A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.
Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.
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, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
Pakkakul
P
Sangsuriya
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Walaiporn
W
Charoensapsri
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand; Program of Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
Sudarat
S
Chomwong
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Saengchan
S
Senapin
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
Anchalee
A
Tassanakajon
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, Klong 1, Klong Luang, Pathumthani 12120, Thailand. Electronic address: piti.amp@biotec.or.th.
Piti
P
Amparyup
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004156:Catechol Oxidase / Q000276:immunology*; D015853:Cysteine Proteinase Inhibitors / Q000276:immunology*; D004792:Enzyme Precursors / Q000276:immunology*; D055785:Gene Knockdown Techniques; D006801:Humans; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000235:genetics / Q000276:immunology*; D011506:Proteins / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D011506:Proteins; C106709:pacifastin; C038451:pro-phenoloxidase; D004156:Catechol Oxidase
Kunitz; Melanization; Pacifastin; Penaeus monodon; Prophenoloxidase; Serine proteinase inhibitor; Shrimp immunity