nas-8

id: D2KBH9
gene_symbol: nas-8
product_type: PROTEIN
status: INITIALIZED
taxon:
  id: NCBITaxon:34508
  label: Steinernema carpocapsae
description: >-
  nas-8 (Sc-AST) is a secreted zinc-dependent astacin metalloendopeptidase
  (EC 3.4.24.21; MEROPS family M12A) from the entomopathogenic nematode
  Steinernema carpocapsae. It is synthesized as a precursor comprising an
  N-terminal signal peptide and a propeptide (zymogen) that are removed to yield
  the mature, catalytically active protease, which contains a peptidase M12A
  (astacin) catalytic domain and a C-terminal ShK-toxin (ShKT) domain. Catalysis
  requires a single active-site zinc ion coordinated by the conserved HExxH motif
  and a downstream histidine, and a methionine-turn; activity is abolished by the
  metal chelators EDTA, EGTA and 1,10-phenanthroline. The mature enzyme hydrolyzes
  protein and peptide substrates (demonstrated against gelatin and azocasein),
  cleaving peptide bonds preferentially with Ala in the P1' position. The protein
  is secreted into the extracellular environment, is expressed in the infective
  and parasitic juvenile stages but not in eggs or adults, and is strongly induced
  by contact with insect host tissues, consistent with a role in degrading host
  proteins during invasion of the insect haemocoel and the parasitic process.
existing_annotations:
- term:
    id: GO:0004222
    label: metalloendopeptidase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Electronic annotation (from EC 3.4.24.21, InterPro M12A astacin domain and
      PANTHER) asserting metalloendopeptidase activity. This is the core
      molecular function of the protein and is independently supported by direct
      experimental characterization of the recombinant enzyme (PMID:20670659),
      which demonstrated zinc-dependent, chelator-inhibited endopeptidase activity
      against gelatin and azocasein. Accept and retain as a core function.
    action: ACCEPT
- term:
    id: GO:0005576
    label: extracellular region
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: >-
      Subcellular location mapping from UniProt (SL-0243, Secreted). The protein
      carries a cleaved N-terminal signal peptide and is described as a secreted
      protease that acts on host tissues during invasion, so localization to the
      extracellular region is well supported. Accept.
    action: ACCEPT
- term:
    id: GO:0006508
    label: proteolysis
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: involved_in
  review:
    summary: >-
      InterPro2GO biological-process annotation for proteolysis. This correctly
      reflects the enzyme's activity (peptide-bond hydrolysis demonstrated against
      gelatin and azocasein) and is the direct biological-process consequence of
      its endopeptidase function. Accurate but generic; retained as a non-core
      process because the more informative statement of function is captured by the
      molecular-function term.
    action: KEEP_AS_NON_CORE
- term:
    id: GO:0008237
    label: metallopeptidase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      InterPro2GO annotation for metallopeptidase activity. This is correct but is
      a less specific parent of GO:0004222 (metalloendopeptidase activity), which
      is the experimentally supported and more informative term for this astacin
      endopeptidase. Marking as over-annotated in favor of the more specific child
      term that is already present.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0008270
    label: zinc ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      InterPro2GO annotation for zinc ion binding. Consistent with the astacin
      mechanism: the active site coordinates one catalytic Zn(2+) ion via the
      conserved HExxHxxGxxH zinc-binding motif, and activity is abolished by metal
      chelators (EDTA, EGTA, o-phenanthroline; PMID:20670659). Accept as a
      supporting molecular function underpinning catalysis.
    action: ACCEPT
- term:
    id: GO:0004222
    label: metalloendopeptidase activity
  evidence_type: EXP
  original_reference_id: PMID:20670659
  qualifier: enables
  review:
    summary: >-
      Experimental (EXP) annotation from PMID:20670659. Recombinant Sc-AST was
      purified and shown to hydrolyze gelatin and azocasein, with activity
      inhibited by the divalent-metal chelators EDTA, EGTA and o-phenanthroline,
      and kinetic parameters (Km, Vmax, kcat) determined. This directly establishes
      zinc-dependent metalloendopeptidase activity and is the core function of the
      gene. Accept and retain as core.
    action: ACCEPT
core_functions:
- description: >-
    Secreted zinc-dependent astacin (M12A) metalloendopeptidase that hydrolyzes
    protein and peptide substrates in the extracellular environment, contributing
    to degradation of host insect proteins during nematode invasion and parasitism.
  molecular_function:
    id: GO:0004222
    label: metalloendopeptidase activity
  supported_by:
  - reference_id: PMID:20670659
    supporting_text: >-
      showed activities against gelatin and azocasein substrates and was inhibited
      by divalent metal-chelating agents
  locations:
  - id: GO:0005576
    label: extracellular region
- description: >-
    Coordination of a single catalytic zinc ion via the conserved astacin
    zinc-binding motif, required for peptide-bond hydrolysis.
  molecular_function:
    id: GO:0008270
    label: zinc ion binding
  supported_by:
  - reference_id: PMID:20670659
    supporting_text: >-
      a zinc-binding motif
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
    vocabulary mapping, accompanied by conservative changes to GO terms applied by
    UniProt
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:20670659
  title: Cloning, characterisation and heterologous expression of an astacin metalloprotease,
    Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Primary characterization paper for Sc-AST (nas-8). Cloned the cDNA,
      identified the astacin family signatures (astacin domain, zinc-binding motif,
      methionine turn, C-terminal ShK domain), expressed recombinant protein, and
      demonstrated zinc-dependent, chelator-inhibited endopeptidase activity
      against gelatin and azocasein with measured kinetics. Also showed parasitic-
      stage-specific expression and induction by insect tissues. Directly supports
      the EC, molecular-function and proteolysis annotations.