Affinage

CA4

Carbonic anhydrase 4 · UniProt P22748

Length
312 aa
Mass
35.0 kDa
Annotated
2026-06-09
54 papers in source corpus 11 papers cited in narrative 11 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CA4 (carbonic anhydrase IV) is a GPI-anchored, extracellular-facing enzyme whose CO2/HCO3− interconversion activity regulates pH at cell surfaces in diverse tissues (PMID:15090652, PMID:2116168). In hippocampal neurons it acts together with the related extracellular isoform CA14 to facilitate AE3-mediated Cl−/HCO3− exchange, thereby controlling cytosolic pH — an activity demonstrated by loss of the effect upon anion-exchange blockade or in AE3-knockout neurons (PMID:19279262). CA4 supports pH-dependent physiology elsewhere: in the male reproductive tract it localizes along the sperm flagellar membrane, and its ablation disrupts luminal acidification, lowers basal sperm intracellular pH, and impairs the pH-sensitive CatSper Ca2+ channel activation required for capacitation, with a regulatory link to the Slo3 K+ channel (PMID:41279408); in aging adipocyte progenitors it sustains intracellular pH and glutathione-mediated ROS resistance to enable beige adipocyte differentiation (PMID:40883493). CA4 expression is broadly distributed and is transcriptionally controlled in a tissue-specific manner by thyroid hormone via TRα1 (PMID:2116168, PMID:26319697). It also serves as a cell-type identity marker for type III taste cells and aerocyte lung capillary endothelial cells (PMID:29099943, PMID:36028133). A signal-sequence R14W mutation causes ER retention of the immature protein, unfolded protein response activation, cell-cycle block and apoptosis selectively in ocular cell types, accounting for RP17 autosomal dominant retinitis pigmentosa while sparing renal tissue (PMID:15090652, PMID:20626030).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 1990 Medium

    Established that CA4 protein is a discrete ~55 kDa species broadly expressed across human fetal and adult tissues, setting the foundation that this is a widely deployed surface enzyme rather than a tissue-restricted one.

    Evidence Western blot with anti-CAIV antibody across multiple human tissues and cell cultures

    PMID:2116168

    Open questions at the time
    • Single method (Western blot) without functional readout
    • No subcellular localization or GPI-anchoring demonstrated in this study
  2. 2004 High

    Identified the disease mechanism of RP17 by showing the signal-sequence R14W mutation reduces enzyme activity, triggers ER stress markers, and induces apoptosis, linking CA4 dysfunction to retinitis pigmentosa.

    Evidence COS-7 transfection, carbonic anhydrase activity assay, ER stress marker Western blots, annexin V and TUNEL apoptosis assays, family co-segregation

    PMID:15090652

    Open questions at the time
    • Mechanism tested in COS-7 surrogate rather than native choriocapillaris endothelium
    • Did not explain why the phenotype is ocular-restricted
  3. 2009 High

    Defined a physiological enzymatic role for CA4 at the cell surface by showing it, with CA14, facilitates AE3-mediated Cl−/HCO3− exchange to regulate neuronal intracellular pH.

    Evidence Intracellular pH imaging with NH4+ challenge, pharmacological and antibody inhibition, AE3-knockout mice, single-cell PCR

    PMID:19279262

    Open questions at the time
    • Functional coupling inferred pharmacologically rather than from direct CA4–AE3 physical interaction
    • Relative contribution of CA4 versus CA14 not separated
  4. 2010 Medium

    Resolved the tissue selectivity of RP17 by showing R14W CAIV is ER-retained and causes cell-cycle arrest and apoptosis in COS-7 and HT-1080 cells but is processed normally in HEK-293, explaining the ocular-but-not-renal phenotype.

    Evidence Immunocytochemistry, processing Western blots, flow cytometry for cell cycle and apoptosis across three cell lines

    PMID:20626030

    Open questions at the time
    • Cell-line-specific factors determining differential processing not identified
    • Surrogate cell lines rather than authentic ocular and renal cells
  5. 2015 Medium

    Showed CA4 expression is under tissue-specific thyroid hormone control, mediated by TRα1 in lung and brain but repressed by thyroid hormone independently of TRα1 in kidney.

    Evidence TRα1-mutant mouse model with mRNA/protein quantification and acute thyroid hormone treatment

    PMID:26319697

    Open questions at the time
    • Direct transcriptional regulatory elements not mapped
    • Single lab, single study
  6. 2017 Medium

    Established CA4 as a marker of a distinct subpopulation of type III presynaptic taste cells, expanding its use as a cell-identity tool.

