Abstract:
A major topic of this work was the subcellular fractionation of different antigen presenting cells using differential centrifugation, percoll density gradient centrifugation and free flow electrophoresis. The combination of these methods allows the separation of different endosomal/lysosomal compartments, which were characterized by fluorogenic substrates, immunoblot, ELISA and HPLC.
Cathepsin B, L and S activities were measured in isolated cell compartments of keratinocytes, with considerable differences between HaCaT-keratinocytes, keratinocytes in primary culture and syngenic B cells. Stimulation with interferon- resulted in the expression of MHC class II molecules and invariant chain, as well as in increased cathepsin S activity. On the mRNA level, cathepsin S was strongly induced in comparison to the control. This also was confirmed on the protein level. MHC class I and II presented peptides were isolated from interferon- stimulated HaCaT-keratinocytes, and 15 peptides have been identified by mass spectrometry.
A further focus was the investigation of asparaginyl endopeptidase (AEP) by cleavage experiments of 20 hexapeptides, designed in such a way that all natural 20 amino acids occur at the P1`position of one of the different peptides. The analysis by HPLC and a competition assay demonstrated cleavage of all peptides. With the exception of the peptide with proline at the P1`position with a slow turnover all peptides are good substrates for AEP. This means that AEP is restricted towards aspargine at the P1 position, whereas the P1`position can be covered by all 20 amino acids. Both myoglobin and myelin basic protein, containing two potential AEP cleavage sites each, revealed no detailed information about cleavage motives in digestion experiments.