{"gene":"ADGB","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2011,"finding":"Androglobin (ADGB) was identified as a chimeric protein with a unique modular architecture comprising an N-terminal calpain-like domain homologous to the catalytic domain II of the large subunit of human calpain-7, an internal circularly permuted globin domain, and an IQ calmodulin-binding motif. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom.","method":"Comparative genomics, recombinant protein expression, UV-vis absorption spectroscopy","journal":"Molecular biology and evolution","confidence":"High","confidence_rationale":"Tier 1 — recombinant protein biochemical characterization with spectroscopic validation of heme hexacoordination; foundational discovery paper with 98 citations","pmids":["22115833"],"is_preprint":false},{"year":2022,"finding":"Adgb knockout male mice are infertile and display impaired maturation of elongating spermatids, abnormal sperm shape, ultrastructural defects in microtubule and mitochondrial organization, multiple flagellar malformations (coiled, bifid, shortened flagella), and erratic acrosomal development. Septin 10 (Sept10) was identified as an interactor of Adgb via immunoprecipitation and mass spectrometry, confirmed by reciprocal co-immunoprecipitation in vivo using testis lysates and in vitro. Absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. In vitro data suggest Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner.","method":"Knockout mouse model, immunoprecipitation, mass spectrometry, reciprocal co-immunoprecipitation, electron microscopy, in vitro proteolysis assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — KO with defined cellular phenotype plus reciprocal Co-IP confirmed in vivo and in vitro, multiple orthogonal methods in a single study","pmids":["35700329"],"is_preprint":false},{"year":2021,"finding":"In the model ciliate Tetrahymena thermophila, Adgb/androglobin is a component of the C1b/C1f supercomplex of the ciliary central apparatus. Loss of Adgb caused only minor alterations in ciliary motility, whereas loss of Spef2A or Cfap69 led to loss of the entire C1b projection and abnormal vortex motion of cilia.","method":"Gene deletion in Tetrahymena thermophila, ciliome proteomics, ciliary motility assays, electron microscopy","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined ciliary phenotype in model organism ortholog, but only minor motility effect attributed to Adgb itself","pmids":["34083607"],"is_preprint":false},{"year":2023,"finding":"ADGB variants identified in infertile men disrupt the binding of ADGB to calmodulin. Mass spectrometry identified 42 candidate interacting proteins involved in sperm assembly and flagella formation; CFAP69 and SPEF2 were confirmed to bind ADGB by co-immunoprecipitation.","method":"Whole-exome sequencing, Sanger sequencing, mass spectrometry, co-immunoprecipitation, electron microscopy","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP confirmation of CFAP69 and SPEF2 interactions; calmodulin binding disruption by pathogenic variants demonstrated functionally","pmids":["36995441"],"is_preprint":false},{"year":2023,"finding":"FOXJ1 activates the ADGB promoter in transactivation assays in vitro, placing ADGB as a downstream transcriptional target of FOXJ1 in ciliated cells. A truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) fails to activate the ADGB promoter.","method":"Transactivation/luciferase reporter assay in vitro, functional variant analysis","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct promoter activation assay with loss-of-function variant as control; single study","pmids":["37158461"],"is_preprint":false},{"year":2024,"finding":"Calmodulin (CaM) interacts with the ADGB globin domain via the IQ motif. Using fluorescently labeled CaM mutants (G41C and G114C), greater fluorescence quenching was observed for the N-lobe label (Cys41) due to energy transfer to the heme group, consistent with structural modeling of the Adgb-CaM complex. Binding of CaM enhanced the nitrite reductase activity of Adgb, as shown by UV-vis kinetic studies.","method":"Recombinant protein expression, fluorescence quenching assay, AlphaFold3/HDOCK structural modeling, UV-vis kinetic assay","journal":"RSC chemical biology","confidence":"Medium","confidence_rationale":"Tier 1/2 — in vitro biochemical assay demonstrating CaM-enhanced enzymatic activity; single lab, moderate evidence","pmids":["39719941"],"is_preprint":false},{"year":2025,"finding":"RFX3 regulates ADGB promoter-driven luciferase activity and endogenous ADGB expression levels, identifying RFX3 as a transcriptional regulator of ADGB. Stable overexpression of ADGB in A549 lung cancer cells leads to transcriptomic changes indicating increased cell motility