    Evidence Car4-IRES-mCherry knock-in reporter mouse with immunofluorescence co-localization against presynaptic markers

    PMID:29099943

    Open questions at the time
    • Functional role of CA4 in taste signaling not tested
    • Marker association does not establish a mechanistic requirement
  7. 2022 Medium

    Defined CA4 as a definitive genetic marker of aerocyte (aCap) lung capillary endothelial cells, distinct from gCap cells, providing a lineage-tracing tool.

    Evidence Car4-CreER knock-in mice with tamoxifen-inducible Cre-loxP lineage tracing in lung

    PMID:36028133

    Open questions at the time
    • Functional contribution of CA4 to aCap biology not addressed
    • Marker identity rather than mechanistic role
  8. 2025 Medium

    Extended CA4 pH-regulatory function to reproduction and metabolism, and to drug-withdrawal synaptic plasticity, while introducing CA4 as a BBB transcytosis receptor.

    Evidence CA4-knockout/knockdown mice and cells with pH imaging, super-resolution localization, electrophysiology, behavior, scRNA-seq, ROS/glutathione rescue, and humanized CA-IV AAV transduction (multiple studies, several preprints)

    PMID:40883493 PMID:41279408 PMID:bio_10.1101_2025.01.23.634619 PMID:bio_10.1101_2025.04.21.649868

    Open questions at the time
    • Several findings are single-lab preprints awaiting peer review
    • Direct CA4 interaction with Slo3, CatSper, ASIC1A, and AAV capsids not all structurally resolved
    • Whether the BBB transcytosis-receptor role reflects a native ligand or engineered interaction is unestablished

Open questions

Synthesis pass · forward-looking unresolved questions
  • How a single catalytic pH-regulating activity is harnessed for such divergent outcomes — anion exchange coupling, channel regulation, ROS resistance, and receptor-mediated transcytosis — and which partners physically engage CA4 in each context remain unresolved.
  • No structural model of CA4 in complex with AE3, Slo3, or CatSper
  • Mechanism by which an extracellular enzyme acts as a transcytosis receptor undefined
  • Direct physical partners largely inferred rather than co-purified

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016787 hydrolase activity 2 GO:0140096 catalytic activity, acting on a protein 1
Localization
GO:0005783 endoplasmic reticulum 2 GO:0005886 plasma membrane 2
Pathway
R-HSA-1643685 Disease 2 R-HSA-382551 Transport of small molecules 2
Partners
AE3CA14CATSPERSLO3

