Pxd (CG3477, DmPO) encodes the chorion peroxidase of Drosophila melanogaster, a secreted 690-residue animal-type heme peroxidase (PANTHER PTHR11475 subfamily SF86) with a signal peptide and a single peroxidase domain that keeps the distal His, transition-state Arg and proximal heme-iron His. Pxd mRNA is expressed in ovarian follicle cells during late oogenesis (stages 11-14), and the protein localises to the eggshell, in the endochorion and the innermost chorionic layer. Recombinant Pxd uses H2O2 to convert tyrosine to di-tyrosine. These findings identify Pxd as the enzyme behind the long-known H2O2-dependent peroxidase activity that hardens the chorion at the end of oogenesis by forming di- and tri-tyrosine cross-links between chorion proteins. No loss-of-function phenotype has been reported, so the in vivo requirement for Pxd in chorion hardening rests on biochemistry and localisation rather than genetics. Pxd has no vertebrate or nematode ortholog; it belongs to an arthropod-specific expansion of peroxidases that also contains CG4009, CG10211, cardinal and CG42331.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004601 peroxidase activity | IBA GO_REF:0000033 | MODIFY | Summary: Peroxidase activity is propagated from the root node PTN000158755 of PTHR11475, from which Pxd descends. Pxd is itself one of the experimental seeds of that node (FB:FBgn0004577), and its own activity was measured on recombinant protein. Reason: The claim is correct and phylogenetically sound: Pxd descends from PTN000158755, it keeps the catalytic residues, and its recombinant protein converts tyrosine to di-tyrosine with H2O2. Pxd appearing among the node's seeds is expected, not circular. PTN000158755 is the only PTHR11475 IBD node that Pxd inherits. The more specific halide, thiocyanate and iodide peroxidase activities sit on vertebrate nodes that Pxd does not descend from, so they are not transferred. The evidence for Pxd itself supports the more specific phenolic-donor term GO:0140825, which the gene already carries by IDA, so this general term is refined to it. GO:0140825 is labelled lactoperoxidase activity, but its definition is the generic phenolic donor reaction of EC 1.11.1.7 and does not imply halide or thiocyanate oxidation. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000158755 · PTN000158755 SUPPORTS TRANSFER Root (LUCA) node of PTHR11475 and the only IBD node Pxd descends from. Pxd (FB:FBgn0004577) is one of its experimental seeds. The general term is correct at the family level; Pxd's own data support the phenolic-donor child term. Proposed replacements: lactoperoxidase activity Supporting Evidence: file:DROME/Pxd/Pxd-bioinformatics/RESULTS.md Of the 15 IBD assertions in PTHR11475, Pxd descends from only one: PTN000158755 GO:0004601 peroxidase activity. PMID:15979004 in the presence of H2O2 was able to convert tyrosine residues to di-tyrosine moieties |
| GO:0004601 peroxidase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the haem peroxidase superfamily signature (IPR010255). Reason: Correct, and consistent with the IBA row for the same term and with the IDA activity. As for the IBA row, the evidence for Pxd supports the more specific phenolic-donor peroxidase term GO:0140825 that the gene already carries, so the general term is refined to it. Proposed replacements: lactoperoxidase activity Supporting Evidence: file:DROME/Pxd/Pxd-uniprot.txt Belongs to the peroxidase family. XPO subfamily. PMID:15979004 in the presence of H2O2 was able to convert tyrosine residues to di-tyrosine moieties |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Mapped from the UniProt subcellular location Secreted. Pxd has a predicted signal peptide (residues 1-20) and is found in the chorion, an extracellular eggshell layer secreted by the follicle cells. Reason: Pxd is a secreted enzyme with a signal peptide, and immunolocalisation places it in the endochorion and innermost chorionic layer, outside the follicle cells. Extracellular region is correct. It is not a GO parent of egg chorion (GO:0042600, under external encapsulating structure), so it is not made redundant by the IDA chorion annotation and is kept. The PAINT extracellular-region node PTN000895746 does not include Pxd, so this row rests on the Pxd-specific signal peptide and chorion localisation, not on family propagation. Supporting Evidence: PMID:15979004 the Pxd peroxidase is selectively localized in the chorion structures and particularly in the endochorion and innermost chorionic layer (ICL) |
