Recombinant Mouse Diacylglycerol kinase epsilon (Dgke)

What is Diacylglycerol kinase epsilon and what are its primary functions?

Diacylglycerol kinase epsilon (DGKE) is a protein encoded by the DGKE gene that plays a crucial role in lipid metabolism. It is highly selective for arachidonate-containing species of diacylglycerol (DAG). The enzyme functions in two primary ways: it may terminate signals transmitted through arachidonoyl-DAG or contribute to the synthesis of phospholipids with defined fatty acid composition . DGKE is expressed in multiple tissues including endothelium, platelets, and podocytes . Understanding these basic functions is essential for designing experiments that investigate its role in cellular signaling pathways and pathological conditions.

What expression systems are available for producing Recombinant Mouse Dgke?

Recombinant Mouse Diacylglycerol kinase epsilon (Dgke) can be produced in multiple expression systems, each offering distinct advantages depending on research objectives:

The choice of expression system should be guided by experimental requirements for protein folding, post-translational modifications, and functional activity .

How can researchers validate the successful expression and purification of Recombinant Mouse Dgke?

Validation of Recombinant Mouse Dgke expression and purification requires multiple complementary approaches. Western blotting using specific antibodies (such as CSB-PA952992) can confirm protein identity and molecular weight . For functional validation, enzymatic activity assays measuring phosphorylation of arachidonate-containing DAG substrates provide critical information. Researchers should assess protein purity through SDS-PAGE and mass spectrometry. For structural integrity, circular dichroism or thermal shift assays can be employed. When reporting results, it is essential to document both protein concentration (determined by Bradford or BCA assay) and specific activity to ensure reproducibility across different experimental settings.

How does DGKE knockdown affect endothelial cell function in experimental models?

DGKE knockdown in endothelial cells produces multiple measurable phenotypic changes that can be quantified in laboratory settings. Studies using siRNA-mediated knockdown in Human Umbilical Vein Endothelial Cells (HUVECs) and Human Microvascular Endothelial Cells (HMECs) have demonstrated:

• Increased ICAM-1 expression (significantly elevated) and E-selectin expression (minimally but significantly increased), indicating endothelial cell activation

• Marked increase in tissue factor (TF) expression at the cell surface with subsequent release of TF into cell supernatants 48-72 hours post-transfection

• Potentiation of TNFα-induced TF expression, suggesting that DGKE absence may amplify procoagulant effects of inflammatory mediators

• Significant increase in platelet adhesion compared to control cells

• Decreased expression of membrane cofactor protein (MCP/CD46), a complement inhibitory protein

These cellular responses collectively indicate that DGKE deficiency promotes endothelial activation and potentially initiates thrombosis—key features observed in atypical hemolytic-uremic syndrome (aHUS) .

What methodologies are most effective for studying DGKE function in mouse models?

When investigating DGKE function in mouse models, researchers should implement a multi-faceted approach:

• Genetic manipulation techniques: CRISPR/Cas9-mediated knockout or knockin strategies are preferred for generating DGKE-deficient or mutant mice. Site-specific mutations corresponding to human pathogenic variants can provide insights into structure-function relationships.

• Phenotypic characterization: Comprehensive assessment should include:

  • Renal function tests (proteinuria, hematuria, BUN, creatinine)

  • Blood pressure monitoring (DGKE-deficient models often show persistent hypertension)

  • Complete blood counts for detection of hemolytic anemia

  • Platelet function assays

• Histopathological analysis: Kidney tissue examination for signs of thrombotic microangiopathy (TMA) and/or membranoproliferative glomerulonephritis (MPGN) patterns .

• Molecular analyses: RNA-seq and proteomics to identify differentially expressed genes and proteins in affected tissues, with particular focus on complement pathway components and coagulation factors.

• Rescue experiments: Introduction of wild-type Recombinant Mouse Dgke to determine if phenotypic abnormalities can be reversed, providing compelling evidence for causality.

When reporting findings, researchers should document both the genotype and phenotypic manifestations with precise age of onset, as DGKE-associated disease typically manifests in the first year of life but shows variable expressivity .

How do DGKE mutations contribute to atypical hemolytic-uremic syndrome (aHUS) pathogenesis?

DGKE is the first gene implicated in aHUS that is not an integral component of the complement cascade, which raises important questions about alternative pathophysiologic mechanisms . Current research indicates that DGKE mutations contribute to aHUS through multiple potential mechanisms:

The disease presents a distinctive Mendelian pattern with recessive inheritance. Affected patients typically present with aHUS before age 1, have persistent hypertension, hematuria, and proteinuria (sometimes in the nephrotic range), and develop chronic kidney disease with age .

What are the recommended protocols for functional assays of Recombinant Mouse Dgke activity?

