CHARACTERIZATION OF THE ENDOPEPTIDASE PC2 ACTIVITY TOWARDS SECRETOGRANIN-II IN STABLY TRANSFECTED PC12 CELLS

To study the processing of secretogranin II (SgII) by the prohormone convertase PC2 we have generated a stable PC12 cell line which expresses mouse PC2. We here present the characteristics of the PC12/PC2 cell line and demonstrate that the exogenous PC2 is sorted and stored in secretory granules in the PC12/PC2 cell line as efficiently as the endogenous granins. By indirect immunofluorescence with antibodies specific for chromogranin B (CgB) and PC2 we were able to establish that the PC2 is stored in secretory granules in the PC12/PC2 cell line. After subcellular fractionation, followed by immunoblotting, the mature 68 kDa form of PC2 was found co-sedimented with SgII in fractions containing secretory granules. Two-dimensional gel electrophoresis was used to characterize a secretory granule fraction obtained from the PC12/PC2 cells, and a comparison was done of the electrophoretic pattern obtained from the PC12/PC2 cells with the parent cell line PC12. The products derived from the processing of SgII by PC2 were identified by immunoblotting with a panel of antibodies directed against SgII. Using [S-35]sulphate to label the newly synthesized SgII, we performed a time course to monitor the appearance of the lower-molecular-mass fragments of SgII: beginning 15 min after a 5 min pulse of [S-35]sulphate we were able to detect the first proteolytic fragment of SgII. Our results demonstrate that SgII is proteolytically processed by PC2 in the immature secretory granule into several lower-molecular-mass proteins, the major ones being an 18 kDa sulphated fragment and a 28 kDa fragment.