and restructuring of the extracellular matrix.","method":"Promoter-luciferase reporter assay, endogenous gene expression measurement, stable overexpression with transcriptome analysis (RNA-seq)","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2/3 — luciferase assay plus endogenous expression validation for RFX3; transcriptomic OE data with no direct mechanistic follow-up for motility","pmids":["41138754"],"is_preprint":false},{"year":2024,"finding":"ADGB localizes to the acrosome and flagella of spermatogenic cells in human and mouse testes. Co-immunoprecipitation confirmed TTC29 and CFAP47 as interacting proteins of ADGB in sperm. Patients with bi-allelic loss-of-function ADGB variants display multiple acrosome and flagellum malformations including acrosome loss, disorganized mitochondrial sheath, and disrupted '9+2' axonemal microtubule structure.","method":"Whole-exome sequencing, immunofluorescence localization, co-immunoprecipitation, transmission electron microscopy","journal":"Andrology","confidence":"Medium","confidence_rationale":"Tier 2/3 — Co-IP confirms novel interactors TTC29 and CFAP47; immunofluorescence localization tied to functional spermatogenesis defects","pmids":["38385883"],"is_preprint":false},{"year":2026,"finding":"Co-immunoprecipitation in sperm from a patient with ADGB loss-of-function confirmed TTC29 and CFAP47 as interacting proteins of ADGB. ADGB was found to be highly expressed after puberty in human and mouse testes and localizes to the acrosome and flagella of spermatogenic cells.","method":"Co-immunoprecipitation, immunofluorescence staining, Western blot, transmission electron microscopy","journal":"Sichuan da xue xue bao. Yi xue ban","confidence":"Low","confidence_rationale":"Tier 3 — single lab, co-IP without reciprocal confirmation; overlaps with prior findings","pmids":["41834962"],"is_preprint":false}],"current_model":"ADGB (androglobin) is a chimeric globin protein with an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; it is essential for male fertility by supporting sperm head shaping, acrosome development, and flagellum formation through interaction with septin 10 (Sept10), CFAP69, SPEF2, TTC29, and CFAP47, and its globin domain possesses calmodulin-regulated nitrite reductase activity and participates in the ciliary central apparatus C1b/C1f supercomplex."},"narrative":{"teleology":[{"year":2011,"claim":"Identification of ADGB as a chimeric globin with unprecedented domain architecture—calpain-like, circularly permuted globin, and IQ motif—established it as a new member of the globin superfamily and raised the question of what biological role such a fusion protein serves.","evidence":"Comparative genomics and recombinant expression with UV-vis spectroscopy confirming hexacoordinated heme","pmids":["22115833"],"confidence":"High","gaps":["No in vivo function assigned","Enzymatic activity of the globin domain not tested","Role of the calpain-like domain unknown"]},{"year":2021,"claim":"Placement of the ADGB ortholog within the C1b/C1f supercomplex of the ciliary central apparatus linked the protein to motile cilia biology, though its individual contribution to motility was minor in Tetrahymena.","evidence":"Gene deletion in Tetrahymena thermophila with ciliome proteomics and motility assays","pmids":["34083607"],"confidence":"Medium","gaps":["Mammalian ciliary role not demonstrated","Precise position within the C1b/C1f supercomplex unresolved","Functional redundancy with other C1b components not tested"]},{"year":2022,"claim":"The Adgb knockout mouse demonstrated that ADGB is essential for male fertility, revealing roles in manchette function, acrosome development, and flagellum formation, and identified septin 10 as a direct interactor whose localization depends on ADGB.","evidence":"Knockout mouse with reciprocal co-IP in vivo and in vitro, electron microscopy, and calmodulin-dependent proteolysis assay","pmids":["35700329"],"confidence":"High","gaps":["Calpain-like domain's catalytic competence in vivo not proven","Mechanism by which ADGB regulates Sept10 localization and proteolysis remains incomplete","Female reproductive or somatic phenotypes not explored"]},{"year":2023,"claim":"Human genetic and biochemical studies expanded the ADGB interactome to include CFAP69 and SPEF2 and showed that pathogenic ADGB variants disrupt calmodulin binding, directly linking calmodulin interaction to human male infertility.","evidence":"Whole-exome sequencing of infertile men, co-immunoprecipitation, and calmodulin-binding assays","pmids":["36995441"],"confidence":"Medium","gaps":["Functional consequence of disrupted CaM binding on enzymatic activity not tested in patient-derived cells","CFAP69 and SPEF2 binding sites on ADGB not mapped","No rescue experiment to confirm