Leishmanolysin (GP63, major surface protease/MSP, EC 3.4.24.36) is the predominant surface glycoprotein of Leishmania promastigotes. It is a zinc-dependent metalloendopeptidase of the peptidase M8 family, built on a metzincin-like catalytic domain with an HEXXH motif in which Glu-265 is the catalytic base and the two histidines coordinate a single zinc ion. The 602-residue precursor carries a signal peptide and an N-terminal activation propeptide, and the mature chain is attached to the outer leaflet of the parasite plasma membrane by a glycosylphosphatidylinositol anchor added at Asn-577, so the enzyme is catalytically active in situ on the cell surface. A soluble pool is released from the surface and GP63 is also exported in parasite exosomes. In Leishmania major the protein is encoded by a tandem gene family whose members are expressed in a stage-specific manner, with the highly expressed promastigote forms distinct from the metacyclic/amastigote form. GP63 is a virulence factor acting at the parasite-host interface. On the parasite surface it accelerates conversion of deposited complement C3b to iC3b, limiting assembly of the terminal complement components and conferring resistance to complement-mediated lysis while leaving the parasite opsonised for uptake through complement receptors; it degrades host antimicrobial peptides and extracellular matrix proteins such as collagen IV and fibronectin, promoting migration through tissue. After internalisation into macrophages it reaches cytoplasmic substrates and cleaves host protein tyrosine phosphatases SHP-1, PTP1B and TCPTP, activating them and thereby damping JAK/STAT and MAPK signalling, cleaves mTOR to inhibit mTORC1 and repress cap-dependent host translation, and degrades synaptotagmin XI, which de-represses macrophage TNF and IL-6 secretion.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004222 metalloendopeptidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro peptidase M8 (IPR001577) to GO mapping. Leishmanolysin is the type member of family M8 and its zinc endopeptidase activity is directly established by enzymology (EC 3.4.24.36) and by the 1.86 A crystal structure, which shows a metzincin-like catalytic domain with a bound zinc ion. Reason: This is the core molecular function of the protein and the electronic family-based assignment is exactly right for P08148, the protein from which the family was defined. Mutating the HEXXH glutamate abolishes catalysis, confirming the assignment is not a fold-only inference. Supporting Evidence: PMID:9739094 Leishmanolysin is a membrane-bound zinc proteinase, active in situ. PMID:9739094 a member of the metzincin class of zinc proteinases. PMID:8626468 the following. 1) Glu-265 in the HEXXH motif is indispensable for the catalytic activity of gp63. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | MODIFY | Summary: UniProt subcellular location mapping from the cell membrane GPI-anchor annotation. GP63 is GPI-anchored at Asn-577 on the outer face of the promastigote plasma membrane and is catalytically active in situ, facing the extracellular space. Reason: The location is correct but less specific than the evidence supports. The structure of the GPI anchor was solved directly for this protein, and mutation of the anchor attachment site releases the protein from the cell, so the supported term is the external-side-of-plasma-membrane descendant rather than the generic plasma membrane parent. The anchored-component terms that would state the GPI attachment directly are obsolete in the current ontology. Proposed replacements: external side of plasma membrane Supporting Evidence: PMID:2145267 contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. PMID:8626468 Asn-577 causes release of all mutant products, indicative of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. PMID:9739094 Leishmanolysin is a membrane-bound zinc proteinase, active in situ. |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro M8-family mapping of the generic proteolysis process term. GP63 is an active endopeptidase whose documented biology is the cleavage of host proteins such as complement C3b, antimicrobial peptides, extracellular matrix proteins, PTPs, mTOR and synaptotagmin XI. Reason: Proteolysis is the process the gene product carries out directly and it is the common denominator of all of its characterised activities, so it is retained as core. The more specific host-directed process terms in this review sit in a different branch (symbiotic interaction) and do not entail this term. Supporting Evidence: PMID:12540585 This correlates with gp63 degradation of extracellular matrix components, such as collagen type IV and fibronectin, and suggests an important role for gp63 in the pathogenesis of leishmaniasis. |
