CG43124 (SPH249) encodes a 245-residue secreted serine-protease homolog (S1A non-peptidase homolog) of Drosophila melanogaster, the single product of a one-transcript gene at 3R 98C3. After a predicted N-terminal signal peptide (residues 1-20) the protein consists of a single trypsin-like (peptidase S1) domain with no clip domain. Two of the three catalytic-triad positions are substituted (the histidine aligns to Ser69 and the aspartate to Asn112) and no canonical GDSG catalytic-serine motif is present, so the protein is predicted to be a noncatalytic pseudoprotease. It belongs to the insect tryptase-related serine-protease lineage whose members act in extracellular protease cascades of innate immunity and development, and phylogenetic inference places it in an immune-associated clade, but no experiment has yet addressed its expression, partners or biological role.
Summary: The positive molecular function of CG43124 is unknown; the ND statement is accurate.
Reason: The ND root records that no positive molecular function has been established, and this re-review confirms that. No CG43124-specific experimental study was found by targeted searches on the gene symbol, synonym SPH249, FlyBase id and UniProt accession. The only molecular-function statements that can be made are negative (loss of peptidase activity, captured by the two NOT rows, which do not count as positive annotations in this aspect). Catalytic-triad substitutions rule out conventional peptidase chemistry but do not identify a binding, cofactor or regulatory activity, and no subfamily-specific activity can be transferred from catalytic relatives such as Snake. The unknown-function statement is therefore correct as it stands.
IKRNOT PMID:30367934 Building a platform for predicting functions of serine prote...
ACCEPT
Summary: Accept the negated peptidase annotation: CG43124 is a noncatalytic serine-protease homolog.
Reason: The annotation is explicitly NOT, and its absence-of-key-residues inference is supported by an independent profile alignment of the exact 245-residue CG43124-PA sequence. The conserved catalytic histidine and aspartate positions align to Ser69 and Asn112 in a significant trypsin-domain segment, while the active trypsin control recovers all three catalytic residues. Cao and Jiang classify SP versus SPH proteins using this triad criterion. The C-terminal catalytic-serine position cannot be mapped reliably, so no third-site replacement is claimed. This is a sequence-based loss-of-catalysis assessment, not a direct biochemical inactivity assay.
Summary: Root cellular component ND annotation: when it was made, no cellular component had been curated for this gene product from literature.
Reason: ACCEPT under the ND acceptance rule (projects/NO_DATA.md). The evidence this review cites for the cellular component is inferred (from orthologs, family membership or sequence features) or comes from other species; none of it is literature on this gene product in this species. This was judged from the review's own content during the NO_DATA re-action pass, not from a new literature search. Earlier action: TOO_GENERAL. Re-checked 2026-10-31: PMID:30367934, cited in core_functions, is a computational survey of serine protease-related proteins. The gene's own literature-search record finds no study of this gene product in this species that identifies a cellular location.
Summary: Extracellular localization is a reasonable conserved-family inference.
Reason: The PAINT IBA is a curated ancestral localization inference, and the exact target sequence independently has a predicted N-terminal signal peptide at residues 1–20. There is no contrary localization evidence or sequence feature suggesting loss of classical secretion. Together these support extracellular localization by inference; neither extracellular-matrix residence nor direct localization experiments are asserted.
Summary: A trypsin-domain mapping alone does not establish proteolysis by this inactive homolog.
Reason: The InterPro-derived IEA transfers a process term from the trypsin fold, but the exact protein lacks essential catalytic residues. Intrinsic proteolysis is therefore unsupported. A noncatalytic serine-protease homolog could still participate in a proteolytic cascade as a cofactor, so catalytic loss does not refute the biological-process term in principle. No CG43124-specific cascade mechanism or partner establishes that participation; the generic domain-to-process mapping exceeds its evidence.
SPHs are enzymatically inactive due to the lack of catalytic residue(s) but some of them (e.g. Masquerade) have known regulatory functions, including somatic muscle attachment, axonal guidance, post-mating responses, and prophenoloxidase (proPO) activation by proPO activating proteases (PAPs)
Summary: Root biological process ND annotation: when it was made, no biological process had been curated for this gene product from literature.
Reason: ACCEPT under the ND acceptance rule (projects/NO_DATA.md). The evidence this review cites for the biological process is inferred (from orthologs, family membership or sequence features) or comes from other species; none of it is literature on this gene product in this species. This was judged from the review's own content during the NO_DATA re-action pass, not from a new literature search. Earlier action: TOO_GENERAL. Re-checked 2026-10-31: PMID:30367934, cited in core_functions, is a computational survey of serine protease-related proteins. The gene's own literature-search record finds no study of this gene product in this species that identifies a biological process.
