| Abstract |
Fish express mammalian-type (M-type) TLRs consisting of leucine-rich repeats (LRRs) and Toll-IL-1R (TIR) homology domain for immunity, whereas invertebrates in deuterostomes appear to have no orthologs of M-type TLRs. Lampetra japonica (lamprey) belongs to the lowest class of vertebrates with little information about its TLRs. We have identified two cDNA sequences of putative TLRs in the lamprey (laTLRs) that contain LRRs and TIR domains. The two laTLRs were 56% homologous to each other, and their TIRs were similar to those of members of the human TLR2 subfamily, most likely orthologs of fish TLR14. We named them laTLR14a and laTLR14b. We raised a rabbit polyclonal Ab against laTLR14b and identified a 85-kDa protein in a human HEK293 transfectant by immunoblotting using the Ab. FACS, histochemical, and confocal analyses showed that laTLR14b is expressed intracellularly in lamprey gill cells and that the overexpressed protein resides in the endoplasmic reticulum of human and fish (medaka) cell lines. Because natural agonists of TLR14 remained unidentified, we made a chimera construct of extracellular CD4 and the cytoplasmic domain of laTLR14. The chimera molecule of laTLR14b, when expressed in HEK293 cells, elicited activation of NF-kappaB and, consequently, weak activation of the IFN-beta promoter. laTLR14b mRNA was observed in various organs and leukocytes. This lamprey species expressed a variable lymphocyte receptor structurally independent of laTLR14 in leukocytes. Thus, the jawless vertebrate lamprey possesses two LRR-based recognition systems, the variable lymphocyte receptor and TLR, and the M-type TLRs are conserved across humans, fish, and lampreys.
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