title: Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.
abstract: Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system.
authors: Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
authors: Miti
authors: M
authors: Jearaphunt
authors: Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong 1, Klong Luang, Pathumthani, Thailand.
authors: Piti
authors: P
authors: Amparyup
authors: Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong 1, Klong Luang, Pathumthani, Thailand.
authors: Pakkakul
authors: P
authors: Sangsuriya
authors: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong 1, Klong Luang, Pathumthani, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Bangkok, Thailand.
authors: Walaiporn
authors: W
authors: Charoensapsri
authors: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong 1, Klong Luang, Pathumthani, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Bangkok, Thailand.
authors: Saengchan
authors: S
authors: Senapin
authors: Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
authors: Anchalee
authors: A
authors: Tassanakajon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D006094:Gram-Positive Bacteria / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D006458:Hemolymph / Q000201:enzymology / Q000382:microbiology; D007814:Larva / Q000254:growth & development; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000382:microbiology; D010457:Peptidoglycan / Q000378:metabolism; D034622:RNA Interference; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D010457:Peptidoglycan; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D057057:Serine Proteases