GO:0031418 L-ascorbic acid binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0031418 L-ascorbic acid binding is a molecular function describing the selective, non-covalent interaction of a protein with L-ascorbic acid (vitamin C), a cofactor and antioxidant in many species.
• L-ascorbic acid binding is essential for the catalytic activity of Fe(II)/2-oxoglutarate-dependent dioxygenases, including HIF prolyl hydroxylases and collagen prolyl hydroxylases.
• The function is mediated by conserved structural motifs that coordinate the ascorbate molecule, often in concert with iron and 2-oxoglutarate.
• Dysregulation of L-ascorbic acid binding contributes to cancer, neurodegeneration, and metabolic disorders, as shown by studies on breast cancer and Friedreich's ataxia.
• SVCT2-mediated ascorbic acid uptake and binding influence DNA hydroxymethylation and stress responses, linking this function to epigenetic regulation.
• Research tools such as CRISPR knockout, point mutation, and knock-in models enable precise dissection of L-ascorbic acid binding in health and disease.
Description
L-ascorbic acid binding (GO:0031418) is a molecular function defined as the selective interaction of a protein with L-ascorbic acid, also known as vitamin C. This binding event is critical for the activity of numerous enzymes that require ascorbate as a cofactor, particularly Fe(II)/2-oxoglutarate-dependent dioxygenases. The function is conserved across species and plays a central role in processes ranging from collagen hydroxylation to epigenetic regulation. Researchers study this term to understand how ascorbate modulates enzyme activity, cellular stress responses, and disease progression. The interaction is not merely a passive binding but often a prerequisite for catalytic turnover, as seen in HIF prolyl hydroxylases where ascorbate acts as a true substrate. Consequently, GO:0031418 is a focal point for investigations into cancer metabolism, neuroprotection, and redox biology [1,4].
L-ascorbic acid binding At A Glance
| GO ID | GO:0031418 |
|---|---|
| GO term | L-ascorbic acid binding |
| Ontology | molecular_function |
| Synonym | L-ascorbate binding, vitamin C binding |
| Definition | Binding to L-ascorbic acid, (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate; L-ascorbic acid is vitamin C and has co-factor and anti-oxidant activities in many species. |
| Major function | Cofactor binding for dioxygenases, antioxidant protection, and modulation of enzyme activity |
| Related diseases | Cancer, Friedreich's ataxia, lead-induced neurotoxicity, microbial infections |
| Research methods | CRISPR knockout, point mutation, knock-in, overexpression, structural biology, biochemical assays |
What Is GO:0031418?
GO:0031418 L-ascorbic acid binding describes the molecular function of selectively binding to L-ascorbic acid, the biologically active form of vitamin C. This binding is typically non-covalent and occurs at specific protein pockets that coordinate the ascorbate molecule, often in conjunction with metal ions such as iron. The definition encompasses both the physical interaction and the functional consequences, as ascorbate binding can modulate enzyme activity, protect against oxidative damage, or serve as a co-substrate in catalytic reactions. The term is used in gene ontology annotations to classify proteins that directly interact with L-ascorbic acid, including enzymes, transporters, and regulatory proteins.
Why Is L-ascorbic acid binding Important in Cell Biology?
L-ascorbic acid binding is fundamentally important because it governs the activity of a wide array of enzymes that depend on ascorbate for catalysis or regulation. Many of these enzymes, such as HIF prolyl hydroxylases, are central to oxygen sensing, metabolism, and stress responses. Disruption of this binding function can lead to impaired collagen synthesis, altered epigenetic marks, and increased susceptibility to oxidative stress, which are hallmarks of diseases like scurvy, cancer, and neurodegeneration [1,4]. Moreover, ascorbate binding influences drug efficacy and microbial virulence, as demonstrated by its anti-microbial effects against Burkholderia cepacia complex. Therefore, understanding GO:0031418 provides mechanistic insights into both normal physiology and pathological states, making it a high-value target for therapeutic development and biomedical research.
