Circumsporozoite protein (CSP) is the GPI-anchored surface coat protein of the Plasmodium falciparum sporozoite, the parasite stage transmitted by Anopheles mosquitoes. It is synthesised in developing oocysts, where it is first cytoplasmic and then redistributes over the surface of the sporoblast and budding sporozoites, and it covers the entire plasma membrane of the mature sporozoite. The mature protein has three parts: an N-terminal region containing the conserved region I (KLKQP) cleavage motif, a long central (NANP)n repeat region, and a C-terminal thrombospondin type-1 repeat (TSR) domain bearing the region II-plus adhesive motif. CSP switches between a non-adhesive conformation, in which the N-terminus masks the TSR, and an adhesive conformation in which the TSR is exposed; unmasking is triggered by contact with highly sulfated heparan sulfate proteoglycans on the hepatocyte surface and involves proteolytic cleavage at region I by a parasite cysteine protease. Through the TSR/region II-plus motif CSP binds the glycosaminoglycan chains of host-cell-surface heparan sulfate proteoglycans, acting as the principal adhesin that attaches the sporozoite to its substrate. This adhesion drives substrate-dependent gliding motility and mediates sporozoite travel through the mosquito hemolymph, invasion of the vector salivary glands, migration through the vertebrate dermis to the liver, and finally invasion of hepatocytes, where the parasite enters inside a parasitophorous vacuole. The central repeat region is the immunodominant B-cell epitope of the sporozoite and the basis of the RTS,S vaccine.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: CSP is cytoplasmic in developing oocysts before it is trafficked to the sporozoite surface, so the cytoplasmic location is real but reflects a transient biosynthetic/developmental stage rather than the site where CSP performs its adhesive function. Reason: The UniProt subcellular-location keyword mapping is faithful to the curated record, which (by similarity to the P. berghei ortholog P23093) places CSP in the cytoplasm of day-6 oocysts before surface redistribution. The claim is correct, and no more specific cytoplasmic compartment is supported, so the term is as precise as the evidence allows; it is peripheral to the core adhesin function carried out at the parasite surface, hence non-core rather than ACCEPT. Supporting Evidence: file:PLAF7/CSP/CSP-uniprot.txt Localizes to the cytoplasm and the |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Same claim as the IEA row, transferred by curator judgment from the P. berghei ortholog P23093, in which CSP is cytoplasmic in developing oocysts. Reason: P23093 (P. berghei CSP) is a close, well-characterised ortholog and the donor observation (cytoplasmic in oocysts at day 6 post infection, then redistributed over the sporoblast and budding sporozoite surface) is a developmental-stage localisation that is expected to be conserved. The row reinforces the IEA row and is given the same, consistent action: correct but not the functional site. Supporting Evidence: file:PLAF7/CSP/CSP-uniprot.txt Cytoplasm |
| GO:0005886 plasma membrane | IDA PMID:16603573 Identification and stoichiometry of glycosylphosphatidylinos... | ACCEPT | Summary: CSP is the GPI-anchored coat of the sporozoite plasma membrane; the location is correct and independently established, although the cited GPI-proteome study was focused on blood-stage parasites. Reason: Plasma-membrane localisation of CSP is firmly established: it is GPI-anchored on the sporozoite surface (PMID:32150583) and UniProt records cell membrane with a GPI lipid anchor. The cited reference is a P. falciparum GPI-anchored-proteome study whose abstract states it focused on the blood stage, and whose eleven proteomically validated GPI proteins are merozoite surface proteins; CSP enters through the study's GPI-HMM ranking of the whole genome. The curator read the full text and the conclusion is correct for this protein regardless, so this is accepted rather than challenged, and the location is core to CSP's function as a surface adhesin. Supporting Evidence: PMID:32150583 PfCSP is GPI-anchored on the sporozoite surface and plays a critical role in sporozoite development, motility and hepatocyte invasion [12, 13]. PMID:16603573 Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Keyword-derived plasma membrane annotation, consistent with the GPI-anchored surface coat localisation of CSP. Reason: The UniProt subcellular-location mapping reproduces the curated "Cell membrane; Lipid-anchor, GPI-anchor" statement. This is the functional site of CSP and reinforces the IDA row; the same action is therefore applied. Supporting Evidence: file:PLAF7/CSP/CSP-uniprot.txt Cell membrane {ECO:0000250|UniProtKB:P19597}; |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from the P. vivax/P. knowlesi-type CSP ortholog P19597; CSP orthologs are uniformly GPI-anchored plasma membrane surface coat proteins. Reason: The donor is a Plasmodium CSP ortholog with the same domain architecture and the same GPI anchor, and the transferred location is confirmed for P. falciparum CSP directly (GPI-anchored on the sporozoite surface, PMID:32150583). Consistent with the other plasma membrane rows. Supporting Evidence: PMID:32150583 PfCSP is GPI-anchored on the sporozoite surface and plays a critical role in sporozoite development, motility and hepatocyte invasion [12, 13]. |
