FUNCTIONAL CHARACTERIZATION OF SNAKE-LIKE SERINE PROTEINASE FROM BLACK TIGER SHRIMP Penaeus monodon

Abstract

Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a clip domain serine proteinase, namely PmSnake, which has previously been identified from the shrimp Penaeus monodon based on subtractive cDNA library of proPO double-stranded RNA (dsRNA) treated hemocytes. An open reading frame of PmSnake contains 1,068 bp encoding a predicted protein of 355 amino acid residues. A putative signal peptide of 22 amino acids and two conserved domains (N-terminual clip domain and C-terminal trypsin-like serine proteinase domain) were identified in PmSnake. Sequence analysis of deduced amino acids revealed that PmSnake shared similarity with ClipSPs in P. monodon and the insect Manduca sexta snake-like hemolymph protease 21. PmSnake is mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection supporting that it is an immune-responsive gene. Suppression of PmSnake transcripts by injection of dsRNA corresponding to the PmSnake gene into shrimp, reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36.1%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. PmSnake with predicted molecular mass of 37 kDa was successfully produced in Escherichia coli BL21 cells and purified by Ni-NTA chromatography. The Western blot analysis using anti-PmSnake showed that PmSnake was detected in hemocytes but not cell-free plasma. In vitro PO activity assay and serine proteinase activity showed that adding rPmSnake could increase PO activity and serine proteinase activity in shrimp hemolymph, suggesting that rPmSnake can activate the proPO system via serine proteinase cascade.