Identifizierung und Charakterisierung von peripheren Membranproteinen der Zymogengranula des exokrinen Pankreas der Ratte durch subgranuläre Proteomanalyse

The exocrine pancreas is a tubuloacinar gland whose acinar cells synthesize the digestive enzymes (zymogenes), sort them into zymogen granules and secrete them by regulated exocytosis. The zymogen granules (ZG) are built at the trans-Golgi-network (TGN), but there is little information about the interaction of the zymogen aggregates with the membrane of the TGN. Missorting and inadequate inactivation of zymogens can lead to proteolytic events within the acinar cells and contribute to severe illness of the pancreas. Knowledge about the molecular composition of the granule membrane and its membrane proteins is therefore of cell biological and clinical relevance.Within the first part of this thesis, peripheral membrane proteins of zymogen granules (socalled „Wash“) of the exocrine pancreas were to be enriched and identified. Therefore, an experimental approach was used which combined granule fractionation, isoelectric focusing, 2D-gelelectrophoretic methods and mass-spectrometric analysis. The proteins of the Wash-fraction show a characteristic and reproducable pattern after 2D-gelelectrophoresis, which is different from other granule fractions. They can be divided into an acidic and a basic group. Altogether, 103 reproducable protein spots were analyzed. From the acidic proteins, only 39% could be identified, maybe because of posttranslational modifications. By the use of glycostain methods I could show, that the acidic proteins but not the basic ones are glycosylated . The identified proteins comprise typical content proteins of the zymogen granules (e.g. RNAse A), lipid-binding proteins (e.g. CEL) as well as peripheral membrane proteins. Some of the identified proteins are of mitochondrial origin. Other working groups also could identify these proteins in zymogen granules. There are some proteins identified, which havent been described in zymogen granules yet. These are the Chymase and the Peptidyl-prolyl-cis/trans-isomerase B (PPIB). Both proteins belong to the basic protein group. Within the second part of this thesis, I verified the membrane localization of RNAse A, CEL, Chymase and PPIB. By the use of specific antibodies after blotting of a 2D-gel, the mast cell specific Chymase could clearly be identified as a peripheral membrane protein. Other mast cell specific proteins could not be detected. Quantitative PCR-studies showed, that the Chymase expression depends on the feeding behaviour of the animals. Morphological studies also showed the Chymase as a peripheral membrane protein of ZG. AR42J cells dont express Chymase, but after transfection with an adequate Chymase construct, immunfluorescence studies showed a sorting of recombinant Chymase in secretory granules of AR42J cells. The PPIB was also identified in the Wash-fraction by the use of a specific antibody. Other ER-resident chaperones were not or hardly detected within this granule fraction. Quantitative PCR-studies also showed a feeding-dependent expression of PPIB in the exocrine pancreas, which supports the localization studies. A potential function of the granule PPIB could include the maintenance of a destinct conformation of the digestive enzymes, which could have an impact on the activity of these proteins. Moreover, PPIB could act within the sorting process of zymogens. Interestingly, all characterized proteins (RNAse A, CEL, Chymase, PPIB) can potentially interact with proteoglycans and therefore underline the hypothesis of the submembranous matrix in zymogen granules.