Abstract
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.
Highlights
Pattern recognition receptors recognize widely-conserved motifs of pathogens and are crucial for initiating immune responses against invading microorganisms
Information on tlr1 and tlr2 genes is available for some fish species but tlr1 orthologues had not yet been identified in carp
We previously reported on the presence of a tlr2 orthologue in carp, given the additional whole genome duplication (WGD) event in the carp lineage, a second copy of the tlr2 gene was expected
Summary
Pattern recognition receptors recognize widely-conserved motifs of pathogens and are crucial for initiating immune responses against invading microorganisms. I.R. Fink et al / Fish & Shellfish Immunology 56 (2016) 70e83 ectodomains of Tlrs are not always highly conserved and sequence information alone cannot infer functional properties [10]. Studies into ligand-binding properties of fish Tlrs are essential to characterize their exact function within the immune system of fish. Studies into ligand-binding properties of fish Tlr and/or Tlr molecules have been scarce. Transfection of human HEK293 cells with carp tlr suggested the ability to bind the prototypical TLR2 ligands LTA, PGN and Pam3CSK4. We compare the gene expression of tlr and tlr in the same tissue samples and purified cell populations and describe our efforts to characterize the function of putative Tlr1/Tlr heterodimers by studying subcellular localization and ligand-binding properties. The organs of interest were aseptically removed and immediately frozen in liquid nitrogen and stored at À80 C until used for RNA isolation
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