Sp7 (CG3066; also called MP2 and PAE1) is a secreted clip-domain serine protease of the S1A (trypsin-like) family in Drosophila melanogaster. It is synthesised as a zymogen with an N-terminal clip domain, an activation propeptide and a C-terminal trypsin-like catalytic domain with an intact His-Asp-Ser catalytic triad. Sp7 is expressed in blood cells, including crystal cells, and is induced by wounding and infection. Once activated, it cleaves prophenoloxidase-1 (PPO1) in the hemolymph, and genetic loss of Sp7 abolishes the induced phenoloxidase activity and the melanization reaction at sites of septic injury. Its activity is inhibited by the serpin Spn27A, and it lies downstream of the Toll-activating proteases ModSP and Grass. The Sp7-dependent melanization is needed for clearance of Staphylococcus aureus and affects survival after infection with several bacteria and fungi. Genetic and biochemical studies disagree on whether Sp7 cleaves PPO1 directly in vivo or acts upstream of the protease MP1.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004252 serine-type endopeptidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBD at PTN001207268 (seeded by easter and vertebrate S1A proteases) propagates serine-type endopeptidase activity to Sp7. Sp7 retains the catalytic triad (His182, Asp244, Ser341) and has been shown biochemically to cleave PPO1 and an IEAR-pNA substrate. Reason: The IBA agrees with direct experimental evidence on Sp7 itself. Activated recombinant MP2 (Sp7) hydrolyses a chromogenic peptide substrate and cleaves PPO1, and the serpin Spn27A inhibits it. Sp7 lies inside the catalytically active clade, with no evidence of triad loss. The activity is the core molecular function of the gene. Supporting Evidence: PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. PMID:24260243 A significant increase in activity (above that of Factor Xa alone) was observed in the presence of MP2Xa that was activated by Factor Xa file:DROME/Sp7/Sp7-uniprot.txt Serine protease that, by cleaving and activating |
| GO:0004252 serine-type endopeptidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the trypsin domain and trypsin His active-site signatures. Reason: The mapping is correct for Sp7, which keeps the catalytic triad and has measured peptidase activity against PPO1. Supporting Evidence: PMID:24260243 The amidase activity of MP2 was inhibited efficiently by Spn27A. |
| GO:0004252 serine-type endopeptidase activity | ISM PMID:12568721 Serine proteases and their homologs in the Drosophila melano... | ACCEPT | Summary: FlyBase sequence-model annotation from the genome-wide survey of Drosophila serine proteases and homologs, which classed catalytic SPs by the presence of the triad. Reason: Sp7 is a clip-domain SP with an intact triad, and its activity was later confirmed biochemically (PMID:24260243). Supporting Evidence: PMID:12568721 A majority of the SPs may be trypsin-like and activated by cleavage after a specific arginine or lysine residue. PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. |
| GO:0004252 serine-type endopeptidase activity | ISM PMID:19546158 Protease gene duplication and proteolytic activity in Drosop... | ACCEPT | Summary: FlyBase sequence-model annotation from a study of secreted serine endoproteases in Drosophila female reproductive tracts. Reason: The paper's sequence-based classification of serine endoproteases applies to Sp7, which has an intact triad and measured activity. The female reproductive tract context of the paper does not bear on the molecular function claim. Supporting Evidence: PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. |
| GO:0004252 serine-type endopeptidase activity | ISM PMID:30367934 Building a platform for predicting functions of serine prote... | ACCEPT | Summary: FlyBase annotation from the insect SP/SPH classification platform, which divides SP genes by the presence of the His-Asp-Ser triad; Sp7 (cSP7/MP2) is classed as a catalytic clip-domain SP. Reason: Consistent with the intact triad and the biochemical data. Supporting Evidence: PMID:30367934 in vitro test indicates that MP2 is a PAP |
