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

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular 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 encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the β‐1,3‐glucan triggered activation of prophenoloxidase in vitro. The C‐terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N‐terminal half contains a cationic glycine‐rich domain, a cationic proline‐rich domain and a clip‐domain, in which the disulfide‐bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian β‐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 change 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 was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram‐positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 µm and 17.9 µm, respectively. These results suggest that the clip‐domains in proppAs may function as antibacterial peptides.