L2HGDH Antibody, HRP conjugated

What is the biological significance of L2HGDH in metabolic regulation?

L2HGDH is a FAD-dependent enzyme that oxidizes L-2-hydroxyglutarate (L-2HG) to alpha-ketoglutarate in various mammalian tissues . The enzyme is critical for preventing pathological accumulation of L-2HG, which can inhibit alpha-ketoglutarate-dependent enzymes involved in diverse biological processes. Mutations in the L2HGDH gene cause L-2-hydroxyglutaric aciduria, a rare autosomal recessive neurometabolic disorder resulting in moderate to severe cognitive disability .

Key biological functions include:

What are the critical specifications for L2HGDH antibodies used in research?

When selecting an L2HGDH antibody for experiments, researchers should consider these specifications:

For optimal experimental design, choose an antibody with validated reactivity against your species of interest and appropriate for your intended application .

How should L2HGDH antibodies be validated before experimental use?

Methodological approach to antibody validation:

• Positive and negative controls: Use tissue or cell lines with known L2HGDH expression levels. Research shows successful detection in A549 cells, rat brain tissue, mouse small intestine tissue, SGC-7901 cells, and MCF-7 cells .

• Knockout validation: Generate L2HGDH knockout cells using CRISPR/Cas9 as demonstrated in studies with HK-2 immortalized renal epithelial cells, which naturally express high levels of L2HGDH .

• Recombinant protein testing: Confirm antibody specificity using purified recombinant L2HGDH protein.

• Cross-reactivity assessment: Test for potential cross-reactivity with related proteins, particularly with D-2-hydroxyglutarate dehydrogenase.

• Dilution optimization: For Western blotting, a dilution range of 1:1000-1:3000 has been validated for certain L2HGDH antibodies .

How does L2HGDH function influence amino acid metabolism in cancer cells?

Recent research has revealed that L2HGDH plays a critical role in regulating amino acid metabolism, particularly in renal cell carcinoma (RCC):

• Transcriptional regulation: Restoration of L2HGDH in RCC cells with reduced L2HGDH expression significantly increased mRNAs related to amino acid metabolism, including biosynthetic enzymes and metabolite transporters .

• Serine biosynthesis pathway: L2HGDH restoration upregulated enzymes involved in de novo serine biosynthesis, particularly phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) .

• ATF4-dependent regulation: L2HGDH catalytic activity increases activating transcription factor 4 (ATF4) protein expression, a master regulator of amino acid metabolism genes .

• Epigenetic mechanisms: L-2HG accumulation due to reduced L2HGDH affects RNA m6A methylation, with specific impacts on the 3'-UTR of PSAT1 mRNA, influencing its stability and expression .

This suggests that when designing experiments to study L2HGDH in cancer models, researchers should include analysis of both epigenetic modifications and downstream amino acid biosynthetic pathways.

What mechanisms drive L-2HG production under different physiological conditions?

L-2HG can be produced through both enzymatic and non-enzymatic mechanisms:

Experimental insight: Purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) enzymes can catalyze stereospecific reduction of α-ketoglutarate to L-2HG. Importantly, gas chromatography-mass spectrometry (GC-MS) analysis confirmed that this reaction produces specifically the L-enantiomer, not D-2HG .

When designing experiments to study L-2HG production, researchers should consider controlling for pH, oxygen levels, and α-ketoglutarate concentration, as these factors significantly influence L-2HG levels independent of L2HGDH activity.

How can I distinguish between L-2HG and D-2HG in experimental samples?

Given the differing biological roles of L-2HG and D-2HG enantiomers, precise analytical methods are crucial:

• Chiral derivatization coupled with GC-MS: This technique has been validated for differentiating between L-2HG and D-2HG. Research has demonstrated that LDH and MDH catalyze stereospecific reduction of α-ketoglutarate to L-2HG, which can be confirmed through comparison to derivatized standards .

• Enantiomer-specific enzymatic assays: Develop assays utilizing the stereospecificity of L2HGDH for L-2HG.

• Liquid chromatography-mass spectrometry (LC-MS): Studies have utilized LC-MS to measure L-2HG levels, showing significant changes in response to L2HGDH expression .

When interpreting results, note that L2HGDH knockout in HK-2 renal epithelial cells increased L-2HG levels without significantly affecting D-2HG levels, confirming the specificity of L2HGDH for the L-enantiomer .

What are optimal protocols for investigating L2HGDH expression and activity?

Western Blot Protocol Optimization:

• Sample preparation: Use standard protein extraction methods for both total cellular and mitochondrial fractions

• Protein loading: 20-40 μg protein per lane

• Antibody dilution: 1:1000-1:3000 for primary antibody

• Detection: Multiple L2HGDH antibodies have shown successful detection at approximately 45 kDa (observed) versus calculated 50 kDa molecular weight

Immunohistochemistry Considerations:

• Antigen retrieval: Use TE buffer pH 9.0 or citrate buffer pH 6.0

• Antibody dilution: 1:50-1:500

• Validated tissues: Human liver cancer tissue and human gliomas tissue

Activity Assays:
To measure L2HGDH activity, design assays that track the conversion of L-2HG to α-ketoglutarate, potentially coupling this to NAD+/NADH conversion for spectrophotometric detection.

