Identifizierung und Charakterisierung von Membranproteinen der Zymogengranula: Eine Beteiligung der kleinen GTPase Rab8A an der Granulabildung im exokrinen Pankreas

Polarized acinar cells of the exocrine pancreas are specialised in the synthesis of digestive enzymes (so called zymogens). The zymogens are stored in zymogen granules (ZG). The process of granule biogenesis in the exocrine pancreas is poorly understood. Of clinical relevance are the pathophysiological changes of the pancreas. The development of new therapy approaches requires the molecular mechanisms of the granule biogenesis and secretion as well as molecular players to be elucidated. Among the molecular players, the proteins of the zymogen granule membrane (ZGM) are of particular interest. So far only few proteins have been identified and characterised on a molecular and functional level. Therefore, it has been the aim of the first part of the present work to identify unknown ZGM proteins by proteomic studies and to characterize them. In order to identify proteins of the ZGM, a doubled SDS-polyacrylamide-gel-electrophoresis (dSDS-PAGE) was established for the first time, allowing an accumulation of ZGM proteins in a polyacrylamide-gel, which were finally analysed by MALDI-TOF mass spectrometry. In the two dimensional Coomassie-stained gels a total of 20-30 protein spots reproducible appeared. In most cases these spots were directly located on a diagonal and only a few spots (normally 3-4) were localized below this diagonal. The dispersal of the protein spots suggested that the proteins of the gel are mainly moderately hydrophobic. The detected protein spots in the gel were distributed irregularly around molecular weight levels of 10-120 kDa. Particularly at the molecular weight level of 20-50 kDa an aggregation of protein spots became visible. The majority (21 spots) was analysed and identified by MALDI-TOF-MS and MALDI-TOF-TOF-MS “peptide mass fingerprinting” using Mascot Software (Matrix Science Ltd.). The identification of the proteins by mass spectrometry revealed that seven proteins were membrane proteins (membranedipeptidase, GP2, pancreatic lipase related protein 2 precursor, ANT2, cell surface antigen RB 13-6, aminopeptidase M, VDAC1) and that some of them were already known ZGM proteins. The majority of these membrane proteins are moderately hydrophobic. This is due to the fact that they are integrated in the ZGM either by a GPI-anchor or by one or two transmembrane domains. Moreover, already known peripheral ZG proteins as well as proteins of the granule content could be identified with the result that a total of 15 already known ZG proteins were detected by dSDS-PAGE. However, hitherto unknown proteins of the ZGM, whose function at the zymogen granule membrane is yet unknown (PPIC, ANT2, VDAC1/porin) were also identified. All in all the results generated by dSDS-PAGE and MALDI-TOF-analysis demonstrate that the dSDS-PAGE is a useful and reproducible method for the separation of ZGM proteins, because both new and already known ZGM as well as ZG content proteins could be detected. In the second part of this thesis the small GTPase Rab8A was identified by antibody screening tests on the ZGM. The studies prove that Rab8A is a new and yet unknown Rab-protein of the ZGM. By immunoblotting Rab8A was detected in isolated ZG fractions of the rat pancreas as well as in ZGM fractions, which were obtained by granule subfractionation. Morphological studies on AR42J cells proved that Rab8A localizes specifically to ZG and to the Golgi complex, because a clearly colocalisation with the ZG marker protein Carboxypeptidase A became visible by immunofluorescence. To determine the putative role of Rab8A in granule formation and/ or granule fusion, we conducted RNA interference experiments to inhibit the expression of Rab8A (to 70-80 %) in pancreatic AR42J cells. Morphological studies (immunofluorescence and electron microscopy) revealed that the silencing of Rab8 resulted in a significant decrease in the number of ZG and in an accumulation of granule marker proteins within the Golgi-complex without affecting the morphology of the Golgi-complex. This was expressed in a reduced amylase secretion after stimulation as well as in a decreased overall secretion of regulated secretory proteins in the cells. Further studies demonstrated that the Golgi-dependent trafficking and sorting of other marker proteins to the endosomal compartment and to the plasma membrane was not affected by the silencing of Rab8A. An accumulation of lysosomal and plasma membrane proteins like Endolyn-78, VSG-SP-GFP and YFP-GL-GPI could not be detected by immunofluorescence. Moreover, a cell-surface immunoprecipitation conducted for VSVG-SP-GFP and YFP-GL-GPI revealed no changes of the protein transport to the plasma membrane. The data provide first evidence that Rab8A is a protein of the ZGM, that plays a role in the early phase of ZG formation at the Golgi-complex of the acinar cells of the exocrine pancreas. It is the first protein identified so far that plays an essential role in the biogenesis of zymogen granules.