Molecular cloning and characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon.

A cDNA encoding shrimp, Penaeus monodon, prophenoloxidase (proPO) was obtained by screening a hemocyte library by plaque hybridization using a proPO cDNA fragment from freshwater crayfish, Pacifastaceus leniusculus, as a probe. The 3,002 bp cDNA contains an open reading frame of 2,121 bp and a 881 bp 3'-untranslated region. The molecular mass of the deduced amino acid sequence (688 amino acids) is 78,700 Da with an estimated pI of 5.8. Two putative copper binding sites are present and they have a highly conserved sequence around these sites. No signal peptide was detected in the shrimp proPO, as has been previously shown to be the case for all arthropod proPOs cloned so far. The cleavage site of zymogen activation is likely to be between Arg 44 and Val 45. A tentative complement-like motif (GCGWPQHM) is also present. Shrimp proPO mRNA is synthesized in the hemocytes and not in the hepatopancreas. Comparison of amino acid sequences showed that shrimp proPO is more closely related to another crustacean proPO, namely crayfish, than to the insect proPOs.

Peroxinectin, a cell adhesive protein associated with the proPO system from the black tiger shrimp, Penaeus monodon.

Upon activation of the prophenoloxidase activating system in the shrimp, Penaeus monodon, a cell adhesion activity in the haemolymph is generated. A cell adhesion assay showed that a high number of granular cells (60%) adhered to coverslips coated with a shrimp haemocyte lysate supernatant, whereas a very low number of cells adhered to coverslips coated with bovine serum albumin. Inhibition of adhesion by an antiserum against crayfish peroxinectin, a cell adhesion protein, revealed that the cell adhesion activity detected in shrimp haemocyte lysate supernatant might result from a peroxinectin-like molecule in shrimp. A cDNA clone encoding shrimp peroxinectin was isolated, which had an open reading frame of 2337 nucleotides, with a polyadenylation sequence and a poly A tail. It encodes a protein of 778 amino acids including a 20 amino acid signal peptide. The mature protein (758 amino acids) has a predicted molecular mass of 84.8kDa and an estimated pI of 9.0. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and KGD (Lys-Gly-Asp), were found in shrimp peroxinectin. Sequence comparison shows that the shrimp protein is similar to crayfish peroxinectin (69%) and to various peroxidases and putative peroxidases from invertebrates and vertebrates. The shrimp peroxinectin cDNA also shows similarity (51%) to both Drosophila peroxinectin-related protein (AAF78217) and peroxidasin (S46224), an extracellular matrix protein combining an active peroxidase domain as well as immunoglobulin domains, leucine rich repeats and procollagen-like motif. However, the sequence similarity to both Drosophila molecules are mostly within the peroxidase domain. Northern blot analysis, using a non-peroxidase region in peroxinectin as a probe, revealed that peroxinectin is constitutively expressed in shrimp haemocyte and was reduced significantly in shrimp injected with a beta-1,3-glucan, laminarin, to mimic an infection with a fungus.