{"gene":"OPALIN","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2008,"finding":"OPALIN (Tmem10) is a transmembrane glycoprotein selectively expressed by oligodendrocytes in the CNS, localized at the cell soma, processes, and myelinated internodes, with concentration at paranodal loops; it is absent from peripheral nervous system myelinating Schwann cells.","method":"In situ hybridization, RT-PCR, developmental immunofluorescence, myelinating spinal cord cultures","journal":"Glia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal localization methods (ISH, RT-PCR, immunofluorescence) in a single study establishing CNS-specific expression and subcellular localization","pmids":["18571792"],"is_preprint":false},{"year":2008,"finding":"OPALIN is a type I transmembrane sialylglycoprotein with a short N-terminal extracellular domain (aa 1–30), a transmembrane domain (aa 31–53), and a long C-terminal intracellular domain (aa 54–143). It contains N-glycans at Asn-6 and Asn-12 and an O-glycan (bearing sialic acids) at Thr-14 in the extracellular domain; site-directed mutation of these glycan sites impaired cell-surface localization of OPALIN. Immunogold electron microscopy confirmed localization to paranodal loop membranes.","method":"Biochemical characterization, enzymatic deglycosylation, site-directed mutagenesis, immunogold electron microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-directed mutagenesis with functional readout (cell surface localization), enzymatic deglycosylation, and immunogold EM in a single rigorous study","pmids":["18490449"],"is_preprint":false},{"year":2007,"finding":"An evolutionarily conserved region in the first intron of the Opalin gene acts as an oligodendrocyte-directed transcriptional enhancer. This enhancer contains binding sites for Myt1 and CREB; overexpression of Myt1, LIF treatment, or cAMP analog (CREB activator) enhanced endogenous Opalin expression. Deletion analysis confirmed subdomains critical for Opalin expression.","method":"Transgenic mice enhancer assay, cotransfection/reporter assay in Oli-neu cells, deletion analysis, Myt1 overexpression","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — enhancer activity validated in transgenic mice and cell line with deletion analysis and cotransfection experiments identifying specific regulatory elements and trans-factors","pmids":["17442045"],"is_preprint":false},{"year":2019,"finding":"TMEM10 (OPALIN) promotes oligodendrocyte terminal differentiation: constitutive overexpression in Oli-neu cells upregulates myelin-associated genes MAG, CNP, and CGT, whereas knockdown in primary OPCs reduces CNP mRNA and decreases the percentage of MBP-positive oligodendrocytes differentiating in vitro. Ectopic TMEM10 expression increases process extension and branching; blocking TMEM10 expression causes abnormal oligodendrocyte morphology.","method":"Overexpression in oligodendroglial cell line (Oli-neu), siRNA knockdown in primary OPCs, qRT-PCR, immunofluorescence","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain- and loss-of-function in two cell systems with defined molecular readouts, single lab","pmids":["30837646"],"is_preprint":false},{"year":2024,"finding":"OPALIN is a receptor for the secreted protein LGI1 on oligodendrocyte membranes. LGI1–OPALIN interaction was identified by LGI1-FLAG affinity chromatography of mouse brain lysates followed by mass spectrometry. Conditional knockout of OPALIN in the oligodendrocyte lineage causes hypomyelination and white matter abnormalities phenocopying LGI1 deficiency, with downregulation of transcription factors Sox10 and Olig2. Virus-mediated re-expression of OPALIN rescues myelination in Opalin cKO mice, whereas re-expression of the LGI1-binding-deficient mutant OPALIN_K23A/D26A fails to rescue hypomyelination.","method":"Affinity chromatography (LGI1-FLAG) + mass spectrometry, conditional knockout mice, biochemical analysis of Sox10/Olig2, viral rescue experiments with OPALIN_K23A/D26A mutant","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — binding partner identified by affinity chromatography + MS, phenotype confirmed by cKO, mechanistic rescue with wild-type vs. binding-deficient mutant in multiple orthogonal experiments","pmids":["39083419"],"is_preprint":false},{"year":2016,"finding":"OPALIN knockout mice show no obvious abnormalities in major myelin protein composition, oligodendrocyte lineage markers, domain organization of myelinated axons, or paranodal loop fine structure by electron microscopy under conventional conditions, indicating OPALIN is not essential for basic CNS myelination. However, Opalin-/- mice display a subtle but significant