• L-ascorbic acid binding is required for the catalytic activity of Fe(II)/2-oxoglutarate-dependent dioxygenases, including HIF prolyl hydroxylases and collagen prolyl hydroxylases.
• It modulates epigenetic regulation through DNA hydroxymethylation, as shown for SVCT2-mediated ascorbic acid uptake affecting S100A4 gene expression.
• Ascorbate binding is linked to cancer suppression, with L-ascorbic acid inhibiting breast cancer growth via endoplasmic reticulum stress and p62/SQSTM1 accumulation.
• It plays a protective role in neurodegeneration, as seen in Friedreich's ataxia models where L-ascorbic acid combinations show therapeutic potential.
• L-ascorbic acid binding contributes to anti-microbial and anti-virulence effects against multi-drug resistant pathogens.
• The function is involved in preventing lead-induced brain damage, as L-ascorbic acid and curcumin mitigate oxidative stress in rat models.
• It is a target for enzyme engineering, such as in the production of 2-O-α-d-glucopyranosyl-L-ascorbic acid using sucrose phosphorylase.
• Structural studies of β-glucosidase-ascorbic acid complexes reveal binding mechanisms relevant to food and pharmaceutical applications.
• Dysregulation of ascorbate binding is implicated in metabolic disorders and oxidative stress-related diseases.
• CRISPR-based models enable precise interrogation of L-ascorbic acid binding in disease contexts, accelerating target validation.
What Happens During L-ascorbic acid binding?
Recognition and Initial Binding
In simple terms: The protein recognizes and grabs onto vitamin C.
The binding process begins with the specific recognition of L-ascorbic acid by a protein binding pocket. This interaction is often mediated by hydrogen bonding and electrostatic interactions with conserved residues, as seen in Fe(II)/2-oxoglutarate-dependent dioxygenases where ascorbate coordinates with the active site iron. The binding is highly selective, distinguishing L-ascorbic acid from other sugars or antioxidants. Structural studies of β-glucosidase-ascorbic acid complexes have elucidated the molecular details of this recognition, showing that ascorbate binds in a pocket that accommodates its dihydroxyethyl and lactone moieties.
Conformational Changes and Cofactor Coordination
In simple terms: The protein changes shape to hold vitamin C and its metal partner.
Upon binding, the protein may undergo conformational changes that stabilize the ascorbate molecule and coordinate with catalytic metal ions such as Fe(II). In HIF prolyl hydroxylases, ascorbate acts as a true substrate, donating electrons to regenerate the active site iron, which is essential for catalytic turnover. This step is critical for the enzyme's function, as without ascorbate, the iron can become oxidized and inactive. The coordination often involves a 2-His-1-carboxylate facial triad that binds iron, with ascorbate occupying a nearby site.
Catalytic Activation and Substrate Turnover
In simple terms: Vitamin C helps the enzyme do its job, like a helper that keeps the machine running.
Once bound, L-ascorbic acid facilitates catalytic cycles by maintaining the metal ion in its reduced state or by participating directly in electron transfer. In prolyl hydroxylases, ascorbate is consumed as a co-substrate, coupling the hydroxylation of proline residues to the reduction of iron. This activity is essential for oxygen sensing and collagen maturation. Similarly, in enzymes involved in epigenetic regulation, ascorbate binding supports DNA demethylation by serving as a cofactor for TET enzymes, influencing hydroxymethylation patterns.
Regulation and Feedback
In simple terms: The cell controls how much vitamin C binds to keep things balanced.
L-ascorbic acid binding is regulated by intracellular ascorbate levels, which are maintained by transporters such as SVCT2. SVCT2-mediated uptake buffers stress responses and reprograms DNA hydroxymethylation, indicating that binding is dynamically regulated by availability. Additionally, oxidative stress can deplete ascorbate, reducing binding and enzyme activity. Feedback mechanisms may involve the expression of ascorbate transporters and enzymes that recycle oxidized ascorbate back to its reduced form.
