Liu Yongjie, Sun Yuhang, Wang Qiai, Hou Fujun, Liu Xiaolin
College of Animal Science and Technology, Northwest A&F University, Shaanxi, Yangling 712100, China.
College of Animal Science and Technology, Northwest A&F University, Shaanxi, Yangling 712100, China.
Fish Shellfish Immunol. 2017 Jan;60:492-501. doi: 10.1016/j.fsi.2016.11.024. Epub 2016 Nov 9.
Serpins have been characterized from varieties of organisms by their inhibitory roles on serine or cysteine proteases. However, research for the functional study of serpins in crustacean is relatively small. To fully clarify the immune characterizations of serpin, a novel serpin (named Lvserpin8) encoding 414 amino acids with a 19-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Sequence analysis indicated that the genomic Lvserpin8 gene contains 5 exons and 4 introns. The P1 residues of the predicted scissile bond in the reactive center loop (RCL) region represented for Lysine (Lys), which is in accordance with Pmserpin8, Dmserpin27A, Ofserpin3, Bmserpin3 and Msserpin3. Quantitative results showed that high mRNA expression of Lvserpin8 was detected in hepatopancreas and testis. Notably, a significant increase of Lvserpin8 was appeared post injection of Vibrio anguillarum, and Micrococcus lysodeikticus. Moreover, Lvserpin8 was knocked down in vivo by double-stranded RNA (dsRNA) mediated RNA interference (RNAi). Suppression of Lvserpin8 led to a significant increase in the transcripts of LvPPAE2 (Prophenoloxidase-activating Enzyme 2) and cumulative mortality. What's more, recombinant Lvserpin8 protein (rLvserpin8) displayed inhibition roles on trypsin activity, and prophenoloxidase activation. Taken together, the results implied that Lvserpin8 may be involved in shrimp innate immunity via the inhibition of prophenoloxidase-activating proteases.
丝氨酸蛋白酶抑制剂（Serpins）已在多种生物体中得到鉴定，因其对丝氨酸或半胱氨酸蛋白酶具有抑制作用。然而，关于甲壳类动物中丝氨酸蛋白酶抑制剂功能研究的报道相对较少。为全面阐明丝氨酸蛋白酶抑制剂的免疫特性，我们从凡纳滨对虾（Litopenaeus vannamei）中鉴定出一种新型丝氨酸蛋白酶抑制剂（命名为Lvserpin8），其编码414个氨基酸，包含一个19个氨基酸的信号肽和一个丝氨酸蛋白酶抑制剂结构域。序列分析表明，基因组Lvserpin8基因包含5个外显子和4个内含子。反应中心环（RCL）区域预测的裂解键的P1残基为赖氨酸（Lys），这与Pmserpin8、Dmserpin27A、Ofserpin3、Bmserpin3和Msserpin3一致。定量结果显示，在肝胰腺和睾丸中检测到Lvserpin8的mRNA高表达。值得注意的是，注射鳗弧菌（Vibrio anguillarum）和溶壁微球菌（Micrococcus lysodeikticus）后，Lvserpin8显著增加。此外，通过双链RNA（dsRNA）介导的RNA干扰（RNAi）在体内敲低了Lvserpin8。抑制Lvserpin8导致LvPPAE2（酚氧化酶原激活酶2）的转录本显著增加和累积死亡率上升。此外，重组Lvserpin8蛋白（rLvserpin8）对胰蛋白酶活性和酚氧化酶原激活具有抑制作用。综上所述，结果表明Lvserpin8可能通过抑制酚氧化酶原激活蛋白酶参与对虾的先天免疫。
