Cloning and characterisation of a prophenoloxidase from the haemocytes of mud crab Scylla serrata

Abstract

A prophenoloxidase (proPO) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full length of 2663bp, with an open reading frame of 2019bp, a 124-bp 5'-untranslated region, and a 520-bp 3'-untranslated region containing a poly A signal. It encodes a protein of 673 amino acids with a predicted molecular weight of 77.5kDa and with an estimated pI of 5.96. It contains two putative tyrosinase copper-binding motifs with six histidine residues (copper A, 185, 189, 211, and copper B, 346, 350, 386). The proPO has thiol-ester-like motif (GCGWPQHM), which showed similar structural features of proPOs from other decapod crustaceans. It also contains five possible glycosylation sites, and a conserved C-terminal region common to all known proPOs. Sequence comparison showed that the proPO-deduced amino acid of mud crab S. serrata has an overall similarity of 78%, 57%, 56%, 51-55%, 54%, 53%, 52%, 52%, and 52% to that of Dungeness crab Cancer magister, American lobster Homarus americanus, European lobster Homarus gammarus, kuruma prawn Marsupenaeus japonicus, crayfish Pacifastacus leniusculus, white shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, green tiger shrimp Penaeus semisulcatus, and giant freshwater prawn Macrobrachium rosenbergii, respectively. The proPO was strongly expressed in haemocytes, but not in heart, eyestalk, gill, muscle, ovary, hepatopancreas, stomach, and intestine. The proPO transcript of mud crab S. serrata increased significantly in 12 and 24h post-lipopolysaccharide (LPS) injection, but returned to the original values in 72h post injection.