Functional characterization of a clip domain serine proteinase PmClipSP2 from the 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 shrimp Penaeus monodon EST database (http://pmonodon.biotec.or.th), a full-length cDNA of PmClipSP2 was identified and characterized. It contains an open reading frame of 1,107 bp encoding a predicted protein of 369 amino acids including a 25 amino acid signal peptide. The predicted molecular mass of the mature protein is 40.18 kDa. PmClipSP2 exhibits a characteristic sequence structure of clip-SPs consisting of an N terminal clip domain and a C terminal SP domain. Knockdown of the PmClipSP2 gene by double-stranded RNA (dsRNA) of PmClipSP2 gene, significantly reduced the PmClipSP2 transcript level and significantly reduced the total phenoloxidase (PO) activity. Silencing of the PmClipSP2 gene led to the 100% mortality rate and high number of bacteria in hemolymph of shrimp systemically infected with Vibrio harveyi. Interestingly, injection of microbial cell wall components had lethal effect on the PmClipSP2 knockdown shrimp that showed significantly decreased in the number of hemocytes. Functional analysis revealed that the recombinant PmClipSP2 could bind to β-1,3-glucan and LPS and activates phenoloxidase (PO) activity and has a proteolytic activity. Western blot analysis using the antibody raised against the rPmClipSP2 revealed that PmClipSP2 was present in hemocytes, but not in cell free plasma. Taken together, these results suggest that PmClipSP2 directly binds to bacterial cell wall components and activates the proPO system through the proteinase cascade and it probably involves in the hemocyte homeostasis by neutralizing LPS toxicity.