| GO:0006508 proteolysis | IMP PMID:25063865 Leishmania promastigotes induce cytokine secretion in macrop... | ACCEPT | Summary: Arango Duque et al. showed that the host protein synaptotagmin XI is directly degraded by GP63 during macrophage infection, using GP63-deficient parasites, which is direct in-cell evidence of proteolysis by this protein. Reason: Same claim as the InterPro-derived proteolysis row, independently supported by mutant-based experimental evidence; actions on the two rows are therefore the same. Supporting Evidence: PMID:25063865 Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. |
| GO:0007155 cell adhesion | IEA GO_REF:0000002 | MODIFY | Summary: Generic cell adhesion term transferred electronically from the peptidase M8 InterPro signature. The adhesion biology actually documented for GP63 is attachment of the parasite to host macrophages, both through the iC3b coat that GP63 generates, which engages Mac-1/CR3, and through direct interaction with macrophage receptors. Reason: GO:0007155 is a broad term whose common usage covers adhesion of cells within a multicellular organism; for a parasite surface protein that mediates attachment to the host cell, the host-interaction branch carries the specific and informative term. Replacing it with adhesion of symbiont to host cell keeps the claim and states which cells adhere to what. Proposed replacements: adhesion of symbiont to host cell Supporting Evidence: PMID:7673725 Organisms expressing wild-type gp63 more rapidly converted C3b on their surface to a form that exhibited the neoantigen of iC3b and interacted avidly with cells expressing Mac-1, the receptor for iC3b. PMID:11849703 Leishmanolysin, the Leishmania surface metalloproteinase of 63 kDa (GP63) has been described as a parasite virulence factor and is involved in the direct interaction of promastigotes and host macrophage receptors and interaction with the complement cascade. |
| GO:0008237 metallopeptidase activity | IMP PMID:25063865 Leishmania promastigotes induce cytokine secretion in macrop... | MODIFY | Summary: Mutant-based evidence that GP63 is an active zinc metalloprotease, from degradation of host synaptotagmin XI by GP63-expressing but not GP63-deficient Leishmania. Reason: The claim is correct but the term is one level more general than the evidence and the enzymology support. GP63 is an endopeptidase (EC 3.4.24.36) with an internal cleavage specificity, and the gene already carries the descendant metalloendopeptidase activity term in this review, so the specific child is the right assertion. Proposed replacements: metalloendopeptidase activity Supporting Evidence: PMID:25063865 Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. PMID:9739094 a member of the metzincin class of zinc proteinases. |
| GO:0016020 membrane | IEA GO_REF:0000002 | TOO GENERAL | Summary: Generic membrane term from the InterPro M8 signature. GP63 is a GPI-anchored plasma membrane protein, a claim asserted at higher resolution elsewhere in this review. Reason: True but uninformative. The plasma membrane assertion in this review, refined to the external side of the plasma membrane, already entails membrane. Supporting Evidence: PMID:2145267 contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. |
| GO:0039514 symbiont-mediated suppression of host JAK-STAT cascade | EXP PMID:22919663 Impact of Leishmania metalloprotease GP63 on macrophage sign... | ACCEPT | Summary: GP63 cleaves the host protein tyrosine phosphatases SHP-1, PTP1B and TCPTP after entering the macrophage through lipid rafts. Cleavage activates the phosphatases, which dephosphorylate JAK and downstream components, attenuating IFN-gamma-driven JAK/STAT signalling and the microbicidal responses it controls. Reason: The suppression is executed by GP63 itself, through its own proteolytic activity on the phosphatases that switch off the cascade, and the mechanism was established with GP63-null and GP63-rescued L. major. The term states the direction of the effect and the pathway correctly for a symbiont protein acting on the host. Supporting Evidence: PMID:22919663 GP63 is responsible for the activation of protein tyrosine phosphatases (PTPs; SHP-1, PTP1B, and TCPTP) that lead to the alteration of JAK, MAP, and IRAK-1 kinase pathways. PMID:19797268 involves the proteolytic activity of the Leishmania surface protease GP63. PMID:19797268 lipid raft-dependent mechanism, resulting in PTP cleavage and stimulation of phosphatase activity. |
