The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

Background  

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.     

Changes in zymogen granule membrane and pancreatic juice after supramaximal stimulation with caerulein in the rat

Pancreatic exocrine secretion was stimulated supramaximally with an intravenous infusion of 10 micrograms/kg of body weight of caerulein for 10 min in four rats. Two animals were killed immediately, and two animals 120 min after the cessation of the caerulein infusion. In specimens fixed immediately after the caerulein infusion, there were irregular electron lucent structures that resembled zymogen granules and were associated with bristle-coated membranes, coated pits and coated vesicles. Two hours after the caerulein infusion there were large vacuoles that contained amorphous or membranous material and cell organelles in acinar cells. The HPLC-analysis of pancreatic juice revealed two new peaks after caerulein injection. It was concluded that supramaximal caerulein stimulation prevents normal maturation and discharge of zymogen granules leading to altered membrane recycling of secretory granules in acinar cells and to the appearance of abnormal secretory products in pancreatic juice. The novel ultrastructural findings of this study, the bristle-coated membranes and the coated pits in the membrane of dilated zymogen granules, may be related to the exceedingly high dose of caerulein administered.     

Changes in zymogen granule membrane and pancreatic juice after supramaximal stimulation with caerulein in the rat

Summary Pancreatic exocrine secretion was stimulated supramaximally with an intravenous infusion of 10 μg/kg of body weight of caerulein for 10 min in four rats. Two animals were killed immediately, and two animals 120 min after the cessation of the caerulein infusion. In specimens fixed immediately after the caerulein infusion, there were irregular electron lucent structures that resembled zymogen granules and were associated with bristle-coated membranes, coated pits and coated vesicles. Two hours after the caerulein infusion there were large vacuoles that contained amorphous or membranous material and cell organelles in acinar cells. The HPLC-analysis of pancreatic juice revealed two new peaks after caerulein injection. It was concluded that supramaximal caerulein stimulation prevents normal maturation and discharge of zymogen granules leading to altered membrane recycling of secretory granules in acinar cells and to the appearance of abnormal secretory products in pancreatic juice. The novel ultrastructural findings of this study, the bristle-coated membranes and the coated pits in the membrane of dilated zymogen granules, may be related to the exceedingly high dose of caerulein administered.     

A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

GP-2, a 75-kDa glycoprotein, was isolated from dog pancreatic zymogen granule membranes (ZGMs). In a carbohydrate-shift strategy, N-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP-2, respectively, by gas-phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library. The sequence of these clones revealed an open reading frame that encodes a protein of 509 amino acids, eight N-linked oligosaccharide attachment sites, and an N-terminal signal sequence absent from the mature form of GP-2 associated with ZGMs. The C terminus shows a 20-residue hydrophobic transmembrane domain preceded by a decapeptide containing potential phosphatidylinositol-glycan attachment sites. GP-2 completely released from ZGMs by exogenous phospholipase C showed similar immunochemical properties and electrophoretic mobilities compared to the form associated with ZGMs. A similar form of GP-2 was released from zymogen granules permeabilized with saponin and incubated in the absence of added phospholipase C. Kinetic analysis of GP-2 release at 0 degrees C and 37 degrees C suggested the presence of a granule enzyme responsible for endogenous release of GP-2 to granule contents and into the apical medium. The data indicate that GP-2 is a phosphatidylinositol-glycan-linked membrane protein released from the membrane of mature zymogen granules by an enzymatic mechanism. The cDNA structure presented here thus encodes both membrane-bound and free forms of GP-2.     

Patch clamped single pancreatic zymogen granules: direct measurements of ion channel activities at the granule membrane.

BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.     

GP2, a GPI-anchored protein in the apical plasma membrane of the pancreatic acinar cell, co-immunoprecipitates with src kinases and caveolin

