Evidence of a Novel Dipeptidyl Aminopeptidase in Mammalian GH3 Cells: New Insights into the Processing of Peptide Hormone Precursors.
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We investigated whether yeast signals could regulate hormone processing in mammalian cells. Chmeric genes coding for the prepro region of yeast α-factor and the functional hormone region of anglerfish somatostatin was expressed in rat pituitary GH3 cells. The nascent prepro-α-factor-somatostatin peptides disappeared from cells with a half-life of 30 min, and about 20% of unprocessed precursors remained intracellular after a 2 h chase period. Disappearance of propeptide was insensitive to lysosomotropic agents, but was inhibited at 15°C or 20°C, suggesting that the hybrid propeptides were not degraded in the secretory pathway to the trans Golgi network or in lysosomes. It appeared that while most unprocessed precursors were constitutively secreted into the medium, a small portion were processed at their paired dibasic sites by prohormone-processing enzymes located in trans Golgi network/secretory vesicles, resulting in the production of mature somatostatin peptides. To test this hypothesis, we investigated the processing pattern of two different hybrid precursors: the 52-1 hybrid precursor, which has a Glu-Ala spacer between the prepro region of α-factor and somatostatin, and the 58-1 hybrid precursor, which lacks the Glu-Ala spacer. Processing of metabolically labeled hybrid propeptides to smaller somatostatin peptides was assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, 68% of the initially synthesized propeptides were secreted constitutively. About 22% of somatostatin-related products were proteolytically processed to mature somatostatin, of which 38.7% were detected intracellularly after 2 h. From N-terminal peptide sequence determination of somatostatin-related products in GH3-52 and GH3-58 cells, we found that both hybrid precursors were accurately cleaved at their dibasic amino acid sites. Notably, we also observed that the Glu-Ala spacer sequence was removed from 52-1 hybrid precursors. The latter result strongly suggests that a novel dipeptidyl aminopeptidase activity a yeast STE13-like enzyme is present in the post-trans Golgi network compartment of GH3 cells. The data from these studies indicate that mechanisms which control protein secretion are conserved between yeast and mammalian cells.
- 日本細胞生物学会の論文
日本細胞生物学会 | 論文
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