| GO:0052028 symbiont-mediated activation of host signal transduction pathway | IMP PMID:25063865 Leishmania promastigotes induce cytokine secretion in macrop... | KEEP AS NON CORE | Summary: GP63-dependent degradation of host synaptotagmin XI removes a brake on macrophage vesicular secretion, so infected cells release TNF and IL-6, and mice injected with GP63-expressing parasites recruit more neutrophils and inflammatory monocytes. Reason: The claim is supported by GP63-deficient parasites and by siRNA epistasis against the substrate, and GP63 performs the cleavage itself. The term is broad, but GO:0052028 has no descendant covering symbiont-driven induction of host cytokine secretion, so it is as precise as the ontology allows; it is a downstream consequence of the protease activity rather than a core function. Supporting Evidence: PMID:25063865 Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. PMID:25063865 Infected macrophages were found to release TNF and IL-6 in a GP63-dependent manner. |
| GO:0052032 symbiont-mediated perturbation of host inflammatory response | IMP PMID:25063865 Leishmania promastigotes induce cytokine secretion in macrop... | ACCEPT | Summary: By degrading synaptotagmin XI, GP63 increases proinflammatory cytokine release from infected macrophages and augments the influx of neutrophils and inflammatory monocytes to the inoculation site, cell types that favour establishment of infection. Reason: Correctly uses the neutral perturbation term rather than the suppression child GO:0052036, because the measured effect of GP63 here is an increase in the inflammatory output, not its inhibition. The effect is traced to a protein that GP63 cleaves directly, so GP63 performs part of the work. Supporting Evidence: PMID:25063865 proinflammatory cytokine release and increases infiltration of inflammatory phagocytes. PMID:25063865 Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. |
| GO:0141115 symbiont-mediated suppression of host complement activation by inactivation of complement proteins | EXP PMID:22442494 Secreted virulence factors and immune evasion in visceral le... | ACCEPT | Summary: Review of secreted Leishmania virulence factors citing GP63 as the surface protease that cleaves complement C3 products and protects the parasite from complement-mediated lysis. The underlying experimental work is in the primary literature, where GP63 accelerates C3b to iC3b conversion and GP63-deficient L. major is complement-sensitive. Reason: The claim is correct and well supported. GP63 itself cleaves surface-deposited C3b, converting it to the inactive iC3b fragment so that the terminal complement components are not assembled. The protease, not the parasite in general, performs the inactivating step, which is exactly what the term describes. Supporting Evidence: PMID:22919663 Leishmania overexpressing proteolytically active GP63 was able to increase the conversion of C3b into C3bi and to reduce the fixation of terminal complement components to Leishmania, consequently increasing their resistance to complement-mediated lysis comparatively to parasite overexpressing inactive GP63 (Brittingham et al., 1995). PMID:9489664 clearly involved in conferring resistance to complement-mediated lysis. The L. major GP63 1-6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. |
| GO:0141115 symbiont-mediated suppression of host complement activation by inactivation of complement proteins | EXP PMID:7673725 Role of the Leishmania surface protease gp63 in complement f... | ACCEPT | Summary: Brittingham et al. compared Leishmania expressing wild-type GP63, a proteolytically inactive mutant, or no GP63. Only parasites with active GP63 converted surface C3b rapidly to iC3b, fixed little of the terminal complement components and resisted lysis; complement fixation itself did not require GP63 proteolysis. Reason: This is the primary experimental basis for the term and it is used correctly. The proteolysis-dependent effect is inactivation of a deposited complement protein and the consequent failure of the lytic pathway, while the opsonic C3b deposition that GP63 does not require proteolysis for is a separate, adhesion-related claim handled by the symbiont-to-host adhesion term in this review. Same term and same claim as the other GO:0141115 row, so the actions agree. Supporting Evidence: PMID:7673725 Organisms expressing wild-type gp63 more rapidly converted C3b on their surface to a form that exhibited the neoantigen of iC3b and interacted avidly with cells expressing Mac-1, the receptor for iC3b. PMID:7673725 However, organisms expressing wild-type gp63 on their surface fixed only small amounts of the terminal complement components and were dramatically more resistant to lysis by complement than were those lacking functional gp63. PMID:11849703 promastigotes recovered from sand flies or from culture showed an increase in sensitivity to complement-mediated lysis and a delay in lesion formation in BALB/c animals. |