causality of identified variants"]},{"year":2023,"claim":"FOXJ1 was identified as a transcriptional activator of ADGB, placing ADGB downstream of the master ciliogenesis regulatory program.","evidence":"Promoter-luciferase transactivation assay with wild-type and truncating FOXJ1 variant","pmids":["37158461"],"confidence":"Medium","gaps":["Endogenous ChIP-based confirmation of FOXJ1 binding at the ADGB promoter lacking","In vivo requirement of FOXJ1 for ADGB expression not shown"]},{"year":2024,"claim":"Calmodulin was shown to bind the ADGB globin domain via the IQ motif and enhance its nitrite reductase activity, providing the first defined enzymatic function for androglobin.","evidence":"Recombinant protein fluorescence quenching and UV-vis kinetic assays with structural modeling","pmids":["39719941"],"confidence":"Medium","gaps":["Physiological relevance of nitrite reductase activity in spermatogenesis not demonstrated","No mutagenesis of catalytic residues to confirm mechanism","Kinetic parameters under physiological conditions not established"]},{"year":2024,"claim":"Localization of ADGB to the acrosome and flagella of human and mouse spermatogenic cells was established, and TTC29 and CFAP47 were confirmed as additional interactors, while bi-allelic ADGB loss-of-function variants in patients recapitulated mouse knockout phenotypes.","evidence":"Immunofluorescence, co-immunoprecipitation, whole-exome sequencing, and transmission electron microscopy of patient sperm","pmids":["38385883"],"confidence":"Medium","gaps":["Direct stoichiometric complex between ADGB and axonemal partners not reconstituted","Whether ADGB acts as a structural scaffold versus enzymatic regulator in the flagellum is unresolved"]},{"year":2025,"claim":"RFX3 was identified as a second transcriptional regulator of ADGB, and overexpression studies linked ADGB to cell motility and ECM remodeling gene programs beyond spermatogenesis.","evidence":"Promoter-luciferase assay, endogenous expression measurement, and RNA-seq of ADGB-overexpressing A549 cells","pmids":["41138754"],"confidence":"Medium","gaps":["Motility and ECM phenotypes inferred from transcriptomics only, not validated functionally","Somatic relevance of ADGB expression outside the testis not confirmed in vivo"]},{"year":null,"claim":"Major open questions remain: the catalytic competence and substrates of the calpain-like domain, the physiological role of nitrite reductase activity during spermiogenesis, and whether ADGB plays any functional role in somatic ciliated tissues or non-ciliated contexts.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Calpain-like domain substrate specificity and catalytic activity not biochemically defined","No structural model of full-length ADGB experimentally validated","In vivo significance of NO/nitrite signaling by ADGB unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,5]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2,7]}],"pathway":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2,7]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[1,3,7]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[2]}],"complexes":["C1b/C1f ciliary central apparatus supercomplex"],"partners":["SEPT10","CFAP69","SPEF2","TTC29","CFAP47","CALM1"],"other_free_text":[]},"mechanistic_narrative":"ADGB (androglobin) is a chimeric globin essential for male fertility, functioning at the intersection of ciliary/flagellar assembly, acrosome biogenesis, and redox biochemistry in spermatogenic cells. The protein contains an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; calmodulin binding to the IQ motif enhances the nitrite reductase activity of the globin domain [PMID:22115833, PMID:39719941]. During spermiogenesis, ADGB interacts with septin 10, CFAP69, SPEF2, TTC29, and CFAP47 to support manchette-mediated sperm head shaping, acrosome development, and flagellum assembly, and its loss in knockout mice or in men with bi-allelic loss-of-function variants causes male infertility with multiple morphological abnormalities of the sperm flagella and acrosome [PMID:35700329, PMID:38385883, PMID:36995441]. ADGB is also a component of the ciliary central apparatus C1b/C1f supercomplex, and its transcription is activated by the ciliogenesis regulators FOXJ1 and RFX3 [PMID:34083607, PMID:37158461, PMID:41138754]."