D2HGDH Antibody

What is D2HGDH and what is its biochemical function?

D2HGDH (D-2-hydroxyglutarate dehydrogenase) is a mitochondrial enzyme that catalyzes the oxidation of D-2-hydroxyglutarate (D-2-HG) to alpha-ketoglutarate (α-KG) . This enzyme plays a critical role in cellular metabolism by regulating α-KG levels, which are essential for numerous dioxygenase-dependent cellular processes. Beyond its primary substrate, D2HGDH also catalyzes the oxidation of other D-2-hydroxyacids, including D-malate (D-MAL) and, to a lesser extent, D-lactate (D-LAC) . The enzyme demonstrates high catalytic activity toward D-2-HG and D-MAL but exhibits considerably weaker activity toward D-LAC .

D2HGDH is encoded by the D2HGDH gene in humans, which may also be known as D2HGD and D-2-hydroxyglutarate dehydrogenase, mitochondrial . Structurally, the protein has a reported molecular mass of approximately 56.4 kilodaltons . Based on gene homology, orthologs are present in multiple species including plants, flies, canines, porcine, primates, mice, and rats .

What are the key applications for D2HGDH antibodies in research?

D2HGDH antibodies are valuable tools in multiple experimental applications, with the most common being:

• Western Blotting (WB): Used to detect and quantify D2HGDH protein levels in cell or tissue lysates .

• Immunohistochemistry (IHC): Applied to visualize the distribution and localization of D2HGDH in tissue sections .

• Immunocytochemistry (ICC)/Immunofluorescence (IF): Employed to determine subcellular localization of D2HGDH in cultured cells .

• Immunoprecipitation (IP): Used to isolate and concentrate D2HGDH from complex biological samples .

When selecting a D2HGDH antibody, researchers should consider:

• Species reactivity (human, mouse, rat are most common)

• Conjugation options (unconjugated, biotin, FITC, HRP, Alexa fluorophores)

• Validated applications for specific experimental needs

• Antibody type (polyclonal vs. monoclonal)

How can researchers validate the specificity of D2HGDH antibodies?

Validation of D2HGDH antibody specificity is critical for reliable experimental results. Recommended methodological approaches include:

• Positive and negative controls: Use cells/tissues with known D2HGDH expression levels. D2HGDH knockout (KO) models serve as excellent negative controls .

• Overexpression validation: Confirm antibody detection using cells transfected with D2HGDH expression constructs. SDS-PAGE/Western blot analysis with D2HGDH-overexpressing cells provides a direct assessment of antibody specificity .

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of D2HGDH to confirm consistent detection patterns.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal specificity.

For Western blot validation specifically, researchers should:

• Include anti-actin and anti-GFP antibodies as controls when working with tagged constructs

• Verify protein band size against the expected 56.4 kDa molecular weight

• Use enhanced chemiluminescent detection systems such as Lumi-Light Plus for optimal visualization

How does D2HGDH expression impact cancer metabolism and tumor progression?

D2HGDH plays a crucial role in cancer metabolism through its regulation of D-2-HG levels, which has significant implications for tumor progression:

• D2HG in IDH-mutant cancers: Mutations in isocitrate dehydrogenase (IDH1/2) result in the production of D-2-HG, which accumulates to millimolar levels in cancers like glioma and leukemia . D2HGDH is the primary enzyme responsible for catabolizing this oncometabolite.

• Epigenetic regulation: D2HGDH elevates α-KG levels, which influences histone and DNA methylation patterns . Conversely, D2HGDH mutations found in diffuse large B-cell lymphoma are enzymatically inactive, resulting in altered epigenetic landscapes that may contribute to oncogenesis .

• HIF1α hydroxylation: Through its effect on α-KG levels, D2HGDH modulates HIF1α hydroxylation, affecting cellular responses to hypoxia and potentially tumor angiogenesis .

• IDH2 regulation: D2HGDH positively modulates mitochondrial IDH activity and induces IDH2 expression, establishing a metabolic feedback loop . This relationship is bidirectional—genetic depletion of IDH2 abrogates D2HGDH effects, while ectopic IDH2 expression rescues D2HGDH-deficient cells .

• Tumorigenic maintenance: D-2-HG has been demonstrated to be essential for maintaining the oncogenic property of mutant IDH-containing cancer cells, though it appears dispensable for basic cell growth and proliferation .

What methodologies are available for measuring D2HG levels and D2HGDH activity?

