Gao Zhan, Qu Baozhen, Yao Lan, Ma Zengyu, Cui Pengfei, Zhang Shicui
Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Qingdao, 266003, China; Department of Marine Biology, Ocean University of China, Qingdao, 266003, China.
Department of Marine Biology, Ocean University of China, Qingdao, 266003, China.
Dev Comp Immunol. 2018 Dec;89:31-43. doi: 10.1016/j.dci.2018.08.005. Epub 2018 Aug 7.
Midkine (MK) and pleiotrophin (PTN) are the only two members of heparin-binding growth factor family. MK/PTN homologues found from Drosophila to humans are shown to have antibacterial activities and their antibacterial domains are conserved during evolution. However, little is known about MK/PTN homologue in the basal chordate amphioxus, and overall, information regarding MK/PTN homologues is rather limited in invertebrates. In this study, we identified a single MK/PTN homologue in Branchiostoma japonicum, termed BjMiple, which has a novel domain structure of PTN-PTNr1-PTNr2, and represents the ancestral form of vertebrate MK/PTN family proteins. BjMiple was expressed mainly in the ovary in a tissue-dependent fashion, and its expression was remarkably up-regulated following challenge with bacteria or their signature molecules LPS and LTA, suggesting its involvement in antibacterial responses. Functional assays revealed that BjMiple had strong antimicrobial activity, capable of killing a panel of Gram-negative and Gram-positive bacteria via a membranolytic mechanism, including interaction with bacterial membrane via LPS and LTA, membrane depolarization and high intracellular levels of ROS. Importantly, strong antibacterial activity was localized in PTN and PTNr1 Additionally, BjMiple and its derived peptides PTN and PTNr1 were not cytotoxic to human RBCs and mammalian cells. Taken together, our study suggests that amphioxus Miple is the ancestral type of vertebrate MK/PTN family homologues, and can play important roles as innate peptide antibiotics, which renders it a promising template for the design of novel peptide antibiotics against multi-drug resistant bacteria.
中期因子（MK）和多效生长因子（PTN）是肝素结合生长因子家族仅有的两个成员。从果蝇到人类发现的MK/PTN同源物显示具有抗菌活性，并且它们的抗菌结构域在进化过程中是保守的。然而，关于基础脊索动物文昌鱼中的MK/PTN同源物知之甚少，总体而言，关于无脊椎动物中MK/PTN同源物的信息相当有限。在本研究中，我们在日本文昌鱼中鉴定出一个单一的MK/PTN同源物，命名为BjMiple，它具有PTN-PTNr1-PTNr2的新型结构域结构，代表脊椎动物MK/PTN家族蛋白的原始形式。BjMiple主要以组织依赖性方式在卵巢中表达，并在受到细菌或其标志性分子脂多糖（LPS）和脂磷壁酸（LTA）攻击后其表达显著上调，表明其参与抗菌反应。功能分析表明BjMiple具有强大的抗菌活性，能够通过膜溶解机制杀死一系列革兰氏阴性和革兰氏阳性细菌，包括通过LPS和LTA与细菌膜相互作用、膜去极化以及细胞内高水平活性氧（ROS）产生。重要的是，强大的抗菌活性定位于PTN和PTNr1。此外，BjMiple及其衍生肽PTN和PTNr1对人红细胞和哺乳动物细胞无细胞毒性。综上所述，我们的研究表明文昌鱼Miple是脊椎动物MK/PTN家族同源物的原始类型，并且可以作为先天性肽抗生素发挥重要作用，这使其成为设计针对多重耐药细菌的新型肽抗生素的有前景的模板。
