CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China; Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China.
Fish Shellfish Immunol. 2019 Jun;89:574-585. doi: 10.1016/j.fsi.2019.04.033. Epub 2019 Apr 14.
Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes.
α-2 巨球蛋白 (A2M) 是一种普遍存在的蛋白酶抑制剂，参与先天宿主防御系统。本文从三疣梭子蟹（Portunus trituberculatus）中分离到两个不同的 A2M 基因（分别命名为 PtA2M-1 和 PtA2M-2）。PtA2M-1 和 PtA2M-2 编码的蛋白质分别含有 1541 或 1516 个氨基酸，含有 A2M 的典型功能结构域。与大多数节肢动物血细胞中高度表达不同，PtA2M-1 和 PtA2M-2 主要在鳃、眼柄和消化道中检测到。在胚胎阶段，发现 PtA2Ms 在受精卵中表达水平最高，表明其母源来源。在受到溶藻弧菌（Vibrio alginolyticus）攻击后，PtA2Ms 的转录物表现出相似的时间依赖性应答表达模式，而 PtA2M-1 对微球菌（Micrococcus luteus）和毕赤酵母（Pichia pastoris）感染的敏感性高于 PtA2M-2。PtA2M-1 或 PtA2M-2 的敲低可显著增强与酚氧化酶原（proPO）相关基因（PtproPO 和 PtPPAF）和丝氨酸蛋白酶相关基因（PtcSP1-3 和 PtSPH）的表达，然而，PtLSZ 和吞噬相关基因（PtMyosin 和 PtRab5）则被有效抑制。这些结果进一步得到了 PtA2M-1 或 PtA2M-2 沉默蟹血液中 PO 和溶菌酶活性的支持。此外，PtA2M-1 和 PtA2M-2 可以通过 Toll 和 NF-κB 途径调节抗菌肽（AMP）基因（PtALF1-3、PtCrustin1 和 PtCrustin3）的表达。我们的研究结果表明，PtA2Ms 可能通过调节 proPO 系统、吞噬作用和 AMP 基因的表达来参与蟹的宿主防御。
