MIC2 is the Toxoplasma gondii member of the thrombospondin-related anonymous protein (TRAP) family of apicomplexan adhesins. It is a type I transmembrane protein with an integrin-like von Willebrand factor A domain followed by six thrombospondin type-1 repeats, and a short acidic cytoplasmic tail ending in a conserved penultimate tryptophan that binds aldolase and so couples the adhesin to the parasite actin-myosin motor. MIC2 associates in the secretory pathway with its escort partner M2AP and is stored in the apical micronemes. On contact with a host cell, micronemes discharge MIC2-M2AP onto the apical parasite surface, where the ectodomain engages sulfated glycosaminoglycans, extracellular matrix and host ICAM-1; motor-driven rearward translocation of these contacts provides traction for helical gliding and the onset of invasion, after which the posterior surface rhomboid protease ROM5 sheds the complex by intramembrane cleavage and the ectodomain is released into the medium. Loss-of-function studies show MIC2 is a major but non-essential attachment factor: depletion impairs substrate and host-cell attachment, helical gliding, invasion efficiency and timely egress, while knockout parasites remain viable and lethal to mice.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic mapping of the UniProt ARBA "Secreted" subcellular location. MIC2 is a transmembrane microneme adhesin displayed on the parasite surface and later shed by intramembrane cleavage, so its ectodomain does end up in the medium. Reason: The term is true of the shed MIC2 ectodomain, which is liberated into the culture supernatant by MPP1/ROM5 cleavage and is routinely recovered in the excreted-secreted antigen fraction. Release into the medium is nevertheless the end of MIC2's working life rather than a site of action; the functional compartments are the microneme and the parasite cell surface, proposed below. Neither of those is a descendant of extracellular region, so refinement by MODIFY does not apply and the term is retained as peripheral. The underlying UniProt call also overlooks the C-terminal membrane anchor. Supporting Evidence: PMID:10799515 the C-terminal domain of MIC2 is removed by a protease, termed MPP1, when MIC2 is released into the culture supernatant. PMID:14982962 MIC2 is shed by intramembrane cleavage within its anchoring domain. PMID:16933991 When levels of MIC2 in cell lysates or in the excreted-secreted antigen fraction are examined by quantitative immunoblotting (Figure 1D), the recombinant strains exhibited incremental changes in MIC2 expression and secretion ranging from 184% and 238%, respectively, in mic2e/mic2i without ATc to only 5% and 2%, respectively, in Ξmic2e/mic2i +ATc. |
| GO:0020009 microneme | IDA PMID:9084040 Molecular characterisation of an expressed sequence tag locu... | NEW | Summary: MIC2 is a marker protein of Toxoplasma micronemes: affinity-purified antisera to the cloned protein label micronemes exclusively by cryo-immunoelectron microscopy, and MIC2 occupies the apical micronemes in immunofluorescence. Reason: Microneme is the storage compartment that defines this protein and it is absent from GOA, which carries only the electronic extracellular-region row. Direct immuno-electron microscopy supports the location. Supporting Evidence: PMID:9084040 exclusively labeled the micronemes of T. gondii by cryo-immunoelectron microscopy. PMID:16933991 both proteins occupied the apical micronemes (Figure 1C, top row). |
| GO:0009986 cell surface | IDA PMID:15753289 A spatially localized rhomboid protease cleaves cell surface... | NEW | Summary: After microneme discharge MIC2 appears on the apical parasite surface and is translocated rearward to the posterior surface, where ROM5 cleaves it; surface MIC2 is the species captured by host ICAM-1. Reason: The parasite surface is where MIC2 exerts its adhesive activity, observed directly by immunofluorescence of secreted MIC2 and by immunoprecipitation of the mature surface form. Supporting Evidence: PMID:15753289 Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. PMID:15760456 human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. |
| GO:0098631 cell adhesion mediator activity | IDA PMID:15003840 Multimerization of the Toxoplasma gondii MIC2 integrin-like ... | NEW | Summary: MIC2 acts as a surface adhesin: its integrin-like A-domain binds heparin and human cells as a multimer, surface MIC2 binds host ICAM-1, and null parasites attach poorly to host cells and to collagen IV. Reason: GO:0098631 covers binding by a cell-surface adhesion protein to a molecule on another cell surface or an external substrate in order to mediate adhesion, which is precisely MIC2's molecular role. It is more informative than protein binding and underlies the attachment, gliding and invasion phenotypes. Supporting Evidence: PMID:15003840 Multimerization of MIC2 may increase the number of interactions with host cell receptors, thereby forming a multivalent adhesive junction during parasite entry. PMID:15760456 human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. PMID:28630943 mic2KO parasites show impaired attachment to host cells and collagen IV. |
| GO:0008201 heparin binding | IDA PMID:15003840 Multimerization of the Toxoplasma gondii MIC2 integrin-like ... | NEW | Summary: Recombinant MIC2 A-domain fusion proteins bind heparin specifically, independently of the MIDAS motif and as a high-molecular-weight multimeric species that mirrors native MIC2 from parasite lysate. Reason: Direct in vitro binding evidence for a defined MIC2 domain, with native protein behaving the same way. Recorded as a specific, non-core ligand-binding activity that underpins the adhesin role; physiological ligands are likely sulfated glycosaminoglycans of the host cell surface and matrix. Supporting Evidence: PMID:15003840 the MIC2 A-domain binds specifically to heparin, a ubiquitous sulfated proteoglycan found in the extracellular matrix (ECM) |
