id: K9IFY6
gene_symbol: K9IFY6
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:9430
  label: Desmodus rotundus
description: >-
  K9IFY6 is the common vampire bat orthologue of CCL28 (mucosae-associated
  epithelial chemokine, MEC), a secreted CC-motif chemokine. The 132-residue
  precursor comprises a signal peptide (residues 1-22), a chemokine
  interleukin-8-like domain (residues 28-87) with the canonical CC cysteine
  motif, and an unusually long, strongly basic C-terminal extension. It is
  highly homologous to human CCL28. Like other chemokines it is secreted and acts
  extracellularly as a receptor ligand, engaging CCR-family G protein-coupled
  receptors (CCR10 and CCR3 for mammalian CCL28) to direct leukocyte chemotaxis;
  in mammals CCL28 is characteristically expressed by mucosal epithelia and
  exocrine glands, where it recruits IgA-producing plasma cells. Human CCL28
  additionally carries a histatin-like basic C-terminus with direct
  broad-spectrum antimicrobial activity, and the bat protein retains the
  equivalent basic extension. It was among the most abundant transcripts of the
  vampire bat submaxillary gland and was confirmed in the gland proteome, making
  it a likely antimicrobial and immune-modulating component of saliva delivered
  to the host bite wound.
existing_annotations:
  - term:
      id: GO:0008009
      label: chemokine activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: >-
        The protein carries a chemokine interleukin-8-like domain (residues
        28-87) with the CC cysteine signature (PROSITE PS00472
        SMALL_CYTOKINES_CC), belongs to the intercrine beta family, and is the
        CCL28 orthologue. Chemokine activity is the informative molecular
        function for this protein.
      action: ACCEPT
      reason: >-
        Domain architecture, family assignment, and orthology to CCL28 all
        converge on chemokine activity. This is the core molecular function.
      supported_by:
        - &id_domain
          reference_id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
          supporting_text: '/note="Chemokine interleukin-8-like"'
        - &id_family
          reference_id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
          supporting_text: 'Belongs to the intercrine beta (chemokine CC) family.'
        - &id_ccl28
          reference_id: PMID:23411029
          supporting_text: >-
            CCL28 is a chemokine signaling via CCR10 and CCR3 that is selectively
            expressed in certain mucosal tissues such as exocrine glands, trachea,
            and colon.
          reference_section_type: DISCUSSION
  - term:
      id: GO:0005125
      label: cytokine activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        Cytokine activity is correct but is the parent of chemokine activity,
        which is already independently annotated and is directly supported by
        the IL8-like domain and the CC cysteine motif.
      action: MODIFY
      reason: >-
        Replace the generic parent with the specific child GO:0008009 chemokine
        activity, which conveys the receptor class and the chemotactic mode of
        action.
      proposed_replacement_terms:
        - id: GO:0008009
          label: chemokine activity
      supported_by:
        - *id_domain
        - *id_family
  - term:
      id: GO:0060326
      label: cell chemotaxis
    evidence_type: IEA
    original_reference_id: GO_REF:0000108
    review:
      summary: >-
        Directing the migration of responding cells is the defining biological
        role of a chemokine, and the UniProt record carries the Chemotaxis
        keyword from the CC chemokine rule. For CCL28 specifically, the
        best-characterised activity is chemoattraction of IgA-producing plasma
        cells into mucosal lamina propria.
      action: ACCEPT
      reason: >-
        Well supported at the family level and specifically for CCL28. This is
        the core biological process.
      supported_by:
        - reference_id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
          supporting_text: 'Chemotaxis {ECO:0000256|RuleBase:RU361150};'
        - &id_iga
          reference_id: PMID:23411029
          supporting_text: >-
            CCL28 is particularly abundant in SGs and plays an important role in
            mucosal immunity as a chemoattractant for IgA-producing plasma cells
            into the mucosal lamina propria
          reference_section_type: DISCUSSION
  - term:
      id: GO:0006935
      label: chemotaxis
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        GO:0006935 chemotaxis is the general parent covering any cell or
        organism moving along a chemical gradient. The specific child GO:0060326
        cell chemotaxis is already annotated and is the correct granularity for
        a leukocyte chemoattractant.
