nas-8

Gene 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 Review

GO Term	Evidence	Action	Reason
GO:0004222 metalloendopeptidase activity	IEA GO_REF:0000120	ACCEPT	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.
GO:0005576 extracellular region	IEA GO_REF:0000044	ACCEPT	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.
GO:0006508 proteolysis	IEA GO_REF:0000002	KEEP AS NON CORE	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.
GO:0008237 metallopeptidase activity	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	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.
GO:0008270 zinc ion binding	IEA GO_REF:0000002	ACCEPT	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.
GO:0004222 metalloendopeptidase activity	EXP PMID:20670659 Cloning, characterisation and heterologous expression of an ...	ACCEPT	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.

Core Functions

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:

metalloendopeptidase activity

Cellular Locations:

extracellular region

Supporting Evidence:

PMID:20670659
showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents

Coordination of a single catalytic zinc ion via the conserved astacin zinc-binding motif, required for peptide-bond hydrolysis.