SRPN2 (Serpin-2) is a secreted serine protease inhibitor that functions as a key negative regulator of the melanization defense response in Anopheles gambiae. It acts in the hemolymph to inhibit CLIP-domain serine proteases (CLIPB9, CLIPB10, CLIPB4) that would otherwise activate prophenoloxidase, thereby preventing excessive melanization. SRPN2 knockdown causes spontaneous melanotic pseudotumors, shortened lifespan, and enhanced killing of Plasmodium parasites through ookinete lysis and melanization.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004867 serine-type endopeptidase inhibitor activity | IBA GO_REF:0000033 | ACCEPT | Summary: SRPN2 is a well-characterized serpin that directly inhibits CLIP-domain serine proteases including CLIPB9, CLIPB10, and CLIPB4. The IBA annotation based on phylogenetic inference is strongly supported by multiple direct experimental studies demonstrating serine protease inhibitor activity [PMID:20953892, PMID:25525260, PMID:33520733]. Reason: This molecular function annotation is the core enzymatic activity of SRPN2. Biochemical studies have demonstrated that SRPN2 forms covalent, SDS-stable inhibitory complexes with its target proteases (CLIPB9, CLIPB10) with stoichiometry consistent with classic serpin inhibition mechanism [PMID:25525260, PMID:33520733]. The IBA annotation is phylogenetically appropriate given serpin family conservation. Supporting Evidence: PMID:20953892 we identified the Anopheles gambiae clip-serine proteinase CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2 PMID:25525260 SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9), which functions in a serine proteinase cascade culminating in the activation of prophenoloxidase and melanization PMID:33520733 The stoichiometry of inhibition was 1.89, indicating that, under the used experimental conditions, SRPN2 acts as an inhibitor and not as substrate for CLIPB10 |
| GO:0050776 regulation of immune response | IBA GO_REF:0000033 | MODIFY | Summary: SRPN2 regulates the melanization arm of the innate immune response by inhibiting proteases that activate prophenoloxidase. However, a more specific term exists (GO:0035009 negative regulation of melanization defense response) that precisely captures SRPN2's function [PMID:16113656, PMID:20953892]. Reason: While technically accurate that SRPN2 is involved in regulation of immune response, this term is too broad. SRPN2 specifically negatively regulates the melanization defense response, not immune responses generally. The more specific term GO:0035009 is already annotated and better represents the biological function. Proposed replacements: negative regulation of melanization defense response Supporting Evidence: PMID:16113656 knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours, which increased in size and number with time, indicating spontaneous melanization PMID:20953892 serpin-2 (SRPN2), which was previously identified as a key negative regulator of melanization |
| GO:0005615 extracellular space | IBA GO_REF:0000033 | ACCEPT | Summary: SRPN2 is a secreted serpin that functions in the hemolymph (the insect equivalent of blood/extracellular fluid). Multiple studies confirm its extracellular localization [PMID:16113656, PMID:20953892]. The IBA annotation aligns with experimental evidence. Reason: The extracellular space annotation is well-supported. SRPN2 contains a signal peptide and is secreted into the hemolymph where it functions to inhibit circulating proteases. This is consistent with the phylogenetic inference and the serpin family biology. Supporting Evidence: PMID:20953892 controlled by an extracellular proteinase cascade and serpin inhibitors |
| GO:0002376 immune system process | IEA GO_REF:0000043 | ACCEPT | Summary: This IEA annotation from UniProt keyword mapping is accurate but very broad. SRPN2 is involved in the melanization branch of innate immunity, specifically as a negative regulator. More specific terms are already annotated [PMID:16113656]. Reason: While broad, this IEA annotation is not incorrect - SRPN2 does function in an immune system process (melanization). More specific annotations are already present (GO:0035009, GO:0045087). IEA annotations providing broader context alongside more specific experimental annotations is acceptable practice. Supporting Evidence: PMID:20953892 Melanization is an innate immune response in arthropods that encapsulates and kills invading pathogens |