reduction in exploratory activity in a novel environment.","method":"Opalin gene knockout mice, electron microscopy (optic nerve), Western blot, immunofluorescence, behavioral testing","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complete knockout with electron microscopy and molecular analysis; negative finding for myelination, subtle positive behavioral phenotype; single lab","pmids":["27855200"],"is_preprint":false},{"year":2021,"finding":"OPALIN protein turnover is regulated by 2-hydroxylated sphingolipids: in Fa2h-/- mice (lacking fatty acid 2-hydroxylase), OPALIN protein accumulates ~6-fold in CNS myelin without change in Opalin mRNA, indicating decreased protein turnover. In CHO cells, OPALIN half-life is reduced when 2-hydroxylated sulfatide is present. OPALIN degradation is inhibited by lysosomal inhibitors but not by proteasome inhibitors, placing OPALIN degradation in the lysosomal pathway.","method":"Quantitative proteomics (tandem mass tag labeling) of purified myelin, Western blot, RT-PCR, CHO cell OPALIN half-life assay with lysosomal and proteasomal inhibitors","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (proteomics, Western blot, cell-based half-life assay, pharmacological inhibition) in a single lab identifying lysosomal degradation mechanism","pmids":["33215680"],"is_preprint":false},{"year":2014,"finding":"OPALIN undergoes age-dependent hypersialylation of O-glycans in the postnatal mouse brain, resulting in an increase in apparent molecular weight with aging. Additionally, immunoreactivity redistributes regionally with age, decreasing in cerebellar white matter relative to corpus callosum in adult mice.","method":"Western blot with enzymatic deglycosylation, immunofluorescence at multiple postnatal time points","journal":"Neuroscience letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, descriptive biochemical characterization with limited functional follow-up","pmids":["25153515"],"is_preprint":false}],"current_model":"OPALIN (TMEM10) is a CNS-specific type I transmembrane sialylglycoprotein localized to oligodendrocyte paranodal loop membranes, where it acts as a receptor for the secreted protein LGI1; LGI1–OPALIN interaction (mediated by residues K23/D26) promotes oligodendrocyte differentiation and myelination by sustaining expression of transcription factors Sox10 and Olig2, while its N- and O-glycosylation (regulated by 2-hydroxylated sphingolipids and subject to age-dependent hypersialylation) controls its cell-surface localization and lysosomal turnover, and its transcription is driven by a first-intron enhancer activated by Myt1, LIF, and cAMP/CREB signaling."},"narrative":{"mechanistic_narrative":"OPALIN (TMEM10) is a CNS-specific type I transmembrane sialylglycoprotein selectively expressed by oligodendrocytes and concentrated at paranodal loop membranes, where it acts in oligodendrocyte differentiation and myelination [PMID:18571792, PMID:18490449]. It has a short N-terminal extracellular domain bearing N-glycans at Asn-6/Asn-12 and a sialylated O-glycan at Thr-14, and these glycosylation sites are required for proper cell-surface localization [PMID:18490449]. OPALIN functions as an oligodendrocyte-membrane receptor for the secreted protein LGI1; this interaction, mediated by extracellular residues K23/D26, sustains the transcription factors Sox10 and Olig2, and conditional deletion of OPALIN causes hypomyelination and white matter abnormalities that phenocopy LGI1 deficiency, with viral re-expression of wild-type but not the LGI1-binding-deficient K23A/D26A mutant rescuing the defect [PMID:39083419]. Consistent with this role, OPALIN promotes oligodendrocyte terminal differentiation, process extension and branching, and induction of myelin-associated genes in gain- and loss-of-function assays [PMID:30837646]. Its abundance is controlled post-translationally through a lysosomal degradation pathway that is accelerated by 2-hydroxylated sphingolipids [PMID:33215680], and its transcription is driven by a first-intron oligodendrocyte enhancer responsive to Myt1, LIF, and cAMP/CREB signaling [PMID:17442045]. Conventional OPALIN knockout mice nonetheless show grossly normal CNS myelin architecture with only a subtle reduction in exploratory behavior, indicating it is not strictly required for basic myelin assembly under standard conditions [PMID:27855200].","teleology":[{"year":2007,"claim":"Before any transcriptional control was known, it was unclear how Opalin achieves oligodendrocyte-restricted expression; identifying a lineage-directed enhancer and its trans-factors established the regulatory logic of its expression.","evidence":"Transgenic mouse enhancer