| GO:0009986 cell surface | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the circumsporozoite protein family signature; CSP coats the external surface of the sporozoite, so cell surface is correct. Reason: IPR003067 is the CSP-specific family signature, not a promiscuous domain, so the mapping is tight rather than an over-propagation. The claim is directly supported: CSP coats the sporozoite surface and is displayed externally on the GPI anchor. Cell surface is complementary to, not redundant with, plasma membrane (it records the externally exposed display of the protein) and is core to its adhesin role. Supporting Evidence: PMID:29554084 which coats the PfSPZ surface and plays a key role in parasite migration out of the skin, entry into the liver parenchyma and invasion of hepatocytes7β12. |
| GO:0043395 heparan sulfate proteoglycan binding | IDA PMID:8478608 Malaria circumsporozoite protein binds to heparan sulfate pr... | ACCEPT | Summary: Frevert et al. identified the hepatocyte receptor for CSP region II-plus as heparan sulfate proteoglycan and showed recognition is via the glycosaminoglycan chains; this is the defining molecular function of CSP. Reason: Direct biochemical demonstration: CSP binding to purified hepatocyte receptors is abolished by heparitinase treatment, identifying highly sulfated heparan sulfate proteoglycans as the ligand and the glycosaminoglycan chains as the recognised moiety. This is the most informative molecular-function term available for CSP and is the core activity underpinning its adhesion, motility and invasion roles. Supporting Evidence: PMID:8478608 We have now identified these receptors as heparan sulfate proteoglycans (HSPG). PMID:8478608 The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. |
| GO:0044409 symbiont entry into host | IDA PMID:12244064 Function of region I and II adhesive motifs of Plasmodium fa... | ACCEPT | Summary: Replacement of the endogenous P. berghei CSP with PfCSP lacking region II abolished motility and dramatically impaired invasion of mosquito salivary glands and infection of the vertebrate host, establishing CSP's requirement for entry into both the vector and the vertebrate host. Reason: Although GO:0044409 is an ancestor of GO:0085017 (also annotated here), it is kept rather than treated as merely redundant because the supporting experiment covers entry events the hepatocyte-vacuole term does not represent: invasion of mosquito salivary glands and entry into the vertebrate host through the dermis. CSP performs the work of these steps itself - it is the surface adhesin that engages the substrate - rather than merely being required for them. The transgenic design (PfCSP expressed in P. berghei) is a legitimate complementation experiment and the authors show explicitly that PfCSP functions in the rodent parasite. Supporting Evidence: PMID:12244064 Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. PMID:12244064 We show here that P. falciparum circumsporozoite protein functions in rodent parasite and that P. berghei sporozoites carrying the P. falciparum CS gene develop normally, are motile, invade mosquito salivary glands, and infect the vertebrate host. Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. These data shed new light on the role of the CS protein in sporozoite motility and infectivity. |
| GO:0046812 host cell surface binding | IDA PMID:8478608 Malaria circumsporozoite protein binds to heparan sulfate pr... | ACCEPT | Summary: The CSP receptors identified by Frevert et al. are proteoglycans tightly associated with the plasma membrane of hepatocytes, i.e. CSP binds a host cell surface component. Reason: This is the host-context counterpart of the heparan sulfate proteoglycan binding annotation from the same study, and the two reinforce rather than duplicate each other: one names the chemical identity of the ligand, the other records that the ligand is displayed on the host cell surface. The direction of the symbiont/host term is correct for a parasite protein acting on host hepatocytes. Retained as a core function. Supporting Evidence: PMID:8478608 the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. PMID:8478608 are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans. |
