K9IIP0 is the common vampire bat orthologue of TSG-6 (TNFAIP6, tumor necrosis factor-inducible gene 6 protein), a secreted, inflammation-inducible glycoprotein of the extracellular matrix. The 277-residue precursor has a signal peptide (residues 1-17) followed by the two modules that define the family: an N-terminal Link module (residues 36-129, with the canonical 58-127 and 82-103 disulfides) that binds hyaluronan, and a C-terminal CUB domain (residues 135-247). Through the Link module TSG-6 binds hyaluronan and non-covalently cross-links it, and it catalyses the covalent transfer of inter-alpha-inhibitor heavy chains onto hyaluronan to build heavy chain- hyaluronan complexes, remodelling matrices at sites of inflammation. The same module binds a wide range of C-X-C and C-C chemokines and thereby blocks their presentation on endothelial glycosaminoglycans, which is the mechanistic basis of the protein's anti-inflammatory action: mammalian TSG-6 suppresses neutrophil transendothelial migration and dampens inflammation in numerous in vivo models. In D. rotundus at least two TSG-6 family members are transcribed in the submaxillary salivary glands, and TSG-6 was confirmed in the principal gland proteome by LC-MS/MS, making it one of the more abundant salivary proteins and a candidate anti-inflammatory component of the bite-site secretion.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005540 hyaluronic acid binding | IEA GO_REF:0000002 | ACCEPT | Summary: Hyaluronan binding is the defining activity of the Link module, and the Link module of this protein is intact and confidently detected (PROSITE PS50963, residues 36-129, with both canonical disulfides annotated). The human orthologue TNFAIP6 carries this term from direct assay. The UniProt CAUTION does not bear on this call: it is scoped to PROSITE ProRule PRU00059, which is the CUB domain rule, not the Link rule (PRU00323) that supports the Link module features here. Reason: Intact, well-detected Link module plus direct experimental support in the human orthologue. The earlier UNDECIDED reflected a misreading of the CAUTION, which concerns the CUB domain. Supporting Evidence: file:DESRO/K9IIP0/K9IIP0-uniprot.txt DOMAIN 36..129 PMID:23411029 The N-terminal domain of TSG-6 represents a structural motif known as the Link module, which confers affinity for hyaluronan. file:DESRO/K9IIP0/K9IIP0-deep-research-falcon.md The Link module mediates binding to hyaluronan (HA) and other glycosaminoglycans |
| GO:0005615 extracellular space | IEA GO_REF:0000118 | ACCEPT | Summary: The precursor has a SignalP-predicted signal peptide (1-17) with a cleaved 18-277 mature chain, TSG-6 is a classical secreted matrix protein, the human orthologue is annotated to the extracellular region from direct assay, and TSG-6 was recovered from the D. rotundus principal submaxillary gland proteome by LC-MS/MS. As with the hyaluronan binding call, the CAUTION concerns the CUB domain and says nothing about localisation. Reason: Signal peptide, family behaviour, and orthologue annotation all agree. The LC-MS/MS detection was made on principal gland tissue, so strictly it is expression evidence rather than localisation evidence; it corroborates that the protein is made in quantity here, while the secreted localisation itself rests on the signal peptide, the classical secreted family, and the direct-assay annotation of the human orthologue. Upgraded from UNDECIDED. Supporting Evidence: file:DESRO/K9IIP0/K9IIP0-uniprot.txt SIGNAL 1..17 PMID:23411029 Of note, expression of TSG-6 was confirmed by the proteome of the PS gland (Figure 2A). file:DESRO/K9IIP0/K9IIP0-deep-research-falcon.md It is secreted into the extracellular space and acts in the ECM and pericellular matrix. |
| GO:0050728 negative regulation of inflammatory response | IEA GO_REF:0000118 | ACCEPT | Summary: Anti-inflammatory action is the best-characterised biology of TSG-6, not a peripheral inference: the Link module binds chemokines and blocks their presentation on endothelial glycosaminoglycans, suppressing neutrophil transendothelial migration, and the protein inhibits inflammation across several in vivo models. The human orthologue carries this exact term both from direct assay (PMID:21569482) and by phylogenetic inference, so the TreeGrafter propagation to the bat protein is a legitimate transfer rather than an over-propagation. An earlier version of this review removed the term; there is no biological argument against it. Reason: Well-supported family function with experimental backing in the human orthologue and an identified mechanism that depends on the Link module, which is intact in this protein. REMOVE is reserved for annotations that can be argued against on biological grounds; this one cannot. Supporting Evidence: PMID:23411029 TSG-6 inhibits inflammation, according to several in vivo models PMID:23411029 TSG-6 also inhibits neutrophil migration to interact with macrophage CD44 |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MODIFY | Summary: This keyword-derived term is uninformative but, contrary to an earlier version of this review, it is not wrong. TSG-6 does catalyse a covalent chemical step: it transfers inter-alpha-inhibitor heavy chains onto hyaluronan via a TSG-6-heavy-chain ester intermediate. The human orthologue TNFAIP6 is annotated to GO:0106435 carboxylesterase activity from two independent direct assays (PMID:16873769, PMID:20463016), and GO:0106435 is a descendant of GO:0016787. The UniProt record for this very protein states that the ITIH2/HC2 interaction is required for transesterification of the heavy chain to hyaluronan. Reason: Replace the root-level hydrolase term with the specific child GO:0106435 carboxylesterase activity, which is the term the GO consortium uses for the experimentally demonstrated heavy-chain transfer activity of the human orthologue. Removing the annotation, as previously proposed, would have discarded a real catalytic