CG4793 (SPH208/cSPH128) encodes a 910-residue secretory-pathway serine protease homolog (SPH) with an N-terminal trypsin-like (S1A) domain and a long repetitive C-terminal region. Glycine replaces the catalytic serine of the S1 fold, so the protein is predicted to be catalytically inactive and to act through a nonenzymatic, regulatory mechanism. It belongs to an insect-specific SPH clade of the tryptase-related family (PANTHER PTHR24256:SF462) whose other members, including mosquito CLIPA-type SPHs, are biochemically uncharacterized. CG4793 is a transcriptional target of JAK/STAT signalling in haemocyte-like Kc167 cells. Depletion in lamellocytes increases encapsulation of parasitoid-wasp eggs, indicating that it restrains this cellular immune response, whereas depletion in a constitutively active JAK (hopTumL) background suppresses melanotic tumour formation. Its direct molecular partners and mechanism are unknown.
Summary: The negated peptidase annotation is consistent with the disrupted catalytic machinery of CG4793.
Reason: This is a NOT annotation, not a positive enzyme assignment. Independent alignment of the exact Q8IP30 sequence to two active trypsin controls identifies glycine at the catalytic serine position, with additional substitutions at the aligned His/Asp positions. The controls retain the intact triad. This supports loss of conventional S1 peptidase activity and agrees with the published SPH classification; it does not exclude noncatalytic regulation of other proteases.
Propagation Review
Root cause:NO FAILURE CORE
Sources checked:
PANTHER:PTN000668109SUPPORTS TRANSFER
GOA WITH/FROM identifies this PAINT node on the NOT IBA; the propagated negation is corroborated by the CG4793-specific loss of the catalytic serine.
PANTHER:PTN001207268SUPPORTS TRANSFER
GOA WITH/FROM also lists the broader serine-protease ancestral node, so the NOT IBA is accepted in the context of the nested PAINT loss call and the exact Q8IP30 residue evidence.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
IKRNOT PMID:12568721 Serine proteases and their homologs in the Drosophila melano...
ACCEPT
Summary: The negated peptidase annotation is consistent with the disrupted catalytic machinery of CG4793.
Reason: This is a NOT annotation, not a positive enzyme assignment. Independent alignment of the exact Q8IP30 sequence to two active trypsin controls identifies glycine at the catalytic serine position, with additional substitutions at the aligned His/Asp positions. The controls retain the intact triad. This supports loss of conventional S1 peptidase activity and agrees with the published SPH classification; it does not exclude noncatalytic regulation of other proteases.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
IKRNOT PMID:30367934 Building a platform for predicting functions of serine prote...
ACCEPT
Summary: The negated peptidase annotation is consistent with the disrupted catalytic machinery of CG4793.
Reason: This is a NOT annotation, not a positive enzyme assignment. Independent alignment of the exact Q8IP30 sequence to two active trypsin controls identifies glycine at the catalytic serine position, with additional substitutions at the aligned His/Asp positions. The controls retain the intact triad. This supports loss of conventional S1 peptidase activity and agrees with the published SPH classification; it does not exclude noncatalytic regulation of other proteases.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
Summary: Extracellular localization is a well-supported sequence and phylogenetic inference.
Reason: The existing PAINT inference is consistent with the independently predicted N-terminal signal peptide in the exact Q8IP30 sequence. The architecture supports entry into the secretory pathway and an extracellular role for this protease homolog. This is inferred localization; the gene-specific RNAi phenotype does not itself demonstrate secretion.
Propagation Review
Root cause:NO FAILURE CORE
Sources checked:
PANTHER:PTN001207268SUPPORTS TRANSFER
PTHR24256 PAINT places the extracellular-region IBD at this ancestor, and the exact Q8IP30 sequence independently carries a predicted N-terminal signal peptide.
Supporting Evidence:
file:DROME/CG4793/CG4793-uniprot.txt
FT SIGNAL 1..18 FT /evidence="ECO:0000256|SAM:SignalP"
Summary: Participation in proteolysis is unresolved for this inactive protease homolog.
Reason: The target lacks the conventional serine nucleophile, so its S1 domain cannot justify intrinsic peptide-bond hydrolysis. Pure regulation of an active protease would belong under GO:0030162 regulation of proteolysis rather than GO:0006508. Proteolysis remains unresolved only because insect SPHs can act as noncatalytic cofactors in prophenoloxidase-activating proteolytic cascades, where the cofactor contributes to a cleavage step without itself being the catalytic protease. The cellular-immunity experiments do not identify or exclude such a cofactor mechanism for CG4793.
Propagation Review
Root cause:UNRESOLVED
Sources checked:
PANTHER:PTN001207268UNRESOLVED
PTHR24256 PAINT places proteolysis and the non-negated GO:0004252 IBD at this broad ancestor, while the nested PTN000668109 IKR prunes GO:0004252 from CG4793's branch. CG4793 catalytic-serine loss refutes peptide-bond hydrolysis by the target itself, but the available papers have not identified or excluded a noncatalytic SPH cofactor role in a proteolytic cascade.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
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: Participation in proteolysis is unresolved for this inactive protease homolog.