},"prefetch_data":{"uniprot":{"accession":"Q8N7X0","full_name":"Androglobin","aliases":[],"length_aa":1667,"mass_kda":189.7,"function":"Probable chimeric globin with a bis-histidyl six-coordinate heme-iron atom through which it could bind dioxygen, carbon monoxide and nitric oxide (PubMed:22115833). Required for sperm flagellum formation and maturation of elongating spermatids, thus playing an essential role in male fertility (PubMed:36995441)","subcellular_location":"Cell projection, cilium, flagellum","url":"https://www.uniprot.org/uniprotkb/Q8N7X0/entry"},"depmap":{"release":"DepMap","has_data":false,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ADGB"},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ADGB","total_profiled":1310},"omim":[{"mim_id":"614630","title":"ANDROGLOBIN; ADGB","url":"https://www.omim.org/entry/614630"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mid piece","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":4.7},{"tissue":"fallopian tube","ntpm":7.1},{"tissue":"testis","ntpm":24.4}],"url":"https://www.proteinatlas.org/search/ADGB"},"hgnc":{"alias_symbol":["FLJ23121","dJ408K24.1","CAPN16"],"prev_symbol":["C6orf103"]},"alphafold":{"accession":"Q8N7X0","domains":[{"cath_id":"-","chopping":"46-62_79-265","consensus_level":"medium","plddt":86.0416,"start":46,"end":265},{"cath_id":"3.90.70.10","chopping":"273-299_413-468_503-515_629-654","consensus_level":"medium","plddt":84.2771,"start":273,"end":654},{"cath_id":"2.60.120.380","chopping":"655-698_707-784","consensus_level":"medium","plddt":87.4215,"start":655,"end":784},{"cath_id":"-","chopping":"785-893_906-982","consensus_level":"medium","plddt":83.3914,"start":785,"end":982},{"cath_id":"2.60.120.380","chopping":"1103-1178_1251-1275_1356-1389","consensus_level":"medium","plddt":88.3827,"start":1103,"end":1389}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N7X0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N7X0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N7X0-F1-predicted_aligned_error_v6.png","plddt_mean":66.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ADGB","jax_strain_url":"https://www.jax.org/strain/search?query=ADGB"},"sequence":{"accession":"Q8N7X0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N7X0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N7X0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N7X0"}},"corpus_meta":[{"pmid":"22115833","id":"PMC_22115833","title":"Androglobin: 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The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom.\",\n      \"method\": \"Comparative genomics, recombinant protein expression, UV-vis absorption spectroscopy\",\n      \"journal\": \"Molecular biology and evolution\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — recombinant protein biochemical characterization with spectroscopic validation of heme hexacoordination; foundational discovery paper with 98 citations\",\n      \"pmids\": [\"22115833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Adgb knockout male mice are infertile and display impaired maturation of elongating spermatids, abnormal sperm shape, ultrastructural defects in microtubule and mitochondrial organization, multiple flagellar malformations (coiled, bifid, shortened flagella), and erratic acrosomal development. Septin 10 (Sept10) was identified as an interactor of Adgb via immunoprecipitation and mass spectrometry, confirmed by reciprocal co-immunoprecipitation in vivo using testis lysates and in vitro. Absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. In vitro data suggest Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner.\",\n      \"method\": \"Knockout mouse model, immunoprecipitation, mass spectrometry, reciprocal co-immunoprecipitation, electron microscopy, in vitro proteolysis assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined cellular phenotype plus reciprocal Co-IP confirmed in vivo and in vitro, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"35700329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In the model ciliate Tetrahymena thermophila, Adgb/androglobin is a component of the C1b/C1f supercomplex of the ciliary central apparatus. Loss of Adgb caused only minor alterations in ciliary motility, whereas loss of Spef2A or Cfap69 led to loss of the entire C1b projection and abnormal vortex motion of cilia.\",\n      \"method\": \"Gene deletion in Tetrahymena thermophila, ciliome proteomics, ciliary motility assays, electron microscopy\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined ciliary phenotype in model organism ortholog, but only minor motility effect attributed to Adgb itself\",\n      \"pmids\": [\"34083607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ADGB variants identified in infertile men disrupt the binding of ADGB to calmodulin. Mass spectrometry identified 42 candidate interacting proteins involved in sperm assembly and flagella formation; CFAP69 and SPEF2 were confirmed to bind ADGB by co-immunoprecipitation.