| GO:0044650 adhesion of symbiont to host cell | IMP PMID:28630943 Parasites lacking the micronemal protein MIC2 are deficient ... | NEW | Summary: Depletion or deletion of MIC2 strongly reduces tachyzoite attachment to host cells and to substrate, and the invasion defect of MIC2-deficient parasites is traced principally to this failure to adhere. Reason: MIC2 performs the adhesion step itself rather than merely being required for it: it is the surface adhesin whose ectodomain engages host ligands while its tail grips the actomyosin motor, so the participation test is met. The comparator check agrees, since other apicomplexan surface adhesins that attach the parasite to host cells carry adhesion-of-symbiont-to-host terms. Two independent genetic systems, a conditional knockdown and a clean null, give the same attachment phenotype. Supporting Evidence: PMID:28630943 mic2KO parasites show impaired attachment to host cells and collagen IV. PMID:28630943 In summary, we find here that MIC2 acts as important, but not essential, attachment factor PMID:16933991 Therefore, the entry defect is principally due to an inability to adhere properly to the target cell, although we cannot rule out an additional defect in the penetration step that follows attachment. |
| GO:0071980 cell surface adhesin-mediated gliding motility | IMP PMID:16933991 Toxoplasma MIC2 is a major determinant of invasion and virul... | NEW | Summary: MIC2-depleted and MIC2-null tachyzoites lose helical gliding and twirling while retaining circular gliding, consistent with MIC2 supplying the substrate attachment that the subpellicular actomyosin motor pulls against. Reason: The term is defined as gliding that results from surface adhesins propelled by membrane-associated motor proteins, which is the mechanism MIC2 serves: it contributes the adhesive engagement that a step of the process depends on, and its cytoplasmic tail binds aldolase to link that engagement to actin. The phenotype is partial rather than absolute, which the later null analysis attributes to defective initiation of motility rather than to its maintenance. Supporting Evidence: PMID:16933991 we suggest that MIC2 is needed to provide sufficient substrate binding to produce both helical and twirling motility. PMID:28630943 reduced invasion and gliding rates are due to a decreased ability to initiate rather than to sustain motility. PMID:12718875 Our findings reveal a potential role for aldolase in connecting TRAP family adhesins with the cytoskeleton, and provide a model linking adhesion with motility in apicomplexan parasites. |
| GO:0085017 entry into host cell by a symbiont-containing vacuole | IMP PMID:16933991 Toxoplasma MIC2 is a major determinant of invasion and virul... | NEW | Summary: Reducing MIC2 expression markedly lowers host-cell invasion, and loss of the MIC2 partner M2AP, which blocks rapid MIC2 secretion, impairs host cell entry by more than 80 per cent; the residual invasion of null parasites shows the contribution is quantitative. Reason: Toxoplasma enters host cells actively and resides in a parasitophorous vacuole, so this is the appropriate entry term. MIC2 participates by providing the adhesive grip at the start of entry rather than by driving penetration, and a clean null is still invasive, so the annotation is recorded as a genuine but non-core contribution and does not duplicate the adhesion term, which covers a distinct step. Supporting Evidence: PMID:16933991 markedly defective host-cell attachment and invasion, the loss of helical gliding motility PMID:12727875 M2AP knockout parasites were >80% impaired in host cell entry. PMID:28630943 We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. |
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Download this section (compressed HTML)Q: Which host cell surface molecules does MIC2 engage during attachment to different cell types, and is sulfated glycosaminoglycan binding by the A-domain the dominant interaction in vivo?
Q: Which adhesins support the residual, MIC2-independent attachment and invasion seen in mic2 knockout parasites?
Q: Is the delayed egress of mic2 knockout parasites a consequence of impaired attachment, or does MIC2 feed into the signalling pathway that triggers egress?
Experiment: Surface plasmon resonance and glycan-array profiling of the recombinant MIC2 ectodomain, in monomeric and multimeric forms, against defined glycosaminoglycans, ICAM-1 and matrix proteins, with affinities compared to attachment efficiency on cells depleted of the corresponding ligands.
Hypothesis: Highly sulfated glycosaminoglycans on the host cell surface are the principal physiological ligands of the MIC2 A-domain, and ICAM-1 engagement is specific to transmigration across cellular barriers.
Experiment: Complementation of the mic2 knockout with point mutants that separate ectodomain ligand binding from aldolase binding by the cytoplasmic tail, followed by quantitative assays of attachment under shear, three-dimensional motility, invasion and egress timing.
Hypothesis: Attachment and gliding defects of mic2 knockout parasites are rescued only when both host ligand binding and motor coupling are restored, whereas delayed egress depends on only one of the two.
Experiment: Determination of the stoichiometry of the native MIC2-M2AP complex on intact parasites by in-cell cross-linking combined with mass spectrometry and by single-molecule imaging of secreted MIC2 at the parasite surface.
Hypothesis: Native surface MIC2-M2AP is multimeric, and the 1:1 ectodomain complex seen by crystallography is the building block of a higher-order adhesive junction.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological host receptor repertoire of the MIC2 A-domain is unresolved: heparin binding was shown with recombinant protein in vitro and ICAM-1 was identified by immunoprecipitation, but which ligands actually mediate attachment to a given host cell type, and with what affinity, is unknown.
BIOLOGY
Gap: How MIC2-null parasites still attach and invade is unknown, that is, which other microneme adhesins provide the redundant attachment activity and whether they act in the same motor-coupled manner.
BIOLOGY
Gap: The oligomeric state of the MIC2-M2AP complex on the parasite surface is unsettled, with native-gel evidence for a hexamer and crystallographic evidence for a 1:1 ectodomain monomer, so the valency of the adhesive junction is unknown.
BIOLOGY
Gap: Why deletion of mic2 delays host cell egress is unexplained; a role for MIC2 in the signalling that triggers egress has been proposed but not demonstrated.
BIOLOGY
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