Drosophila melanogaster prophenoloxidases respond inconsistently to Cu2+ and have different activity in vitro

Dipteran insects, like mosquitoes, possess more than two prophenoloxidase (PPO) genes, but it is unclear whether their gene products differ in biochemical properties and physiological functions. Here, we used three Drosophila melanogaster PPOs as models to study their properties through expression in S2 cells. Our data revealed that the PPOs were expressed in the ethanol-activatable conformation: rPPO1 and rPPO2 needed additional Cu2+ in the medium, but rPPO3 did not. rPPO1 bound Cu2+ within minutes; rPPO2 did that in hours when Cu2+ were present at a higher concentration. Thus, rPPO1 and rPPO2 were expressed as apo-rPPO and became holo-PPO upon Cu2+ binding; rPPO3 was holo-PPO immediately after expression. Surprisingly, in the absence of ethanol, the apparently intact rPPO3 catalyzed dopamine oxidation and melanization. The successful method for rPPO expression in S2 cells described in this paper will provide us with an opportunity to study the properties of a specific PPO gene in a small insect like mosquitoes in the future.

► Drosophila rPPOs expressed in S2 cells have different properties.
► rPPO1 and rPPO2 became holo-rPPOs after physical association with Cu2+.
► rPPO3 was expressed as holo-rPPO3 that has auto-activation property.