\",\n      \"method\": \"Whole-exome sequencing, Sanger sequencing, mass spectrometry, co-immunoprecipitation, electron microscopy\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP confirmation of CFAP69 and SPEF2 interactions; calmodulin binding disruption by pathogenic variants demonstrated functionally\",\n      \"pmids\": [\"36995441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FOXJ1 activates the ADGB promoter in transactivation assays in vitro, placing ADGB as a downstream transcriptional target of FOXJ1 in ciliated cells. A truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) fails to activate the ADGB promoter.\",\n      \"method\": \"Transactivation/luciferase reporter assay in vitro, functional variant analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter activation assay with loss-of-function variant as control; single study\",\n      \"pmids\": [\"37158461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Calmodulin (CaM) interacts with the ADGB globin domain via the IQ motif. Using fluorescently labeled CaM mutants (G41C and G114C), greater fluorescence quenching was observed for the N-lobe label (Cys41) due to energy transfer to the heme group, consistent with structural modeling of the Adgb-CaM complex. Binding of CaM enhanced the nitrite reductase activity of Adgb, as shown by UV-vis kinetic studies.\",\n      \"method\": \"Recombinant protein expression, fluorescence quenching assay, AlphaFold3/HDOCK structural modeling, UV-vis kinetic assay\",\n      \"journal\": \"RSC chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro biochemical assay demonstrating CaM-enhanced enzymatic activity; single lab, moderate evidence\",\n      \"pmids\": [\"39719941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFX3 regulates ADGB promoter-driven luciferase activity and endogenous ADGB expression levels, identifying RFX3 as a transcriptional regulator of ADGB. Stable overexpression of ADGB in A549 lung cancer cells leads to transcriptomic changes indicating increased cell motility and restructuring of the extracellular matrix.\",\n      \"method\": \"Promoter-luciferase reporter assay, endogenous gene expression measurement, stable overexpression with transcriptome analysis (RNA-seq)\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — luciferase assay plus endogenous expression validation for RFX3; transcriptomic OE data with no direct mechanistic follow-up for motility\",\n      \"pmids\": [\"41138754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ADGB localizes to the acrosome and flagella of spermatogenic cells in human and mouse testes. Co-immunoprecipitation confirmed TTC29 and CFAP47 as interacting proteins of ADGB in sperm. Patients with bi-allelic loss-of-function ADGB variants display multiple acrosome and flagellum malformations including acrosome loss, disorganized mitochondrial sheath, and disrupted '9+2' axonemal microtubule structure.\",\n      \"method\": \"Whole-exome sequencing, immunofluorescence localization, co-immunoprecipitation, transmission electron microscopy\",\n      \"journal\": \"Andrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — Co-IP confirms novel interactors TTC29 and CFAP47; immunofluorescence localization tied to functional spermatogenesis defects\",\n      \"pmids\": [\"38385883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Co-immunoprecipitation in sperm from a patient with ADGB loss-of-function confirmed TTC29 and CFAP47 as interacting proteins of ADGB. ADGB was found to be highly expressed after puberty in human and mouse testes and localizes to the acrosome and flagella of spermatogenic cells.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence staining, Western blot, transmission electron microscopy\",\n      \"journal\": \"Sichuan da xue xue bao. Yi xue ban\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, co-IP without reciprocal confirmation; overlaps with prior findings\",\n      \"pmids\": [\"41834962\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ADGB (androglobin) is a chimeric globin protein with an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; it is essential for male fertility by supporting sperm head shaping, acrosome development, and flagellum formation through interaction with septin 10 (Sept10), CFAP69, SPEF2, TTC29, and CFAP47, and its globin domain possesses calmodulin-regulated nitrite reductase activity and participates in the ciliary central apparatus C1b/C1f supercomplex.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ADGB (androglobin) is a chimeric globin essential for male fertility, functioning at the intersection of ciliary/flagellar assembly, acrosome biogenesis, and redox biochemistry in spermatogenic cells. The protein contains an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; calmodulin binding to the IQ motif enhances the nitrite reductase activity of the globin domain [PMID:22115833, PMID:39719941]. During spermiogenesis, ADGB interacts with septin 10, CFAP69, SPEF2, TTC29, and CFAP47 to support manchette-mediated sperm head shaping, acrosome development, and flagellum assembly, and its loss in knockout mice or in men with bi-allelic loss-of-function variants causes male infertility with multiple morphological abnormalities of the sperm flagella and acrosome [PMID:35700329, PMID:38385883, PMID:36995441]. ADGB is also a component of the ciliary central apparatus C1b/C1f supercomplex, and its transcription is activated by the ciliogenesis regulators FOXJ1 and RFX3 [PMID:34083607, PMID:37158461, PMID:41138754].