assay and reporter cotransfection in Oli-neu cells with deletion analysis and Myt1 overexpression/LIF/cAMP stimulation","pmids":["17442045"],"confidence":"High","gaps":["Does not link enhancer activity to a downstream functional output of OPALIN","Direct binding of Myt1 and CREB to the enhancer not biochemically resolved beyond site presence"]},{"year":2008,"claim":"The cellular and subcellular distribution of OPALIN was undefined; demonstrating CNS-restricted oligodendrocyte expression with paranodal concentration and absence from Schwann cells positioned it as a candidate CNS myelin component.","evidence":"In situ hybridization, RT-PCR, developmental immunofluorescence, and myelinating spinal cord cultures","pmids":["18571792"],"confidence":"Medium","gaps":["Localization alone does not establish a molecular function","No binding partner or pathway identified"]},{"year":2008,"claim":"The topology and post-translational modification of OPALIN were unknown; defining it as a type I transmembrane sialylglycoprotein and showing that defined N-/O-glycosylation sites are required for surface delivery established the structural basis of its membrane presentation.","evidence":"Biochemical characterization, enzymatic deglycosylation, site-directed mutagenesis with surface-localization readout, and immunogold electron microscopy","pmids":["18490449"],"confidence":"High","gaps":["The functional role of the cytoplasmic C-terminal domain not addressed","Did not connect glycosylation to a signaling or differentiation function"]},{"year":2014,"claim":"Whether OPALIN modification changes over development was unexamined; documenting age-dependent O-glycan hypersialylation and regional redistribution suggested its glycoform is dynamically regulated with maturation.","evidence":"Western blot with enzymatic deglycosylation and immunofluorescence across postnatal time points in mouse brain","pmids":["25153515"],"confidence":"Low","gaps":["Descriptive only with limited functional follow-up","Single lab; functional consequence of hypersialylation not established"]},{"year":2016,"claim":"It was unknown whether OPALIN is essential for myelination; a complete knockout showing grossly normal myelin but reduced exploratory behavior established that it is dispensable for basic myelin assembly while hinting at a non-structural role.","evidence":"Opalin knockout mice analyzed by electron microscopy, Western blot, immunofluorescence, and behavioral testing","pmids":["27855200"],"confidence":"Medium","gaps":["Negative myelin phenotype may reflect compensation or non-stressed conditions","Mechanistic basis of the behavioral phenotype not determined"]},{"year":2019,"claim":"The cell-autonomous contribution of OPALIN to oligodendrocyte maturation was unclear; reciprocal gain- and loss-of-function showed it promotes terminal differentiation, morphology, and myelin gene expression, establishing a pro-differentiation role.","evidence":"Overexpression in Oli-neu cells and siRNA knockdown in primary OPCs with qRT-PCR and immunofluorescence","pmids":["30837646"],"confidence":"Medium","gaps":["Mechanism downstream of OPALIN not defined at this stage","In vitro systems; relationship to in vivo dispensability in knockout unresolved"]},{"year":2024,"claim":"OPALIN lacked a defined molecular partner and signaling mechanism; identifying it as the oligodendrocyte receptor for LGI1 and showing K23/D26-dependent rescue of hypomyelination established a ligand-receptor axis sustaining Sox10/Olig2 expression.","evidence":"LGI1-FLAG affinity chromatography plus mass spectrometry, conditional knockout mice, Sox10/Olig2 analysis, and viral rescue with wild-type versus K23A/D26A binding-deficient OPALIN","pmids":["39083419"],"confidence":"High","gaps":["Intracellular signal transduction linking LGI1-OPALIN binding to Sox10/Olig2 not delineated","Reconciliation with the mild conventional knockout phenotype not fully resolved"]},{"year":null,"claim":"How LGI1-OPALIN binding is transduced through the cytoplasmic domain to regulate Sox10/Olig2 transcription, and how glycosylation/lysosomal turnover tunes receptor availability, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No downstream signaling effector of OPALIN identified","Link between glycoform/lysosomal turnover and LGI1 receptor function untested","No structural model of the LGI1-OPALIN interface"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[4]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["LGI1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96PE5","full_name":"Opalin","aliases":["Oligodendrocytic myelin paranodal and inner loop protein","Transmembrane protein 