Several methodological approaches have been developed to measure D2HG levels and assess D2HGDH activity:

• Single-cell D2HG detection: A resazurin-based fluorescence reporter system has been developed that adapts cascade enzymatic reactions to detect D2HG at the single-cell level . This method involves:

  • Immobilization of resazurin probes to sensing surfaces via biotin-streptavidin interaction

  • Optimization of surface chemistry to translate D2HG levels to sensitive fluorescence readouts

  • Integration with single-cell barcode chip (SCBC) technology for simultaneous profiling of multiple parameters

• Coupled enzymatic reactions: D2HGDH activity can be measured through a two-step enzymatic reaction:

  • D2HGDH catalyzes the conversion of D-2-HG to α-KG while converting NAD+ to NADH

  • The generated NADH fuels diaphorase-catalyzed redox reactions, reducing resazurin analogs to highly fluorescent resorufin analogs

• Mass spectrometry: Provides high specificity and sensitivity for D2HG measurements in bulk samples .

• Magnetic resonance spectroscopy: Non-invasive detection of D2HG in clinical samples and tissues .

The most sensitive approach for single-cell analysis combines D2HGDH-mediated reactions with fluorescent detection systems, allowing researchers to investigate metabolic heterogeneity within tumor cell populations .

What is the relationship between D2HGDH, IDH mutations, and cancer immunotherapy?

Research has revealed complex interactions between D2HGDH, IDH mutations, and cancer immunotherapy responses:

How do D2HGDH missense variants affect enzyme function?

Analysis of D2HGDH missense variants provides insight into structure-function relationships and potential pathogenic mechanisms:

• Functional analysis approaches:

  • SDS-PAGE/Western blot analysis using rabbit polyclonal anti-D2HGDH primary antibodies to detect protein expression levels

  • Use of polyclonal goat anti-rabbit immunoglobulins/HRP secondary antibodies and enhanced chemiluminescent detection

  • Control antibodies (anti-actin, anti-GFP) to normalize protein expression

  • In silico prediction tools (MaxEntScan, NNsplice, Human Splicing Finder) to evaluate potential effects on mRNA splicing

• Enzymatic consequences:

  • Certain D2HGDH mutants found in diffuse large B-cell lymphoma are enzymatically inert, lacking the ability to oxidize D-2-HG to α-KG

  • Loss of enzymatic function results in D-2-HG accumulation and reduced α-KG levels, affecting numerous downstream α-KG-dependent processes

• Secondary effects:

  • Altered regulation of mitochondrial IDH activity and IDH2 expression

  • Disrupted epigenetic remodeling due to changes in α-KG-mediated dioxygenase function

  • Potential impacts on HIF1α hydroxylation and hypoxic response mechanisms

What are the current applications of D2HGDH-modified cells in cancer therapy research?

Research has identified several promising applications for D2HGDH-modified cells in cancer therapy research:

These findings highlight the potential of D2HGDH-modified cell therapies as a novel approach to overcome the immunosuppressive effects of oncometabolites like D2HG in the tumor microenvironment of IDH-mutant cancers .

What are the key factors to consider when selecting D2HGDH antibodies for specific applications?

When selecting D2HGDH antibodies for research applications, several technical factors should be considered:

• Antibody type and origin:

  • Polyclonal antibodies (e.g., rabbit polyclonal) offer broad epitope recognition but may have batch-to-batch variation

  • The immunogen used for antibody production should target relevant regions of D2HGDH (e.g., amino acids 50-250 of human D2HGDH)

• Species reactivity:

  • Verify cross-reactivity with your species of interest (commonly available for human, mouse, and rat samples)

  • Consider potential differences in D2HGDH structure between species when interpreting results

• Application-specific validation:

  • Western Blot (WB): Confirm the antibody detects the expected 56.4 kDa band

  • Immunohistochemistry (IHC): Verify antibodies are suitable for paraffin-embedded tissues if applicable

  • ICC/IF: Ensure antibodies can detect the protein under fixation conditions used

• Conjugation options:

  • Unconjugated antibodies for flexible detection methods

  • Conjugated versions (biotin, FITC, HRP, Alexa fluorophores) for direct detection

  • Consider downstream applications when selecting conjugation type

• Concentration and format:

  • Available quantities typically range from 50 μl to 1 ml

  • Consider the number of experiments planned when selecting quantity

What are the recommended protocols for using D2HGDH antibodies in Western blotting?