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2004 The R14W mutation in the CA4 signal sequence (at position -5 relative to the cleavage site) causes ER stress and apoptosis: in transfected COS-7 cells, R14W CAIV (i) reduced steady-state carbonic anhydrase IV enzymatic activity by 28% due to decreased synthesis and accelerated turnover, (ii) upregulated ER stress markers (BiP/GRP78, PERK, CHOP), and (iii) induced apoptosis (annexin V binding, TUNEL staining) in most cells expressing the mutant but not wild-type protein. This identifies ER stress-induced apoptosis in choriocapillaris endothelial cells as the mechanism of RP17 retinitis pigmentosa. COS-7 cell transfection, carbonic anhydrase activity assay, Western blot (ER stress markers), annexin V binding, TUNEL staining Proceedings of the National Academy of Sciences of the United States of America High 15090652
2010 Cell-type-specific processing of the R14W CAIV mutant: in COS-7 and HT-1080 cells, R14W CAIV is retained in the ER (unlike WT which reaches the plasma membrane), remains in its immature form by Western blot, and causes S and G2/M cell-cycle block followed by apoptosis. Strikingly, HEK-293 cells are unaffected by R14W CAIV and process mutant and WT protein equally effectively, providing a mechanistic explanation for the exclusively ocular (not renal) phenotype of RP17. Immunocytochemistry (subcellular localization), Western blot (processing), flow cytometry (cell cycle and apoptosis) in three cell lines Journal of cellular biochemistry Medium 20626030
2009 CA4 and CA14, the extracellular carbonic anhydrases in the brain, facilitate AE3-mediated Cl−/HCO3− exchange in hippocampal neurons to regulate intracellular pH. Inhibition of extracellular CAs (by benzolamide or isoform-specific antibodies against CA4 or CA14) enhanced NH4+-induced cytosolic alkalinization; this effect was abolished when anion exchange was blocked or Cl− removed, and was absent in AE3-knockout neurons. Single-cell PCR confirmed AE3 (Slc4a3) as the predominant exchanger isoform. Intracellular pH measurement with NH4+ challenge, pharmacological inhibition (benzolamide, DIDS), inhibitory antibodies (CA4- and CA14-specific), AE3-knockout mice, quantitative PCR, single-cell PCR The Journal of neuroscience : the official journal of the Society for Neuroscience High 19279262
1990 CA4 (CAIV) protein is expressed as a single ~55 kDa band in a wide range of human fetal and adult tissues including lung, pancreatic tumour cells, and skin cell cultures, as detected by Western blotting, establishing its broad tissue distribution. Western blot with anti-CAIV antibody across fetal and adult human tissues and cell cultures Biochimica et biophysica acta Medium 2116168
2015 CA4 expression in lung and brain of adult mice depends on intact thyroid hormone receptor α1 (TRα1) signaling (upregulated by functional TRα1), whereas in kidney, CA4 mRNA and protein are not affected by mutant TRα1 but are acutely repressed by thyroid hormone, demonstrating tissue-specific transcriptional regulation of CA4 by thyroid hormone. Mouse model with TRα1 mutation, mRNA and protein quantification in lung, brain, and kidney; acute thyroid hormone treatment Molecular and cellular endocrinology Medium 26319697
2017 CA4-expressing cells in taste buds are a subpopulation of type III (presynaptic) taste cells. Using a Car4-mCherry knock-in mouse, CA4+ cells co-localize with presynaptic markers (AADC, SNAP25, GAD67) in fungiform papillae and palate, but represent only a subset of presynaptic cells in posterior tongue papillae, establishing CA4 as a marker of a distinct subpopulation of type III taste cells. Genetic knock-in reporter mouse (Car4-IRES-mCherry), immunofluorescence co-localization with presynaptic cell markers in lingual papillae and taste buds Chemical senses Medium 29099943
2022 CAR4 (CA4) marks aerocyte (aCap) capillary endothelial cells in the mammalian lung, a distinct subpopulation from PLVAP+ general capillary (gCap) cells. Car4-CreER knock-in mice mediate specific and efficient Cre-loxP recombination in CAR4+ aCap cells upon tamoxifen induction, establishing CA4 as a definitive genetic marker and tool for studying this lung capillary subpopulation. Knock-in mouse generation (Car4-CreER), tamoxifen-inducible Cre-loxP recombination, lineage tracing in lung Journal of genetics and genomics = Yi chuan xue bao Medium 36028133
2025 CA4 is required for oxycodone withdrawal-induced synaptic adaptations in nucleus accumbens core (NAcC) medium spiny neurons (MSNs): genetic disruption of CA4 prevented oxycodone withdrawal-induced increases in AMPAR/NMDAR ratio and synaptic recruitment of calcium-permeable AMPARs. Pharmacological inhibition with acetazolamide reversed these synaptic changes in vitro and in vivo in a CA4-dependent manner and also required acid-sensing ion channel-1A (ASIC1A). CA4 disruption and acetazolamide reduced oxycodone-seeking behavior after 30 days of forced abstinence in a self-administration paradigm. Genetic CA4 knockout mice, pharmacological inhibition (acetazolamide), electrophysiology (AMPAR/NMDAR ratio, rectification index), oxycodone self-administration with extinction/reinstatement bioRxivpreprint Medium bio_10.1101_2025.01.23.634619
2025 CAIV (CA4), a GPI-anchored extracellular enzyme, is located along the entire flagellar membrane of spermatozoa, while CAII is within the flagellum near CatSper in the principal piece. Genetic ablation of CAIV in mice disrupts normal luminal acidification in the male reproductive tract and lowers basal intracellular pH (pHi) of sperm, which reduces effectiveness of subsequent alkalinization and diminishes activation of pH-sensitive CatSper Ca2+ channels required for capacitation. A direct link between CAIV and the Slo3 K+ channel was established: Slo3-deficient sperm showed reduced pHi and decreased CAIV protein levels. CA4 knockout mice, intracellular pH measurement, super-resolution imaging (CAIV and CAII localization), Slo3-knockout sperm analysis (Western blot, pHi), CatSper-knockout sperm analysis bioRxivpreprint Medium 41279408
2025 CA4-positive adipocyte progenitor cells (APCs) increase in number with age and cold exposure. Car4 knockdown mitigated intracellular pH reduction and significantly suppressed beige adipocyte differentiation. Car4 KD cells showed reduced expression of glutathione pathway genes and increased susceptibility to reactive oxygen species (ROS), which was alleviated by glutathione supplementation, indicating CA4 supports ROS resistance in aging APCs through regulation of intracellular pH and glutathione metabolism. Single-cell RNA sequencing (identification of Car4+ APCs), Car4 siRNA knockdown, intracellular pH measurement, beige adipocyte differentiation assay, ROS susceptibility assay, glutathione supplementation rescue Scientific reports Medium 40883493
2025 CA4 (CA-IV) functions as a blood-brain barrier (BBB) transcytosis receptor in brain endothelial cells. AAVs engineered to bind human CA-IV achieved ~100-fold greater brain transduction than AAV9 in humanized mice expressing human CA-IV in brain endothelial cells, demonstrating that CA-IV mediates transcytosis-dependent AAV entry across the BBB. In vitro receptor-based AAV library selection, in vivo selection in humanized CA-IV knock-in mice, brain transduction quantification vs. AAV9 control bioRxivpreprint Medium bio_10.1101_2025.04.21.649868

