Jearaphunt Miti, Amparyup Piti, Sangsuriya Pakkakul, Charoensapsri Walaiporn, Senapin Saengchan, Tassanakajon Anchalee
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
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.
PLoS One. 2015 Mar 24;10(3):e0121073. doi: 10.1371/journal.pone.0121073. eCollection 2015.
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.
由酚氧化酶原（proPO）激活系统介导的黑化作用是无脊椎动物抵御入侵病原体的一种快速免疫反应。几种伪装样和丝氨酸蛋白酶同源物（SPHs）已被证明在昆虫和甲壳类动物的proPO激活中起重要作用。在先前的一项研究中，我们将黑虎虾凡纳滨对虾中的伪装样SPH——PmMasSPH1鉴定为一种多功能免疫蛋白，基于其对革兰氏阴性菌哈维氏弧菌的识别和抗菌活性。在本研究中，我们鉴定了一种新型SPH，称为PmMasSPH2，它由一个N端夹子结构域和一个C端SP样结构域组成，与其他昆虫和甲壳类动物的SPHs具有高度相似性。我们证明，PmMasSPH1和PmMasSPH2的基因沉默显著降低了PO活性，导致血淋巴中哈维氏弧菌数量增多。有趣的是，敲低PmMasSPH1不仅抑制了其基因转录，还抑制了proPO系统中的其他免疫相关基因（如PmPPAE2）和抗菌肽（如PenmonPEN3、PenmonPEN5、crustinPm1和Crus样Pm）。PmMasSPH1和PmMasSPH2还显示出与革兰氏阳性菌的肽聚糖（PGN）的结合活性。通过酵母双杂交分析和免疫共沉淀，我们证明PmMasSPH1与proPO级联反应的最终蛋白酶PmPPAE2特异性相互作用。此外，PmMasSPH1和PmPPAE2的同时存在增强了体外PGN诱导的PO活性。综上所述，这些结果表明PmMasSPHs在对虾proPO系统激活中的重要性。
