Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp, Litopenaeus vannamei.

cDNA encoding prophenoloxidase (proPO) of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus (accession no.: AF521949) and kuruma shrimp Marsupenaeus japonicus (accession no.: AB0733223). proPO of L. vannamei was constitutively expressed. The 2471-bp cDNA contained an open reading frame (ORF) of 2058 bp, a 96-bp 5'-untranslated region, and a 317-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (686 amino acids) was 78.1 kDa with an estimated pI of 6.02. It contained putative copper binding sites, a complement-like motif (GCGWPQHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in white shrimp proPO. Comparison of amino acid sequences showed that white shrimp proPO is more closely related to the proPO of another penaeid than to that of a freshwater crayfish. White shrimp proPO mRNA was synthesized in haemocytes and not in the hepatopancreas or muscle. The activation responses of the proPO of the white shrimp to an exogenous protease (trypsin), a detergent (sodium dodecyl sulphate), and algal and microbial cell wall components (laminarin, sodium alginate, zymosan, and lipopolysaccharide), and its susceptibility to protease inhibitors in vitro resemble the proPO activation system of other crustaceans. These facts suggest that the proPO system in haemocytes of the white shrimp Litopenaeus vannamei serves an important function in non-self recognition and host immune reactions.