Fink Inge R, Pietretti Danilo, Voogdt Carlos G P, Westphal Adrie H, Savelkoul Huub F J, Forlenza Maria, Wiegertjes Geert F
Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, PO Box 338, 6700 AH, Wageningen, The Netherlands.
Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands.
Fish Shellfish Immunol. 2016 Sep;56:70-83. doi: 10.1016/j.fsi.2016.06.049. Epub 2016 Jun 29.
Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs.
Toll样受体（TLRs）是天然免疫的基本组成部分，在抵御病原体入侵中发挥着重要作用。在本研究中，我们展示了鲤（Cyprinus carpio）tlr1、tlr2a和tlr2b全长编码序列的分子特征。每个基因都由单个外显子编码，包含数量保守的富含亮氨酸重复序列、一个跨膜区域和一个用于信号传导的细胞内TIR结构域。实际上，鲤tlr1、tlr2a和tlr2b的序列、系统发育和共线性分析支持这些基因是哺乳动物TLR1和TLR2的直系同源物。tlr基因在各种免疫器官和细胞类型中表达。此外，鲤的序列与人类TLR1-TLR2异二聚体结构具有良好的三维匹配，包括与配体Pam3CSK4结合的潜力。这支持鲤Tlr1-Tlr2异二聚体可能的形成。然而，尽管显示了Tlr1和Tlr2的表达及共定位，但我们无法通过NF-κB激活在转染细胞系中证明Tlr1/Tlr2介导的配体结合。我们讨论了在人类和鱼类细胞系中表达Tlr1和/或Tlr2后研究NF-κB配体特异性激活时可能存在的局限性，并提出了研究鱼类Tlr配体结合特性的未来替代策略。