function. Caveat to record explicitly: carboxylesterase activity is defined as ester hydrolysis, whereas TSG-6 performs two sequential transesterifications with no net hydrolysis, the heavy chain moving from the chondroitin sulfate of inter-alpha-inhibitor onto hyaluronan through a covalent TSG-6-heavy-chain ester intermediate. GO:0106435 is used here because it is the term the consortium applies to the human orthologue, not because the chemistry is a good fit; a transferase-branch term would describe the reaction better (see suggested_questions). Proposed replacements: carboxylesterase activity Supporting Evidence: file:DESRO/K9IIP0/K9IIP0-uniprot.txt is required for transesterification of the HC to hyaluronan. file:DESRO/K9IIP0/K9IIP0-deep-research-falcon.md enzymatic transesterase activity transferring I PMID:20463016 In concert with HC2, TSG-6 have a unique catalytic activity transferring HCs from bikunin proteins to hyaluronan (HA). PMID:20463016 The reaction involves two divalent cation-dependent transesterifications and a covalent intermediate composed of TSG-6 and the HC to be transferred PMID:26468290 TSG-6 was shown to play a direct role in the transfer of HCs from IΞ±I onto HA via the formation of covalent intermediates |
| GO:0007155 cell adhesion | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: This comes from the UniProt Cell adhesion keyword. Even in the human orthologue the term has never risen above electronic evidence, and the documented matrix biology of TSG-6 is hyaluronan binding, cross-linking, and heavy-chain transfer rather than adhesion between cells. Any effect on adhesion is indirect, through the altered hyaluronan matrix, and is better captured by the hyaluronan terms. Reason: Keyword-derived, unsupported by experiment in any species, and attributing to the protein a role that is a downstream consequence of its matrix remodelling rather than its own activity. Supporting Evidence: file:DESRO/K9IIP0/K9IIP0-uniprot.txt Cell adhesion {ECO:0000256|ARBA:ARBA00022889}; file:DESRO/K9IIP0/K9IIP0-deep-research-falcon.md The Link module mediates binding to hyaluronan (HA) and other glycosaminoglycans |
| GO:0030212 hyaluronan metabolic process | ISS PMID:20463016 The TSG-6/HC2-mediated transfer is a dynamic process shuffli... | NEW | Summary: Proposed new annotation. The heavy-chain transfer reaction covalently modifies hyaluronan to form heavy chain-hyaluronan complexes, which is a hyaluronan metabolic process; the human orthologue carries this term from two direct assays (PMID:20463016, PMID:26468290). This is the biological process counterpart of the carboxylesterase activity proposed above, and the GOA record for this protein has no term covering it. Reason: Completes the molecular function to biological process link for the heavy-chain transfer activity, and mirrors the experimentally supported annotation of the human orthologue. Anchored to the orthologue experimental evidence (PMID:20463016, PMID:26468290) rather than to the deep-research survey, since the justification is orthologue transfer. Supporting Evidence: file:DESRO/K9IIP0/K9IIP0-uniprot.txt is required for transesterification of the HC to hyaluronan. PMID:20463016 can be transferred to different hyaluronan (HA) molecules by TSG-6/HC2 PMID:26468290 both HC1 and HC2 of IΞ±I can be covalently transferred onto the C6-hydroxyls of the N-acetyl glucosamine sugars in HA |
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Download this section (compressed HTML)Q: Is the abundant salivary TSG-6 of Desmodus rotundus delivered to the host bite wound, and does it suppress host neutrophil recruitment there?
Suggested experts: Ivo M. B. Francischetti, JosΓ© M. C. Ribeiro
Q: Does salivary TSG-6 interact functionally with the bat's own salivary plasminogen activator, given that mammalian TSG-6 potentiates the antiplasmin activity of inter-alpha-inhibitor?
Q: Is GO:0106435 carboxylesterase activity the right term for the TSG-6 heavy-chain transfer reaction, or is a transferase-branch term warranted? The reaction is two transesterifications with no net hydrolysis, so the hydrolase-branch term used for the human orthologue describes the chemistry poorly even though it is the established consortium precedent.
Suggested experts: Anthony J. Day, Jan J. Enghild
Q: What distinguishes the two TSG-6 family members transcribed in vampire bat salivary gland, one of which carries C-terminal deletions - are they functionally redundant or specialised?
Suggested experts: Anthony J. Day
Experiment: Express recombinant full-length bat TSG-6 and its isolated Link module, incubate each with inter-alpha-inhibitor and hyaluronan in the presence of magnesium, and detect heavy chain-hyaluronan complex formation by Western blot against inter-alpha-inhibitor heavy chains, using human TSG-6 as the positive control.
Hypothesis: Vampire bat salivary TSG-6 retains the heavy-chain transferase activity of mammalian TSG-6 despite the flagged CUB domain divergence.
Type: In vitro heavy-chain transfer assay
Experiment: Measure binding of recombinant bat TSG-6 to a panel of host C-X-C and C-C chemokines by surface plasmon resonance, then test whether it inhibits chemokine-driven neutrophil transendothelial migration across a host endothelial monolayer.
Hypothesis: Salivary TSG-6 suppresses host leukocyte recruitment at the bite site.
Type: Surface plasmon resonance and transendothelial migration assay
Experiment: Align the bat CUB domain against characterised CUB domains to identify which conserved residues are missing, model the domain with AlphaFold, and compare full-length bat TSG-6 with a CUB-deleted construct in fibronectin binding and heavy-chain transfer assays.
Hypothesis: The C-terminal CUB domain of the bat protein is degenerate, specialising it toward Link-module functions.
Type: Comparative sequence and structure analysis with domain-deletion assays
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