The following methodological approach is recommended for optimal results when using D2HGDH antibodies in Western blotting:

• Sample preparation:

  • For cell lysates: Use standard SDS-PAGE sample preparation procedures

  • For detection of overexpressed D2HGDH: Follow established protocols for transfection and lysis

• Gel electrophoresis and transfer:

  • Use standard SDS-PAGE procedures, typically with 10-12% polyacrylamide gels

  • Transfer to appropriate membrane (PVDF or nitrocellulose)

• Antibody incubation:

  • Primary antibody: Use rabbit polyclonal anti-D2HGDH antibody at manufacturer-recommended dilution

  • Secondary antibody: Polyclonal goat anti-rabbit immunoglobulins/HRP is recommended

• Detection:

  • Enhanced chemiluminescent detection using Lumi-Light Plus Western blot substrate is effective

  • Image acquisition using systems like ChemiDoc MP Imager provides optimal results

• Controls:

  • Include anti-actin antibody as a loading control

  • When working with tagged D2HGDH constructs, include anti-tag antibodies (e.g., anti-GFP)

  • For validating D2HGDH-specific bands, include positive controls (overexpression) and negative controls (knockdown/knockout) when possible

How can researchers incorporate D2HGDH antibodies in single-cell analysis workflows?

Single-cell analysis of D2HGDH and related metabolites requires specialized approaches:

• Single-cell barcode chip (SCBC) technology:

  • Enables simultaneous analysis of D2HGDH protein, D2HG levels, and other signaling proteins

  • Integrates surface-based D2HG assays with protein detection

  • Allows investigation of relationships between metabolism, oncogenic signaling, and cell proliferation

• Surface-based D2HG assay integration:

  • Use 16-well PDMS slabs on microscope glass slides with patterned single-strand DNA oligomers

  • Immobilize streptavidin-DNA (100 nM) and BP2Rz probe (10 μM) onto the surface

  • This approach enables detection of D2HG at the single-cell level through cascade enzymatic reactions

• Antibody-based detection:

  • Use D2HGDH antibodies for immunofluorescence to visualize protein expression and localization

  • Combine with metabolite detection for multiparametric analysis

  • Correlate D2HGDH levels with D2HG production and downstream cellular effects

• Workflow considerations:

  • Single-cell isolation should be optimized for the cell type of interest

  • Fixation and permeabilization protocols need validation for D2HGDH antibody compatibility

  • Multiplexed detection may require antibodies from different host species to avoid cross-reactivity

This integrated approach allows researchers to dissect heterogeneity in tumor cell populations and better understand the complex interplay between metabolic pathways and oncogenic signaling axes .

How does D2HGDH function in regulating the interconversion between D2HG and α-KG impact tumor biology?

The regulation of D2HG and α-KG interconversion by D2HGDH has profound implications for tumor biology:

What emerging research approaches are advancing our understanding of D2HGDH in disease?

Several cutting-edge research approaches are advancing our understanding of D2HGDH's role in disease:

• Genetic modification technologies:

  • CRISPR-Cas9 engineering of D2HGDH knockout and overexpression models

  • Development of engineered CAR-T cells with D2HGDH modifications for therapeutic applications

  • Generation of cell lines with varying levels of D2HGDH expression to study dose-dependent effects

• Single-cell metabolic profiling:

  • Single-cell barcode chip technology allowing simultaneous analysis of D2HG, glucose uptake, and signaling proteins

  • Surface-based D2HG assays compatible with single-cell resolution

  • Integration of metabolic and proteomic data to reveal complex interplays between pathways

• Functional genomics:

  • Systematic analysis of D2HGDH missense variants to assess their impact on enzyme function

  • Evaluation of variants using in silico splice prediction tools (MaxEntScan, NNsplice, Human Splicing Finder)

  • Correlation of genetic variations with disease phenotypes and severity

• Therapeutic targeting strategies:

  • Development of D2HGDH-OE CAR-T cells that catabolize D2HG in the tumor microenvironment

  • Exploration of combination therapies targeting both D2HGDH and related metabolic pathways

  • Investigation of D2HGDH modulation as a strategy to overcome immunotherapy resistance in IDH-mutant cancers

These emerging approaches are providing unprecedented insights into the biological roles of D2HGDH and opening new avenues for therapeutic intervention in diseases characterized by D2HG accumulation or D2HGDH dysfunction.