LasB (elastase B, pseudolysin, EC 3.4.24.26) is the most abundant secreted protease of P. aeruginosa and the founding Pseudomonas member of the M4 thermolysin family of neutral zinc metalloendopeptidases. It is made as a 498-residue pre-pro-protein: the signal peptide is removed during export across the inner membrane, the 174-residue propeptide is cleaved in the periplasm (largely autocatalytically, at the 197/198 bond), and the 33 kDa mature enzyme is secreted across the outer membrane by the Xcp type II secretion system. The catalytic domain has the thermolysin fold with one catalytic Zn(2+) (His337, His341, Glu361) and one structural Ca(2+), and His420 as the proton donor; it is inhibited by phosphoramidon. Efficient production and processing of the mature enzyme require both zinc and calcium in the growth medium. Extracellularly it cleaves peptide bonds with a bulky hydrophobic residue in P1', degrading host elastin, collagen types III and IV, fibronectin, immunoglobulins and complement components, and it also matures other P. aeruginosa exoproteins, removing the propeptides of the chitin-binding protein CbpD and of the secreted aminopeptidase PaAP. It is a major virulence factor in acute and early chronic lung infection, and lasB mutants are also impaired in swarming and in biofilm formation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006508 proteolysis | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN002138658 (PTHR33794, "bacillolysin"), whose only seed is LasB itself. The term is the broadest possible correct claim for a family of M4 neutral metalloproteinases. Reason: LasB in its own With/From is the expected marker of experimental grounding on the target, not circularity. The 21 leaves are all M4 thermolysin-family proteases (Bacillus bacillolysins and neutral metalloproteinases, Listeria Mpl, Vibrio HA/protease, Leptospira thermolysins, Streptomyces neutral metalloproteinases), for which proteolysis is sound and conservative; the specific substrate preferences and the virulence context were correctly not propagated. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002138658 · PTN002138658 (PTHR33794) SUPPORTS TRANSFER 21 leaf IBAs in 10 taxa in the PAINT leaf file, every one a named or predicted M4 metalloprotease. The node term is an aspect-appropriate family-level claim; even the few leaves whose own activity is uncharacterised retain the HEXXH zinc-protease motif. UniProtKB:P14756 · lasB (self; sole IBD seed) SUPPORTS TRANSFER LasB is one of the best characterised bacterial metalloproteases (EC 3.4.24.26, MEROPS M04.005, 19 PDB entries), so a single seed is not weak support here. Supporting Evidence: file:PSEAE/lasB/lasB-uniprot.txt Belongs to the peptidase M4 family. |
| GO:0006508 proteolysis | IDA PMID:1597429 Efficient production and processing of elastase and LasA by ... | ACCEPT | Summary: PseudoCAP IDA and the sole seed of the IBD at PTN002138658. Olson and Ohman measured proteolytic and elastolytic activity of culture supernatants as a function of zinc and calcium, correlating it with the appearance of the mature 33 kDa elastase. Reason: The claim is the gene product's own activity, and it is correct beyond doubt for LasB (EC 3.4.24.26, thermolysin-like active site, 19 structures, phosphoramidon-inhibited). The specific reference is a supernatant activity assay rather than an assay on purified LasB, which makes it a weaker choice of evidence than PMID:1899664 or PMID:1744034, but the attribution to elastase is supported in the same experiment by the zinc-dependent recovery of activity from the 33 kDa species, and the curator read the full text. So the seed is sound and the IBD is justified. Supporting Evidence: PMID:1597429 High levels of proteolytic and elastolytic activity were exhibited by all strains when cultured in the presence of both zinc and calcium, and this was associated with the production of mature 33-kDa elastase and 21-kDa LasA. PMID:1597429 Proteolytic activity could be recovered from 33-kDa elastase produced in the presence of calcium by inclusion of zinc in the enzymatic assay. |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the M4 peptidase signatures, which LasB matches (IPR023612, IPR013856, IPR001570, PROSITE ZINC_PROTEASE). Reason: The signature assignment is correct and the term is the demonstrated process for this gene, so the row reinforces the IDA. Supporting Evidence: file:PSEAE/lasB/lasB-uniprot.txt DR InterPro; IPR023612; Peptidase_M4. |
| GO:0004222 metalloendopeptidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: The core molecular function: a zinc-dependent endopeptidase of the thermolysin type, with the catalytic Zn(2+) and the active-site residues resolved in the 1.5 A structure. Reason: Although the row is electronic, the claim is as specific as the evidence supports and is directly established: Zn(2+) ligands His337, His341 and Glu361 and the proton donor His420 are recorded from PDB 1EZM, the His420 (His223 of the mature chain) substitution abolishes proteolytic and elastolytic activity, and UniProt assigns EC 3.4.24.26. Supporting Evidence: PMID:1899664 Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. PMID:1744034 No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. file:PSEAE/lasB/lasB-uniprot.txt /ligand_note="catalytic" |
