One of the two Toll-like receptor 5 proteins produced by the duplicated tlr5 genes of zebrafish, and the flagellin-sensing receptor whose ectodomain provided the reference crystal structure of a TLR5-flagellin complex. It has a signal peptide, a leucine-rich-repeat ectodomain that contacts the D1 helices of bacterial flagellin, a single transmembrane segment and a cytoplasmic TIR domain. Unlike TLR5 of mammals, birds and reptiles, which signals as a homodimer, the zebrafish receptor is an obligate heterodimer: this protein and its paralogue together confer flagellin-induced NF-kappa-B activation, and neither works alone even when the trafficking chaperone UNC93B1 is supplied, which improves signalling and redistributes both proteins to shared vesicle-like compartments. Both the ectodomain and the cytoplasmic domain must be in heterodimeric configuration for activation, indicating that the two paralogues have subfunctionalised. Flagellin recognition itself is conserved with mammalian TLR5, but the contact residues are species-specific: transplanting the zebrafish binding-site residues into human TLR5 abolishes its activation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0002224 toll-like receptor signaling pathway | IEA GO_REF:0000002 | MODIFY | Summary: Correct, and the receptor-specific TLR5 pathway term applies, since flagellin recognition is conserved in this paralogue. Reason: The InterPro-based family assertion is right, and the specific child is justified: this gene product is a genuine tlr5 paralogue and flagellin-induced NF-kappa-B activation through it has been demonstrated directly, with the crystal structure of its ectodomain bound to flagellin defining the interaction. The ligand-specific term therefore does transfer from mammalian TLR5, which is the question the project asked; what does not transfer is the receptor configuration, since the zebrafish receptor is an obligate heterodimer with its paralogue. That is recorded in the core function rather than by withholding the pathway term, because GO:0034146 describes signals initiated by ligand binding to toll-like receptor 5 without asserting a homodimer. There is also in vivo support in the animal itself: knocking down this gene in zebrafish embryos reduces the flagellin-induced induction of il1b, partially when this paralogue alone is targeted and completely when both are, which is the phenotype expected of one subunit of an obligate heterodimer. Proposed replacements: toll-like receptor 5 signaling pathway Supporting Evidence: PMID:29555749 zebrafish ( Danio rerio ) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a PMID:22344444 of zebrafish TLR5 (as a variable lymphocyte receptor hybrid protein) in complex PMID:26208853 Our results revealed that abrogation of both tlr5a and tlr5b effectively prevented the il1b up-regulation observed in control embryos upon flagellin stimulation PMID:26208853 a complete block of induction of il1b by injection with flagellin and a partial effect of the tlr5b morpholino |
| GO:0004888 transmembrane signaling receptor activity | IEA GO_REF:0000002 | MODIFY | Summary: The receptor binds bacterial flagellin directly, so the specific pattern recognition receptor activity term applies. Reason: GO:0004888 is correct but generic. Flagellin is a pathogen-associated molecular pattern, and binding by this gene product is established structurally: the reference TLR5-flagellin complex was solved with the zebrafish TLR5b ectodomain, which contacts the flagellin D1 helices. GO:0038187 is also the conventional molecular function for characterised TLRs, held by human TLR5 by IDA. Because neither zebrafish paralogue signals as a homodimer, the activity is recorded in core_functions as a contribution to the heterodimer rather than as an activity of this subunit alone. Proposed replacements: pattern recognition receptor activity Supporting Evidence: PMID:22344444 of zebrafish TLR5 (as a variable lymphocyte receptor hybrid protein) in complex PMID:22344444 TLR5 interacts primarily with the three helices of the FliC D1 PMID:29555749 serves as a model structure for the homodimeric TLR5 PMID:29555749 Even after expression of drUNC93B1, which led to redistribution and more robust NF-ΞΊB activation by the heterodimeric receptor, individual drTLR5a and drTLR5b did not operate as homodimers file:DANRE/tlr5b/tlr5b-deep-research-falcon.md However, this dual-ligand capability has not been directly demonstrated for zebrafish TLR5b |
| GO:0006955 immune response | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: True but generic. Reason: Sound for a flagellin receptor, but the informative process statement is the proposed GO:0034146, a descendant of the innate arm of this term. |
| GO:0007165 signal transduction | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: True but generic. Reason: Sound TIR-domain inference; superseded by the proposed pathway term. |
| GO:0016020 membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Correct: signal peptide plus a single predicted transmembrane segment. Reason: Membrane localisation is well founded for this family and for this entry's features. It is left at this generic level deliberately: the paralogues were observed in vesicle-like compartments that redistributed on UNC93B1 co-expression rather than at a cleanly defined surface or endosomal location, so a more specific location term would go beyond the published observations. What was observed is worth recording precisely, because it is the basis of the open question below: both paralogues associated more strongly with LAMP-1-positive than with EEA-1-positive vesicles, and co-expression of UNC93B1 shifted them further toward the LAMP-1 compartment while increasing flagellin responsiveness. That is suggestive of a late endosomal or lysosomal site of action, but it comes from overexpression in a heterologous cell line, so no specific compartment term is proposed. Supporting Evidence: PMID:29555749 Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the PMID:29555749 drUNC93B1 enhanced relocalization of both receptors toward LAMP-1βspecific compartments |
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Download this section (compressed HTML)Q: Does zebrafish tlr5b respond to double-stranded RNA as a homodimer, as has been reported for the grass carp protein? If so, this gene product would have an interferon-directed antiviral function in addition to its flagellin function, and the two would use different receptor configurations.
Q: Where does the zebrafish TLR5 heterodimer engage flagellin? Both paralogues were seen in vesicle-like compartments that redistributed with UNC93B1, which is unusual for a receptor whose mammalian counterpart works at the cell surface, and no location term beyond generic membrane is currently justified.
Q: How should GO express an obligate heterodimeric receptor whose subunits are separate gene products? Neither paralogue has the activity alone, so the honest molecular-function statement is a contribution to a complex, but no complex term exists for this heterodimer and the pathway term is written in terms of a single receptor.
Q: Do the two paralogues differ in expression rather than only in biochemistry? Subfunctionalisation of a duplicated receptor would predict that the heterodimer can only form where both genes are expressed, which would bound the tissues in which flagellin sensing is possible.
Experiment: Test dsRNA responsiveness of zebrafish tlr5b directly: express it alone and with its paralogue in zebrafish cells, stimulate with poly(I:C) and with an RNA virus, and read both NF-kappa-B and interferon-stimulated response element reporters, with knockdown of the endogenous gene as a control.
Hypothesis: The dsRNA-sensing capacity reported for grass carp TLR5b is general to teleost TLR5b and is a homodimeric function distinct from heterodimeric flagellin sensing.
Type: receptor activation assay
Experiment: Localise the two paralogues at endogenous levels using epitope-tagged knock-in alleles, with and without flagellin stimulation, and determine whether flagellin recognition occurs at the plasma membrane or after internalisation.
Hypothesis: The heterodimer engages flagellin in an intracellular compartment, which would explain the UNC93B1 dependence and would justify a specific location annotation.
Type: endogenous localisation
Experiment: Quantify the flagellin affinity and signalling output of defined ectodomain combinations in vitro, comparing the tlr5b homodimer, the tlr5a homodimer and the heterodimer against flagellins from several fish pathogens.
Hypothesis: The heterodimer, not either homodimer, is the functional flagellin receptor across ligand variants, and the two paralogues contribute unequal binding surfaces.
Type: quantitative binding and signalling comparison
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