Key Genes Involved in GO:0031418 L-ascorbic acid binding
The following genes and proteins are directly involved in or regulated by L-ascorbic acid binding, as supported by published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| EGLN1 (PHD2) | HIF prolyl hydroxylase that binds L-ascorbic acid as a co-substrate | Oxygen sensing, cancer, and ischemia research |
| EGLN2 (PHD1) | Prolyl hydroxylase requiring ascorbate for activity | Metabolic regulation and hypoxia response |
| EGLN3 (PHD3) | Prolyl hydroxylase with ascorbate-dependent catalysis | Neuronal survival and cancer |
| P4HA1 | Collagen prolyl hydroxylase that binds ascorbate | Fibrosis and connective tissue disorders |
| P4HA2 | Collagen prolyl hydroxylase requiring ascorbate | Cancer metastasis and extracellular matrix remodeling |
| TET1 | DNA demethylase that may bind ascorbate as a cofactor | Epigenetic regulation and cancer |
| TET2 | DNA demethylase influenced by ascorbate availability | Leukemia and epigenetic therapy |
| SLC23A2 (SVCT2) | Sodium-dependent vitamin C transporter 2 | Ascorbate uptake and stress response |
| S100A4 | Calcium-binding protein regulated by ascorbate-mediated hydroxymethylation | Metastasis and inflammation |
| HIF1A | Hypoxia-inducible factor 1-alpha, regulated by ascorbate-dependent hydroxylation | Cancer and angiogenesis |
| VHL | E3 ubiquitin ligase that recognizes hydroxylated HIF1A | Von Hippel-Lindau disease and cancer |
| BGLB | β-glucosidase that binds ascorbic acid | Enzyme engineering and food science |
| SP | Sucrose phosphorylase engineered for ascorbic acid derivatives | Biocatalysis and industrial production |
| BCL2 | Apoptosis regulator influenced by ascorbate-induced ER stress | Breast cancer research |
| SQSTM1 (p62) | Autophagy receptor accumulated upon ascorbate treatment | Cancer and stress response |
| FXN | Frataxin, involved in Friedreich's ataxia and oxidative stress | Neurodegeneration and ascorbate therapy |
| NFE2L2 (NRF2) | Transcription factor regulating antioxidant response | Oxidative stress and neuroprotection |
How Is L-ascorbic acid binding Regulated?
L-ascorbic acid binding is regulated at multiple levels. Intracellular ascorbate concentration is controlled by transporters such as SVCT2, which mediates uptake and influences DNA hydroxymethylation reprogramming. Oxidative stress can deplete ascorbate, reducing binding and enzyme activity, while antioxidant recycling systems restore ascorbate levels. Additionally, the expression of ascorbate-dependent enzymes like prolyl hydroxylases is regulated by hypoxia and metabolic signals, indirectly affecting the extent of ascorbate binding. In cancer, ascorbate treatment induces endoplasmic reticulum stress and p62/SQSTM1 accumulation, suggesting that binding is linked to stress-responsive pathways.
L-ascorbic acid binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| EGLN1 | Cancer, hypoxia-related disorders | Knockout and point mutation in cancer cell lines |
| SLC23A2 | Stress response, epigenetic regulation | Overexpression and knockout in neuronal cells |
| FXN | Friedreich's ataxia | Knock-in of patient mutations in iPSCs |
| SQSTM1 | Breast cancer, autophagy | Knockout and overexpression in breast cancer cells |
| BGLB | Microbial virulence, enzyme function | Point mutation in bacterial β-glucosidase |
Cancer
L-ascorbic acid binding influences cancer progression through multiple mechanisms. In breast cancer, L-ascorbic acid inhibits growth by inducing IRE/JNK/CHOP-related endoplasmic reticulum stress and promoting p62/SQSTM1 accumulation in the nucleus. Ascorbate-dependent prolyl hydroxylases regulate HIF1A stability, affecting angiogenesis and metabolic adaptation in tumors. Moreover, ascorbate-mediated DNA hydroxymethylation can alter the expression of genes like S100A4, which is implicated in metastasis. These findings highlight the therapeutic potential of targeting ascorbate binding in oncology.