| GO:0004175 endopeptidase activity | IDA PMID:33510733 Pseudomonas aeruginosa Elastase Contributes to the Establish... | MODIFY | Summary: CACAO row from the murine chronic-colonisation study, which measured active LasB produced by clinical isolates and compared wild type with a lasB deletion. Reason: Correct but less specific than the evidence supports. LasB is a zinc-dependent endopeptidase, and the more specific descendant GO:0004222 metalloendopeptidase activity is already carried by this gene; the metal dependence is established structurally and by the inhibitor profile. Proposed replacements: metalloendopeptidase activity Supporting Evidence: PMID:33510733 The zinc metalloprotease elastase B (also called LasB or pseudolysin) is the most abundant protease in the P. |
| GO:0004175 endopeptidase activity | IDA PMID:30227935 Anti-quorum sensing and antibiofilm potential of Alternaria ... | MODIFY | Summary: CACAO row from a study of an Alternaria alternata extract that reduced quorum-sensing-dependent virulence factors, including LasB elastase activity in culture supernatants. Reason: Same claim as the other GO:0004175 row and refined the same way: the specific descendant GO:0004222 metalloendopeptidase activity is already carried by this gene and is what LasB is. The evidence in this particular paper is an indirect supernatant assay in a quorum-sensing inhibition screen, so it adds little, but the claim is true and must not be removed just because a better-supported row for the same term exists. Proposed replacements: metalloendopeptidase activity Supporting Evidence: PMID:30227935 significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase |
| GO:0005576 extracellular region | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping of the UniProt Secreted location, which rests on three experimental papers. Reason: LasB is the major protein of P. aeruginosa culture supernatants; the electronic row agrees with the three experimental rows for the same term. Supporting Evidence: PMID:1744034 The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. |
| GO:0005576 extracellular region | EXP PMID:1597429 Efficient production and processing of elastase and LasA by ... | ACCEPT | Summary: Mature 33 kDa elastase and its activity were detected in culture supernatants of PAO1, DG1 and FRD2. Reason: Direct detection of the protein in the extracellular medium; the location where the enzyme acts. Supporting Evidence: PMID:1597429 When grown under metal ion-deficient conditions, P. aeruginosa culture supernatants were found to exhibit a low level of elastolytic activity, which coincided with production of low levels of the 51-kDa proelastase and no detectable LasA. |
| GO:0005576 extracellular region | EXP PMID:3141383 Synthesis, processing, and transport of Pseudomonas aerugino... | ACCEPT | Summary: Kessler and Safrin followed synthesis, periplasmic processing and transport of elastase to the extracellular medium. Reason: Reinforces the other extracellular-region rows; the paper is the source of UniProt's Secreted and PTM statements. Supporting Evidence: file:PSEAE/lasB/lasB-uniprot.txt SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:1597429, file:PSEAE/lasB/lasB-uniprot.txt SUBCELLULAR LOCATION, INDUCTION, AND PROTEOLYTIC CLEAVAGE. |
| GO:0005576 extracellular region | EXP PMID:9642203 Secretion of elastinolytic enzymes and their propeptides by ... | ACCEPT | Summary: Braun et al. showed that both the mature enzyme and its propeptide are secreted, the propeptide then being degraded extracellularly. Reason: Direct demonstration of the extracellular location, and the correct way to record what this paper shows about LasB (as opposed to the two secretion-process rows below). Supporting Evidence: PMID:9642203 We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. |
| GO:0015628 protein secretion by the type II secretion system | IDA PMID:9642203 Secretion of elastinolytic enzymes and their propeptides by ... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IDA from Braun et al. 1998. LasB does cross the outer membrane through the Xcp T2SS, but as cargo. Reason: The biology is accepted (UniProt: secreted in an Xcp-dependent fashion), but a secreted substrate does none of the work of the secretion process; GO annotates the Xcp/Gsp machinery to this term, not its cargo. The fact belongs on the transporter side and, for LasB, in the extracellular-region rows above. Full text of PMID:9642203 was not available, so the precise experiment behind the row could not be checked and I do not REMOVE. The same pattern was recorded for the other Braun-et-al. cargo, PaAP (genes/PSEAE/lap). Supporting Evidence: file:PSEAE/lasB/lasB-uniprot.txt Note=Secreted PMID:9642203 We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. |
| GO:0043952 protein transport by the Sec complex | IDA PMID:9642203 Secretion of elastinolytic enzymes and their propeptides by ... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IDA from the same paper; what is shown for LasB is that its signal sequence is removed during transport across the inner membrane. Reason: Two problems, as for PaAP. First, LasB is a cargo and does not perform Sec-dependent translocation (SecYEG/SecA do). Second, signal-peptide cleavage shows inner-membrane export but does not by itself identify the Sec translocon rather than Tat. Full text unavailable, so whether the paper has Sec-specific evidence is unchecked; no REMOVE, and a probable PseudoCAP defect is recorded on the cargo point. Supporting Evidence: PMID:9642203 The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. |
