Proteoglykane und die Verpackung von Exportproteinen: Interaction von Serglycin und ZG 16 in den Zymogengranula des exokrinen Rattenpankreas

In this thesis the interaction of the mainly membrane-associated secretory lectin ZG 16 and the proteoglycan serglycin should be analysed to gain deeper insight in the mechanisms of sorting and packaging of the zymogens into secretory granules. Moreover further proteoglycans should be isolated and identified from the content fraction of the zymogen granules. The following results were obtained: 1. By cloning and expressing the unmodified N- and C-terminal parts of serglycin it could be demonstrated in various protein-protein-interaction studies that serglycin interacts with ZG 16 via its unmodified N-terminal part (SGN) and is so linked to the granule membrane. 2. By analysing several point- and deletion-mutants of the N-terminal part of serglycin (SGN), the binding sequence could be restricted to 9 amino acids (ARYQWVRCK) near the N-terminus of serglycin. 3. Modelling the hypothetical secondary structure of serglycin (ExPASy Molecular Biology Server) it was revealed that the binding motif of serglycin to ZG 16 forms a b-strand. This interaction represents a sorting mechanism by which the zymogens, that are attached to the glycosaminoglycans of serglycin (as previously shown by Biederbick et al., 2003) are transported into the zymogen granules. 4. Further proteoglycans in the granule content could only be shown indirectly. In a special proteoglycan precipitation assay (Blyscan Assay) proteoglycans could be detected by extinction measurement. The highest extinction was obtained after incubating the content fraction with NaHCO3.In this thesis the interaction of the mainly membrane-associated secretory lectin ZG 16 and the proteoglycan serglycin should be analysed to gain deeper insight in the mechanisms of sorting and packaging of the zymogens into secretory granules. Moreover further proteoglycans should be isolated and identified from the content fraction of the zymogen granules. The following results were obtained: 1. By cloning and expressing the unmodified N- and C-terminal parts of serglycin it could be demonstrated in various protein-protein-interaction studies that serglycin interacts with ZG 16 via its unmodified N-terminal part (SGN) and is so linked to the granule membrane. 2. By analysing several point- and deletion-mutants of the N-terminal part of serglycin (SGN), the binding sequence could be restricted to 9 amino acids (ARYQWVRCK) near the N-terminus of serglycin. 3. Modelling the hypothetical secondary structure of serglycin (ExPASy Molecular Biology Server) it was revealed that the binding motif of serglycin to ZG 16 forms a b-strand. This interaction represents a sorting mechanism by which the zymogens, that are attached to the glycosaminoglycans of serglycin (as previously shown by Biederbick et al., 2003) are transported into the zymogen granules. 4. Further proteoglycans in the granule content could only be shown indirectly. In a special proteoglycan precipitation assay (Blyscan Assay) proteoglycans could be detected by extinction measurement. The highest extinction was obtained after incubating the content fraction with NaHCO3.