Neurodegeneration
In Friedreich's ataxia, a neurodegenerative disorder caused by frataxin deficiency, L-ascorbic acid in combination with N-acetylcysteine and dimethyl fumarate shows therapeutic benefits in in vitro models. Ascorbate binding protects against oxidative stress and mitochondrial dysfunction, which are central to neurodegeneration. Additionally, L-ascorbic acid prevents lead-induced brain damage by mitigating oxidative stress and inflammation, as demonstrated in rat models. These studies suggest that maintaining proper ascorbate binding is neuroprotective.
Infectious Diseases
L-ascorbic acid exerts anti-microbial and anti-virulence effects against multi-drug resistant Burkholderia cepacia complex, potentially through binding to bacterial proteins and disrupting virulence factors. This highlights a role for ascorbate binding in host-pathogen interactions and suggests that ascorbate-based therapies could combat antibiotic-resistant infections.
From L-ascorbic acid binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of EGLN1 affect HIF1A stability? | CRISPR knockout of EGLN1 in HeLa cells |
| How does a point mutation in the ascorbate-binding pocket alter enzyme activity? | CRISPR point mutation knock-in in HEK293T cells |
| Can overexpression of SVCT2 enhance ascorbate uptake and DNA hydroxymethylation? | Overexpression of SLC23A2 in neuroblastoma cells |
| What is the effect of tagged EGLN1 on protein interactions? | Knock-in of FLAG-tagged EGLN1 in cancer cells |
| Does knockout of SQSTM1 abolish ascorbate-induced ER stress? | CRISPR knockout of SQSTM1 in breast cancer cells |
| Can a disease-associated mutation in FXN be corrected? | Knock-in of wild-type FXN in patient-derived iPSCs |
How to Study the L-ascorbic acid binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Isothermal titration calorimetry (ITC) | Binding affinity and thermodynamics | Characterizing ascorbate binding to purified proteins |
| Surface plasmon resonance (SPR) | Real-time binding kinetics | Screening for ascorbate-binding proteins |
| X-ray crystallography | Three-dimensional structure of protein-ascorbate complex | Elucidating binding pocket and metal coordination |
| CRISPR knockout screening | Gene essentiality and pathway identification | Discovering regulators of ascorbate binding |
| hMeDIP-seq | DNA hydroxymethylation patterns | Linking ascorbate binding to epigenetic changes |
| Western blot | Protein expression and stability | Assessing HIF1A levels upon ascorbate treatment |
| Cell viability assays | Proliferation and survival | Testing ascorbate effects on cancer cells |
| Microbial virulence assays | Pathogen virulence and antimicrobial susceptibility | Evaluating ascorbate against Burkholderia cepacia |
Biochemical Binding Assays
Direct measurement of L-ascorbic acid binding can be performed using isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or fluorescence quenching. These methods quantify binding affinity and stoichiometry, as demonstrated in structural studies of β-glucosidase-ascorbic acid complexes. Such assays are essential for validating candidate ascorbate-binding proteins and for characterizing mutant variants.
Structural Biology
X-ray crystallography and cryo-electron microscopy provide atomic-level insights into the ascorbate-binding pocket and its coordination with metal ions. For example, structural studies of HIF prolyl hydroxylases have revealed how ascorbate binds and participates in catalysis. These techniques guide the design of point mutations to probe function.
CRISPR Screening
Genome-wide CRISPR knockout screens can identify genes whose loss affects ascorbate-dependent phenotypes, such as cell growth under oxidative stress or hypoxia. This approach has been used to uncover pathways related to ascorbate metabolism and binding. Hits can be validated with targeted knockouts and point mutations.
Epigenomic Profiling
DNA hydroxymethylation and methylation patterns can be assessed by bisulfite sequencing or hydroxymethylated DNA immunoprecipitation (hMeDIP) following modulation of ascorbate levels. SVCT2-mediated ascorbate uptake alters hydroxymethylation of the S100A4 gene, demonstrating a direct link between binding and epigenetic regulation.
