Abstract
The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells.
Highlights
The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors
Because mouse PC2 requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. Drosophila PC2 (dPC2) and Drosophila 7B2 (d7B2) cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls
In this paper we present the characterization of an important prohormone converting enzyme in Drosophila, dPC2
Summary
The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. Expression and secretion of dPC2 enzyme activity appears to require insect cell-specific posttranslational processing events. Many of the proteases involved in these processing steps have been identified and are related to the yeast subtilisin-like endoprotease Kex2 These proprotein or prohormone convertases (PCs) cleave proproteins at pairs of basic amino acids or occasionally at monobasic sites Seven vertebrate PCs have been reported, namely furin, PACE 4 (paired amino acid-cleaving enzyme 4), PC1/PC3, PC2, PC4, PC5/PC6, and PC7/lymphoma PC/PC8 A homolog of PC2 from Drosophila melanogaster has been identified and termed amontillado; expression of this gene was found to be essential in the production of the movements that result in hatching [53], presumably to generate bioactive peptides required for this behavior. We characterize the intracellular interaction of Drosophila PC2 (dPC2) with d7B2 and demonstrate specific differences in the cellular handling of the Drosophila enzyme as compared with a vertebrate homolog
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