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of ADGB as a chimeric globin with unprecedented domain architecture—calpain-like, circularly permuted globin, and IQ motif—established it as a new member of the globin superfamily and raised the question of what biological role such a fusion protein serves.\",\n      \"evidence\": \"Comparative genomics and recombinant expression with UV-vis spectroscopy confirming hexacoordinated heme\",\n      \"pmids\": [\"22115833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No in vivo function assigned\",\n        \"Enzymatic activity of the globin domain not tested\",\n        \"Role of the calpain-like domain unknown\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placement of the ADGB ortholog within the C1b/C1f supercomplex of the ciliary central apparatus linked the protein to motile cilia biology, though its individual contribution to motility was minor in Tetrahymena.\",\n      \"evidence\": \"Gene deletion in Tetrahymena thermophila with ciliome proteomics and motility assays\",\n      \"pmids\": [\"34083607\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mammalian ciliary role not demonstrated\",\n        \"Precise position within the C1b/C1f supercomplex unresolved\",\n        \"Functional redundancy with other C1b components not tested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The Adgb knockout mouse demonstrated that ADGB is essential for male fertility, revealing roles in manchette function, acrosome development, and flagellum formation, and identified septin 10 as a direct interactor whose localization depends on ADGB.\",\n      \"evidence\": \"Knockout mouse with reciprocal co-IP in vivo and in vitro, electron microscopy, and calmodulin-dependent proteolysis assay\",\n      \"pmids\": [\"35700329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Calpain-like domain's catalytic competence in vivo not proven\",\n        \"Mechanism by which ADGB regulates Sept10 localization and proteolysis remains incomplete\",\n        \"Female reproductive or somatic phenotypes not explored\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Human genetic and biochemical studies expanded the ADGB interactome to include CFAP69 and SPEF2 and showed that pathogenic ADGB variants disrupt calmodulin binding, directly linking calmodulin interaction to human male infertility.\",\n      \"evidence\": \"Whole-exome sequencing of infertile men, co-immunoprecipitation, and calmodulin-binding assays\",\n      \"pmids\": [\"36995441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of disrupted CaM binding on enzymatic activity not tested in patient-derived cells\",\n        \"CFAP69 and SPEF2 binding sites on ADGB not mapped\",\n        \"No rescue experiment to confirm causality of identified variants\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"FOXJ1 was identified as a transcriptional activator of ADGB, placing ADGB downstream of the master ciliogenesis regulatory program.\",\n      \"evidence\": \"Promoter-luciferase transactivation assay with wild-type and truncating FOXJ1 variant\",\n      \"pmids\": [\"37158461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Endogenous ChIP-based confirmation of FOXJ1 binding at the ADGB promoter lacking\",\n        \"In vivo requirement of FOXJ1 for ADGB expression not shown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Calmodulin was shown to bind the ADGB globin domain via the IQ motif and enhance its nitrite reductase activity, providing the first defined enzymatic function for androglobin.\",\n      \"evidence\": \"Recombinant protein fluorescence quenching and UV-vis kinetic assays with structural modeling\",\n      \"pmids\": [\"39719941\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Physiological relevance of nitrite reductase activity in spermatogenesis not demonstrated\",\n        \"No mutagenesis of catalytic residues to confirm mechanism\",\n        \"Kinetic parameters under physiological conditions not established\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Localization of ADGB to the acrosome and flagella of human and mouse spermatogenic cells was established, and TTC29 and CFAP47 were confirmed as additional interactors, while bi-allelic ADGB loss-of-function variants in patients recapitulated mouse knockout phenotypes.