10"],"length_aa":141,"mass_kda":15.7,"function":"Central nervous system-specific myelin protein that increase myelin genes expression during oligodendrocyte differentiation. Promotes oligodendrocyte terminal differentiation","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q96PE5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OPALIN","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/OPALIN","total_profiled":1310},"omim":[{"mim_id":"617200","title":"OLIGODENDROCYTIC MYELIN PARANODAL AND INNER LOOP PROTEIN; OPALIN","url":"https://www.omim.org/entry/617200"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":139.3}],"url":"https://www.proteinatlas.org/search/OPALIN"},"hgnc":{"alias_symbol":["TMP10","HTMP10"],"prev_symbol":["TMEM10"]},"alphafold":{"accession":"Q96PE5","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96PE5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96PE5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96PE5-F1-predicted_aligned_error_v6.png","plddt_mean":62.47},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OPALIN","jax_strain_url":"https://www.jax.org/strain/search?query=OPALIN"},"sequence":{"accession":"Q96PE5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96PE5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96PE5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96PE5"}},"corpus_meta":[{"pmid":"18571792","id":"PMC_18571792","title":"Identification of Tmem10/Opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablation.","date":"2008","source":"Glia","url":"https://pubmed.ncbi.nlm.nih.gov/18571792","citation_count":50,"is_preprint":false},{"pmid":"18490449","id":"PMC_18490449","title":"Opalin, a transmembrane sialylglycoprotein located in the central nervous system myelin paranodal loop membrane.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18490449","citation_count":45,"is_preprint":false},{"pmid":"30837646","id":"PMC_30837646","title":"TMEM10 Promotes Oligodendrocyte Differentiation and is Expressed by Oligodendrocytes in Human Remyelinating Multiple Sclerosis Plaques.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30837646","citation_count":37,"is_preprint":false},{"pmid":"17442045","id":"PMC_17442045","title":"An oligodendrocyte enhancer in a phylogenetically conserved intron region of the mammalian myelin gene Opalin.","date":"2007","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17442045","citation_count":21,"is_preprint":false},{"pmid":"24391449","id":"PMC_24391449","title":"Identification of Tmem10 as a novel late-stage oligodendrocytes marker for detecting hypomyelination.","date":"2013","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/24391449","citation_count":16,"is_preprint":false},{"pmid":"39083419","id":"PMC_39083419","title":"OPALIN is an LGI1 receptor promoting oligodendrocyte differentiation.","date":"2024","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/39083419","citation_count":14,"is_preprint":false},{"pmid":"27855200","id":"PMC_27855200","title":"Mammalian-Specific Central Myelin Protein Opalin Is Redundant for Normal Myelination: Structural and Behavioral Assessments.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27855200","citation_count":14,"is_preprint":false},{"pmid":"33215680","id":"PMC_33215680","title":"Decreased turnover of the CNS myelin protein Opalin in a mouse model of hereditary spastic paraplegia 35.","date":"2021","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33215680","citation_count":9,"is_preprint":false},{"pmid":"25153515","id":"PMC_25153515","title":"Age-dependent redistribution and hypersialylation of the central myelin paranodal loop membrane protein Opalin in the mouse brain.","date":"2014","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/25153515","citation_count":6,"is_preprint":false},{"pmid":"41053007","id":"PMC_41053007","title":"Repeated ablations of mature Tmem10+ oligodendrocytes recapitulates key pathological features of multiple sclerosis with prolonged demyelination.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41053007","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7088,"output_tokens":2399,"usd":0.028625,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9519,"output_tokens":2804,"usd":0.058847,"stage2_stop_reason":"end_turn"},"total_usd":0.087472,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"OPALIN (Tmem10) is a transmembrane glycoprotein selectively expressed by oligodendrocytes in the CNS, localized at the cell soma, processes, and myelinated internodes, with concentration at paranodal loops; it is absent from peripheral nervous system myelinating Schwann cells.