| GO:0085017 entry into host cell by a symbiont-containing vacuole | IMP PMID:29554084 A public antibody lineage that potently inhibits malaria inf... | ACCEPT | Summary: Antibodies against PfCSP block P. falciparum sporozoite invasion of human hepatocytes in vitro and reduce liver burden in vivo by up to 98.3%, showing that CSP function is required for hepatocyte invasion, which proceeds by formation of a parasitophorous vacuole. Reason: The term is the correct parasite-specific specific term: Plasmodium sporozoites enter hepatocytes inside a parasitophorous vacuole, so this is not an animal-centric or mis-transferred term. CSP is the adhesin that engages the hepatocyte surface and whose region I cleavage licenses productive invasion, so it executes part of the entry process rather than merely being needed for it. Antibody blockade is a direct perturbation of the protein's function and the assays used P. falciparum sporozoites with a human hepatocyte line. This is a core function. Supporting Evidence: PMID:29554084 we tested a panel of IgG antibodies in vitro for their capacity to inhibit PfSPZ traversal and invasion of a human hepatocyte cell line (Fig. 2a). PMID:29554084 which is involved in the cleavage of the N-terminus to allow PfSPZ invasion of hepatocytes |
| GO:0085017 entry into host cell by a symbiont-containing vacuole | IMP PMID:32150583 Structure and mechanism of monoclonal antibody binding to th... | ACCEPT | Summary: Monoclonal antibodies to the PfCSP junctional/NANP epitopes gave greater than 90% reduction in parasite liver burden in vivo, independently supporting the requirement for CSP in hepatocyte infection. Reason: Second, independent antibody-blockade line of evidence for the same claim; its action is kept consistent with the other GO:0085017 row. Caveat recorded for completeness: the in vivo protection experiments used transgenic P. berghei sporozoites displaying the PfCSP epitope, so this row supports the claim somewhat more indirectly than PMID:29554084, which used P. falciparum sporozoites. The paper also states plainly that PfCSP has a critical role in hepatocyte invasion. Supporting Evidence: PMID:32150583 Both antibodies are potent, especially at high concentrations (300 and 600 ΞΌg/mouse) with >90% reduction in liver burden load as compared to naΓ―ve infected mice (Fig 5). PMID:32150583 PfCSP is GPI-anchored on the sporozoite surface and plays a critical role in sporozoite development, motility and hepatocyte invasion [12, 13]. |
| GO:0044650 adhesion of symbiont to host cell | IDA PMID:8478608 Malaria circumsporozoite protein binds to heparan sulfate pr... | NEW | Summary: Proposed new annotation: CSP is the sporozoite adhesin that attaches the parasite to hepatocytes by binding heparan sulfate proteoglycans on the host plasma membrane. Reason: GOA records the binding activity (GO:0043395, GO:0046812) and the downstream invasion process (GO:0085017), but not the adhesion step itself, which is a distinct biological process and the one CSP directly performs: it is the adhesin, not a factor merely required for adhesion by something else. Comparator check against other P. falciparum invasion adhesins: RH5 (Q8IFM5) carries GO:0044650, propagated automatically from its host cell surface receptor binding activity, whereas AMA1 carries only host cell surface binding and symbiont entry into host. The term is therefore applied inconsistently across this role rather than withheld by convention, and CSP - which is itself the attaching adhesin, with the host ligand directly identified - fits it. The term is neither an ancestor nor a descendant of GO:0085017 (adhesion versus entry), so it adds coverage rather than redundancy. Supporting Evidence: PMID:8478608 the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. PMID:8478608 We have now identified these receptors as heparan sulfate proteoglycans (HSPG). |
| GO:0071976 cell gliding | IMP PMID:12244064 Function of region I and II adhesive motifs of Plasmodium fa... | NEW | Summary: Proposed new annotation: deletion of the region II adhesive motif of CSP abolished sporozoite gliding motility, while deletion of region I had no effect, showing that the CSP adhesive motif is required for the substrate-dependent gliding locomotion of the sporozoite. Reason: Sporozoite locomotion is gliding motility - smooth movement along a solid surface driven by surface adhesins coupled to the subpellicular actomyosin motor - which matches the GO:0071976 definition. CSP participates directly: as a surface adhesin it provides the substrate engagement that the motor pulls against, so it contributes the adhesive activity a step of the process depends on rather than merely being required for an outcome. The evidence is a specific motif deletion with a clean internal control (region I deletion gives no phenotype), which argues against a generic loss-of-fitness explanation. No existing annotation covers motility. Supporting Evidence: PMID:12244064 Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. PMID:12244064 Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. |
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Download this section (compressed HTML)Q: Which parasite papain-like cysteine protease cleaves CSP at region I, and is its activity directly stimulated by CSP engagement of highly sulfated heparan sulfate proteoglycans?