| GO:0004867 serine-type endopeptidase inhibitor activity | IEA GO_REF:0000120 | ACCEPT | Summary: Redundant with the IBA and IDA annotations for the same term but from automated InterPro/ARBA mapping. The annotation is correct and consistent with experimental evidence. Reason: This IEA annotation is accurate. SRPN2 belongs to the serpin family (IPR000215) and exhibits serine-type endopeptidase inhibitor activity as demonstrated experimentally. The IEA provides supporting evidence from sequence/domain analysis consistent with experimental findings. Supporting Evidence: PMID:25525260 SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9) |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation from UniProt subcellular location mapping. SRPN2 is secreted into the hemolymph, making this annotation accurate. The more specific child term GO:0005615 (extracellular space) is also annotated. Reason: This broader cellular component term is accurate - SRPN2 is indeed located in the extracellular region (secreted into hemolymph). Having both this broader term and the more specific extracellular space term is acceptable. Supporting Evidence: PMID:20953892 controlled by an extracellular proteinase cascade and serpin inhibitors |
| GO:0005615 extracellular space | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation from InterPro2GO mapping for serpin family. Redundant with IBA and IDA annotations but consistent with the established extracellular localization of SRPN2. Reason: Accurate annotation derived from serpin domain presence. Serpins of this type are typically secreted, and SRPN2 has been experimentally confirmed to be secreted into the hemolymph. Supporting Evidence: PMID:20953892 extracellular proteinase cascade and serpin inhibitors |
| GO:0030414 peptidase inhibitor activity | IEA GO_REF:0000043 | ACCEPT | Summary: IEA annotation from UniProt keyword mapping. This is a parent term of GO:0004867 (serine-type endopeptidase inhibitor activity). While accurate, it is less specific than what is known about SRPN2. Reason: This parent-level annotation is technically correct but adds limited information beyond the more specific serine-type endopeptidase inhibitor activity annotation. However, it is not wrong to have both parent and child terms annotated when the evidence supports it. Supporting Evidence: PMID:25525260 SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9) |
| GO:0045087 innate immune response | IEA GO_REF:0000043 | ACCEPT | Summary: IEA annotation from UniProt keyword mapping (Innate immunity). SRPN2 functions in the melanization arm of innate immunity. This is an appropriate level of specificity for the immune process aspect of SRPN2 function. Reason: Melanization is a component of the innate immune response in arthropods. SRPN2's role in regulating melanization means it participates in innate immunity. The annotation is appropriate. Supporting Evidence: PMID:20953892 Melanization is an innate immune response in arthropods that encapsulates and kills invading pathogens |
| GO:0004867 serine-type endopeptidase inhibitor activity | IMP PMID:33520733 CLIPB10 is a Terminal Protease in the Regulatory Network Tha... | ACCEPT | Summary: IMP annotation based on mutant phenotype evidence from PMID:33520733. The study demonstrates that SRPN2 knockdown causes spontaneous melanization, which is partially rescued by CLIPB10 co-knockdown, supporting SRPN2's role as a serine protease inhibitor. Reason: The study shows that SRPN2 knockdown leads to uncontrolled melanization (due to loss of protease inhibition), and this is partially rescued by depleting the target protease CLIPB10. This genetic evidence supports the serine protease inhibitor function. Supporting Evidence: PMID:33520733 The kd of SRPN2 caused severe melanotic tumors on the abdomen of mosquitoes, which were partially reversed by CLIPB10 kd in SRPN2-depleted mosquitoes |
| GO:0005515 protein binding | IPI PMID:33520733 CLIPB10 is a Terminal Protease in the Regulatory Network Tha... | MODIFY | Summary: IPI annotation based on physical interaction with CLIPB10 (UniProtKB:Q7QBP4). While the interaction is real and biologically meaningful, "protein binding" is too vague and does not capture the nature of the serpin-protease inhibitory complex formation. Reason: Per curation guidelines, "protein binding" is too uninformative when more specific MF terms are available. The SRPN2-CLIPB10 interaction is specifically an inhibitory serpin-protease complex. A better annotation would be the specific inhibitor activity term or a more informative binding term that indicates the functional outcome. Proposed replacements: serine-type endopeptidase inhibitor activity Supporting Evidence: PMID:33520733 formed a SDS-stable protein complex with activated recombinant CLIPB10 |
| GO:0010951 negative regulation of endopeptidase activity | IMP PMID:33520733 CLIPB10 is a Terminal Protease in the Regulatory Network Tha... | ACCEPT | Summary: IMP annotation based on SRPN2 knockdown leading to increased endopeptidase activity (evidenced by increased melanization due to uncontrolled PPO activation). This is a direct consequence of SRPN2's inhibitor function. Reason: SRPN2 knockdown results in enhanced protease activity (CLIPB10, CLIPB9) leading to spontaneous melanization. This genetic evidence supports the annotation that SRPN2 negatively regulates endopeptidase activity in vivo. Supporting Evidence: PMID:33520733 CLIPB10 activity decreased linearly with increasing concentrations of SRPN2 |