      action: MODIFY
      reason: >-
        Replace with GO:0060326 cell chemotaxis, which is what a chemokine
        actually mediates.
      proposed_replacement_terms:
        - id: GO:0060326
          label: cell chemotaxis
      supported_by:
        - *id_iga
  - term:
      id: GO:0005615
      label: extracellular space
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        The precursor has a signal peptide (1-22) with a cleaved mature chain
        (23-132), UniProt records the subcellular location as Secreted, and
        salivary CCL28 was recovered from the D. rotundus submaxillary gland
        proteome, so the mature protein is a soluble extracellular species.
      action: ACCEPT
      reason: >-
        Secretion is supported by signal peptide prediction, the UniProt
        subcellular location, and direct proteomic detection of CCL28 in bat
        salivary gland.
      supported_by:
        - &id_secreted
          reference_id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
          supporting_text: 'SUBCELLULAR LOCATION: Secreted'
        - &id_abundant
          reference_id: PMID:23411029
          supporting_text: >-
            In our analysis, bat salivary CCL28 was found to be abundant at the
            transcriptional (Table 4) and proteome (Figure 2A) levels.
          reference_section_type: RESULTS
  - term:
      id: GO:0005576
      label: extracellular region
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        Extracellular region is the broad parent of extracellular space, which
        is already annotated and better describes a small soluble secreted
        chemokine.
      action: MODIFY
      reason: >-
        Use the more specific GO:0005615 extracellular space for a soluble
        secreted protein.
      proposed_replacement_terms:
        - id: GO:0005615
          label: extracellular space
      supported_by:
        - *id_secreted
  - term:
      id: GO:0006955
      label: immune response
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: >-
        Immune response is correct for a CC chemokine and is directly
        appropriate for CCL28, which is a mucosal immunity effector. The term is
        general, but the more specific descendants that would apply to CCL28
        (mucosal IgA plasma cell recruitment, antibacterial humoral response)
        have not been demonstrated for the vampire bat protein, so the general
        term is the honest level of granularity here.
      action: ACCEPT
      reason: >-
        Correct, if broad. Retained at this level rather than replaced with a
        speculative descendant, because no bat CCL28 has been assayed.
      supported_by:
        - *id_iga
        - *id_family
  - term:
      id: GO:0007165
      label: signal transduction
    evidence_type: IEA
    original_reference_id: GO_REF:0000108
    review:
      summary: >-
        This annotation comes from an inter-ontology logical inference from
        cytokine activity, not from evidence about this protein. Signal
        transduction is the process carried out by the responding cell after
        receptor engagement; a secreted ligand initiates that process rather
        than performing it. Its informative content for this protein is already
        captured by chemokine activity and cell chemotaxis.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Root-level, ligand-inappropriate biological process term produced by
        automatic inter-ontology inference. It adds no information beyond the
        retained chemokine activity and cell chemotaxis annotations.
      supported_by:
        - *id_domain
  - term:
      id: GO:0048020
      label: CCR chemokine receptor binding
    evidence_type: ISS
    original_reference_id: PMID:23411029
    review:
      summary: >-
        Proposed new annotation. CCL28 signals through the CCR-family G
        protein-coupled receptors CCR10 and CCR3. K9IFY6 is the D. rotundus
        CCL28 orthologue (UniProt cross-references to NCBI Gene 112315258 and to
        PANTHER subfamily PTHR12015:SF205 C-C motif chemokine 28), and the
        Vampirome authors report that bat salivary CCL28 is highly homologous to
        the human protein. Receptor binding is the mechanistic basis of the
        accepted chemokine activity annotation and is worth stating explicitly.
      action: NEW
      reason: >-
        Adds mechanism to the generic chemokine activity call. The conservative
        parent GO:0048020 is proposed rather than GO:0031735 (CCR10 chemokine
        receptor binding), because the specific receptor has not been tested for
        the bat protein and mammalian CCL28 engages both CCR10 and CCR3.
      supported_by:
        - *id_ccl28
        - reference_id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
          supporting_text: 'PANTHER; PTHR12015:SF205; C-C MOTIF CHEMOKINE 28; 1.'
