Characterization and expression analysis of the prophenoloxidase activating factor from the mud crab Scylla...

Prophenoloxidase activating factors (PPAFs) are a group of clip domain serine proteinases that can convert prophenoloxidase (pro-PO) to the active form of phenoloxidase (PO), causing melanization of pathogens. Here, two full-length PPAF cDNAs from Scylla paramamosain (SpPPAF1 and SpPPAF2) were cloned and characterized. The full-length SpPPAF1 cDNA was 1677 bp in length, including a 5'-untranslated region (UTR) of 52 bp, an open reading frame (ORF) of 1131 bp coding for a polypeptide of 376 amino acids, and a 3'-UTR of 494 bp. The full-length SpPPAF2 cDNA was 1808 bp in length, including a 5'-UTR of 88 bp, an ORF of 1125 bp coding for a polypeptide of 374 amino acids, and a 3'-UTR of 595 bp. The estimated molecular weight of SpPPAF1 and SpPPAF2 was 38.43 and 38.56 kDa with an isoelectric point of 7.54 and 7.14, respectively. Both SpPPAF1 and SpPPAF2 proteins consisted of a signal peptide, a characteristic structure of clip domain, and a carboxyl-terminal trypsin-like serine protease domain. Expression analysis by qRT-PCR showed that SpPPAF1 mRNA was mainly expressed in the gill, testis, and hemocytes, and SpPPAF2 mRNA was mainly expressed in hemocytes. In addition, SpPPAF1 and SpPPAF2 mRNA was expressed in a time-dependent manner after Vibrio parahaemolyticus challenge. The results showed that expression of both SpPPAF1 and SpPPAF2 was related to the bacterial challenge but the expression patterns differed. These findings suggest that SpPPAF is a serine proteinase and may be involved in the pro-PO activation pathway of the crab innate immune system.