Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus
2001 (English)In: European Journal of Biochemistry, ISSN 0014-2956, Vol. 268, no 4, 895-902 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2001. Vol. 268, no 4, 895-902 p.
antibacterial, clip-domain, freshwater crayfish, prophenoloxidase activating enzyme, serine proteinase, PRO-PHENOL-OXIDASE, HOLOTRICHIA-DIOMPHALIA LARVAE, HORSESHOE-CRAB HEMOCYTES, SERINE-PROTEASE, CDNA CLONING, PROTEOLYTIC-ENZYMES, BLOOD-CELLS
IdentifiersURN: urn:nbn:se:uu:diva-38346DOI: 10.1046/j.1432-1327.2001.01945.xOAI: oai:DiVA.org:uu-38346DiVA: diva2:66245
Addresses: Soderhall K, Uppsala Univ, Dept Comparat Physiol, Evolutionary Biol Ctr, Norbyvagen 18 A, S-75236 Uppsala, Sweden. Uppsala Univ, Dept Comparat Physiol, Evolutionary Biol Ctr, S-75236 Uppsala, Sweden.2006-10-162006-10-162009-04-21Bibliographically approved