We previously showed that endocytosis at the apical plasma membrane (APM) of the pancreatic acinar cell is activated by the cleavage of GP2, a GPI-linked protein, from the apical cell surface. This endocytic process, as measured by horseradish peroxidase uptake into pancreatic acinar cells, is blocked by the tyrosine kinase inhibitors genistein and tyrphostin B42 as well as by disruption of actin filaments with cytochalasin. This suggests that the cleavage of GP2 from the cell membrane may activate endocytosis through a tyrosine kinase-regulated pathway. However, the mechanism by which GP2 and tyrosine kinases act together to activate endocytosis at the APM remains unknown. In this study, we demonstrate that pp60, p62yes, caveolin, and annexin, which have previously been implicated in endocytosis in other cell lines, were present in high abundance in GPI-enriched membranes by Western blot analysis. pp60, p62yes, and caveolin all co-immunoprecipitated with GP2 except annexin. An 85-kDa protein whose tyrosine-dependent phosphorylation is correlated with the activation of endocytosis in intact acinar cells also was present in these immunoprecipitates. This suggests that in pancreatic acini, GP2 may exist in a complex with src kinases, caveolin, and an 85-kDa phosphorylated substrate to regulate endocytosis at the APM.     

Acetylcholine-induced zymogen granule exocytosis: comparison between acini and single pancreatic acinar cells

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.     

Alcohol and zymogen activation in the pancreatic acinar cell

SUMMARY: Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiologic concentrations of cholecystokinin (CCK) cause intrapancreatic zymogen activation and pancreatitis. Supraphysiologic concentrations of CCK also cause zymogen activation in isolated pancreatic acini. This activation first occurs in a nonzymogen granule compartment that contains lysosomal markers. A low pH environment may also be needed for activation. To examine the ability of alcohols to sensitize the acinar cell to CCK, the conversion of zymogens to active enzymes in isolated acini was assayed. Alcohols, including 35 mmol/L ethanol, sensitized acini to CCK induced activation. The sensitization increased with chain length and was less in branched compared with unbranched alcohols. The relationship of alcohol's structure to sensitization may be related to the mechanism of sensitization.     

Expression of GP73, a resident Golgi membrane protein,in viral and nonviral liver disease

GP73 is a novel type II Golgi membrane protein of unknown function that is expressed in the hepatocytes of patients with adult giant-cell hepatitis (Gene 2000;249:53-65). Its expression pattern in human liver disease and the regulation of its expression in hepatocytes have not been systematically studied. The aims of the present study were to compare GP73 protein levels in viral and nonviral human liver disease and in normal livers, to identify its cellular sources, and to study the regulation of its expression in hepatoma cells in vitro. GP73 protein levels were quantitated in explant livers of patients with well-defined disease etiologies and compared with the levels in normal donor livers. GP73-expressing cells were identified immunohistochemically. GP73 expression in vitro was studied by Western blotting and immunofluorescence microscopy in HepG2 and SK-Hep-1 cells and in the HepG2-derived, hepatitis B virus (HBV)-transfected HepG2215 and HepG2T14.1 cell lines. Whole organ levels of GP73 were low in normal livers. Significant increases were found in liver disease due to viral causes (HBV, HCV) or nonviral causes (alcohol-induced liver disease, autoimmune hepatitis). In normal livers, GP73 was constitutively expressed by biliary epithelial cells but not by hepatocytes. Hepatocyte expression of GP73 was dramatically up-regulated in diseased livers, regardless of the etiology, whereas biliary epithelial cell expression did not change appreciably. GP73 was present at high levels in HepG2215 cells (a cell line that supports active HBV replication), but was absent in HepG2T14.1 cells (an HBV-transfected cell line that does not support HBV replication) and in HBV-free HepG2 cells. In SK-Hep-1 cells, GP73 expression was increased in response to interferon gamma (IFN-gamma), and inhibited by tumor necrosis factor alpha (TNF-alpha). In conclusion, increased expression of GP73 in hepatocytes appears to be a general feature of advanced liver disease, and may be regulated via distinct pathways that involve hepatotropic viruses or cytokines.     

In vitro synthesis of the major lens membrane protein.

The biosynthetic activity of a polyribosomal fraction isolated from the lens fiber plasma membrane-cytoskeleton complex by DNase I treatment has been assayed. After translation of these polyribosomes in a reticulocyte cell-free system and analysis of the products by electrophoresis in sodium dodecyl sulfate gels, the preferential synthesis of a protein with an apparent molecular weight of 26,000 was observed. By means of immunochemical characterization we showed that this protein, which seems not to be synthesized by "free" polyribosomes, is identical with the major intrinsic plasma membrane protein MP26 of lens fibers. Upon storage, the molecular weight of the newly synthesized protein decreases to about 22,000, a phenomenon that has previously been observed for MP26 in isolated plasma membranes and that may be caused by the presence of a specific proteolytic cleaving site in the protein.     

Chronic ethanol consumption increases the fragility of rat pancreatic zymogen granules.

Intracellular activation of pancreatic digestive enzymes by lysosomal hydrolases is thought to be an early event in the pathogenesis of pancreatic injury. As ethanol excess is an important association of pancreatitis, experimental work has been directed towards exploring possible mechanisms whereby ethanol may facilitate contact between inactive digestive enzyme precursors and lysosomal enzymes. The aim of this study was to find out if chronic ethanol administration increases the fragility of rat pancreatic zymogen granules. Sixteen male Sprague-Dawley rats were pair fed ethanol and control liquid diets for four weeks. Zymogen granule fragility was then assessed in pancreatic homogenate by determination of (a) latency and (b) per cent supernatant enzyme after sedimentation of zymogen granules. Amylase was used as a zymogen granule marker enzyme. Latency was significantly reduced in pancreatic homogenates of ethanol fed animals suggesting increased zymogen granule fragility. In support of this finding, there was a trend towards increased supernatant enzyme after ethanol feeding. In conclusion, administration of ethanol increases the fragility of pancreatic zymogen granules in the absence of morphological evidence of pancreatic injury. It is proposed that zymogen granule fragility may play an early part in the pathogenesis of alcoholic pancreatitis by permitting contact between digestive and lysosomal enzymes.     

Chitin synthetase zymogen is attached to the yeast plasma membrane.

Pretreatment of yeast protoplasts with concanavalin A, according to the method used by G. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3H]concanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found In this fraction; in the zymogen form. About 20% of the activity sedimented in a plasma-membrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a lysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation.     

Evidence for secretion of human eosinophil granule major basic protein and Charcot-Leyden crystal protein during eosinophil maturation

Prior electron microscopic studies have suggested that immature eosinophils degranulate during normal maturation in human bone marrow. This hypothesis was tested by measuring levels of eosinophil granule major basic protein (MBP) and Charcot-Leyden crystal (CLC) protein in bone marrow sinusoidal blood. MBP and CLC protein levels were elevated initially in bone marrow sinusoidal blood from normal volunteers when compared with peripheral blood, and levels of both proteins decreased during serial sampling; CLC protein levels remained significantly elevated, while MBP levels declined to equal those of peripheral blood. To investigate whether MBP and CLC protein were secreted during eosinophil maturation, bone marrow cells were cultured in soft agar; MBP and CLC protein levels were measured in culture supernatants by RIA. There was a significant positive correlation between eosinophil colony growth and levels of each protein. Electron micrographs of cells in soft agar colonies provided ultrastructural evidence for secretion of granule products by immature eosinophils. These results support prior observations that eosinophil promyelocytes degranulate during maturation.     

Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition.

Cytotoxic lymphocytes are characterized by their inclusion of cytoplasmic granules that fuse with the plasma membrane following target cell recognition. We previously identified a cytotoxic granule membrane protein designated p15-TIA-1 that is immunochemically related to an RNA-recognition motif (RRM)-type RNA-binding protein designated p40-TIA-1. Although it was suggested that p15-TIA-1 might be derived from p40-T1A-1 by proteolysis, N-terminal amino acid sequencing of p15-TIA-1 immunoaffinity purified from a natural killer (NK) cell line by using monoclonal antibody (mAb) 2G9 revealed that p15-T1A-1 is identical to the deduced amino acid sequence of NKG7 and GIG-1, cDNAs isolated from NK cells and granulocyte-colony-stimulating factor-treated mononuclear cells, respectively. Epitope mapping revealed that mAb 2G9 recognizes the C terminus of p15-T1A-1 and p40-T1A-1. The deduced amino acid sequence of p15-T1A-1/NKG7/GIG-1 predicts that the protein possesses four transmembrane domains, and immuno-electron microscopy localizes the endogenous protein to the membranes of cytotoxic granules in NK cells. Given its subcellular localization, we propose to rename-this protein GMP-17, for granule membrane protein of 17 kDa. Immunofluorescence microscopy of freshly isolated NK cells confirms this granular localization. Target cell-induced NK cell degranulation results in translocation of GMP-17 from granules to the plasma membrane, suggesting a possible role for GMP-17 in regulating the effector function of lymphocytes and neutrophils.