id: A0A8C2TBA7 gene_symbol: A0A8C2TBA7 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:93934 label: Coturnix japonica description: >- Peptidylglycine alpha-amidating monooxygenase (PAM) of the Japanese quail, a bifunctional type I membrane enzyme of the regulated secretory pathway that generates C-terminally alpha-amidated neuropeptides and peptide hormones from their glycine-extended precursors. Its luminal region holds two catalytic domains. The N-terminal PHM domain (peptidylglycine alpha-hydroxylating monooxygenase) is a copper type II ascorbate-dependent monooxygenase: it binds two copper ions and uses molecular oxygen and ascorbate to hydroxylate the alpha-carbon of the C-terminal glycine. The PAL domain (peptidyl-alpha-hydroxyglycine alpha-amidating lyase) is a six-bladed NHL-repeat beta-propeller with a zinc active site; it cleaves the hydroxyglycine intermediate to release the amidated peptide and glyoxylate. An N-terminal signal peptide directs the protein into the secretory pathway, and a single transmembrane helix plus a disordered C-terminal cytosolic tail anchor it in the membranes of the trans-Golgi network and secretory granules. The tail carries the trafficking information that returns the protein through the endocytic pathway after exocytosis. This gene model lacks the linker between PHM and PAL that mammalian PAM-1 carries (exon 16), giving it the domain arrangement of the mammalian PAM-2 splice form, in which the two catalytic domains are rarely separated by cleavage. Soluble PHM and PAL, which arise from endoproteolysis of PAM, are released with the peptide products during regulated secretion. existing_annotations: - term: id: GO:0001519 label: peptide amidation evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in review: summary: Peptide amidation is the defining process of PAM, which performs both chemical steps of the conversion of a peptidylglycine to an amidated peptide. action: ACCEPT reason: The quail protein belongs to PANTHER subfamily PTHR10680:SF14, the same subfamily as the experimentally characterized human (P19021), rat (P14925), mouse and bovine PAM. In mammals PAM is the only known peptide-amidating enzyme. Both catalytic cores of the quail model are intact, and all PHM copper ligands and PAL zinc ligands are conserved (file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md). The gene product does the catalytic work of the process, so the participation test is met. The term is ARBA-derived, but the claim is independently supported. supported_by: - reference_id: PMID:26667899 supporting_text: "Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential" - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md supporting_text: "A0A8C2TBA7 is a full-length bifunctional PAM gene model that lacks exon 16" - term: id: GO:0003824 label: catalytic activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: The term is correct but uninformative. PAM's two specific activities are known and are already annotated. action: MODIFY reason: The InterPro PHM/PAL signature (IPR000720) correctly identifies PAM as an enzyme, but catalytic activity is a root-level term. Following the refinement rule, the replacements are the two specific descendant activities that the gene already carries, GO:0004504 (PHM step) and GO:0004598 (PAL step). Both are listed because the bifunctional protein has both. proposed_replacement_terms: - id: GO:0004504 label: peptidylglycine monooxygenase activity - id: GO:0004598 label: peptidylamidoglycolate lyase activity supported_by: - reference_id: PMID:26667899 supporting_text: "peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate" - term: id: GO:0004497 label: monooxygenase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: The term is correct for the PHM domain but is a general ancestor of peptidylglycine monooxygenase activity. action: MODIFY reason: The PHM domain (Cu2_ascorb_mOase N- and C-terminal domains, IPR000323 and IPR024548) is a copper monooxygenase. Its substrate is the C-terminal glycine of peptidylglycines, and the corresponding specific term, GO:0004504, is already on this gene. The previous review kept this row as non-core while arguing that it was too general; the action is now MODIFY to match that reason. proposed_replacement_terms: - id: GO:0004504 label: peptidylglycine monooxygenase activity supported_by: - reference_id: PMID:9360928 supporting_text: "The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states." - term: id: GO:0004504 label: peptidylglycine monooxygenase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: This is the core activity of the PHM domain, the first step of peptide amidation. action: ACCEPT reason: The annotation combines an EC mapping with PANTHER placement. Its phylogenetic grounding is the PAINT IBD for GO:0004504 at PTN000073733 (Deuterostomia), whose seeds are human PAM (P19021), bovine PAM (P10731), mouse, rat