The first homolog of pacifastin-related precursor in the swimming crab ( Portunus trituberculatus): Characterization and potential role in immune response to bacteria and fungi

Abstract

Among all the serine proteinase inhibitor families (SPIs), the pacifastin-related inhibitor is seldom isolated. A pacifastin-related SPI named as PtPLC was identified from the swimming crab Portunus trituberculatus in this study. The full-length of PtPLC was cloned from haemocytes cDNA library by the combination of homology cloning and rapid amplification of cDNA ends (RACE). The PtPLC contained an open reading frame (ORF) of 1098 bp encoding a putative pacifastin-related precursor of 365 amino acids, a 5′-untranslated region (UTR) of 33 bp, and a 3′-UTR of 524 bp. The estimated molecular weight of mature PtPLC was 40.51 kDa and its isoelectric point was 5.04. Eight PLD domains in PtPLC shared a common characteristic of conserved array of six cysteine residues (Cys–Xaa 9–12–Cys–Asn–Xaa–Cys–Xaa– Cys–Xaa 2–3–Gly–Xaa 3–4–Cys–Thr–Xaa 3–Cys). The mRNA expression of PtPLC transcripts was highly detected in haemocytes, gill, hepatopancreas and stomach. The temporal expression levels of PtPLC transcripts in haemocytes showed different expression patterns after challenged by Gram-negative bacteria Vibrio alginolyticus, Gram-positive bacteria Micrococcus luteus and fungus Pichia pastoris. There were two peaks in the mRNA expression profile after M. luteus stimulation. And after V. alginolyticus and P. pastoris stimulation, there were three peaks in the mRNA expression profiles. These findings suggest that PtPLC is involved in the antibacterial defense mechanism of P. tritubercualtus.