Secreted zinc-dependent alkaline metalloendopeptidase (serralysin / aeruginolysin) of the peptidase M10B (RTX-metalloprotease) family. The mature enzyme is a two-domain protein comprising an N-terminal catalytic metallopeptidase domain bearing a single catalytic zinc ion (coordinated by an HEXXH-type motif) and a C-terminal parallel beta-roll domain built from hemolysin-type (RTX) calcium-binding repeats; calcium binding drives folding and activation and stabilises the fold. It lacks a cleavable Sec signal peptide and is exported across both membranes by a dedicated type I secretion system (the AprDEF transporter), acting in the extracellular environment. Catalytically it preferentially cleaves peptide bonds N-terminal to hydrophobic residues and degrades a broad, immune-biased set of substrates. In the host it cleaves monomeric flagellin, complement components (notably C2, plus C1q, C1s, C3, C5a), cytokines, and the epithelial sodium channel, contributing to innate-immune evasion and modulation of host epithelial physiology.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004222 metalloendopeptidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: Core molecular function. AprA is a zinc-dependent metalloendopeptidase (serralysin, EC 3.4.24.40) of the peptidase M10B family, with a catalytic zinc ion coordinated by an HEXXH-type motif (His185, His189, His195; active-site Glu186) and an endopeptidase mechanism cleaving internal peptide bonds. Reason: Strongly supported by the solved crystal structures, the assigned EC number, MEROPS family classification (M10.056), and the catalytic zinc-binding active site. This is the central, defining molecular function of the protein. Supporting Evidence: UniProtKB:Q03023 RecName: Full=Serralysin; EC=3.4.24.40. Active site His185/His189/His195 coordinate catalytic Zn(2+); Belongs to the peptidase M10B family. |
| GO:0005509 calcium ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: Structural calcium binding. The C-terminal beta-roll is formed by hemolysin-type (RTX) calcium-binding repeats and the protein binds 8 Ca(2+) ions per subunit; the calcium-loaded beta-roll is required for folding, activation, and type I secretion. Reason: Directly demonstrated by X-ray crystallography (calcium-binding parallel beta-roll motif) and recorded as a UniProt cofactor (binds 8 Ca(2+) per subunit). This is a genuine, structurally essential function that contributes to rather than constitutes the core catalytic function. Supporting Evidence: UniProtKB:Q03023 COFACTOR Name=Ca(2+); Note=Binds 8 Ca(2+) ions per subunit. Hemolysin-type calcium-binding repeats form a parallel beta-roll. |
| GO:0005576 extracellular region | IEA GO_REF:0000120 | ACCEPT | Summary: Correct localization. AprA is a secreted protein exported by a dedicated type I secretion system (AprDEF) and acts extracellularly on host and microbial substrates. Reason: UniProt records the subcellular location as "Secreted", and all characterised activities (flagellin/complement/ENaC cleavage) occur extracellularly. The more precise child term GO:0005615 (extracellular space) would also be appropriate, but extracellular region is correct. Supporting Evidence: UniProtKB:Q03023 SUBCELLULAR LOCATION: Secreted. |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: Core biological process. AprA is a proteolytic enzyme; proteolysis is the process directly carried out by its metalloendopeptidase activity on host and microbial protein substrates. Reason: Proteolysis is the fundamental process executed by this protease and is well supported by biochemical characterisation of its catalytic activity and substrate specificity. Supporting Evidence: UniProtKB:Q03023 CATALYTIC ACTIVITY: Preferential cleavage of bonds with hydrophobic residues in P1'.; EC=3.4.24.40 |
| GO:0008233 peptidase activity | IEA GO_REF:0000117 | ACCEPT | Summary: Correct but uninformatively broad. Peptidase activity is a high-level parent of the specific metalloendopeptidase activity (GO:0004222) already annotated for this enzyme. Reason: The annotation is not wrong, but GO:0004222 (metalloendopeptidase activity) already captures this function at the appropriate level of specificity. Retained as a correct broad parent; it is not the core (most informative) molecular-function term. Supporting Evidence: UniProtKB:Q03023 RecName: Full=Serralysin; EC=3.4.24.40; Belongs to the peptidase M10B family. |
| GO:0008237 metallopeptidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: Correct but broad. Metallopeptidase activity is a parent of the specific metalloendopeptidase activity (GO:0004222) already annotated. Reason: AprA is genuinely a zinc metallopeptidase, so the term is accurate, but it is subsumed by the more informative GO:0004222. Retained as a correct broad parent rather than the core molecular-function term. Supporting Evidence: UniProtKB:Q03023 Binds 1 zinc ion per subunit; catalytic Zn(2+) coordinated by His185/His189/His195; peptidase M10B (serralysin) family. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: Catalytic zinc binding. AprA binds one catalytic Zn(2+) ion in its active site, coordinated by the HEXXH-derived histidines (His185, His189, His195). Reason: Directly supported by crystallography and UniProt cofactor/binding-site annotations; the catalytic zinc is essential for the metalloendopeptidase mechanism. Supporting Evidence: UniProtKB:Q03023 COFACTOR Name=Zn(2+); Note=Binds 1 zinc ion per subunit. BINDING 185/189/195 ligand=Zn(2+) ligand_note=catalytic. |