| GO:0005576 extracellular region | EXP PMID:24260243 Serine protease MP2 activates prophenoloxidase in the melani... | ACCEPT | Summary: Sp7 has a 27-residue secretion signal and acts on PPO1 in the hemolymph; UniProt records it as secreted based on this paper. Reason: Sp7 is a hemolymph protease. It has a signal peptide, and its substrate PPO1 and its inhibitor Spn27A are extracellular hemolymph proteins. Supporting Evidence: PMID:24260243 Drosophila MP2 consists of 391 amino acids residues including a predicted 27-residue secretion signal peptide. file:DROME/Sp7/Sp7-uniprot.txt SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:24260243}. |
| GO:0005576 extracellular region | HDA PMID:15466469 Proteomic analysis of the Drosophila larval hemolymph clot. | ACCEPT | Summary: High-throughput proteomic identification in cell-free larval hemolymph (clot proteomics). Reason: The abstract does not name Sp7, but the study mapped cell-free hemolymph proteins, and detection there is consistent with the secreted nature of Sp7. Deferred to the curator, who had the full protein list. Supporting Evidence: PMID:15466469 our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph |
| GO:0005576 extracellular region | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBD at PTN001207268 for extracellular region. Sp7 (FBgn0037515) is itself one of the experimental seeds, which marks experimental grounding on the target. Reason: Consistent with the signal peptide and the experimental localization of Sp7. Sp7's presence among the seeds is expected and is not circular. Supporting Evidence: PMID:24260243 Drosophila MP2 consists of 391 amino acids residues including a predicted 27-residue secretion signal peptide. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Secreted). Reason: The UniProt location is experimentally based (ECO:0000269, PMID:24260243). Supporting Evidence: file:DROME/Sp7/Sp7-uniprot.txt SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:24260243}. |
| GO:0006508 proteolysis | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBD at PTN001207268 for proteolysis. Reason: Sp7 catalyses limited proteolysis of PPO1, so it does perform proteolysis. The term is broad, but it is the family-level process that PAINT placed. The specific biological process is captured by the melanization terms. Supporting Evidence: PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from trypsin-domain signatures. Reason: Correct for an active S1A protease; consistent with the IBA row. Supporting Evidence: PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. |
| GO:0006508 proteolysis | ISM PMID:12568721 Serine proteases and their homologs in the Drosophila melano... | ACCEPT | Summary: FlyBase sequence-model annotation from the Drosophila SP/SPH genome survey. Reason: Consistent with the intact triad and measured PPO1 cleavage. Supporting Evidence: PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. |
| GO:0006508 proteolysis | ISM PMID:19546158 Protease gene duplication and proteolytic activity in Drosop... | ACCEPT | Summary: FlyBase sequence-model annotation from the female reproductive tract protease study. Reason: Consistent with the intact triad and measured PPO1 cleavage. Supporting Evidence: PMID:24260243 Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. |
| GO:0035006 melanization defense response | IMP PMID:16861233 Two proteases defining a melanization cascade in the immune ... | ACCEPT | Summary: Genetic characterization of MP2 (Sp7) as a protease in the Spn27A-regulated melanization cascade, mainly required during fungal infection. Reason: Sp7 is a catalytic component of the protease cascade that generates active phenoloxidase, so it does some of the work of the melanization response and is not merely required for it. Several independent loss-of-function studies (RNAi and the PAE1 mutant) show loss of induced PO activity and melanization. This is the core biological process. Supporting Evidence: PMID:16861233 Here, we have described the genetic characterization of two immune inducible serine proteases, MP1 and MP2, which act in a melanization cascade regulated by Spn27A. PMID:16256951 Silencing of the gene impairs the melanization reaction upon injury. PMID:16322759 PAE1 mutants fail to activate phenoloxidase in the haemolymph following microbial challenge |
