A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon.
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 snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
Warunthorn
W
Monwan
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong1, Klong Luang, Pathumthani 12120, Thailand.
Piti
P
Amparyup
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand. Electronic address: anchalee.k@chula.ac.th.
Anchalee
A
Tassanakajon
D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D004156:Catechol Oxidase / Q000378:metabolism; D003001:Cloning, Molecular; D004330:Drosophila / Q000276:immunology; D029721:Drosophila Proteins / Q000235:genetics; D004792:Enzyme Precursors / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006434:Hemocytes / Q000276:immunology*; D006458:Hemolymph / Q000378:metabolism; D007109:Immunity; D033561:Penaeidae / Q000276:immunology*; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*; D014733:Vibrio / Q000276:immunology*; D014735:Vibrio Infections / Q000276:immunology*
D060829:Arthropod Proteins; D029721:Drosophila Proteins; D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D057057:Serine Proteases; D012697:Serine Endopeptidases; C081508:snk protein, Drosophila
Peneaus monodon; Prophenoloxidase; Shrimp; Snake-like serine proteinase