\",\n      \"evidence\": \"Immunofluorescence, co-immunoprecipitation, whole-exome sequencing, and transmission electron microscopy of patient sperm\",\n      \"pmids\": [\"38385883\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct stoichiometric complex between ADGB and axonemal partners not reconstituted\",\n        \"Whether ADGB acts as a structural scaffold versus enzymatic regulator in the flagellum is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RFX3 was identified as a second transcriptional regulator of ADGB, and overexpression studies linked ADGB to cell motility and ECM remodeling gene programs beyond spermatogenesis.\",\n      \"evidence\": \"Promoter-luciferase assay, endogenous expression measurement, and RNA-seq of ADGB-overexpressing A549 cells\",\n      \"pmids\": [\"41138754\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Motility and ECM phenotypes inferred from transcriptomics only, not validated functionally\",\n        \"Somatic relevance of ADGB expression outside the testis not confirmed in vivo\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major open questions remain: the catalytic competence and substrates of the calpain-like domain, the physiological role of nitrite reductase activity during spermiogenesis, and whether ADGB plays any functional role in somatic ciliated tissues or non-ciliated contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Calpain-like domain substrate specificity and catalytic activity not biochemically defined\",\n        \"No structural model of full-length ADGB experimentally validated\",\n        \"In vivo significance of NO/nitrite signaling by ADGB unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [1, 3, 7]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"C1b/C1f ciliary central apparatus supercomplex\"\n    ],\n    \"partners\": [\n      \"SEPT10\",\n      \"CFAP69\",\n      \"SPEF2\",\n      \"TTC29\",\n      \"CFAP47\",\n      \"CALM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I need to fix the pathway entries - they should use Reactome IDs, not GO IDs. Let me correct:\n\n```json\n{\n  \"mechanistic_narrative\": \"ADGB (androglobin) is a chimeric globin essential for male fertility, functioning at the intersection of ciliary/flagellar assembly, acrosome biogenesis, and redox biochemistry in spermatogenic cells. The protein contains an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; calmodulin binding to the IQ motif enhances the nitrite reductase activity of the globin domain [PMID:22115833, PMID:39719941]. During spermiogenesis, ADGB interacts with septin 10, CFAP69, SPEF2, TTC29, and CFAP47 to support manchette-mediated sperm head shaping, acrosome development, and flagellum assembly, and its loss in knockout mice or in men with bi-allelic loss-of-function variants causes male infertility with multiple morphological abnormalities of the sperm flagella and acrosome [PMID:35700329, PMID:38385883, PMID:36995441]. ADGB is also a component of the ciliary central apparatus C1b/C1f supercomplex, and its transcription is activated by the ciliogenesis regulators FOXJ1 and RFX3 [PMID:34083607, PMID:37158461, PMID:41138754].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of ADGB as a chimeric globin with unprecedented domain architecture—calpain-like, circularly permuted globin, and IQ motif—established it as a new member of the globin superfamily and raised the question of what biological role such a fusion protein serves.\",\n      \"evidence\": \"Comparative genomics and recombinant expression with UV-vis spectroscopy confirming hexacoordinated heme\",\n      \"pmids\": [\"22115833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No in vivo function assigned\",\n        \"Enzymatic activity of the globin domain not tested\",\n        \"Role of the calpain-like domain unknown\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placement of the ADGB ortholog within the C1b/C1f supercomplex of the ciliary central apparatus linked the protein to motile cilia biology, though its individual contribution to motility was minor in Tetrahymena.\",\n      \"evidence\": \"Gene deletion in Tetrahymena thermophila with ciliome proteomics and motility assays\",\n      \"pmids\": [\"34083607\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mammalian ciliary role not demonstrated\",\n        \"Precise position within the C1b/C1f supercomplex unresolved\",\n        \"Functional redundancy with other C1b components not tested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The Adgb knockout mouse demonstrated that ADGB is essential for male fertility, revealing roles in manchette function, acrosome development, and flagellum formation, and identified septin 10 as a direct interactor whose localization depends on ADGB.