| GO:0010955 negative regulation of protein processing | IMP PMID:33045027 The CLIP-domain serine protease CLIPC9 regulates melanizatio... | ACCEPT | Summary: IMP annotation from PMID:33045027. SRPN2 knockdown results in enhanced cleavage/processing of downstream substrates (CLIPA28, CLIPC9) in the melanization cascade, demonstrating that SRPN2 negatively regulates protein processing. Reason: The study shows that SRPN2 silencing enhances CLIPC9 cleavage, indicating SRPN2 normally restrains proteolytic processing in the cascade. This is consistent with its role as a protease inhibitor that prevents cascade activation. Supporting Evidence: PMID:33045027 CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2) |
| GO:0004867 serine-type endopeptidase inhibitor activity | IDA PMID:20953892 Characterization of a regulatory unit that controls melaniza... | ACCEPT | Summary: IDA annotation based on direct biochemical demonstration that SRPN2 inhibits CLIPB9. This is the first study to identify a specific SRPN2 target protease. Reason: Direct biochemical evidence shows SRPN2 forms an inhibitory complex with CLIPB9 and blocks its protease activity. This is core experimental evidence for SRPN2's molecular function. Supporting Evidence: PMID:20953892 we identified the Anopheles gambiae clip-serine proteinase CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2 |
| GO:0004867 serine-type endopeptidase inhibitor activity | IDA PMID:25525260 Structural and inhibitory effects of hinge loop mutagenesis ... | ACCEPT | Summary: IDA annotation based on detailed biochemical and structural characterization of SRPN2-CLIPB9 interaction. The study includes kinetic analysis and structure-function studies of the inhibitory mechanism. Reason: High-quality biochemical study demonstrating the inhibitory mechanism of SRPN2 against CLIPB9 with kinetic parameters and structural insights into the hinge region function. Supporting Evidence: PMID:25525260 Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9) |
| GO:0005515 protein binding | IPI PMID:20953892 Characterization of a regulatory unit that controls melaniza... | MODIFY | Summary: IPI annotation for interaction with CLIPB9 (UniProtKB:F5HKX0). As with the CLIPB10 interaction, "protein binding" is too vague for this specific inhibitory interaction. Reason: The SRPN2-CLIPB9 interaction is an inhibitory serpin-protease complex formation. The more informative serine-type endopeptidase inhibitor activity term better captures this functional interaction. Proposed replacements: serine-type endopeptidase inhibitor activity Supporting Evidence: PMID:20953892 clip-serine proteinase CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2 |
| GO:0005515 protein binding | IPI PMID:25525260 Structural and inhibitory effects of hinge loop mutagenesis ... | MODIFY | Summary: IPI annotation for SRPN2-CLIPB9 interaction from structural/biochemical study. This is redundant with the PMID:20953892 annotation and similarly too vague. Reason: Same rationale as other protein binding annotations - the interaction represents a specific inhibitory complex formation that is better captured by the serine-type endopeptidase inhibitor activity term. Proposed replacements: serine-type endopeptidase inhibitor activity Supporting Evidence: PMID:25525260 SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9) |
| GO:0005615 extracellular space | IDA PMID:20953892 Characterization of a regulatory unit that controls melaniza... | ACCEPT | Summary: IDA annotation for extracellular localization based on detection of SRPN2 in hemolymph samples and its function in the extracellular protease cascade. Reason: SRPN2 is detected in hemolymph and functions to inhibit extracellular proteases. This experimental evidence supports the extracellular space localization. Supporting Evidence: PMID:20953892 The molecular composition of this system is largely unknown in mosquitoes with the exception of serpin-2 (SRPN2) |
| GO:0035009 negative regulation of melanization defense response | IDA PMID:20953892 Characterization of a regulatory unit that controls melaniza... | ACCEPT | Summary: IDA annotation for SRPN2's core biological function - negative regulation of melanization. This is a highly specific and appropriate term for SRPN2's role in the melanization cascade. Reason: This is the most specific and informative biological process annotation for SRPN2. SRPN2 knockdown causes spontaneous melanization, demonstrating it is a key negative regulator of this immune response. This represents a core function of the gene. Supporting Evidence: PMID:20953892 serpin-2 (SRPN2), which was previously identified as a key negative regulator of melanization PMID:16113656 knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours |
| GO:0045861 negative regulation of proteolysis | IDA PMID:20953892 Characterization of a regulatory unit that controls melaniza... | ACCEPT | Summary: IDA annotation based on SRPN2's inhibition of proteolytic cascade enzymes. This is an accurate but somewhat redundant annotation given the more specific negative regulation of melanization defense response annotation. Reason: SRPN2 inhibits CLIPB9 and other proteases, thereby negatively regulating proteolysis in the melanization cascade. While this is a parent term of more specific regulatory processes, it accurately describes SRPN2's biochemical effect. Supporting Evidence: PMID:20953892 CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2 |
| GO:0045861 negative regulation of proteolysis | IDA PMID:25525260 Structural and inhibitory effects of hinge loop mutagenesis ... | ACCEPT | Summary: IDA annotation from the structural/biochemical study showing SRPN2 inhibits CLIPB9 proteolytic activity. Reason: The study provides detailed biochemical evidence for SRPN2's inhibition of CLIPB9 proteolytic activity, including kinetic analysis. Supports negative regulation of proteolysis. Supporting Evidence: PMID:25525260 SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9) |
| GO:0005615 extracellular space | IDA PMID:16113656 Anopheles gambiae SRPN2 facilitates midgut invasion by the m... | ACCEPT | Summary: IDA annotation from the original study characterizing SRPN2 function. SRPN2 is secreted into the hemolymph. Reason: This foundational study established SRPN2's function and localization. The extracellular space annotation is appropriate for this secreted serpin. Supporting Evidence: PMID:20953892 controlled by an extracellular proteinase cascade and serpin inhibitors |
| GO:0035009 negative regulation of melanization defense response | IDA PMID:16113656 Anopheles gambiae SRPN2 facilitates midgut invasion by the m... | ACCEPT | Summary: IDA annotation from the foundational study demonstrating SRPN2 knockdown causes spontaneous melanization. This is core evidence for SRPN2's function. Reason: The original Michel et al. 2005 study provides the primary evidence that SRPN2 is a negative regulator of melanization - knockdown results in melanotic pseudotumors. This is the defining biological function of SRPN2. Supporting Evidence: PMID:16113656 knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours, which increased in size and number with time, indicating spontaneous melanization |
| GO:0042832 defense response to protozoan | IDA PMID:16113656 Anopheles gambiae SRPN2 facilitates midgut invasion by the m... | MODIFY | Summary: IDA annotation based on SRPN2's effect on Plasmodium berghei infection outcomes. SRPN2 knockdown results in enhanced ookinete killing through melanization, suggesting SRPN2 modulates defense against the parasite. Reason: This annotation is problematic. SRPN2 actually FACILITATES parasite survival by suppressing the melanization response that would otherwise kill ookinetes. Knockdown of SRPN2 enhances defense, meaning the wild-type protein acts as a negative regulator of defense response to the protozoan, not a positive contributor to defense. The annotation should be to negative regulation of defense response to protozoan or removed. Proposed replacements: negative regulation of defense response Supporting Evidence: PMID:16113656 knockdown of SRPN2 strongly interfered with the invasion of A. gambiae midguts by the rodent malaria parasite Plasmodium berghei. It did not affect ookinete formation, but markedly reduced oocyst numbers, by 97%, as a result of increased ookinete lysis and melanization |
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Download this section (compressed HTML)Q: Are there additional CLIPB proteases that are SRPN2 targets? The incomplete rescue of SRPN2-knockdown phenotype by single CLIPB knockdowns suggests additional targets.
Q: What is the physiological role of the partial hinge insertion in SRPN2? The structural studies suggest it may represent a trade-off between RCL accessibility and inhibitory efficiency.
Q: How does SRPN2 expression and activity change during Plasmodium infection to permit localized melanization while preventing systemic pathology?
Experiment: Proteomics analysis of SRPN2 complexes in hemolymph to identify the complete set of SRPN2 target proteases under different immune challenge conditions.
Experiment: Time-course analysis of SRPN2 protein levels during Plasmodium infection to understand how melanization is permitted at the ookinete invasion site.
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