Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori - Purification, characterization, and cDNA cloning

Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as ju dged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixt ure of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at diff erent retention times in reversed-phase high performance liquid chromatogra phy, PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage( s) and glycosylated serine proteases, The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesize d as prepro-PPAE with 441 amino acid residues and is activated from pro-PPA E: by cleavage of a peptide bond between Lys(152) and Ile(153), The homolog y search showed 36.9% identity of PPAE to easter, which is a serine proteas e involved in dorsoventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light ch ain. A single copy of the PPAE gene was suggested to be present in the silk worm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or m id gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was tra nsiently detected in the extract of eggs between 1 and 3 h after they were laid.