How CRISPR Can Be Used to Study GO:0031418 L-ascorbic acid binding
Knockout
CRISPR knockout of genes encoding L-ascorbic acid-binding proteins, such as EGLN1 or SLC23A2, allows researchers to assess loss-of-function phenotypes. For example, knocking out EGLN1 stabilizes HIF1A and alters hypoxia responses. Knockout of SLC23A2 reduces ascorbate uptake and affects DNA hydroxymethylation. These models are invaluable for target validation.
Point Mutation
Introducing point mutations in the ascorbate-binding pocket can dissect the contribution of specific residues to binding affinity and catalysis. For instance, mutating iron-coordinating residues in prolyl hydroxylases abolishes ascorbate-dependent activity. Such models help distinguish binding from catalytic function.
Knock-in
Knock-in of tagged or disease-associated variants enables tracking of protein localization and interaction. For example, FLAG-tagged EGLN1 can be knocked into cells to study its interactome under ascorbate treatment. Knock-in of patient mutations in FXN models Friedreich's ataxia for therapeutic testing.
Overexpression
Overexpression of ascorbate-binding proteins or transporters can enhance cellular responses. Overexpressing SVCT2 increases ascorbate uptake and buffers stress responses via DNA hydroxymethylation. This approach is useful for gain-of-function studies and for producing recombinant proteins for biochemical assays.
How EDITGENE Supports L-ascorbic acid binding Research
Researchers studying L-ascorbic acid binding-related genes often need to determine whether a candidate gene is causally involved in a phenotype or whether a specific mutation alters binding affinity. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research, from knockout to precise point mutations and knock-in models.
Contact EDITGENE today to design your custom CRISPR model for L-ascorbic acid binding research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| EGLN1 Knockout HEK293 Cell Line | EDJ-KQ1495 | Human | 54583 | Details Get a Quote |
| EGLN3 Knockout HEK293 Cell Line | EDJ-KQ1497 | Human | 112399 | Details Get a Quote |
| EGLN2 Knockout HEK293 Cell Line | EDJ-KQ1498 | Human | 112398 | Details Get a Quote |
| DBH Knockout HEK293 Cell Line | EDJ-KQ2134 | Human | 1621 | Details Get a Quote |
| PLOD1 Knockout HEK293 Cell Line | EDJ-KQ3159 | Human | 5351 | Details Get a Quote |
| P4HA1 Knockout HEK293 Cell Line | EDJ-KQ3363 | Human | 5033 | Details Get a Quote |
| PHYH Knockout HEK293 Cell Line | EDJ-KQ5454 | Human | 5264 | Details Get a Quote |
| PLOD2 Knockout HEK293 Cell Line | EDJ-KQ5484 | Human | 5352 | Details Get a Quote |
| P4HA2 Knockout HEK293 Cell Line | EDJ-KQ6417 | Human | 8974 | Details Get a Quote |
| PLOD3 Knockout HEK293 Cell Line | EDJ-KQ6421 | Human | 8985 | Details Get a Quote |
| P3H1 Knockout HEK293 Cell Line | EDJ-KQ11360 | Human | 64175 | Details Get a Quote |
| P4HTM Knockout HEK293 Cell Line | EDJ-KQ11483 | Human | 54681 | Details Get a Quote |
| OGFOD1 Knockout HEK293 Cell Line | EDJ-KQ14555 | Human | 55239 | Details Get a Quote |
| OGFOD2 Knockout HEK293 Cell Line | EDJ-KQ14557 | Human | 79676 | Details Get a Quote |
| P3H2 Knockout HEK293 Cell Line | EDJ-KQ14655 | Human | 55214 | Details Get a Quote |
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Frequently Asked Questions About L-ascorbic acid binding
What is L-ascorbic acid binding?
L-ascorbic acid binding (GO:0031418) is a molecular function where a protein selectively binds to vitamin C, often as a cofactor for enzymatic reactions.
What genes are involved in L-ascorbic acid binding?
