Structurally diverse genes encode Tlr2 in rainbow trout: The conserved receptor cannot be stimulated by classical ligands to activate NF-κB in vitro.
The mammalian toll-like receptor 2 (TLR2) is a dominant receptor for the recognition of Gram-positive bacteria. Its structure and functional properties were unknown in salmonid fish. In RT-PCR and RACE experiments, we obtained the full-length cDNA sequence encoding Tlr2 from rainbow trout (Oncorhynchus mykiss) as well as a copy of an unspliced nonsense message from a highly segmented gene. The primary structure of the encoded receptor complies with the domain structure and ligand-binding sites known from mammals and other fish species and sorts well into the evolutionary tree of teleostean Tlr2s. We retrieved a gene version encoding the receptor on a single exon (tlr2a) and also a partial sequence of a second gene variant being segmented into multiple exons (tlr2b). Surprisingly, the abundances of both transcript variants accounted only for ∼10% of all Tlr2-encoding transcripts in various tissues and cell types of healthy fish. This suggests the expression of several distinct tlr2 gene variants in rainbow trout. We expressed tlr2a in HEK-293 cells, but were unable to demonstrate its functionality through NF-κB activation. Neither synthetic lipopeptides known to stimulate mammalian TLR2 nor different bacterial challenges induced OmTLR2-mediated NF-κB activation, not in HEK-293 or in salmon CHSE-214 cells. Positive demonstration of TLR2-MYD88 interaction excluded that its functional impairment caused the failure of NF-κB activation. We discuss impaired heterodimerization with a necessary Tlr partner as one from among several alternatives to explain the dysfunction of Tlr2a in the interspecies reconstitution system of TLR signaling.
Leibniz Institute for Farm Animal Biology (FBN), Institute of Genome Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
Andreas
A
Brietzke
University of Oslo, School of Pharmacy, Department of Pharmaceutical Biosciences, PO Box 1068 Blindern, NO-0316 Oslo, Norway.
Marianne
M
Arnemo
University of Oslo, School of Pharmacy, Department of Pharmaceutical Biosciences, PO Box 1068 Blindern, NO-0316 Oslo, Norway.
Tor
T
Gjøen
Rostock University Medical Center, Department of Cell Biology, Schillingallee 69, 18057 Rostock, Germany.
Henrike
H
Rebl
Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Institute of Immunology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
Tomáš
T
Korytář
Leibniz Institute for Farm Animal Biology (FBN), Institute of Genome Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
Tom
T
Goldammer
Leibniz Institute for Farm Animal Biology (FBN), Institute of Genome Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. Electronic address: rebl@fbn-dummerstorf.de.
Alexander
A
Rebl
Leibniz Institute for Farm Animal Biology (FBN), Institute of Genome Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
Hans-Martin
HM
Seyfert
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000276:immunology* / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000276:immunology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D051195:Toll-Like Receptor 2 / Q000235:genetics* / Q000276:immunology; D014162:Transfection
D008024:Ligands; D016328:NF-kappa B; D051195:Toll-Like Receptor 2
Innate immunity; Pattern recognition receptors; Salmonid; TLR2; Teleost