and zebrafish PAM. Quail is a deuterostome and shares the SF14 subfamily with these seeds. Rat PHM structures place the substrate hydroxylation at a pair of copper sites. Both of those sites, with all six histidine and methionine ligands, are conserved in the quail model. supported_by: - reference_id: PMID:9360928 supporting_text: "The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states." - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md supporting_text: "All six PHM copper ligands are conserved (human H102, H103, H167, H237, H239, M309 -> quail H103, H104, H168, H238, H240, M310)." - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-deep-research-falcon.md supporting_text: "The N-terminal PHM domain catalyzes the stereospecific hydroxylation of the α-carbon of the C-terminal glycine residue. This rate-limiting step requires three essential cofactors: (1) copper ions (two copper atoms per PHM domain), (2) molecular oxygen, and (3) reduced ascorbate as an electron donor" - term: id: GO:0004598 label: peptidylamidoglycolate lyase activity evidence_type: IEA original_reference_id: GO_REF:0000003 qualifier: enables review: summary: This is the core activity of the PAL domain, the second and final step of peptide amidation. action: ACCEPT reason: The annotation comes from an EC 4.3.2.5 mapping. It is supported because the quail protein has a complete NHL-repeat PAL domain (four NHL repeats, CDD NHL_PAL_like) with conserved zinc ligands, and because the structure of the PAL catalytic core of rat PAM, an SF14 subfamily member, establishes a Zn(II)-histidine active site. PAINT has no separate IBD for GO:0004598 in this family, so the support rests on the domain and residue conservation and on the shared subfamily with characterized mammalian PAM, not on an IBA. supported_by: - reference_id: PMID:19604476 supporting_text: "The enzyme peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-amidated peptide and glyoxylate." - reference_id: PMID:19604476 supporting_text: "The active site is formed by a Zn(II) ion coordinated by three histidine residues" - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md supporting_text: "All three PAL zinc ligands are conserved (human H582, H687, H783 -> quail H453, H557, H654)" - term: id: GO:0005507 label: copper ion binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: The PHM domain binds two catalytic Cu(2+) ions. Copper binding is correct but is a cofactor property of the monooxygenase, not a separate function. action: KEEP_AS_NON_CORE reason: Copper binding is real and required. PIRSR places six catalytic copper ligands (H103, H104, H168, H238, H240, M310), and all of them are conserved relative to human PAM. The term is as precise as GO allows for a metal ligand. It describes the cofactor of the PHM activity, not the enzyme's role, so it is kept as non-core rather than accepted as a core function. supported_by: - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: "Binds 2 Cu(2+) ions per subunit" - reference_id: PMID:9360928 supporting_text: "peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states" - term: id: GO:0005576 label: extracellular region evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: located_in review: summary: Soluble PHM and PAL derived from PAM are released with peptide products during regulated secretion and are found in serum and cerebrospinal fluid. This is a secondary location. action: KEEP_AS_NON_CORE reason: The TreeGrafter annotation comes from PTN000840922. The family PAINT table also has an IBD for GO:0005576 at PTN000840899 (taxon 6072, Eumetazoa), seeded by fly and rat genes. Release of soluble PHM and PAL is documented in mammals. This gene model lacks the exon-16 linker, so PAM-1-style separation of PHM from PAL is unlikely. Soluble bifunctional or PHM-containing products can still arise from cleavage near the membrane, and the term applies to the gene product rather than to one splice form. The protein amidates peptides in granules, not outside the cell, so the location is kept as non-core. The previous review accepted this term while calling it a secondary location; the action now matches that reasoning. supported_by: - reference_id: PMID:26667899 supporting_text: "Both in neurons and endocrine cells, soluble PHM and PAL are secreted along with neuropeptides and peptide hormones upon stimulation; PHM and PAL can be detected in serum and cerebrospinal fluid" - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-deep-research-falcon.md supporting_text: "Soluble PAM forms generated through endoproteolytic processing can be secreted into the extracellular space in active form" - term: id: GO:0006518 label: peptide metabolic process evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in review: summary: The term is correct but general. Its is_a descendant, peptide amidation (GO:0001519), is already on this gene and states the actual