\",\n      \"evidence\": \"Knockout mouse with reciprocal co-IP in vivo and in vitro, electron microscopy, and calmodulin-dependent proteolysis assay\",\n      \"pmids\": [\"35700329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Calpain-like domain's catalytic competence in vivo not proven\",\n        \"Mechanism by which ADGB regulates Sept10 localization and proteolysis remains incomplete\",\n        \"Female reproductive or somatic phenotypes not explored\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Human genetic and biochemical studies expanded the ADGB interactome to include CFAP69 and SPEF2 and showed that pathogenic ADGB variants disrupt calmodulin binding, directly linking calmodulin interaction to human male infertility.\",\n      \"evidence\": \"Whole-exome sequencing of infertile men, co-immunoprecipitation, and calmodulin-binding assays\",\n      \"pmids\": [\"36995441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of disrupted CaM binding on enzymatic activity not tested in patient-derived cells\",\n        \"CFAP69 and SPEF2 binding sites on ADGB not mapped\",\n        \"No rescue experiment to confirm causality of identified variants\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"FOXJ1 was identified as a transcriptional activator of ADGB, placing ADGB downstream of the master ciliogenesis regulatory program.\",\n      \"evidence\": \"Promoter-luciferase transactivation assay with wild-type and truncating FOXJ1 variant\",\n      \"pmids\": [\"37158461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Endogenous ChIP-based confirmation of FOXJ1 binding at the ADGB promoter lacking\",\n        \"In vivo requirement of FOXJ1 for ADGB expression not shown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Calmodulin was shown to bind the ADGB globin domain via the IQ motif and enhance its nitrite reductase activity, providing the first defined enzymatic function for androglobin.\",\n      \"evidence\": \"Recombinant protein fluorescence quenching and UV-vis kinetic assays with structural modeling\",\n      \"pmids\": [\"39719941\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Physiological relevance of nitrite reductase activity in spermatogenesis not demonstrated\",\n        \"No mutagenesis of catalytic residues to confirm mechanism\",\n        \"Kinetic parameters under physiological conditions not established\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Localization of ADGB to the acrosome and flagella of human and mouse spermatogenic cells was established, and TTC29 and CFAP47 were confirmed as additional interactors, while bi-allelic ADGB loss-of-function variants in patients recapitulated mouse knockout phenotypes.\",\n      \"evidence\": \"Immunofluorescence, co-immunoprecipitation, whole-exome sequencing, and transmission electron microscopy of patient sperm\",\n      \"pmids\": [\"38385883\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct stoichiometric complex between ADGB and axonemal partners not reconstituted\",\n        \"Whether ADGB acts as a structural scaffold versus enzymatic regulator in the flagellum is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RFX3 was identified as a second transcriptional regulator of ADGB, and overexpression studies linked ADGB to cell motility and ECM remodeling gene programs beyond spermatogenesis.\",\n      \"evidence\": \"Promoter-luciferase assay, endogenous expression measurement, and RNA-seq of ADGB-overexpressing A549 cells\",\n      \"pmids\": [\"41138754\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Motility and ECM phenotypes inferred from transcriptomics only, not validated functionally\",\n        \"Somatic relevance of ADGB expression outside the testis not confirmed in vivo\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major open questions remain: the catalytic competence and substrates of the calpain-like domain, the physiological role of nitrite reductase activity during spermiogenesis, and whether ADGB plays any functional role in somatic ciliated tissues or non-ciliated contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Calpain-like domain substrate specificity and catalytic activity not biochemically defined\",\n        \"No structural model of full-length ADGB experimentally validated\",\n        \"In vivo significance of NO/nitrite signaling by ADGB unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [1, 3, 7]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"C1b/C1f ciliary central apparatus supercomplex\"\n    ],\n    \"partners\": [\n      \"SEPT10\",\n      \"CFAP69\",\n      \"SPEF2\",\n      \"TTC29\",\n      \"CFAP47\",\n      \"CALM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}