How can I investigate the relationship between L2HGDH and epigenetic regulation?

Recent research demonstrates that L-2HG levels significantly impact the epigenome through modulation of α-ketoglutarate-dependent dioxygenases:

• RNA m6A methylation analysis:

  • Dot blot assays using antibodies specific to the m6A mark have shown that L2HGDH restoration significantly reduces mRNA m6A levels

  • LC-MS can provide quantitative measurement of m6A levels

  • Transcriptome-wide profiling of RNA methylation (m6A-Seq) can determine the location and intensity of m6A peaks in mRNA

• Histone methylation assessment:

  • Western blotting for various histone methylation marks

  • ChIP-seq to analyze genome-wide distribution of histone modifications

• ALKBH5 and FTO inhibition studies:

  • Research has shown that L-2HG inhibits ALKBH5 activity in a dose-dependent manner on methylated RNA substrates

  • Knockdown of either FTO or ALKBH5 increases m6A levels in cells expressing L2HGDH

These approaches allow for comprehensive investigation of how L2HGDH impacts epigenetic regulation through control of L-2HG levels.

What are the cellular consequences of L2HGDH dysfunction in cancer models?

L2HGDH dysfunction leads to L-2HG accumulation, which has several important consequences in cancer cells:

• Altered amino acid metabolism: Reduced L2HGDH expression leads to decreased levels of amino acid biosynthetic enzymes including PHGDH, PSAT1, and asparagine synthetase (ASNS) .

• Dysregulated ATF4 expression: L-2HG accumulation suppresses ATF4, a master regulator of amino acid metabolism genes. Conversely, restoring L2HGDH increases ATF4 protein expression in multiple RCC lines .

• mTOR-ATF4 axis activation: In colorectal cancer, L-2HG has been shown to activate the mTOR-ATF4 axis, ameliorating nutritional stress and potentially serving as a therapeutic target .

• Epigenetic alterations: L-2HG accumulation increases RNA m6A methylation throughout the transcriptome, with enrichment in the 3'-UTR of mRNAs .

• HIF-1α stabilization: pH-dependent induction of L-2HG acts as a potent stabilizer of HIF-1α even under normoxia, representing a pathway of HIF-1α stabilization with potential relevance to human disease states .

These findings highlight the importance of considering both metabolic and epigenetic consequences when studying L2HGDH dysfunction in cancer models.

What are the best model systems for studying L-2-hydroxyglutaric aciduria?

L-2-hydroxyglutaric aciduria is a rare neurometabolic disorder caused by mutations in L2HGDH. For effective research:

• Cellular models:

  • CRISPR/Cas9-engineered L2HGDH knockout cell lines, such as the HK-2 immortalized renal epithelial cells model

  • Patient-derived fibroblasts or iPSCs carrying L2HGDH mutations

  • Cells expressing patient-derived L2HGDH mutants (e.g., A241G) that retain partial enzymatic activity

• Animal models:

  • L2HGDH knockout or knockin mice carrying disease-specific mutations

  • Naturally occurring animal models with L2HGDH mutations

• Treatment approaches:

  • Rescue experiments using wild-type L2HGDH cDNA

  • Metabolic interventions targeting pathways affected by L-2HG accumulation

  • Epigenetic modifiers to counteract the effects of L-2HG on histone and RNA methylation

When designing research on L-2-hydroxyglutaric aciduria, it's important to include both enzymatic activity assays and downstream pathway analyses to fully characterize the disease mechanisms.

How does the function of L2HGDH differ from enzymes involved in D-2HG metabolism?

Understanding the distinctions between L-2HG and D-2HG metabolism is crucial for accurate experimental design:

While both metabolites inhibit α-ketoglutarate-dependent enzymes, they have distinct origins and biological effects. Unlike D-2HG, which is prominently produced by mutant IDH enzymes in gliomas and AML, L-2HG can be produced by "promiscuous" activity of LDH and MDH enzymes under specific conditions like hypoxia and acidic pH .

Research has shown that while (R)-2-HG (D-2HG) stimulates the growth of non-transformed cells, it also displays antitumor activity by suppressing the growth of tumors harboring wild-type IDH2 . This contrasting behavior highlights the importance of distinguishing between these enantiomers in experimental design and interpretation.

What are emerging methods for targeting L2HGDH in therapeutic applications?

Based on current understanding of L2HGDH biology, several therapeutic approaches warrant investigation:

• Enzyme restoration strategies: Gene therapy approaches to restore functional L2HGDH in deficient cells.

• Targeting L-2HG production: Development of specific inhibitors for the promiscuous activity of LDH and MDH that produces L-2HG, particularly under acidic or hypoxic conditions.

• Epigenetic modulators: Given L-2HG's effects on RNA methylation and potentially histone modifications, epigenetic modulators might counteract the downstream effects of L-2HG accumulation.

• mTOR-ATF4 axis intervention: Research indicates that L-2HG activates the mTOR-ATF4 axis in colorectal cancer , suggesting this pathway as a potential therapeutic target.

• Metabolic reprogramming: Strategies to counteract the amino acid metabolic alterations caused by L-2HG accumulation, potentially through supplementation of key amino acids or metabolites.