There is no retrieved knockout phenotype, infection-survival result, expression-induction measurement, biochemical interaction, cleavage assay, or epistasis experiment connecting this locus to Toll, phenoloxidase, development, digestion, fertility, or wound repair.
Summary: Accept the negated peptidase annotation: CG43124 is a noncatalytic serine-protease homolog.
Reason: The annotation is explicitly NOT, and its absence-of-key-residues inference is supported by an independent profile alignment of the exact 245-residue CG43124-PA sequence. The conserved catalytic histidine and aspartate positions align to Ser69 and Asn112 in a significant trypsin-domain segment, while the active trypsin control recovers all three catalytic residues. Cao and Jiang classify SP versus SPH proteins using this triad criterion. The C-terminal catalytic-serine position cannot be mapped reliably, so no third-site replacement is claimed. This is a sequence-based loss-of-catalysis assessment, not a direct biochemical inactivity assay. FlyBase independently places the gene in its S1A non-peptidase homolog gene group, and the sequence carries no GDSG catalytic-serine motif.
Summary: Retain the curated phylogenetic inference of an innate-immune role, at its broad level.
Reason: PAINT placed this process at ancestral node PTN000667087, seeded by experimental annotations of Toll-pathway serine proteases and homologues (Persephone, Spirit, MP1, SPE, Grass, ModSP, Sphinx1/2). CG43124 descends from that node, so the IBA is a curated node-level judgement rather than a pairwise transfer. The node placement is deep: in PANTHER 19.0 those donors fall in several subfamilies of PTHR24260 (SF134, SF135, SF136, SF140, SF144), whereas CG43124 is classified in SF147 together with the developmental protease Snake and the immune-cell-regulating CG10764. Catalytic loss does not argue against the inheritance, because the node's own seeds include the noncatalytic homologues Sphinx1 and Sphinx2 and SPHs are known to act as cascade regulators (e.g. CG4793 in hemocyte encapsulation). There is no CG43124-specific evidence of loss or divergence, so the IBA is accepted at its broad process level. It is not refined to Toll signalling, melanization or encapsulation, and no catalytic or cascade mechanism is inferred.
SPHs are enzymatically inactive due to the lack of catalytic residue(s) but some of them (e.g. Masquerade) have known regulatory functions, including somatic muscle attachment, axonal guidance, post-mating responses, and prophenoloxidase (proPO) activation by proPO activating proteases (PAPs)
Secreted, noncatalytic trypsin-fold serine-protease homolog (pseudoprotease) inferred by phylogeny to act in innate immunity; its positive molecular activity, partners and the cascade it may regulate are unknown.
SPHs are enzymatically inactive due to the lack of catalytic residue(s) but some of them (e.g. Masquerade) have known regulatory functions, including somatic muscle attachment, axonal guidance, post-mating responses, and prophenoloxidase (proPO) activation by proPO activating proteases (PAPs)
Q: Which extracellular partners and molecular activity mediate the inferred innate-immune role of CG43124?
Q: Does CG43124 serve as a noncatalytic cofactor in a defined protease cascade?
Q: Is CG43124 expressed in immune tissues (fat body, hemocytes) or induced by infection, and does its tandem neighbour CG43125 (same PANTHER 19.0 subfamily) share or diverge from its catalytic-site substitutions?
Suggested Experiments
Experiment: Generate a CG43124 null (CRISPR) and test survival and Toll/Imd target-gene induction after Gram-positive bacterial or fungal challenge, and encapsulation of parasitoid wasp eggs.
Experiment: Express tagged CG43124 in S2 cells or fat body to confirm secretion into the medium/hemolymph, and identify hemolymph binding partners by co-immunoprecipitation and mass spectrometry.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
The predicted serine-type endopeptidase activity is contradicted by loss of essential catalytic-triad residues in the exact CG43124 polypeptide. The trypsin-like sequence supports a noncatalytic homolog, not an active serine protease.
Review rationale: The exact target is the single 245-residue CG43124-PA polypeptide. Reproducible PF00089 profile alignment maps the essential trypsin catalytic histidine and aspartate to target Ser69 and Asn112 within a significant homologous segment; active bovine trypsin provides a successful positive control. The catalytic-serine region is poorly aligned, so a third substitution is not asserted. These two replacements contradict conventional serine endopeptidase chemistry and support a noncatalytic homolog. The original prediction has model score 0.98 and phmmer donor Q90WD8, toad ovochymase-2; fold similarity to this protease does not restore the missing target catalytic residues. Target GOA contains an explicit NOT annotation for the same activity, consistent with this independent analysis. Opposite polarity is not an equivalent existing positive annotation or CNN. Pseudoenzyme overannotation is supported; prediction-time sequence identity and the native noncatalytic function remain unestablished.
PMID:30367934: "The SP-related sequences in each species were divided into SPs or SPHs based on the presence or absence of the His-Asp-Ser catalytic triad"