Facing the challenge of a functional characterization of toll-like receptor (TLR)1 and TLR2 in common carp

Abstract

Toll-like receptors are a family of germline-encoded pattern recognition receptors which activate rapid inflammatory responses upon detection of their cognate ligands. In mammals, TLR2 can form a heterodimer with TLR1, 6, or 10, and recognize lipoproteins/lipopeptides from Gram-positive bacteria. Of these TLRs, only tlr1 and tlr2 are present in fish genomes. In the carp genome, a single full-length tlr1 gene could be identified, whereas two functional genes for carp tlr2 exist. High expression of tlr1 and both tlr2 genes was found in immune organs and leukocytes of both myeloid and lymphoid origin. Of the two tlr2 genes, tlr2a was always higher expressed than tlr2b. Three-dimensional modelling of the carp Tlr1 and Tlr2 proteins appear to confirm the ability to form a heterodimer, with a pocket that can accommodate the tri-acylated lipopeptide ligand Pam3CSK4, similar to the heterodimer structure of human TLR1-TLR2 on which the model was built. In cell lines of human as well as fish origin transfected to overexpress the carp Tlr proteins, co-localization of carp Tlr1 and Tlr2 could be revealed with confocal microscopy. We were unable to confirm Tlr localization to the cell surface, the expected sub-cellular localization of Tlr1 and Tlr2. Further experimental evidence would be needed to unequivocally prove molecular interaction between the two Tlr proteins. In previous work, we have described ligand-specific activation of carp Tlr2 overexpressed in human cells (HEK), via measurement of increased phosphorylation levels of the MAP kinase p38 by Western blot. Instead of this semi-quantitative method, here we used a read-out system based on NF-κB activation and subsequent measurement of luminescence. This quantitative method could not confirm our initial ligand binding studies, not using human cell lines (HEK, HeLa), nor using a fish cell line (EPC). We will discuss possible explanations for the unsuccessful ligand-specific activation of NF-κB after overexpression of Tlr1 and/or Tlr2 in human, but also fish cell lines, to propose alternative future strategies for studying ligand binding properties of fish Tlrs.