| GO:0006979 response to oxidative stress | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Generic InterPro2GO mapping from the haem peroxidase superfamily signature. Reason: Pxd uses H2O2 as a co-substrate to cross-link chorion proteins in the eggshell. The H2O2 is a programmed product of the follicle cells at the end of oogenesis, not a stressor. No study links Pxd to a response to oxidative stress, and its expression is developmentally programmed in late follicle cells. The mapping reflects the superfamily signature, not anything known about Pxd. Supporting Evidence: PMID:18620142 It is concluded that hydrogen peroxide is an endogenous, programmed product of the follicle cells, responsible for the action of peroxidase in order to oxidize the tyrosyl residues producing di-tyrosine and tri-tyrosine bonds between the chorion polypeptides. PMID:15979004 the Pxd mRNA is specifically expressed in follicle cells during the late stages of oogenesis 11-14 |
| GO:0007306 egg chorion assembly | IC PMID:15979004 The enzymatic component of Drosophila melanogaster chorion i... | ACCEPT | Summary: FlyBase inferred this from the chorion localisation of Pxd and its di-tyrosine forming activity. Pxd is the enzyme that hardens the chorion by cross-linking chorion proteins through di- and tri-tyrosine bonds. Reason: Pxd does part of the work of chorion assembly: it catalyses the final cross-linking step that hardens the chorion. Its mRNA is in late follicle cells, the protein is in the endochorion and innermost chorionic layer, and the recombinant enzyme forms di-tyrosine with H2O2. Older work showed that the chorion contains an endogenous peroxidase activity that cross-links chorion proteins in late stage 14 and is blocked by peroxidase inhibitors, and that peroxidase and H2O2 sit in the same chorion layers where Pxd is found. This is participation as the catalyst, not merely a requirement. The support is biochemical and positional; no Pxd mutant or RNAi eggshell phenotype has been published, so the in vivo requirement has not been tested genetically. Supporting Evidence: PMID:15979004 This specific peroxidase is involved in the chorion hardening process, through protein crosslinking mediated by the formation of di- and tri-tyrosine bonds. PMID:28305174 TheDrosophila chorion contains an endogenous peroxidase activity which remains inactive until late stage 14 when it catalyzes the crosslinking of the chorionic proteins. PMID:18620142 it was found that the enzyme exists in two out of the five egg-shell layers: the innermost choronic layer and the endochorion |
| GO:0020037 heme binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the haem peroxidase superfamily. UniProt records heme b as cofactor by similarity, and the proximal heme-iron ligand His437 is conserved. Reason: Animal haem peroxidases need a bound heme for catalysis, and Pxd keeps the proximal His that ligates the heme iron as well as the distal His and Arg. Heme binding is very likely correct. It is a cofactor-binding property that serves the peroxidase activity rather than a separate function, so it is kept as non-core. Heme binding has not been measured directly for Pxd. Supporting Evidence: file:DROME/Pxd/Pxd-uniprot.txt Binds 1 heme b (iron(II)-protoporphyrin IX) group per subunit. file:DROME/Pxd/Pxd-bioinformatics/RESULTS.md Q01603 retains His185 (distal His, proton acceptor), Arg331 (transition-state stabiliser) and His437 (proximal heme-iron ligand) at the PROSITE-ProRule PRU00298 positions. |