        - reference_id: PMID:23411029
          supporting_text: >-
            It is highly homologous to the human counterpart according to a
            Clustal analysis (Figure 13A) and phylogenetic tree (Figure 13B).
          reference_section_type: DISCUSSION
references:
  - id: GO_REF:0000002
    title: Gene Ontology annotation through association of InterPro records with GO
      terms
    findings:
      - statement: >-
          InterPro signatures for the IL8-like chemokine domain (IPR001811) and
          the CC chemokine conserved site (IPR000827) were used to infer
          chemokine activity and immune response for K9IFY6.
  - id: GO_REF:0000043
    title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
    findings:
      - statement: >-
          Keyword-based mapping is the source of three K9IFY6 annotations. Per
          the GOA WITH/FROM column, the Cytokine keyword (UniProtKB-KW:KW-0202)
          drives both the cytokine activity and the extracellular space calls,
          and the Chemotaxis keyword (UniProtKB-KW:KW-0145) drives the
          chemotaxis call.
  - id: GO_REF:0000108
    title: Automatic assignment of GO terms using logical inference, based on on inter-ontology
      links
    findings:
      - statement: >-
          Cell chemotaxis and signal transduction were assigned by logical
          inference from the molecular function terms rather than from direct
          evidence about this protein; the signal transduction call in
          particular is an artefact of applying a ligand's activity term to the
          responding cell's process. Per the GOA WITH/FROM column the inference
          sources are GO:0005125 (signal transduction) and GO:0008009 (cell
          chemotaxis).
  - id: GO_REF:0000120
    title: Combined Automated Annotation using Multiple IEA Methods
    findings:
      - statement: >-
          The extracellular region annotation derives from combined automated
          methods; per the GOA WITH/FROM column these are the InterPro
          chemokine signatures (IPR000827, IPR001811, IPR036048) together with
          the UniProt Secreted subcellular location (UniProtKB-SubCell:SL-0243).
  - id: PMID:23411029
    title: 'The "Vampirome": Transcriptome and proteome analysis of the principal
      and accessory submaxillary glands of the vampire bat Desmodus rotundus, a
      vector of human rabies.'
    findings:
      - statement: >-
          The abundant CC chemokine of the D. rotundus submaxillary gland is
          CCL28, detected at both transcript and protein level and highly
          homologous to human CCL28. This is the study whose EMBL submission
          (JAA45050.1, salivary gland) underlies the K9IFY6 accession.
        supporting_text: >-
          In our analysis, bat salivary CCL28 was found to be abundant at the
          transcriptional (Table 4) and proteome (Figure 2A) levels.
        reference_section_type: RESULTS
      - statement: >-
          CCL28 signals through CCR10 and CCR3 and is selectively expressed in
          mucosal tissues and exocrine glands, which is consistent with its
          abundance in a salivary gland.
        supporting_text: >-
          CCL28 is a chemokine signaling via CCR10 and CCR3 that is selectively
          expressed in certain mucosal tissues such as exocrine glands, trachea,
          and colon.
        reference_section_type: DISCUSSION
      - statement: >-
          The defining immunological role of CCL28 is chemoattraction of
          IgA-producing plasma cells into the mucosal lamina propria.
        supporting_text: >-
          CCL28 is particularly abundant in SGs and plays an important role in
          mucosal immunity as a chemoattractant for IgA-producing plasma cells
          into the mucosal lamina propria
        reference_section_type: DISCUSSION
      - statement: >-
          Independently of receptor signalling, human CCL28 has direct
          broad-spectrum antimicrobial activity against Candida albicans and
          both Gram-negative and Gram-positive bacteria, attributed to a
          histatin-5-like basic C-terminus. The authors propose the same role
          for the bat salivary protein. This is a hypothesis for D. rotundus,
          not a measurement.
        supporting_text: >-
          More recently, it has been shown that CCL28 had a potent antimicrobial
          activity against Candida albicans, Gram-negative bacteria, and
          Gram-positive bacteria.
        reference_section_type: DISCUSSION
    reference_review:
      relevance: HIGH
      correctness: VERIFIED
      review_notes: >-
        PubMed-verified. This is the source publication for the EMBL entry
        (JAA45050.1) behind K9IFY6 and the only study to identify the D.
        rotundus salivary CC chemokine as CCL28. Its statements about CCL28
        receptor usage and antimicrobial activity are citations to the human
        CCL28 literature, not new bat measurements, and are treated as such
        here.