Source papers

Stage 0 corpus · 54 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2019 CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA>p Formation Terminating RNase Activity. Molecular cell 107 31326273
1998 Linkage analysis and comparative mapping of canine progressive rod-cone degeneration (prcd) establishes potential locus homology with retinitis pigmentosa (RP17) in humans. Proceedings of the National Academy of Sciences of the United States of America 98 9501213
2004 Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa. Proceedings of the National Academy of Sciences of the United States of America 83 15090652
1986 Hippocampal pyramidal cells and aging in the human: a quantitative study of neuronal loss in sectors CA1 to CA4. Experimental neurology 81 3758285
1994 The cAMP receptor CAR4 regulates axial patterning and cellular differentiation during late development of Dictyostelium. Genes & development 79 7958880
2009 Carbonic anhydrases CA4 and CA14 both enhance AE3-mediated Cl--HCO3- exchange in hippocampal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 62 19279262
1997 Disproportionate loss of CA4 parvalbumin-immunoreactive interneurons in patients with Ammon's horn sclerosis. Journal of neuropathology and experimental neurology 59 9291940
2007 Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells. Apoptosis : an international journal on programmed cell death 53 17143747
2015 Proteome-wide characterization of signalling interactions in the hippocampal CA4/DG subfield of patients with Alzheimer's disease. Scientific reports 52 26059363
1990 Membrane specific carbonic anhydrase (CAIV) expression in human tissues. Biochimica et biophysica acta 46 2116168
2016 Design, Synthesis and Antitumor Activity of Novel link-bridge and B-Ring Modified Combretastatin A-4 (CA-4) Analogues as Potent Antitubulin Agents. Scientific reports 41 27138035
2008 Novel A-ring and B-ring modified combretastatin A-4 (CA-4) analogues endowed with interesting cytotoxic activity. Journal of medicinal chemistry 41 18783207
2019 Second Messenger cA4 Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path. Molecular cell 40 31326272
2009 Increased D-amino acid oxidase expression in the bilateral hippocampal CA4 of schizophrenic patients: a post-mortem study. Journal of neural transmission (Vienna, Austria : 1996) 36 19823762
2020 Discovery of novel CA-4 analogs as dual inhibitors of tubulin polymerization and PD-1/PD-L1 interaction for cancer treatment. European journal of medicinal chemistry 33 33280898
2021 Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model. Molecular therapy oncolytics 28 35118195
2017 Genetic Labeling of Car4-expressing Cells Reveals Subpopulations of Type III Taste Cells. Chemical senses 26 29099943
1999 Delayed apoptotic pyramidal cell death in CA4 and CA1 hippocampal subfields after a single intraseptal injection of kainate. Neuroscience 26 10625049
1998 N-methyl-D-aspartate receptor-mediated, calcium-induced calcium release in rat dentate gyrus/CA4 in vivo. Journal of neuroscience research 26 9452311
2005 Genetic stability of live, cold-adapted influenza virus components of the FluMist/CAIV-T vaccine throughout the manufacturing process. Vaccine 24 16413951
2022 Anticancer Activity of Thiophene Carboxamide Derivatives as CA-4 Biomimetics: Synthesis, Biological Potency, 3D Spheroid Model, and Molecular Dynamics Simulation. Biomimetics (Basel, Switzerland) 23 36546947
2020 Carbonic anhydrases CA1 and CA4 function in atmospheric CO2-modulated disease resistance. Planta 23 32146566
2003 Influenza virus vaccine live intranasal--MedImmune vaccines: CAIV-T, influenza vaccine live intranasal. Drugs in R&D 23 12952502
2016 Identification of two novel Chlorotoxin derivatives CA4 and CTX-23 with chemotherapeutic and anti-angiogenic potential. Scientific reports 22 26831010
2013 Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway. Toxicology and applied pharmacology 22 24321340
2019 Dual-targeting antitumor conjugates derived from platinum(IV) prodrugs and microtubule inhibitor CA-4 significantly exhibited potent ability to overcome cisplatin resistance. Bioorganic chemistry 21 31494328
2020 Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR-Cas that degrades the second messenger cA4. The Journal of biological chemistry 20 32826317
