PRO-PHENOL OXIDASE ACTIVATING PROTEINASE FROM AN INSECT, MANDUCA-SEXTA - A BACTERIA-INDUCIBLE PROTEIN SIMILAR TO DROSOPHILA EASTER

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymoge n is activated by a specific proteolytic cleavage. PO oxidizes phenoli c compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsula te parasites. We have isolated from the tobacco hornworm, Manduca sext a, a serine proteinase that activates proPO, and have cloned its cDNA The isolated proPO activating proteinase (PAP) hydrolyzed artificial s ubstrates but required other protein factors for proPO activation, sug gesting that proPO-activating enzyme may exist as a protein complex, o ne component of which is PAP, PAP (44 kDa) is composed of two disulfid e-linked polypeptide chains (31 kDa and 13 kDa), A cDNA for PAP was is olated from a hemocyte library, by using a PCR-generated probe based o n the aminoterminal 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'' do main. 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 m uch more abundant in fat body after insects were injected with Escheri chia coli, Sequence data and H-3-diisopropyl fluorphosphate labeling r esults suggest that the same PAP exists in hemolymph and cuticle.