process. action: MODIFY reason: GO:0001519 peptide amidation is_a GO:0006518 peptide metabolic process. Under the refinement rule, a correct but general term is MODIFY with the specific descendant, not KEEP_AS_NON_CORE as in the previous review. proposed_replacement_terms: - id: GO:0001519 label: peptide amidation supported_by: - reference_id: PMID:26667899 supporting_text: "Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential" - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: located_in review: summary: PAM is a type I single-pass membrane protein. The specific membrane is the secretory granule membrane, a descendant of membrane. action: MODIFY reason: The quail model has a SignalP signal peptide (1-21) and a Phobius transmembrane helix (734-758), and the juxtamembrane, transmembrane and cytosolic segments of mammalian PAM are all present. Mammalian PAM is a granule membrane protein whose cytosolic domain directs it to the trans-Golgi network and secretory granules. GO:0030667 secretory granule membrane is_a (via vesicle membrane) GO:0016020. proposed_replacement_terms: - id: GO:0030667 label: secretory granule membrane supported_by: - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-uniprot.txt record_section: ["FT:TRANSMEM"] supporting_text: "TRANSMEM 734..758" - reference_id: PMID:26667899 supporting_text: "Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein." - reference_id: PMID:26667899 supporting_text: "an epitope-tagged protein consisting of the transmembrane and cytosolic domains of PAM localizes to the trans Golgi network and secretory granules" - term: id: GO:0016715 label: oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced ascorbate as one donor, and incorporation of one atom of oxygen evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: The term correctly describes the ascorbate-dependent PHM chemistry and is an ancestor of peptidylglycine monooxygenase activity. action: MODIFY reason: GO:0004504 peptidylglycine monooxygenase activity is_a GO:0016715, and the gene already carries GO:0004504. The previous review kept this row as non-core while arguing that it was too general; the action now matches the reason. proposed_replacement_terms: - id: GO:0004504 label: peptidylglycine monooxygenase activity supported_by: - reference_id: PMID:26667899 supporting_text: "peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate" - term: id: GO:0030658 label: transport vesicle membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: The term comes from UniProt's "secretory vesicle membrane" subcellular location, but the vesicles PAM resides in are dense-core secretory granules, for which GO has the more accurate term secretory granule membrane. action: MODIFY reason: The UniProt subcellular-location statement on this unreviewed entry is ARBA-derived. It maps through GO_REF:0000044 to transport vesicle membrane. The independent evidence places membrane PAM in regulated secretory granules and the trans-Golgi network, from which it is retrieved through the endocytic pathway after exocytosis. GO:0030667 secretory granule membrane is the better-supported term. It is not an is_a descendant of GO:0030658 in the current ontology, so this is a lateral correction to the term that the core function already uses. proposed_replacement_terms: - id: GO:0030667 label: secretory granule membrane supported_by: - reference_id: PMID:26667899 supporting_text: "Unlike soluble granule content proteins, granule membrane proteins are not secreted; after exocytosis and insertion into the plasma membrane, membrane PAM can be retrieved, traveling through the endocytic pathway in a regulated manner." - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-deep-research-falcon.md supporting_text: "PAM is packaged into regulated secretory granules and neurosecretory vesicles, where it exists in both membrane-associated and soluble forms. In atrial cardiomyocytes, PAM represents a major membrane protein of secretory granules" core_functions: - description: The PHM domain catalyzes the first step of peptide C-terminal amidation, the copper- and ascorbate-dependent alpha-hydroxylation of the C-terminal glycine of peptidylglycine hormone and neuropeptide precursors in the lumen of the secretory pathway. molecular_function: id: GO:0004504 label: peptidylglycine monooxygenase activity directly_involved_in: - id: GO:0001519 label: peptide amidation locations: - id: GO:0030667 label: secretory granule membrane supported_by: - reference_id: PMID:9360928 supporting_text: "The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states." - reference_id: PMID:26667899 supporting_text: "peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate" - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md supporting_text: "All six PHM copper ligands are conserved (human H102, H103, H167, H237, H239, M309 -> quail H103, H104, H168, H238, H240, M310)." - description: The PAL domain catalyzes the second step of peptide C-terminal amidation. Its zinc-dependent active site cleaves the peptidyl-alpha-hydroxyglycine intermediate made by PHM, releasing the alpha-amidated peptide and glyoxylate. molecular_function: id: GO:0004598 label: peptidylamidoglycolate lyase activity directly_involved_in: - id: GO:0001519 label: peptide amidation locations: - id: GO:0030667 label: secretory granule membrane supported_by: - reference_id: PMID:19604476 supporting_text: "The active site is formed by a Zn(II) ion coordinated by three histidine residues" - reference_id: PMID:26667899 supporting_text: "peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate" - reference_id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md supporting_text: "All three PAL zinc ligands are conserved (human H582, H687, H783 -> quail H453, H557, H654)" references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: - statement: InterPro domain signatures (IPR000720, IPR008977, IPR000323, IPR036939, IPR014783, IPR014784, IPR020611) identify PAM as a copper type II ascorbate-dependent monooxygenase with catalytic, membrane-associated, and copper-binding properties. - id: GO_REF:0000003 title: Gene Ontology annotation based on Enzyme Commission mapping findings: - statement: EC 4.3.2.5 maps to GO:0004598 (peptidylamidoglycolate lyase activity), correctly identifying the PAL domain enzymatic activity. - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: - statement: UniProt subcellular location "Cytoplasmic vesicle, secretory vesicle membrane" maps to GO:0030658 (transport vesicle membrane). - id: GO_REF:0000117 title: Electronic Gene Ontology annotations created by ARBA machine learning models findings: - statement: ARBA rule ARBA00093736 identifies PAM as involved in peptide amidation (GO:0001519). - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: - statement: PANTHER family assignment (PTN000840922) supports extracellular region localization for PAM based on phylogenetic inference. - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: - statement: Combined evidence from EC:1.14.17.3 and PANTHER:PTN000840922 supports peptidylglycine monooxygenase activity (GO:0004504). - id: PMID:9360928 title: 'Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase.' findings: - statement: Crystal structure of the rat PHM catalytic core shows two copper sites and supports substrate activation by a copper-bound oxygen species. reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed esummary confirms the title (Science 1997). Abstract-only cache. It concerns rat PAM, a member of the same PANTHER subfamily (SF14) as the quail protein. - id: PMID:19604476 title: 'Amidation of bioactive peptides: the structure of the lyase domain of the amidating enzyme.' findings: - statement: The PAL catalytic core is a six-bladed beta-propeller with a three-histidine Zn(II) active site that catalyzes N-dealkylation of the peptidyl-alpha-hydroxyglycine. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached. It concerns rat PAL, a member of the same PANTHER subfamily as the quail protein. - id: PMID:26667899 title: '60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C.' findings: - statement: PAM is the only known peptide-amidating enzyme, a type 1 membrane protein with PHM and PAL in its luminal domain. Splice forms PAM-1, PAM-2 (no linker between PHM and PAL) and PAM-3 (soluble) exist. Soluble PHM and PAL are co-secreted with peptides, and the cytosolic domain directs trafficking to the TGN and secretory granules. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Review by the Eipper/Mains group, full text cached. Supports the gene-level claims and the PAM-2 interpretation of this exon-16-lacking model. - id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md title: Quail A0A8C2TBA7 vs human PAM P19021 alignment (re-review) findings: - statement: The quail model lacks the 107-residue exon-16 linker (PAM-2-like) but keeps the transmembrane and cytosolic segments and every Cu, Zn and Ca ligand of human PAM. - id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-deep-research-falcon.md title: Falcon deep research on quail PAM reference_review: relevance: MEDIUM correctness: UNVERIFIED review_notes: LLM-generated literature summary without PMIDs. It is used only for corroborating quotes; the load-bearing claims are quoted from primary PMIDs. - id: file:COTJA/A0A8C2TBA7/A0A8C2TBA7-uniprot.txt title: UniProtKB A0A8C2TBA7 record reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Only PIRSR cofactor statements and Phobius/SignalP features are quoted. The ARBA-derived catalytic-activity, subcellular-location and EC lines are no longer cited as support. suggested_questions: - question: Which PAM splice forms are expressed in quail neuroendocrine tissues, and is the exon-16-skipped (PAM-2-like) model A0A8C2TBA7 a major transcript?