To accurately assess Recombinant Mouse Dgke activity, researchers should consider these methodological approaches:

• Kinase activity assay: Measure the phosphorylation of arachidonoyl-DAG to phosphatidic acid using radiolabeled ATP ([γ-32P]ATP) or non-radioactive alternatives. The assay should be performed at physiological pH (7.2-7.4) and temperature (37°C).

• Substrate specificity analysis: Compare phosphorylation rates of different DAG species (varying in fatty acid composition) to confirm the preference for arachidonate-containing DAGs. This is critical for validating the functional integrity of the recombinant protein.

• Inhibitor studies: Test known DGK inhibitors (such as R59022 or R59949) to establish inhibition profiles and IC50 values.

• Cell-based functional reconstitution: Introduce Recombinant Mouse Dgke into DGKE-knockout cells and measure:

  • Restoration of normal DAG levels

  • Normalization of PKC signaling

  • Reduction in cell activation markers (ICAM-1, E-selectin)

  • Restoration of normal complement regulatory protein expression

• Protein-protein interaction studies: Investigate binding partners using pull-down assays or surface plasmon resonance with the recombinant protein.

Data should be normalized to protein concentration and presented with appropriate statistical analysis, including technical and biological replicates to ensure reproducibility.

How does the phenotypic spectrum of DGKE-associated nephropathy vary in research models?

The phenotypic spectrum of DGKE-associated nephropathy in research models reflects the variable presentation observed in humans. Most patients present with hemolytic-uremic syndrome (HUS) accompanied by proteinuria, while a subset exhibits clinical and histologic patterns of membranoproliferative glomerulonephritis (MPGN) without thrombotic microangiopathy (TMA) .

Key observations from patient studies that inform research models include:

• Variable histopathology: Even siblings with identical DGKE mutations (e.g., p.W322*/p.W322*) can show different histologic features (MPGN/TMA versus TMA) and outcomes (preserved renal function versus rapid ESRD development) .

• Age-related progression: DGKE-HUS typically manifests in the first year of life but is not exclusively limited to infancy. Research models should account for this developmental timeline .

• Environmental triggers: Viral infections frequently precede HUS episodes, suggesting that research models should incorporate inflammatory challenges to fully recapitulate the disease .

• Complement activation: Some patients with DGKE mutations show signs of complement activation, while others do not, indicating heterogeneity in pathogenic mechanisms .

• Long-term outcomes: Approximately 80% of patients do not develop end-stage renal disease within 10 years of diagnosis, but most develop slowly progressive proteinuric nephropathy .

When designing mouse models of DGKE-associated nephropathy, researchers should consider incorporating these variables through conditional knockouts, inducible systems, or environmental challenges to capture the full spectrum of disease manifestations.

What are the technical challenges in using Recombinant Mouse Dgke for complement-related research?

Using Recombinant Mouse Dgke in complement-related research presents several technical challenges that researchers should address:

• Maintaining protein stability: DGKE is a membrane-associated enzyme, making it challenging to maintain proper folding and activity in recombinant form. Researchers should consider using detergent screening to identify optimal solubilization conditions.

• Expression system selection: While E. coli-expressed protein (CSB-CF865640MO) is readily available, it may lack post-translational modifications potentially relevant to complement interactions. Mammalian expression systems (CSB-MP865640MO1) may provide more physiologically relevant protein for complement studies .

• Experimental design complexity: Since DGKE is not directly part of the complement cascade, studying its effects on complement activation requires carefully designed experiments that can distinguish direct versus indirect effects.

• In vitro versus in vivo discrepancies: Complement activation observed in cell culture studies may not fully recapitulate the complexity of in vivo complement regulation. Researchers should validate findings across multiple experimental systems.

• Species differences: Mouse complement system differs from human in several aspects, potentially affecting translational relevance. Consider using humanized mice or complementary human cell systems when studying DGKE-complement interactions.

To address these challenges, researchers should incorporate appropriate controls, including both wild-type DGKE and catalytically inactive mutants, and measure multiple parameters of complement activation (C3b deposition, MCP expression, etc.) as demonstrated in previous studies .

How can researchers distinguish between DGKE-dependent and complement-dependent mechanisms in aHUS models?

Distinguishing between DGKE-dependent and complement-dependent mechanisms in aHUS models requires systematic experimental approaches:

• Genetic manipulation strategies: Generate:

  • DGKE knockout models

  • Complement component knockout models (C3, CFH, etc.)

  • Double knockout models
    This approach allows assessment of whether DGKE deficiency requires intact complement for disease manifestation.

• Pharmacological interventions: Treat DGKE-deficient models with:

  • Complement inhibitors (e.g., eculizumab)

  • PKC inhibitors

  • Anticoagulants
    Differential responses help delineate pathogenic mechanisms.