| GO:0035006 melanization defense response | IMP PMID:17356067 Crystal cell rupture after injury in Drosophila requires the... | ACCEPT | Summary: Study of crystal cell rupture and PPO release after injury. The abstract does not mention Sp7, so the Sp7 mutant data must be in the full text, which was not available. Reason: The cached abstract does not show the Sp7 experiment, but the function is well established for Sp7 by independent studies. Deferred to the FlyBase curator, who read the full text. Supporting Evidence: PMID:17356067 Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. PMID:16256951 Our data demonstrate that Sp7 is required for phenoloxidase activation |
| GO:0035008 positive regulation of melanization defense response | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning rule assigning positive regulation of melanization. Reason: The ARBA rule itself is not evidence, but the term matches experimental IMP and IGI annotations on Sp7 from two independent studies. Sp7 activates PPO1, the melanization effector enzyme, so it positively regulates the response. Supporting Evidence: PMID:24260243 MP2 functions as a prophenoloxidase-activating protease, and that this function is inhibited by Spn27A |
| GO:0035008 positive regulation of melanization defense response | IGI PMID:31018123 More Than Black or White: Melanization and Toll Share Regula... | ACCEPT | Summary: Compound-mutant analysis showing that Sp7 activates an alternate melanization reaction, downstream of ModSP and Grass. Reason: Sp7 is a positive activator of melanization. Genetic interaction data place it downstream of the Toll-activating proteases ModSP and Grass. Supporting Evidence: PMID:31018123 We find that two SPs, Hayan and Sp7, activate the melanization response in different manners PMID:31018123 We present evidence that Sp7 is regulated by SPs activating the Toll NF-ΞΊB pathway, namely ModSP and Grass. |
| GO:0035008 positive regulation of melanization defense response | IMP PMID:16256951 The serine protease Sp7 is expressed in blood cells and regu... | ACCEPT | Summary: Inducible RNAi of Sp7 impairs melanization upon injury and blocks phenoloxidase activation. Reason: Loss of Sp7 abolishes PO activation. Its enzymatic role is to generate the active effector enzyme, so it positively regulates melanization. Supporting Evidence: PMID:16256951 Our data demonstrate that Sp7 is required for phenoloxidase activation and its activity is restricted to a subclass of blood cells, the crystal cells. |
| GO:0050830 defense response to Gram-positive bacterium | IMP PMID:31018123 More Than Black or White: Melanization and Toll Share Regula... | KEEP AS NON CORE | Summary: Sp7-dependent melanization is needed for clearance of Staphylococcus aureus. Reason: Sp7 acts in the melanization effector arm that kills S. aureus, so the term describes a real defense context, and Ayres and Schneider independently found reduced survival of Sp7 mutants after S. aureus infection. The core process is melanization. This broader organismal defense term is one context of it, and the effect depends on the microbe (Sp7 mutants survive S. pneumoniae better than wild type). Supporting Evidence: PMID:31018123 Sp7 regulates an alternate melanization reaction responsible for the clearance of Staphylococcus aureus PMID:19071960 S. aureus infected CG3066 mutants exhibited the most striking difference in survival |
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Download this section (compressed HTML)Q: Is Sp7 the terminal PPO1-activating protease in vivo (as in vitro cleavage suggests), or does it act upstream of MP1, as the genetic data indicate?
Q: Should PPO1 cleavage by Sp7 be captured as GO:0031638 zymogen activation (or a has_input PPO1 extension on the activity)? This needs a comparator check against other PPO-activating proteases such as Hayan, Manduca PAP1 and Holotrichia PPAF1.
Q: Which protease cleaves the Sp7 zymogen (between the activation propeptide and Ile137), and is it ModSP/Grass-dependent?
Experiment: Determine the in vivo PPO1 cleavage site and kinetics in Sp7 and MP1 single and double mutants, and test whether catalytically dead Sp7 (S341A) rescues PO activation in Sp7 nulls.
Experiment: Identify the activating protease of proSp7 by N-terminal sequencing of activated Sp7 from infected hemolymph combined with ModSP, Grass and Hayan mutant backgrounds.
Serine protease 7 (Sp7) encodes a hemolymphatic serine protease involved in the activation of the melanization cascade.
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