Key genes include EGLN1, EGLN2, EGLN3, P4HA1, P4HA2, TET1, TET2, SLC23A2, and SQSTM1, among others [6,7].
How does L-ascorbic acid binding affect cancer?
It can inhibit breast cancer growth by inducing ER stress and p62/SQSTM1 accumulation, and it regulates HIF1A stability in tumors [4,6].
What diseases are associated with L-ascorbic acid binding?
Diseases include cancer, Friedreich's ataxia, lead-induced neurotoxicity, and infections by multi-drug resistant bacteria [1,2,5].
What is the role of ascorbate in epigenetics?
Ascorbate binding supports DNA hydroxymethylation, influencing gene expression such as S100A4, via TET enzymes.
How can I study L-ascorbic acid binding in the lab?
Use biochemical assays like ITC or SPR, structural biology, CRISPR knockout/knock-in models, and epigenomic profiling [6,7,8].
What are the research methods for L-ascorbic acid binding?
Common methods include isothermal titration calorimetry, X-ray crystallography, CRISPR screening, and hMeDIP-seq [6,7,8].
Why is L-ascorbic acid binding important for drug discovery?
It is a target for cancer therapy, neuroprotection, and anti-microbial strategies, as ascorbate modulates key enzymes and stress responses [4,5].
Can CRISPR be used to study L-ascorbic acid binding?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models enable precise interrogation of genes involved in ascorbate binding [6,7].
What cell models are available for L-ascorbic acid binding research?
EDITGENE offers knockout, point mutation, knock-in, tagged knock-in, and overexpression cell models for genes like EGLN1, SLC23A2, and SQSTM1.
Conclusion
L-ascorbic acid binding (GO:0031418) is a fundamental molecular function that underpins diverse biological processes, from oxygen sensing and collagen synthesis to epigenetic regulation and stress responses. Its dysregulation is implicated in cancer, neurodegeneration, and infectious diseases, making it a compelling target for therapeutic intervention. By leveraging CRISPR-based models and advanced biochemical techniques, researchers can dissect the precise roles of ascorbate-binding proteins and translate these insights into clinical applications.
References
- 1. Edzeamey FJ et al.. 2025. Therapeutic combination of L-ascorbic acid, N-acetylcysteine, and dimethyl fumarate in Friedreich's ataxia: insights from in vitro models.. Redox Rep 30(1):2505303 PMID: 40375363
- 2. Alhusaini AM et al.. 2022. L-Ascorbic Acid and Curcumin Prevents Brain Damage Induced via Lead Acetate in Rats: Possible Mechanisms.. Dev Neurosci 44(2):59-66 PMID: 34942627
- 3. Shen Y et al.. 2025. Production of 2-O-α-d-glucopyranosyl-l-ascorbic acid using sucrose phosphorylase by semi-rational design.. Int J Biol Macromol 284(Pt 2):138213 PMID: 39617240
- 4. Choi YK et al.. 2020. L-Ascorbic Acid Inhibits Breast Cancer Growth by Inducing IRE/JNK/CHOP-Related Endoplasmic Reticulum Stress-Mediated p62/SQSTM1 Accumulation in the Nucleus.. Nutrients 12(5) PMID: 32397306
- 5. Bhujbal SR et al.. 2026. L-ascorbic acid exerts anti-microbial and anti-virulence effects against multi-drug resistant Burkholderia cepacia complex.. Microb Pathog 214:108409 PMID: 41763415
- 6. Osipyants AI et al.. 2018. L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?. Biochimie 147:46-54 PMID: 29289682
- 7. Han QQ et al.. 2022. SVCT2-mediated ascorbic acid uptake buffers stress responses via DNA hydroxymethylation reprogramming of S100 calcium-binding protein A4 gene.. Redox Biol 58:102543 PMID: 36436457
- 8. Ding X et al.. 2027. Structural and molecular insights into the binding mechanisms of β-glucosidase-ascorbic acid-rutin/isoquercetin ternary systems.. Spectrochim Acta A Mol Biomol Spectrosc 364(Pt 2):128623 PMID: 42660054
