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Characterization of the endopeptidase PC2 activity towards secretogranin II in stably transfected PC12 cells.

ABSTRACT: 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 [35S]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 [35S]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.

SUBMITTER: Dittie AS

PROVIDER: S-EPMC1135966 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Project description:Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.

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Characterization of the endopeptidase PC2 activity towards secretogranin II in stably transfected PC12 cells.

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