| GO:0008270 zinc ion binding | IDA PMID:9739094 The crystal structure of the Leishmania major surface protei... | NEW | Summary: The 1.86 A crystal structure of L. major leishmanolysin was solved with a bound zinc ion in a metzincin-type active site. UniProt records three explicit BINDING features for the catalytic Zn(2+) in P08148 itself, at His-264, His-268 and His-334, each evidenced to that structure (PubMed:9739094). Substituting either histidine of the HEXXH motif destabilises the protein in vivo, consistent with a structural as well as catalytic role for zinc binding. Reason: Zinc coordination is annotated residue-by-residue on this very protein, so the claim rests on observed ligand positions rather than on membership of the metzincin class, and it is not entailed by the metalloendopeptidase activity term, which is about the hydrolysis rather than the metal. Proposed conservatively as a molecular function the protein itself performs. Supporting Evidence: file:LEIMA/gp63/gp63-uniprot.txt BINDING 264 /ligand="Zn(2+)" /ligand_id="ChEBI:CHEBI:29105" /ligand_note="catalytic" file:LEIMA/gp63/gp63-uniprot.txt BINDING 268 /ligand="Zn(2+)" /ligand_id="ChEBI:CHEBI:29105" /ligand_note="catalytic" file:LEIMA/gp63/gp63-uniprot.txt BINDING 334 /ligand="Zn(2+)" /ligand_id="ChEBI:CHEBI:29105" /ligand_note="catalytic" |
| GO:0039604 symbiont-mediated suppression of host translation | IMP PMID:21501832 Leishmania repression of host translation through mTOR cleav... | NEW | Summary: Jaramillo et al. showed that GP63 cleaves host mTOR in infected macrophages, inhibiting mTORC1 and activating the translational repressor 4E-BP1, which represses cap-dependent host translation and promotes parasite proliferation. GP63-deficient parasites do not produce the effect. Reason: GP63 performs the step itself, by proteolysing the kinase that controls the host translational repressor, so this is participation and not merely a downstream consequence of infection. The effect is on host translation specifically and is not covered by the existing JAK-STAT, inflammatory-response or signal-transduction terms. Supporting Evidence: PMID:21501832 we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. PMID:21501832 mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. |
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Download this section (compressed HTML)Q: Which of the seven L. major gp63/msp genes contributes each documented activity? P08148 is one member of a stage-regulated tandem array, and annotations derived from experiments on parasites expressing the whole family cannot by themselves assign a function to a single paralog.
Q: Is there a suitable GO term for a symbiont protease that degrades host antimicrobial peptides? Leishmanolysin-null parasites are hypersensitive to defensins, magainins and cathelicidins because wild-type parasites degrade the peptides, but no existing child of the symbiont-mediated defence-suppression branch captures this.
Q: Likewise, should degradation of host extracellular matrix by a symbiont protease (collagen IV, fibronectin) be annotatable, given that it promotes parasite migration through tissue?
Q: How does surface-anchored, extracellularly oriented GP63 reach cytoplasmic host substrates such as PTP1B, mTOR and synaptotagmin XI? Lipid-raft-dependent entry is reported, but the translocation mechanism and the form of the enzyme that crosses the membrane are not established.
Q: Does GP63 have endogenous parasite substrates, or is its substrate repertoire entirely host-derived? No intra-parasite substrate has been reported, yet the protein is the most abundant surface protein of the promastigote.
Experiment: Reconstitute single gp63 paralogs in the L. major whole-locus (gp63 1-7) null background and assay each for C3b conversion, PTP cleavage, mTOR cleavage and antimicrobial peptide degradation side by side.
Hypothesis: The documented activities are not evenly distributed across the paralog family; the stage-specific metacyclic/amastigote form carries the activities relevant to intracellular survival.
Experiment: Use an active-site-directed, cell-permeable zinc protease probe together with proximity labelling in infected macrophages to identify GP63 substrates and the host compartments it occupies over the course of infection.
Hypothesis: GP63 accesses a defined set of cytoplasmic substrates from a lipid-raft entry route rather than acting promiscuously throughout the host cell.
Experiment: Compare catalytically dead (E265D) and GPI-anchorless (N577 substituted) GP63 in the null background for each host-directed phenotype.
Hypothesis: Surface anchoring is required for complement evasion and adhesion, whereas the released enzyme accounts for extracellular matrix degradation and for delivery to intracellular host substrates.
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