  - id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
    title: UniProtKB record for K9IFY6 (K9IFY6_DESRO)
    findings:
      - statement: >-
          The protein is a secreted CC-motif chemokine of the intercrine beta
          family with a signal peptide (1-22), a cleaved mature chain (23-132),
          and an IL8-like chemokine domain (28-87).
        supporting_text: 'Belongs to the intercrine beta (chemokine CC) family.'
      - statement: >-
          Cross-references identify the protein as CCL28: NCBI Gene 112315258
          (Desmodus rotundus CCL28), CTD 56477 (human CCL28), RefSeq
          XP_024427703.1, and PANTHER subfamily PTHR12015:SF205 "C-C motif
          chemokine 28".
        supporting_text: 'PANTHER; PTHR12015:SF205; C-C MOTIF CHEMOKINE 28; 1.'
      - statement: >-
          The mature chain carries a long disordered C-terminal region (83-132)
          with a strongly basic compositional bias (92-114), the architectural
          counterpart of the histatin-like antimicrobial tail of human CCL28.
        supporting_text: '/note="Basic residues"'
  - id: file:DESRO/K9IFY6/K9IFY6-deep-research-falcon.md
    title: Deep research report on K9IFY6
    findings:
      - statement: >-
          No peer-reviewed study directly characterising D. rotundus K9IFY6 was
          found; functional assignment rests on CC chemokine family features.
        supporting_text: >-
          no peer-reviewed study directly characterizing Desmodus rotundus K9IFY6
          was found in the searched literature
    reference_review:
      relevance: LOW
      correctness: UNVERIFIED
      review_notes: >-
        LLM-generated survey that did not identify the protein as CCL28 despite
        the UniProt cross-references, and instead reasoned only at the level of
        the CC chemokine family plus unrelated vampire bat serum proteomics. Two
        of its five sources are flagged in the report itself as
        non-peer-reviewed or low quality. Used here only for generic
        family-level statements about secretion and CCR engagement.
core_functions:
  - description: >-
      Secreted CC chemokine (CCL28 orthologue) that acts in the extracellular
      space as a ligand for CCR-family G protein-coupled receptors, directing
      the chemotactic migration of responding leukocytes. In mammals this is the
      mucosal/exocrine-gland chemokine that recruits IgA-producing plasma cells,
      consistent with its abundance in the vampire bat submaxillary gland.
    supported_by:
      - reference_id: file:DESRO/K9IFY6/K9IFY6-uniprot.txt
        supporting_text: 'Belongs to the intercrine beta (chemokine CC) family.'
      - reference_id: PMID:23411029
        supporting_text: >-
          CCL28 is particularly abundant in SGs and plays an important role in
          mucosal immunity as a chemoattractant for IgA-producing plasma cells
          into the mucosal lamina propria
        reference_section_type: DISCUSSION
      - reference_id: PMID:23411029
        supporting_text: >-
          In our analysis, bat salivary CCL28 was found to be abundant at the
          transcriptional (Table 4) and proteome (Figure 2A) levels.
        reference_section_type: RESULTS
    molecular_function:
      id: GO:0008009
      label: chemokine activity
    directly_involved_in:
      - id: GO:0060326
        label: cell chemotaxis
      - id: GO:0006955
        label: immune response
    locations:
      - id: GO:0005615
        label: extracellular space
    knowledge_gaps:
      - gap_statement: >-
          It is undetermined whether vampire bat CCL28 retains the direct,
          receptor-independent antimicrobial activity of its human orthologue,
          and therefore whether an antibacterial humoral response annotation is
          warranted.
        boundary: >-
          What is established is that human CCL28 kills Candida albicans and
          both Gram-negative and Gram-positive bacteria through a
          histatin-5-like basic C-terminus, and that the bat protein carries the
          corresponding long basic C-terminal extension (residues 92-114 basic
          compositional bias). What is not established is any measured
          antimicrobial activity for the bat protein; the Vampirome authors
          state this only as a possibility.
