Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2 - Insect cell-specific processing and secretion

The prohormone convertases (PCs) are an evolutionarily ancient group of pro teases required for the maturation of neuropeptide and peptide hormone prec ursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunobl otting data indicate that flies express 80- and 75-kDa forms of this protei n. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive m aturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open re ading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expre ssion vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were us ed as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpress ion of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synth esized but not secreted from HEK-293 cells. However, when dPC2 and either d 7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactiv e 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 signi ficant 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 ide a that the Drosophila enzyme has specific requirements for maturation and s ecretion that can be met only in insect cells.