin, acinar cells synthesize, for 40% of the proteins. In the past decade, package and secrete a mixture of nearly 20 several studies have been carried out to different enzymes and isoenzymes including characterize ZG membrane proteins using two- amylase, trypsinogens, chymotrypsinogens, dimensional gel electrophoresis. These efforts led carboxypeptidases, esterases, lipases and to the identifications of two major ZG membrane ribonucleases. Most pancreatic proteases are components, GP3 (75) and membrane synthesized as inactive precursors, zymogens, dipeptidase (33), by N-terminal amino acid which only become activated by a cascade of sequence analysis. However, due to the lack of limited proteolysis within the intestinal lumen. sensitive tools for protein identification, the Because of the importance of ZG to digestive identities of many spots resolved on the 2D gels enzyme storage and regulated secretion and as a remained unknown. In another study, fourteen general model for secretory vesicles, the spots corresponding to small GTP binding identification and characterization of both the proteins were resolved on a 2D gel of ZG soluble and membrane proteins of ZGs have been membrane proteins by [35S]GTPγS blotting of great interest in the field. In an early pioneering analysis (26). However, the identities of the spots study, the secreted ZG contents from the guinea were undetermined. Different from the above pig exocrine pancreas were analyzed by two- abundant ZG membrane proteins, a number of dimensional gel electrophoresis which resolved low abundant regulatory proteins were identified 19 distinct high molecular weight proteins. on ZG membrane by immunoblotting and Thirteen of the 19 proteins were identified by immunocytochemistry. Examples of these actual or potential enzymatic activity (66). In more proteins included small GTPase, Rab3D (52, 73), recent studies using modern mass spectrometry- and SNARE proteins, VAMP2 (23) and syntaxin 3 based proteomics analyses, the identities of these (23, 32). More recently, VAMP 8 was found on ZG enzymes were confirmed and additional isoforms membrane and to play a major physiological role of these digestive enzymes were found (11, 60). in regulated exocytosis (77). Despite of the The ZG content contains primarily the digestive significant amount of knowledge on ZG enzymes and associated proteins which make up membrane proteins accumulated in the past the major protein components of the pancreatic decades on an individual basis, a comprehensive juice secreted into the duodenum. Therefore, the characterization of the membrane protein identification of ZG content proteins also has a components of this organelle was not achieved significant impact on biomarker studies in the until the recent decade. The more recent pancreatic juice (15). organellar proteomic analyses using modern mass spectrometry revealed a much more Components and topology of the ZG complex makeup of the ZG membrane (10, 11, membrane proteins 60). 2 Organellar proteomics represents an analytical strategy that combines biochemical fractionation and comprehensive protein identification. Initial purification of organelles leads to reduced sample complexity and links proteomics data to functional analysis (5, 76, 86). In the past decade, organellar proteomic analysis has been carried out for virtually every subcellular compartment in the mammalian secretory pathway including Golgi, Figure 1. Outline of ZG membrane purification. ER and secretory granules (76). The first Left, rat pancreata were homogenized and then comprehensive analysis of rat ZG membrane centrifuged in two consecutive low speed steps to were carried out as a natural combination of generate a crude particulate fraction (P2) enriched in ZGs. The particulate was resuspended, mixed with modern mass spectrometry-based proteomics equal volume of Percoll, and ultracentrifuged. The technologies and a well-established protocol of dense white ZG band was then collected and washed. ZG purification (11). Using this protocol (outlined To purify ZG membrane, the isolated ZGs were lysed in Figure 1, left), a crude granule pellet (P2) was with nigericin and ultracentrifuged to separate contents and membranes. The membrane pellet was prepared by two consecutive low speed then washed sequentially with 250 mM KBr and 0.1 M centrifugations and then further purified by an Na2CO3 (pH 11.0). Right, Top shows a cartoon to ultracentrifugation in a self-forming Percoll illustrate the Percoll gradient ultracentrifugation; at the gradient. A heavy white band, containing highly bottom are Nomarski and fluorescent images of purified ZGs to demonstrate the purity of ZGs and the purified ZGs, was observed and collected just positive staining of a ZG marker, Rab3D. Reproduced above the bottom of the tube. The ZG membrane from reference (11). and content proteins were then separated by dynactin2 (45) and VAMP2 which are involved in osmotic lysis of ZGs with the ionophore, nigericin, ZG trafficking and exocytosis. A large number of followed by ultracentrifugation. The membrane novel ZG membrane proteins were also identified, pellet was washed first with 0.25M KBr and then including the SNARE protein, SNAP 29, the small with 0.1M Na2CO3 (pH 11.0) to remove soluble G proteins Rab27B, Rab11A and Rap1 and the content proteins and loosely associated proteins. molecular motor protein, myosin Vc. Indicative of The known ZG membrane marker such as Rab3D the interest in understanding the ZG membrane was highly enriched