Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzin g the conversion of prophenoloxidase to an active phenoloxidase, has a mole cular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs en codes a zymogenic protein (proppA) of 468 amino acids. An antibody raised a gainst a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta -1,3-glucan triggered activation of prophenol oxidase in vitro. The C-terminal half of the proppA is composed of a typica l serine proteinase domain, with a sequence similar to other invertebrate a nd vertebrate serine proteinases. The N-terminal half contains a cationic g lycine-rich domain, a cationic proline-rich domain and a clip-domain, in wh ich the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta -defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational c hange takes place upon reduction that allows the anti-N to react with the N -terminal half of proppA. The recombinant clip-domain in crayfish proppA wa s overexpressed in Escherichia coli and the resulting peptide exhibited ant ibacterial activity against Gram-positive bacterial strains such as Microco ccus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory co ncentrations of 1.43 muM and 17.9 muM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.