| GO:0031012 extracellular matrix | IEA GO_REF:0000002 | REMOVE | Summary: Incorrect localization from an over-propagated electronic inference. This InterPro-to-GO mapping (peptidase M10 / matrixin signature) places AprA in the extracellular matrix, but AprA is a freely secreted bacterial protease, not a structural component of or resident in a host extracellular matrix. Reason: Extracellular matrix (GO:0031012) is part_of extracellular region, so this is a more specific localization claim than GO:0005576, not a looser one, and the specific claim is unsupported. The annotation derives from InterPro IPR001818 (peptidase M10), a signature shared with eukaryotic matrix metalloproteinases (matrixins/MMPs) that genuinely reside in and act on the ECM; for a bacterial secreted serralysin this is an over-propagated IEA. AprA is secreted via the type I secretion system (AprDEF) and acts on soluble host substrates (complement C2/C1q/C1s, cytokines, monomeric flagellin); there is no evidence it localizes to or is a component of the extracellular matrix. Its correct localization is already captured by GO:0005576 (extracellular region). This is the demonstrably-wrong, argue-against-on-biological-grounds case for which REMOVE (rather than MARK_AS_OVER_ANNOTATED) is appropriate. Supporting Evidence: UniProtKB:Q03023 SUBCELLULAR LOCATION: Secreted. |
| GO:0141141 symbiont-mediated evasion of recognition by host pattern recognition receptor | EXP PMID:22131330 Pseudomonas aeruginosa alkaline protease blocks complement a... | ACCEPT | Summary: Well-supported virulence function. AprA enables P. aeruginosa to evade host pattern-recognition-receptor detection. Its best-characterised mechanism is degradation of free monomeric flagellin, the ligand for the PRRs TLR5 (mammals) and FLS2 (plants), while flagellar (polymeric) flagellin and the receptors themselves are spared, preserving motility. Reason: The term is genuinely correct for AprA and experimentally established. The most apt primary reference is the flagellin/TLR5/FLS2 study (PMID:21901099), which directly demonstrates evasion of PRR recognition; the cited complement paper (PMID:22131330) also bears on innate-immune evasion (lectin-pathway initiators MBL/ficolins are soluble PRRs). Added the flagellin reference as supporting evidence. Supporting Evidence: PMID:21901099 monomeric flagellin, while polymeric flagellin (involved in bacterial motility) PMID:21901099 and TLR5 itself resist degradation. PMID:21901099 This was due to decreased activation of the receptor FLS2 and |
| GO:0001869 negative regulation of complement activation, lectin pathway | IDA PMID:22131330 Pseudomonas aeruginosa alkaline protease blocks complement a... | ACCEPT | Summary: AprA blocks lectin-pathway complement activation. It degrades complement C1s and C2 and, by cleaving C2, prevents formation of the C4b2a C3 convertase and C3b deposition via the lectin (and classical) pathway, while the alternative pathway is unaffected. Reason: Directly demonstrated experimentally (IDA). Serum degradation and repletion assays show the inhibitory mechanism is cleavage of C2, blocking lectin-pathway C3b deposition. This is a specific, downstream (non-core) virulence consequence of the protease activity. Supporting Evidence: PMID:22131330 deposition via the classical and lectin pathways, whereas the alternative PMID:22131330 of action for complement inhibition is cleavage of C2. In summary, we showed |
| GO:0008233 peptidase activity | IDA PMID:22859302 Activation of the epithelial sodium channel (ENaC) by the al... | ACCEPT | Summary: Experimental confirmation of proteolytic activity. Purified AprA (AP) was shown to be folded and proteolytically active and to activate ENaC by cleavage. Reason: Direct experimental evidence (IDA) of peptidase activity for purified AprA. The activity is more specifically a zinc metalloendopeptidase activity (GO:0004222), but the experimental peptidase annotation is correct as recorded. Supporting Evidence: PMID:22859302 further characterize this AP-induced ENaC activation, AP was purified, and its PMID:22859302 folding, activity, and ability to activate ENaC were assessed. |
| GO:0010765 positive regulation of sodium ion transport | IDA PMID:22859302 Activation of the epithelial sodium channel (ENaC) by the al... | ACCEPT | Summary: AprA proteolytically activates the epithelial sodium channel (ENaC). Apically applied AprA increased basal ENaC current consistent with sustained channel activation in human bronchial epithelia (CF and non-CF), with activation mapped to the channel's gamma-subunit. Reason: Directly demonstrated experimentally (IDA) by short-circuit current measurements on polarized epithelial monolayers. A specific, downstream (non-core) effect of the protease on host ion transport, relevant to airway pathophysiology in cystic fibrosis. Supporting Evidence: PMID:22859302 trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To PMID:22859302 ENaC in polarized monolayers indicated that AP activated ENaC in immortalized |
| GO:0045959 negative regulation of complement activation, classical pathway | IDA PMID:22131330 Pseudomonas aeruginosa alkaline protease blocks complement a... | ACCEPT | Summary: AprA blocks classical-pathway complement activation. By degrading C1s and cleaving C2 it prevents C3b deposition via the classical (and lectin) pathway, inhibiting opsonization, C5a formation, and complement-dependent neutrophil phagocytosis/killing. Reason: Directly demonstrated experimentally (IDA); the C2-cleavage mechanism blocks classical-pathway complement activation while sparing the alternative pathway. A specific, downstream (non-core) virulence consequence of the protease activity. Supporting Evidence: PMID:22131330 both human C1s and C2. However, repletion assays demonstrated that the mechanism PMID:22131330 complement activation via cleavage of C2. file:PSEAE/aprA/aprA-deep-research-falcon.md AprA was identified as the first bacterial protease demonstrated to cleave C2 |
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