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Published on: July 28, 2016
PmPPAF is a pro-phenoloxidase activating factor involved in innate immunity response of the shrimp Penaeus monodon
Tracy H T Ma1, John A H Benzie2, Jian-Guo He3
1Fisheries College, Guangdong Ocen University, Zhanjiang, PR China; School of Biological Sciences, The University of Hong Kong, PR China.
Abstract:
One of the major steps in the innate immune response of shrimp includes the activation of serine proteinases of the pro-phenoloxidase pathway by the prophenoloxidase activation enzyme (PPAF). In this study, the cDNA encoding a serine proteinase homologue (SPH) with prophenoloxidase activating activity of Penaeus monodon (PmPPAF) was cloned and characterized. PmPPAF cDNA consists of 1444 nucleotides encoding a protein with 394 amino acid residues. The estimated molecular weight of PmPPAF is 43.5 kDa with an isoelectric point of 5.19. PmPPAF consists of a signal peptide, a CLIP domain and a carboxyl-terminal trypsin-like serine protease domain. It is highly similar to the masquerade-like protein 2A (61% similarity) of the crayfish Pacifastacus leniusculus, other serine proteases (42.9-67% identity) of P. monodon, and the PPAF of the crab (61% similarity). Unlike other SPH of P. monodon, which express mainly in the hemocytes, PmPPAF transcripts were detected in the hemocytes, eyestalk, hypodermis, gill, swimming leg and brain. Similar to the crab PPAF, PmPPAF transcript level is high in shrimp at the premolt stages and PmPPAF expression is up-regulated in shrimp infected with white spot syndrome virus (WSSV). Gene silencing of PmPPAF decreased expression of a prophenoloxidase-like gene and injection of Anti-PmPPAF antibody causes a decrease in PO activity. Taken together, these results provided evidence that PmPPAF is a serine proteinase homologue, and is involved in the pro-PO activation pathway of the shrimp innate immune system.
Insights
This study identifies a key shrimp immune protein, prophenoloxidase activating enzyme (PPAF), crucial for innate immunity. Its activation is vital for shrimp defense against pathogens like white spot syndrome virus (WSSV).
Area of Science:
- Marine Biology
- Immunology
- Biochemistry
Background:
- The innate immune response in shrimp relies on the pro-phenoloxidase (pro-PO) activation pathway.
- Serine proteinases play a critical role in this pathway, with prophenoloxidase activating enzyme (PPAF) being a key activator.
Purpose of the Study:
- To clone and characterize the cDNA encoding a serine proteinase homologue (SPH) with prophenoloxidase activating activity from Penaeus monodon (PmPPAF).
- To investigate the role of PmPPAF in the shrimp innate immune system and its response to white spot syndrome virus (WSSV).
Main Methods:
- Cloning and sequencing of PmPPAF cDNA.
- Bioinformatic analysis of the PmPPAF protein structure and homology.
- Gene expression analysis using quantitative PCR in various tissues and developmental stages.
- Gene silencing and antibody-based functional assays to determine the role of PmPPAF in pro-PO activation.
Main Results:
- PmPPAF cDNA encodes a 394-amino acid protein with a molecular weight of 43.5 kDa, featuring a signal peptide, CLIP domain, and trypsin-like serine protease domain.
- PmPPAF exhibits high similarity to known PPAF and serine proteases from other species.
- Unlike other SPHs, PmPPAF transcripts are widely distributed in shrimp tissues and are upregulated during premolt stages and upon WSSV infection.
- Gene silencing of PmPPAF and antibody inhibition experiments demonstrated its essential role in the pro-PO activation pathway.
Conclusions:
- PmPPAF is a functional serine proteinase homologue involved in the pro-PO activation pathway of shrimp innate immunity.
- PmPPAF plays a significant role in shrimp defense mechanisms, particularly against viral infections like WSSV.
- The expression patterns suggest PmPPAF is regulated by developmental stages and immune challenges.
