Activation of pro-
phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and
infection. The proPO
zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce
quinones, which may help to kill pathogens and can also be used for synthesis of
melanin to seal
wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a
serine proteinase that activates proPO, and have cloned its
cDNA. The isolated
proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other
protein factors for proPO activation, suggesting that proPO-activating
enzyme may exist as a
protein complex, one component of which is PAP. PAP (44 kDa) is composed of two
disulfide-linked
polypeptide chains (31 kDa and 13 kDa). A
cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod
serine proteinases containing a carboxyl-terminal
proteinase domain and an amino-terminal "
clip" domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP
mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.