2006 Treatment of coxsackievirus-B3-infected BALB/c mice with the soluble coxsackie adenovirus receptor CAR4/7 aggravates cardiac injury. Journal of molecular medicine (Berlin, Germany) 20 16924471
2011 FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways. Journal of cellular biochemistry 19 21268067
2017 Influence of knockout of At4g20990 gene encoding α-CA4 on photosystem II light-harvesting antenna in plants grown under different light intensities and day lengths. Protoplasma 18 28643084
1997 Retinitis pigmentosa locus on 17q (RP17): fine localization to 17q22 and exclusion of the PDEG and TIMP2 genes. Human genetics 17 9385361
1999 Refinement of the RP17 locus for autosomal dominant retinitis pigmentosa, construction of a YAC contig and investigation of the candidate gene retinal fascin. European journal of human genetics : EJHG 15 10234509
1994 Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4. FEMS microbiology letters 15 8001765
2021 Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus. Biomolecules 14 34944496
2014 Combretastatin (CA)-4 and its novel analogue CA-432 impair T-cell migration through the Rho/ROCK signalling pathway. Biochemical pharmacology 14 25450669
2022 Generation of Plvap-CreER and Car4-CreER for genetic targeting of distinct lung capillary populations. Journal of genetics and genomics = Yi chuan xue bao 10 36028133
2022 Fine Mapping of a Vigor QTL in Chickpea (Cicer arietinum L.) Reveals a Potential Role for Ca4_TIFY4B in Regulating Leaf and Seed Size. Frontiers in plant science 8 35283931
2022 Advances in antitumor research of CA-4 analogs carrying quinoline scaffold. Frontiers in chemistry 8 36385996
1999 Refined mapping of the gene for autosomal dominant retinitis pigmentosa (RP17) on chromosome 17q22. Human genetics 7 10071195
2024 Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production. Nucleic acids research 6 38989619
2022 Singlet Oxygen Activatable Prodrugs of Paclitaxel, SN-38, MMC and CA4: Nonmitochondria-Targeted Prodrugs. Photochemistry and photobiology 6 34970997
2022 Discovery of pyrimidine-bridged CA-4 CBSIs for the treatment of cervical cancer in combination with cisplatin with significantly reduced nephrotoxicity. European journal of medicinal chemistry 6 35339837
2008 Antimultidrug-resistant effect and mechanism of a novel CA-4 analogue MZ3 on leukemia cells. Die Pharmazie 6 18717489
2015 Morphometric Parameters of Pyramidal Cells in CA1-CA4 Fields in the Hippocampus of Arctic Fox (Vulpes lagopus). Folia biologica 3 26975141
2010 Cell-specific differences in the processing of the R14W CAIV mutant associated with retinitis pigmentosa 17. Journal of cellular biochemistry 3 20626030
2025 A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4. The Biochemical journal 2 41190788
2025 Protective Effect of α-Carbonic Anhydrase CAH3 Against Photoinhibition and Thermal Inactivation of Photosystem II in Membrane Preparations as Compared with α-Carbonic Anhydrase CA4. Biochemistry. Biokhimiia 1 40759418
2015 Thyroid hormone drives the expression of mouse carbonic anhydrase Car4 in kidney, lung and brain. Molecular and cellular endocrinology 1 26319697
2007 [Genotyping and CA4 gene analysis in a Chinese family with retinitis pigmentosa]. Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 1 18067080
2026 Design, synthesis, and biological evaluation of Indazole-based CA-4 analogues as tubulin polymerization inhibitors. Bioorganic chemistry 0 42085806
2026 Spiro-Linked Polyketides from Cultures of Westerdykella dispersa Ca4-13 as Inhibitors of iNOS-Associated Neuroinflammation. Journal of natural products 0 42208015
2025 Car4-positive adipocyte progenitor cells adapt to the aging environment and work as protection against ROS via glutathione metabolism. Scientific reports 0 40883493
2025 Novel inducible nitric oxide synthase-inhibiting cytochalasins from an oyster-derived fungus Westerdykella dispersa Ca4-13: structural insights and molecular docking analysis. Botanical studies 0 41055802
2025 Coordinated and compartmentalized functions of CAII and CAIV establish a dual pH-regulatory mechanism essential for sperm maturation and capacitation. bioRxiv : the preprint server for biology 0 41279408

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