| GO:0044010 single-species biofilm formation | IMP PMID:18245294 Swarming of Pseudomonas aeruginosa is a complex adaptation l... | KEEP AS NON CORE | Summary: Four independent lasB mutants formed about 50% less biofilm biomass in microtitre assays, and the defect was complemented by a chromosomally integrated lasB. Reason: The genetic requirement is solid and complemented, so this is not a spurious row, but the contribution is peripheral and almost certainly indirect: LasB is an extracellular protease, and the authors themselves attribute the phenotype to the same route that T2SS mutants take. Keep it as a non-core process rather than part of the gene's core function, and do not refine it further, since no mechanism is identified. Supporting Evidence: PMID:18245294 Moreover, all four tested lasB mutants also showed a strong impairment in biofilm formation with about 50% less biomass in the respective microtiter plate assays (Fig. PMID:18245294 The swarming as well as the biofilm phenotype could be completely restored by the expression of the chromosomally integrated lasB + gene (Fig. |
| GO:0071978 bacterial-type flagellum-dependent swarming motility | IMP PMID:18245294 Swarming of Pseudomonas aeruginosa is a complex adaptation l... | KEEP AS NON CORE | Summary: lasB was identified among swarming-induced genes and lasB mutants fail to swarm normally; the phenotype is complemented. Reason: Real and complemented requirement, but LasB performs none of the work of flagellum-dependent motility; the dependence is that of a secreted protease whose loss changes surface behaviour, as for T2SS mutants. Keep as non-core. I do not MARK_AS_OVER_ANNOTATED because the complementation makes the gene-specific requirement clear, and involved_in with IMP is the conventional way GO records such a requirement. Supporting Evidence: PMID:18245294 Elastase LasB and PvdQ are essential for normal swarming behavior. PMID:18245294 Our results are consistent with the secretion of LasB by the type II secretion system, since mutants with defects in this secretion apparatus also exhibit strong swarming and biofilm defects ( 41 ). |
| GO:0008270 zinc ion binding | IDA PMID:1899664 Three-dimensional structure of the elastase of Pseudomonas a... | NEW | Summary: One catalytic Zn(2+) per subunit, resolved in the 1.5 A structure with His337, His341 and Glu361 as ligands. Reason: Directly demonstrated metal binding, recorded by UniProt as catalytic binding sites, and the chemical basis of the core metalloendopeptidase activity. GOA has no metal-binding row for this gene. Supporting Evidence: PMID:1899664 Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. file:PSEAE/lasB/lasB-uniprot.txt Note=Binds 1 Zn(2+) ion per subunit. |
| GO:0005509 calcium ion binding | IDA PMID:1899664 Three-dimensional structure of the elastase of Pseudomonas a... | NEW | Summary: One structural Ca(2+) per subunit (ligands Asp333, Asp369, Glu372, Asp380, Asp382 in the precursor numbering), resolved in PDB 1EZM and the later inhibitor complexes. Reason: Directly demonstrated metal binding with mapped ligands, and physiologically meaningful: calcium in the growth medium is required for efficient production and for full activity of the mature enzyme. Supporting Evidence: file:PSEAE/lasB/lasB-uniprot.txt Note=Binds 1 Ca(2+) ion per subunit. PMID:1597429 Supplementing cultures with calcium stimulated the production of elastase but had no effect on LasA production. |
| GO:0016540 protein autoprocessing | IMP PMID:1744034 Substitution of active-site His-223 in Pseudomonas aeruginos... | NEW | Summary: Pro-elastase is matured to the 33 kDa enzyme autocatalytically: in E. coli the wild-type protein appears as the correctly cleaved mature form, whereas the active-site His substitutions accumulate the 51 kDa pro-enzyme, which exogenous purified elastase can then process in trans. Reason: Here the gene product genuinely performs the step, which is the condition for a process term: the bond cleaved is its own (recorded by UniProt as SITE 197..198, "Cleavage; by autolysis") and the catalytic residue needed is its own active site. The claim is independent of the general proteolysis row and is absent from GOA. Supporting Evidence: PMID:1744034 The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. PMID:1744034 The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase. |
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Download this section (compressed HTML)Q: Is the swarming and biofilm requirement for lasB a consequence of any identified LasB substrate (for example a matrix or surface protein), or of the general loss of extracellular proteolysis?
Q: Should GO record cargo relationships for type II secretion substrates on the Xcp machinery (has_input) so that rows like GO:0015628 on LasB and PaAP can be retired without losing the information?
Experiment: Compare a lasB deletion with a catalytically dead point mutant (His420 substitution expressed from the chromosome) for swarming and biofilm formation, to separate loss of protease activity from loss of the (secreted) protein itself.
Experiment: Define the extracellular substrate repertoire of mature LasB by N-terminomics on wild-type versus delta-lasB supernatants and infected airway surface liquid, which would let the generic proteolysis annotation be replaced by substrate-specific statements.
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