Q: Does CSP recognise a heparan-sulfate-like ligand on the mosquito salivary gland, or a chemically distinct vector receptor, and does the TSR adopt the same adhesive conformation in both hosts?
Q: Should the GPI-anchored display of CSP be captured by a more specific cellular component term (anchored component of plasma membrane) rather than plasma membrane, given that the GPI anchor is at present a sequence-based prediction in the P. falciparum entry and experimental only for orthologs?
Q: Is the central (NANP)n repeat region purely an immunodominant decoy, or does repeat length and sequence contribute measurably to adhesion avidity or to the conformational masking of the TSR?
Experiment: Conditional depletion or region-II point mutagenesis of CSP in P. falciparum (rather than transgenic P. berghei) sporozoites, with quantitative gliding motility, hepatocyte attachment and invasion assays in human hepatocytes, to confirm directly in the species annotated here the motility and invasion phenotypes established in the rodent surrogate.
Hypothesis: Loss of the region II-plus adhesive motif in P. falciparum sporozoites abolishes gliding motility and hepatocyte attachment without affecting sporozoite development.
Experiment: Surface plasmon resonance and glycan-array profiling of recombinant PfCSP TSR against heparan sulfate oligosaccharides of defined sulfation patterns, and against mosquito salivary gland extracts, to define the minimal ligand determinant and test whether vector and vertebrate ligands are chemically related.
Hypothesis: CSP binding requires a minimum sulfation density on the glycosaminoglycan chain, and the salivary gland ligand shares that determinant.
Experiment: Activity-based protein profiling of cysteine proteases in salivary gland sporozoites combined with a cell-contact-triggered CSP cleavage reporter, to identify the region I protease and test whether HSPG engagement is sufficient to activate it.
Hypothesis: A single sporozoite papain-family cysteine protease is responsible for region I cleavage and is activated downstream of HSPG binding.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The identity of the parasite papain-like cysteine protease that cleaves CSP at region I during hepatocyte invasion is not established, and nor is how contact with highly sulfated heparan sulfate proteoglycans is transduced into activation of that cleavage.
BIOLOGY MF_DARK
What is known: That region I cleavage occurs at the parasite cell membrane, is triggered by contact with highly sulfated host HSPGs, and is required for productive invasion but not for adhesion, motility or cell traversal, is established (recorded in the curated UniProt PTM annotation, by similarity to P. berghei CSP).
Significance: The protease and its activation mechanism are the molecular switch that commits a sporozoite to invasion rather than continued migration, and would be a pre-erythrocytic drug or vaccine target distinct from the antibody-targeted repeat region.
What would resolve it: Chemical-genetic or conditional-knockdown screening of P. falciparum sporozoite cysteine proteases combined with an in vitro cleavage assay on recombinant CSP and a hepatocyte-contact-dependent cleavage readout.
Provenance (the field's own admissions):
Gap: It is not known what, if anything, CSP binds on the mosquito salivary gland surface, nor whether salivary gland invasion uses the same TSR/HSPG-type recognition as hepatocyte invasion or a distinct vector-side receptor.
BIOLOGY MF_DARK
What is known: That the region II motif of CSP is required for salivary gland invasion is firmly established by motif-deletion experiments in transgenic parasites; the hepatocyte ligand (heparan sulfate proteoglycan) is likewise established.
Significance: Determines whether one conserved adhesive activity serves both hosts or whether CSP has a second, vector-specific binding specificity - and therefore whether transmission-blocking strategies aimed at the vector stage can target the same epitope as pre-erythrocytic ones.
What would resolve it: Affinity purification or glycan-array/receptor-capture profiling of the mosquito salivary gland basal lamina ligands bound by recombinant PfCSP TSR, with validation by region II point mutants in transgenic sporozoites.
Provenance (the field's own admissions):
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