\",\n      \"method\": \"In situ hybridization, RT-PCR, developmental immunofluorescence, myelinating spinal cord cultures\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal localization methods (ISH, RT-PCR, immunofluorescence) in a single study establishing CNS-specific expression and subcellular localization\",\n      \"pmids\": [\"18571792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"OPALIN is a type I transmembrane sialylglycoprotein with a short N-terminal extracellular domain (aa 1–30), a transmembrane domain (aa 31–53), and a long C-terminal intracellular domain (aa 54–143). It contains N-glycans at Asn-6 and Asn-12 and an O-glycan (bearing sialic acids) at Thr-14 in the extracellular domain; site-directed mutation of these glycan sites impaired cell-surface localization of OPALIN. Immunogold electron microscopy confirmed localization to paranodal loop membranes.\",\n      \"method\": \"Biochemical characterization, enzymatic deglycosylation, site-directed mutagenesis, immunogold electron microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-directed mutagenesis with functional readout (cell surface localization), enzymatic deglycosylation, and immunogold EM in a single rigorous study\",\n      \"pmids\": [\"18490449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"An evolutionarily conserved region in the first intron of the Opalin gene acts as an oligodendrocyte-directed transcriptional enhancer. This enhancer contains binding sites for Myt1 and CREB; overexpression of Myt1, LIF treatment, or cAMP analog (CREB activator) enhanced endogenous Opalin expression. Deletion analysis confirmed subdomains critical for Opalin expression.\",\n      \"method\": \"Transgenic mice enhancer assay, cotransfection/reporter assay in Oli-neu cells, deletion analysis, Myt1 overexpression\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enhancer activity validated in transgenic mice and cell line with deletion analysis and cotransfection experiments identifying specific regulatory elements and trans-factors\",\n      \"pmids\": [\"17442045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM10 (OPALIN) promotes oligodendrocyte terminal differentiation: constitutive overexpression in Oli-neu cells upregulates myelin-associated genes MAG, CNP, and CGT, whereas knockdown in primary OPCs reduces CNP mRNA and decreases the percentage of MBP-positive oligodendrocytes differentiating in vitro. Ectopic TMEM10 expression increases process extension and branching; blocking TMEM10 expression causes abnormal oligodendrocyte morphology.\",\n      \"method\": \"Overexpression in oligodendroglial cell line (Oli-neu), siRNA knockdown in primary OPCs, qRT-PCR, immunofluorescence\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain- and loss-of-function in two cell systems with defined molecular readouts, single lab\",\n      \"pmids\": [\"30837646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OPALIN is a receptor for the secreted protein LGI1 on oligodendrocyte membranes. LGI1–OPALIN interaction was identified by LGI1-FLAG affinity chromatography of mouse brain lysates followed by mass spectrometry. Conditional knockout of OPALIN in the oligodendrocyte lineage causes hypomyelination and white matter abnormalities phenocopying LGI1 deficiency, with downregulation of transcription factors Sox10 and Olig2. Virus-mediated re-expression of OPALIN rescues myelination in Opalin cKO mice, whereas re-expression of the LGI1-binding-deficient mutant OPALIN_K23A/D26A fails to rescue hypomyelination.\",\n      \"method\": \"Affinity chromatography (LGI1-FLAG) + mass spectrometry, conditional knockout mice, biochemical analysis of Sox10/Olig2, viral rescue experiments with OPALIN_K23A/D26A mutant\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — binding partner identified by affinity chromatography + MS, phenotype confirmed by cKO, mechanistic rescue with wild-type vs. binding-deficient mutant in multiple orthogonal experiments\",\n      \"pmids\": [\"39083419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"OPALIN knockout mice show no obvious abnormalities in major myelin protein composition, oligodendrocyte lineage markers, domain organization of myelinated axons, or paranodal loop fine structure by electron microscopy under conventional conditions, indicating OPALIN is not essential for basic CNS myelination. However, Opalin-/- mice display a subtle but significant reduction in exploratory activity in a novel environment.\",\n      \"method\": \"Opalin gene knockout mice, electron microscopy (optic nerve), Western blot, immunofluorescence, behavioral testing\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complete knockout with electron microscopy and molecular analysis; negative finding for myelination, subtle positive behavioral phenotype; single lab\",\n      \"pmids\": [\"27855200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OPALIN protein turnover is regulated by 2-hydroxylated sphingolipids: in Fa2h-/- mice (lacking fatty acid 2-hydroxylase), OPALIN protein accumulates ~6-fold in CNS myelin without change in Opalin mRNA, indicating decreased protein turnover. In CHO cells, OPALIN half-life is reduced when 2-hydroxylated sulfatide is present. OPALIN degradation is inhibited by lysosomal inhibitors but not by proteasome inhibitors, placing OPALIN degradation in the lysosomal pathway.\",\n      \"method\": \"Quantitative proteomics (tandem mass tag labeling) of purified myelin, Western blot, RT-PCR, CHO cell OPALIN half-life assay with lysosomal and proteasomal inhibitors\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (proteomics, Western blot, cell-based half-life assay, pharmacological inhibition) in a single lab identifying lysosomal degradation mechanism\",\n      \"pmids\": [\"33215680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"OPALIN undergoes age-dependent hypersialylation of O-glycans in the postnatal mouse brain, resulting in an increase in apparent molecular weight with aging. Additionally, immunoreactivity redistributes regionally with age, decreasing in cerebellar white matter relative to corpus callosum in adult mice.\",\n      \"method\": \"Western blot with enzymatic deglycosylation, immunofluorescence at multiple postnatal time points\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, descriptive biochemical characterization with limited functional follow-up\",\n      \"pmids\": [\"25153515\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OPALIN (TMEM10) is a CNS-specific type I transmembrane sialylglycoprotein localized to oligodendrocyte paranodal loop membranes, where it acts as a receptor for the secreted protein LGI1; LGI1–OPALIN interaction (mediated by residues K23/D26) promotes oligodendrocyte differentiation and myelination by sustaining expression of transcription factors Sox10 and Olig2, while its N- and O-glycosylation (regulated by 2-hydroxylated sphingolipids and subject to age-dependent hypersialylation) controls its cell-surface localization and lysosomal turnover, and its transcription is driven by a first-intron enhancer activated by Myt1, LIF, and cAMP/CREB signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"OPALIN (TMEM10) is a CNS-specific type I transmembrane sialylglycoprotein selectively expressed by oligodendrocytes and concentrated at paranodal loop membranes, where it acts in oligodendrocyte differentiation and myelination [#0, #1]. It has a short N-terminal extracellular domain bearing N-glycans at Asn-6/Asn-12 and a sialylated O-glycan at Thr-14, and these glycosylation sites are required for proper cell-surface localization [#1]. OPALIN functions as an oligodendrocyte-membrane receptor for the secreted protein LGI1; this interaction, mediated by extracellular residues K23/D26, sustains the transcription factors Sox10 and Olig2, and conditional deletion of OPALIN causes hypomyelination and white matter abnormalities that phenocopy LGI1 deficiency, with viral re-expression of wild-type but not the LGI1-binding-deficient K23A/D26A mutant rescuing the defect [#4]. Consistent with this role, OPALIN promotes oligodendrocyte terminal differentiation, process extension and branching, and induction of myelin-associated genes in gain- and loss-of-function assays [#3]. Its abundance is controlled post-translationally through a lysosomal degradation pathway that is accelerated by 2-hydroxylated sphingolipids [#6], and its transcription is driven by a first-intron oligodendrocyte enhancer responsive to Myt1, LIF, and cAMP/CREB signaling [#2]. Conventional OPALIN knockout mice nonetheless show grossly normal CNS myelin architecture with only a subtle reduction in exploratory behavior, indicating it is not strictly required for basic myelin assembly under standard conditions [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Before any transcriptional control was known, it was unclear how Opalin achieves oligodendrocyte-restricted expression; identifying a lineage-directed enhancer and its trans-factors established the regulatory logic of its expression.