        gap_kind:
          - BIOLOGY
        dark_aspect: RESIDUAL_SUBGAP
        status: OPEN
        significance: >-
          Vampire bats ingest a microbially rich fluid through an open wound;
          whether their most abundant salivary chemokine doubles as a
          broad-spectrum antimicrobial determines how saliva protects both the
          bat and the bite site.
        resolution: >-
          Radial diffusion and minimum inhibitory concentration assays with
          recombinant bat CCL28 and with a synthetic peptide corresponding to
          its basic C-terminus, against oral and blood-borne bacteria and yeast,
          under the low-salt conditions in which CCL28 is active.
        provenance:
          - reference_id: PMID:23411029
            supporting_text: >-
              CCL28 may function as a broad-spectrum antimicrobial protein in the
              saliva.
            reference_section_type: DISCUSSION
      - gap_statement: >-
          The receptor actually engaged by vampire bat CCL28 is undetermined:
          neither CCR10 nor CCR3 usage has been tested for this protein, and it
          is unknown whether it acts on bat leukocytes, on host leukocytes at
          the bite site, or both.
        boundary: >-
          Orthology to CCL28 is firm (NCBI Gene 112315258, PANTHER
          PTHR12015:SF205), and mammalian CCL28 is known to signal through CCR10
          and CCR3. What is missing is any binding or functional assay with the
          bat protein.
        gap_kind:
          - BIOLOGY
        dark_aspect: MF_DARK
        status: OPEN
        significance: >-
          Whether this chemokine is a self-directed mucosal immunity effector or
          a host-directed saliva component changes its biological process
          assignment entirely.
        resolution: >-
          Calcium flux and beta-arrestin recruitment assays on cells expressing
          bat and human CCR10 or CCR3, plus transwell chemotaxis of bat and host
          leukocytes toward recombinant bat CCL28.
        provenance:
          - reference_id: file:DESRO/K9IFY6/K9IFY6-deep-research-falcon.md
            supporting_text: >-
              no peer-reviewed study directly characterizing Desmodus rotundus
              K9IFY6 was found in the searched literature
suggested_questions:
  - question: >-
      Is the abundant salivary CCL28 of Desmodus rotundus acting on the bat's
      own mucosal immunity, or is it delivered to the host bite wound where it
      would modulate host leukocyte recruitment?
    experts:
      - Ivo M. B. Francischetti
      - José M. C. Ribeiro
  - question: >-
      Does the basic C-terminal extension of bat CCL28 have the direct
      antimicrobial activity described for the histatin-like tail of human
      CCL28, and is it under positive selection as vampire bat lysozyme is?
  - question: >-
      Does bat CCL28 engage CCR10, CCR3, or both, and does it cross-react with
      the receptors of the mammalian hosts on which vampire bats feed?
suggested_experiments:
  - hypothesis: >-
      Vampire bat CCL28 is a broad-spectrum antimicrobial protein, not only a
      chemoattractant.
    description: >-
      Express recombinant mature bat CCL28 and synthesise a peptide covering its
      basic C-terminal extension (approximately residues 88-132); measure
      killing of Candida albicans, Escherichia coli, and Staphylococcus aureus
      by radial diffusion and MIC assays at low ionic strength, alongside human
      CCL28 as a positive control and a C-terminally truncated bat construct as
      the structure-function test.
    experiment_type: Antimicrobial activity assay with truncation mutants
  - hypothesis: >-
      Bat CCL28 signals through CCR10 and/or CCR3 to recruit leukocytes.
    description: >-
      Assay recombinant bat CCL28 on HEK293 cells expressing bat or human CCR10
      and CCR3 using calcium mobilisation and beta-arrestin recruitment
      readouts, and confirm functional consequences with transwell chemotaxis of
      primary leukocytes.
    experiment_type: Receptor pharmacology and chemotaxis assay
  - hypothesis: >-
      CCL28 is transferred to the host during feeding rather than merely being
      stored in the gland.
    description: >-
      Use targeted parallel reaction monitoring mass spectrometry on saliva
      expectorated by feeding bats, and immunohistochemistry of principal versus
      accessory submaxillary gland sections, to establish that CCL28 reaches the
      bite site.
    experiment_type: Targeted proteomics and immunohistochemistry