title: Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus.
abstract: 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 beta-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 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 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 microM and 17.9 microM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.
authors.affiliation: Department of Comparative Physiology, Evolutionary Biology Centre, Uppsala University, Sweden.
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.forename: S Y
authors.initials: SY
authors.lastname: Lee
authors.forename: L
authors.initials: L
authors.lastname: Cerenius
authors.forename: K
authors.initials: K
authors.lastname: Söderhäll
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D023082:Defensins / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D015870:Gene Expression; D006094:Gram-Positive Bacteria / Q000187:drug effects; D019476:Insect Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D010455:Peptides / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology
chemical_list: D023082:Defensins; D004792:Enzyme Precursors; D019476:Insect Proteins; D010455:Peptides; D012333:RNA, Messenger; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme