id: Q7ZUS3 gene_symbol: gad1b product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:7955 label: Danio rerio description: gad1b (glutamate decarboxylase 1b; formerly gad1 or GAD67) is one of two zebrafish co-orthologs of vertebrate GAD1, retained from the teleost-specific genome duplication; its paralog is gad1a. It encodes a pyridoxal 5'-phosphate-dependent group II decarboxylase that converts L-glutamate to the inhibitory neurotransmitter GABA in the cytoplasm of GABAergic neurons. The protein is 83% identical to human GAD1 and keeps its PLP-binding lysine and GABA-binding residues. gad1b is the dominant, pan-GABAergic gad1 copy, expressed from pharyngula stages in GABAergic neurons throughout the brain, retina and spinal cord (including Kolmer-Agduhr neurons, Purkinje cells and lateral-line efferent neurons), and it is the classic zebrafish GAD67 marker. gad1b null mutants and morphants show abnormal, seizure-like brain activity and increased locomotion, consistent with reduced GABA synthesis. existing_annotations: - term: id: GO:0004351 label: glutamate decarboxylase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - FB:FBgn0004516 - MGI:MGI:95632 - PANTHER:PTN002903772 - RGD:2652 - RGD:2653 - UniProtKB:Q7PNL7 - UniProtKB:Q99259 review: summary: Glutamate decarboxylase activity propagated from the GAD node PTN002903772, seeded by fly Gad1, rodent Gad1/Gad2 and human GAD1. action: ACCEPT reason: gad1b is a GAD1 co-ortholog inside the node's clade, keeps the PLP-binding lysine and GABA-binding residues, and its expression coincides with GABA immunoreactivity in the embryo. The zebrafish enzyme has not been assayed in vitro, but the null mutant phenotype (seizure-like brain activity, like the GABA-A antagonist PTZ) fits loss of GABA synthesis. supported_by: - reference_id: PMID:9634146 supporting_text: Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. - reference_id: PMID:30200754 supporting_text: The electrophysiological traces of the cMO morphants were comparable to those obtained from larvae null for gad1b and wild-type fish exposed to PTZ, which causes seizures in zebrafish. - reference_id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md supporting_text: Both copies keep the PLP lysine and the GABA-binding residues of human GAD1. - term: id: GO:0005737 label: cytoplasm evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - MGI:MGI:95632 - MGI:MGI:95634 - PANTHER:PTN000242648 - RGD:2653 - RGD:621030 - WB:WBGene00006762 review: summary: Cytoplasm propagated from the group II decarboxylase node PTN000242648. action: ACCEPT reason: GAD1 is a soluble cytoplasmic enzyme; Gad1b has no signal peptide or transmembrane segment. supported_by: - reference_id: PMID:27461130 supporting_text: While both GADs synthesize GABA and are co-expressed in most vertebrate GABAergic neurons, GAD1 synthesizes cytoplasmic GABA that is used for extrasynaptic and metabolic purposes and GAD2 regulates the vesicular pool for release - term: id: GO:0009449 label: GABA biosynthetic process evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - FB:FBgn0004516 - PANTHER:PTN002903772 - RGD:2652 - ZFIN:ZDB-GENE-030909-3 - ZFIN:ZDB-GENE-030909-9 review: summary: GABA biosynthetic process propagated from PTN002903772; gad1b itself (ZDB-GENE-030909-3) is one of the donors. action: ACCEPT reason: gad1b catalyses the step that makes GABA. Its own experimental annotation (the IEP row from Martin et al. 1998) is among the descendant evidence for this node, which is expected and shows that the function is both inherited and grounded on this gene. supported_by: - reference_id: PMID:9634146 supporting_text: The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo. - reference_id: PMID:17384644 supporting_text: GAD67 is constitutively active and is responsible for basal GABA production. - term: id: GO:0009449 label: GABA biosynthetic process evidence_type: IEP original_reference_id: PMID:9634146 qualifier: acts_upstream_of_or_within review: summary: GABA biosynthetic process from Martin et al. 1998, where the zebrafish GAD67 cDNA was cloned and its in situ pattern compared with GABA immunostaining. action: ACCEPT reason: The GAD67 cDNA of this paper (GenBank AF017266) is gad1b by sequence (99.6% identical to Gad1b over the partial CDS, vs 90.9% to Gad1a), so the row is on the right paralog. GABA was found at all sites of GAD expression. The evidence is correlative (expression pattern), but the process is the correct one for the enzyme and is supported by the gad1b null mutant. supported_by: - reference_id: PMID:9634146 supporting_text: Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. - reference_id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md supporting_text: The classic GAD67 cDNA AF017266 is gad1b by sequence. - term: id: GO:0016830 label: carbon-carbon lyase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR002129 review: summary: Carbon-carbon lyase activity from the InterPro PLP-dependent decarboxylase domain (IPR002129). action: ACCEPT reason: Correct but very general; glutamate decarboxylase activity (GO:0004351) is the informative term and is also present. Kept as a correct domain-level inference. supported_by: - reference_id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md supporting_text: Both copies keep the PLP lysine and the GABA-binding residues of human GAD1. - term: id: GO:0016831 label: carboxy-lyase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR021115 review: summary: Carboxy-lyase activity from the InterPro pyridoxal-phosphate attachment site signature (IPR021115). action: ACCEPT reason: Correct parent of glutamate decarboxylase activity; the PLP attachment lysine is present. supported_by: - reference_id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md supporting_text: Both copies keep the PLP lysine and the GABA-binding residues of human GAD1. - term: id: GO:0030170 label: pyridoxal phosphate binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR002129 review: summary: Pyridoxal phosphate binding from the InterPro PLP-dependent decarboxylase domain. action: ACCEPT reason: PLP is the covalently bound cofactor of GAD enzymes, attached to the active-site lysine that Gad1b keeps. supported_by: - reference_id: PMID:17384644 supporting_text: Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). - reference_id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md supporting_text: Both copies keep the PLP lysine and the GABA-binding residues of human GAD1. - term: id: GO:0048786 label: presynaptic active zone evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - MGI:MGI:95632 - PANTHER:PTN002903810 - RGD:2652 review: summary: Presynaptic active zone propagated from node PTN002903810, seeded by mouse and rat Gad1. action: KEEP_AS_NON_CORE reason: gad1b is inside the GAD1 clade that carries this node, and ZFIN records Gad1b antibody staining at presynaptic active zones in the adult cerebellum (Bae et al. 2009), which I could not read beyond the abstract. The location is plausible for a GABA-synthesizing enzyme in inhibitory terminals, but GAD1 is mainly cytoplasmic and terminal association is more characteristic of GAD2, so the term is kept as non-core. supported_by: - reference_id: PMID:27461130 supporting_text: While both GADs synthesize GABA and are co-expressed in most vertebrate GABAergic neurons, GAD1 synthesizes cytoplasmic GABA that is used for extrasynaptic and metabolic purposes and GAD2 regulates the vesicular pool for release core_functions: - description: Pyridoxal 5'-phosphate-dependent glutamate decarboxylase (GAD67-type) that synthesizes GABA from L-glutamate in the cytoplasm of GABAergic neurons; the dominant, pan-GABAergic gad1 copy in zebrafish embryos and larvae. Loss of gad1b gives seizure-like brain activity. molecular_function: id: GO:0004351 label: glutamate decarboxylase activity directly_involved_in: - id: GO:0009449 label: GABA biosynthetic process locations: - id: GO:0005737 label: cytoplasm supported_by: - reference_id: PMID:9634146 supporting_text: Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. - reference_id: PMID:30200754 supporting_text: The gad1bgav2303/gav2303 allele used in these studies harbored a 10 bp deletion in exon 4 and was a functional null mutation. - reference_id: PMID:42466317 supporting_text: At 5 dpf, as shown previously, gad1b expression occupies all regions of the larval brain (Figure 4A). - reference_id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md supporting_text: gad1b is about 20-fold more abundant than gad1a in whole larvae (84 vs 4 TPM at day 5). references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: PMID:9634146 title: Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Abstract only; cloning and in situ of zebrafish GAD65 and GAD67 with GABA immunostaining. The GAD67 cDNA (AF017266) is gad1b by sequence (gad1a-bioinformatics/probe_identity.py). - id: PMID:17384644 title: GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Abstract only; human GAD67 constitutively active; PLP enzyme. - id: PMID:27461130 title: Three Distinct Glutamate Decarboxylase Genes in Vertebrates. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: GAD family evolution; background on GAD1 vs GAD2 roles. - id: PMID:30200754 title: Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text; gad1b translation-blocking MO (craniofacial defects not seen in mutants), photoactivatable MO, gad1b null allele gav2303 with abnormal brain field potentials. - id: PMID:34650032 title: Increased locomotor activity via regulation of GABAergic signalling in foxp2 mutant zebrafish-implications for neurodevelopmental disorders. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text; gad1b splice MO increases locomotor activity; gad1b reduced in foxp2 heterozygotes. - id: PMID:41950195 title: Single-cell analysis of inhibitory efferent neurons of the zebrafish lateral line. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text; efferent neurons express gad1b and gad2, not gad1a. - id: PMID:42466317 title: Gene expression mapping of conserved GABAergic interneuron markers in developing zebrafish. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text; gad1b HCR as pan-GABAergic marker at 3 and 5 dpf. - id: PMID:24374659 title: vglut2 and gad expression reveal distinct patterns of dual GABAergic versus glutamatergic cotransmitter phenotypes of dopaminergic and noradrenergic neurons in the zebrafish brain. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Full text; gad1b and gad2 in catecholaminergic groups; gad1b alone in locus coeruleus. - id: PMID:26896392 title: Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2-independent mechanism. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Methods note that the old gad67a/gad67b probes target gad1b. - id: file:DANRE/gad1a/gad1a-bioinformatics/RESULTS.md title: gad1a / gad1b protein, expression and synteny comparison reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Local pair analysis directly supports GAD1-family active-site retention in both copies and resolves the classic AF017266 GAD67 probe as gad1b; the report also flags later probe-identity ambiguity where the sequence was not published. findings: [] suggested_questions: - question: Does gad1a expression rise in gad1b null mutants (transcriptional adaptation), and how much GABA remains in gad1b mutant brains? - question: Why does the gad1b translation-blocking morpholino cause craniofacial defects that gad1b null mutants lack - off-target effects, knockdown of gad1a, or compensation in the mutant? suggested_experiments: - hypothesis: gad1b supplies most GABA in the larval brain, with gad1a and gad2 providing a residual pool. description: Measure brain GABA by HPLC or mass spectrometry in gad1b, gad2, gad1a single and combined mutants, and record gad1a and gad2 mRNA levels in gad1b mutants. experiment_type: genetic interaction - hypothesis: Zebrafish Gad1b is constitutively active like mammalian GAD67. description: Express recombinant Gad1b and measure glutamate decarboxylase kinetics and PLP saturation, alongside Gad1a. experiment_type: biochemical assay