Cloning and characterization of clip domain serine proteinase, PmClipSP1, from black tiger shrimp Penaeus monodon

Abstract

Clip domain serine proteinases (clip-SPs) are the essential components of signaling cascades in the innate immune system of invertebrates. From the Penaeus monodon EST database (http://pmonodon.biotec.or.th), a full-length cDNA of PmClipSP1 was characterized. It contains an open reading frame (ORF) of 1,101 bp encoding a predicted protein of 364 amino acids including a 25 amino acid signal peptide. The mature protein of PmClipSP1 exhibits a characteristic sequence structure of clip-SPs consisting of an N terminal clip domain and a C terminal SP domain. The mature protein and SP domain of PmClipSP1 were cloned into the pET22b(+) vector with an N terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli as 37 kDa and 28 kDa proteins, respectively. The recombinant proteins were successfully purified by Ni-NTA chromatography. Functional analysis revealed that the recombinant proteins lack a proteolytic activity and could not activate of phenoloxidase (PO) activity. Western blot analysis using the antibody raised against the SP domain of PmClipSP1 revealed that PmClipSP1 was present in hemocytes, but not in cell free plasma. Knockdown of the PmClipSP1 gene by double-stranded RNA (dsRNA) of PmClipSP1 gene, significantly reduced PmClipSP1 transcript levels, but did not significantly reduced the total PO enzyme activity, suggesting that PmClipSP1 might not involved in the proPO system. However, silencing of the PmClipSP1 gene led to a significant increase in the number of viable bacteria in the hemolymph (~2.4 fold) and in the mortality rate (59%) of shrimp systemically infected with Vibrio harveyi. These findings suggest that PmClipSP1 plays a role in the antibacterial defense mechanism of P. monodon shrimp.