Reason: The target lacks the conventional serine nucleophile, so its S1 domain cannot justify intrinsic peptide-bond hydrolysis. Pure regulation of an active protease would belong under GO:0030162 regulation of proteolysis rather than GO:0006508. Proteolysis remains unresolved only because insect SPHs can act as noncatalytic cofactors in prophenoloxidase-activating proteolytic cascades, where the cofactor contributes to a cleavage step without itself being the catalytic protease. The cellular-immunity experiments do not identify or exclude such a cofactor mechanism for CG4793.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
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: The negated peptidase annotation is consistent with the disrupted catalytic machinery of CG4793.
Reason: This is a NOT annotation, not a positive enzyme assignment. Independent alignment of the exact Q8IP30 sequence to two active trypsin controls identifies glycine at the catalytic serine position, with additional substitutions at the aligned His/Asp positions. The controls retain the intact triad. This supports loss of conventional S1 peptidase activity and agrees with the published SPH classification; it does not exclude noncatalytic regulation of other proteases.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
GO:0035208 positive regulation of hemocyte proliferation
IGI PMID:20168330 Transcriptional targets of Drosophila JAK/STAT pathway signa...
KEEP AS NON CORE
Summary: Retain the genetic interaction in the activated-JAK haemocyte tumour model as context-dependent biology.
Reason: Bina et al. identify CG4793 in an RNAi screen for effectors of hopTumL-induced melanotic tumours; its depletion suppresses the tumour index. This supports the existing FlyBase IGI inference in that overproliferation model, rather than demonstrating a universal positive effect on normal haemocyte proliferation. Direct circulating-haemocyte counts in the article concern baz, not CG4793. The physiological parasitoid assay instead establishes a negative effect on lamellocyte-mediated encapsulation, a distinct outcome and context.
Conversely, knockdown of CG4793 in lamellocytes resulted in a significant increase in the encapsulation rate.
GO:0140540 negative regulation melanotic encapsulation of foreign target
IMP PMID:33644706 The S1A protease family members CG10764 and CG4793 regulate ...
NEW
Summary: CG4793 restrains lamellocyte-mediated encapsulation of parasitoid eggs.
Reason: Two independent RNAi constructs increase encapsulation when CG4793 is depleted in lamellocytes, with no effect after plasmatocyte depletion. This directly supports negative regulation of the melanotic encapsulation response in the tested Drosophila–Leptopilina clavipes infection model. The experiment is gene-level; it does not establish which CG4793 isoform or molecular partner mediates the effect.
Conversely, knockdown of CG4793 in lamellocytes resulted in a significant increase in the encapsulation rate.
Core Functions
Restrains lamellocyte-mediated melanotic encapsulation of parasitoid eggs through an unresolved noncatalytic mechanism. The molecular activity is not established, so no specific molecular-function term is asserted.
Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA.
Q: Which extracellular binding partner or protease-regulatory interaction mediates CG4793-dependent control of lamellocyte encapsulation?
Q: Which CG4793 isoform and tissue source mediate the distinct parasitoid-infection and hopTumL tumour phenotypes?
Suggested Experiments
Experiment: Express tagged CG4793 in haemocytes (or S2 cells) and identify extracellular binding partners by co-immunoprecipitation and mass spectrometry, testing in particular whether it associates with active clip-domain or melanization proteases.
Hypothesis: CG4793 restrains lamellocyte encapsulation by binding and modulating an active immune serine protease or its substrate, as other noncatalytic SPHs do.
Experiment: Restore the catalytic serine (G290S) in a UAS rescue construct and compare its ability with the wild-type construct to rescue the increased encapsulation of lamellocyte-specific CG4793 knockdown larvae, using an RNAi-resistant coding sequence.
Hypothesis: The regulatory function of CG4793 is independent of, or incompatible with, peptidase catalysis.
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 intrinsic serine-type endopeptidase prediction is contradicted by loss of the catalytic serine in the exact Q8IP30 protease-like domain. CG4793 has experimentally supported immune-regulatory biology, but its direct molecular mechanism is unresolved.
Review rationale: The exact 910-residue Q8IP30 sequence has a protease-like domain with glycine replacing the catalytic serine, supported by substantial-coverage alignments to two active trypsin controls. The controls agree on the additional His-to-Glu and Asp-to-Asn site mappings, although the histidine region is more divergent. These residue observations, rather than the SPH name or an automated domain caution alone, refute conventional serine endopeptidase catalysis. Four existing GOA NOT annotations are concordant negative annotations, not evidence that the positive prediction is correct or non-novel. The source gives model score 0.99 and phmmer donor Q7Z410 with score 226.0; donor similarity cannot restore the missing nucleophile. Gene-specific RNAi establishes cellular immune regulation, without assaying purified-protein proteolysis or resolving a direct mechanism.
Supporting Evidence:
file:DROME/CG4793/CG4793-bioinformatics/RESULTS.md: "Global domain alignments to active trypsins P00760 and P00761 map the catalytic His, Asp and Ser to Q8IP30 Glu145, Asn191 and Gly290, respectively. In particular, the catalytic serine is replaced by glycine in the conserved local segment TCKGDGGAPLA."
PMID:33644706: "CG4793 is an SPH gene and encodes a protein that is predicted to be catalytically inactive."