\",\n      \"evidence\": \"Transgenic mouse enhancer assay and reporter cotransfection in Oli-neu cells with deletion analysis and Myt1 overexpression/LIF/cAMP stimulation\",\n      \"pmids\": [\"17442045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not link enhancer activity to a downstream functional output of OPALIN\", \"Direct binding of Myt1 and CREB to the enhancer not biochemically resolved beyond site presence\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The cellular and subcellular distribution of OPALIN was undefined; demonstrating CNS-restricted oligodendrocyte expression with paranodal concentration and absence from Schwann cells positioned it as a candidate CNS myelin component.\",\n      \"evidence\": \"In situ hybridization, RT-PCR, developmental immunofluorescence, and myelinating spinal cord cultures\",\n      \"pmids\": [\"18571792\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Localization alone does not establish a molecular function\", \"No binding partner or pathway identified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The topology and post-translational modification of OPALIN were unknown; defining it as a type I transmembrane sialylglycoprotein and showing that defined N-/O-glycosylation sites are required for surface delivery established the structural basis of its membrane presentation.\",\n      \"evidence\": \"Biochemical characterization, enzymatic deglycosylation, site-directed mutagenesis with surface-localization readout, and immunogold electron microscopy\",\n      \"pmids\": [\"18490449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The functional role of the cytoplasmic C-terminal domain not addressed\", \"Did not connect glycosylation to a signaling or differentiation function\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether OPALIN modification changes over development was unexamined; documenting age-dependent O-glycan hypersialylation and regional redistribution suggested its glycoform is dynamically regulated with maturation.\",\n      \"evidence\": \"Western blot with enzymatic deglycosylation and immunofluorescence across postnatal time points in mouse brain\",\n      \"pmids\": [\"25153515\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Descriptive only with limited functional follow-up\", \"Single lab; functional consequence of hypersialylation not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"It was unknown whether OPALIN is essential for myelination; a complete knockout showing grossly normal myelin but reduced exploratory behavior established that it is dispensable for basic myelin assembly while hinting at a non-structural role.\",\n      \"evidence\": \"Opalin knockout mice analyzed by electron microscopy, Western blot, immunofluorescence, and behavioral testing\",\n      \"pmids\": [\"27855200\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative myelin phenotype may reflect compensation or non-stressed conditions\", \"Mechanistic basis of the behavioral phenotype not determined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The cell-autonomous contribution of OPALIN to oligodendrocyte maturation was unclear; reciprocal gain- and loss-of-function showed it promotes terminal differentiation, morphology, and myelin gene expression, establishing a pro-differentiation role.\",\n      \"evidence\": \"Overexpression in Oli-neu cells and siRNA knockdown in primary OPCs with qRT-PCR and immunofluorescence\",\n      \"pmids\": [\"30837646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism downstream of OPALIN not defined at this stage\", \"In vitro systems; relationship to in vivo dispensability in knockout unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"OPALIN lacked a defined molecular partner and signaling mechanism; identifying it as the oligodendrocyte receptor for LGI1 and showing K23/D26-dependent rescue of hypomyelination established a ligand-receptor axis sustaining Sox10/Olig2 expression.\",\n      \"evidence\": \"LGI1-FLAG affinity chromatography plus mass spectrometry, conditional knockout mice, Sox10/Olig2 analysis, and viral rescue with wild-type versus K23A/D26A binding-deficient OPALIN\",\n      \"pmids\": [\"39083419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intracellular signal transduction linking LGI1-OPALIN binding to Sox10/Olig2 not delineated\", \"Reconciliation with the mild conventional knockout phenotype not fully resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How LGI1-OPALIN binding is transduced through the cytoplasmic domain to regulate Sox10/Olig2 transcription, and how glycosylation/lysosomal turnover tunes receptor availability, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No downstream signaling effector of OPALIN identified\", \"Link between glycoform/lysosomal turnover and LGI1 receptor function untested\", \"No structural model of the LGI1-OPALIN interface\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"LGI1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}