| GO:0042600 egg chorion | IDA PMID:15979004 The enzymatic component of Drosophila melanogaster chorion i... | ACCEPT | Summary: Immunolocalisation showed Pxd in chorion structures, mainly the endochorion and innermost chorionic layer, and a single 55 kDa band in purified chorions. Reason: Direct experimental localisation of Pxd to the chorion. Its location matches the layers where peroxidase activity and H2O2 had been detected cytochemically. The antibody used was raised against the Aedes aegypti peroxidase (anti-rAePO), so the signal could in principle include cross-reacting paralogs; the curator read the full text and the localisation fits the expression of Pxd in late follicle cells. Supporting Evidence: PMID:15979004 the Pxd peroxidase is selectively localized in the chorion structures and particularly in the endochorion and innermost chorionic layer (ICL) PMID:18620142 it was found that the enzyme exists in two out of the five egg-shell layers: the innermost choronic layer and the endochorion |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Inferred automatically from the peroxidase activity annotations. Reason: Consuming H2O2 is part of any peroxidase reaction, but Pxd works outside the cell, in the chorion, where the follicle cells produce H2O2 on purpose to drive tyrosine cross-linking. Its role is to build the eggshell, not to detoxify oxidants at the cellular level, and no study connects Pxd to oxidant detoxification. The inference rule applies a process that is typical of intracellular antioxidant peroxidases to an extracellular cross-linking enzyme. Supporting Evidence: PMID:18620142 It is concluded that hydrogen peroxide is an endogenous, programmed product of the follicle cells, responsible for the action of peroxidase in order to oxidize the tyrosyl residues producing di-tyrosine and tri-tyrosine bonds between the chorion polypeptides. |
| GO:0140825 lactoperoxidase activity | IDA PMID:15979004 The enzymatic component of Drosophila melanogaster chorion i... | ACCEPT | Summary: Recombinant mature Pxd converted tyrosine residues to di-tyrosine in the presence of H2O2. Tyrosine is a phenolic donor, so this is the reaction defined by GO:0140825 (2 a phenolic donor + H2O2 = 2 a phenolic radical donor + 2 H2O; EC 1.11.1.7). Reason: This is the core, experimentally established activity of Pxd. The term label refers to lactoperoxidase, but its definition is the generic phenolic-donor peroxidase reaction, which fits the measured activity. Neither this annotation nor the phylogeny supports the halide or thiocyanate oxidation of vertebrate lactoperoxidase (GO:0036393 sits on vertebrate PAINT nodes that Pxd does not descend from); only the phenolic-donor reaction should be transferred to relatives. Supporting Evidence: PMID:15979004 in the presence of H2O2 was able to convert tyrosine residues to di-tyrosine moieties file:DROME/Pxd/Pxd-bioinformatics/RESULTS.md The halide and thiocyanate oxidising activities of vertebrate myeloperoxidase, lactoperoxidase and eosinophil peroxidase are therefore not phylogenetically assigned to Pxd. |
| GO:0140825 lactoperoxidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Mapped from the UniProt catalytic activity (RHEA:56136, EC 1.11.1.7). Reason: Agrees with the IDA annotation for the same term. The phenolic-donor peroxidase reaction is supported by the di-tyrosine forming activity of recombinant Pxd. Supporting Evidence: PMID:15979004 in the presence of H2O2 was able to convert tyrosine residues to di-tyrosine moieties |
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Download this section (compressed HTML)Q: Is Pxd required in vivo for chorion hardening? A Pxd null or follicle-cell RNAi has not been reported, and CG4009, its closest paralog (same subfamily SF86), could share the role.
Q: Which chorion proteins does Pxd cross-link, and does it also act on vitelline membrane proteins, whose cross-linking during egg activation is peroxidase-dependent?
Q: Where a substrate relationship needs to be machine-readable, should Pxd's activity carry a has_input to chorion proteins in a GO-CAM, rather than a process term on the substrates?
Experiment: Make a Pxd null (or follicle-cell-specific RNAi) and test eggshell hardening by bleach resistance, di-tyrosine fluorescence and chorion protein solubility; compare with CG4009 knockdown and the double knockdown.
Type: genetic loss-of-function
Experiment: Assay recombinant Pxd with iodide, bromide, chloride and thiocyanate as well as phenolic substrates, to determine whether it has any of the halide or pseudohalide activities of vertebrate peroxidases or is restricted to phenolic donors.
Type: biochemical enzyme assay
Peroxidase (Pxd) encodes an enzyme that converts hydrogen peroxide to water and molecular oxygen, in the process oxidizing glutathione to glutathione disulfide. It is involved in the cellular response to oxidative stress.
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