• Biomarker profiling: Measure:

  • Complement activation products (C3a, C5a, C5b-9)

  • Endothelial activation markers (ICAM-1, E-selectin)

  • Coagulation parameters

  • PKC activity
    Temporal relationships between these markers can suggest causality.

• Cell-specific conditional knockouts: Target DGKE deletion to specific cell types (endothelium, platelets, podocytes) to determine which cellular compartment initiates disease processes.

• Rescue experiments: Test whether:

  • Complement inhibition rescues DGKE-deficient phenotypes

  • DGKE reconstitution normalizes complement activation

What experimental designs best evaluate the efficacy of Recombinant Mouse Dgke supplementation in disease models?

To evaluate the efficacy of Recombinant Mouse Dgke supplementation in disease models, researchers should implement rigorous experimental designs:

• Dose-response studies: Administer graduated doses of Recombinant Mouse Dgke to determine the minimal effective concentration for:

  • Restoring normal DAG metabolism

  • Normalizing PKC signaling

  • Reversing endothelial activation markers

  • Improving renal function parameters

• Timing optimization: Compare early versus late intervention to determine:

  • Preventive efficacy (pre-disease manifestation)

  • Therapeutic efficacy (post-disease manifestation)

  • Required duration of treatment

• Delivery methods assessment:

  • Systemic administration (intravenous, intraperitoneal)

  • Targeted delivery approaches (nanoparticles, cell-type specific targeting)

  • Gene therapy approaches (AAV-mediated DGKE expression)
    Each approach should be evaluated for tissue distribution, cellular uptake, and functional enzyme activity.

• Combinatorial approaches: Test Recombinant Mouse Dgke in combination with:

  • Complement inhibitors

  • Anti-inflammatory agents

  • Anticoagulants
    This helps determine whether Dgke supplementation alone is sufficient or if multimodal therapy is required.

• Long-term efficacy and safety: Monitor treated animals for:

  • Duration of therapeutic effect

  • Development of antibodies against recombinant protein

  • Off-target effects

  • Impact on disease progression markers

Outcome measures should include both biochemical parameters (DAG levels, PKC activity) and clinically relevant endpoints (proteinuria, renal function, histopathology). Statistical design should include adequate sample sizes with age-matched and sex-matched controls to account for potential variability.

What are the critical quality control parameters for Recombinant Mouse Dgke in experimental applications?

When using Recombinant Mouse Dgke for experimental applications, researchers must establish rigorous quality control parameters:

• Purity assessment:

  • SDS-PAGE should demonstrate >90% purity

  • Mass spectrometry confirmation of protein identity

  • Endotoxin testing (<1 EU/mg protein for cell culture applications)

• Functional validation:

  • Specific enzyme activity (nmol phosphate transferred/min/mg protein)

  • Substrate selectivity (preferential activity toward arachidonate-containing DAG)

  • Kinetic parameters (Km, Vmax, and catalytic efficiency)

• Stability monitoring:

  • Thermal stability profile

  • Activity retention after freeze-thaw cycles

  • Shelf-life determination under recommended storage conditions

• Batch consistency:

  • Lot-to-lot variation should be <15% for critical parameters

  • Documentation of expression system and purification method

  • Certificate of analysis with standardized testing results

• Application-specific validation:

  • For cell-based assays: absence of cytotoxicity at working concentrations

  • For in vivo studies: sterility and pyrogenicity testing

  • For binding studies: confirmation of proper folding through circular dichroism

Researchers should maintain detailed records of these parameters and report them in publications to ensure reproducibility. When comparing results across studies, differences in protein source (e.g., E. coli-derived CSB-CF865640MO versus mammalian-derived CSB-MP865640MO1) should be explicitly considered as these may affect experimental outcomes.

How can researchers address variability in transfection efficiency when conducting DGKE knockdown experiments?

Addressing variability in transfection efficiency during DGKE knockdown experiments requires systematic optimization and rigorous controls:

• Standardized transfection protocols:

  • Optimize siRNA concentration: Previous studies successfully used 10-20 nM DGKE siRNA in HUVECs and HMECs, achieving 50-60% knockdown

  • Control cell density: Maintain consistent confluence (70-80%) at transfection

  • Minimize passage number variation: Use cells within similar passage ranges (±2 passages)

• Quantitative knockdown assessment:

  • Perform quantitative PCR to measure DGKE mRNA levels (normalized to housekeeping genes like HPRT)

  • Confirm protein reduction through western blot analysis with densitometry

  • Establish acceptance criteria (e.g., >50% reduction in DGKE expression)

• Individual siRNA validation:

  • Test individual siRNAs from pooled sets to confirm specificity

  • Include scrambled siRNA controls

  • Consider rescue experiments with siRNA-resistant DGKE constructs

• Reporter systems:

  • Co-transfect with fluorescent reporters to identify successfully transfected cells

  • Consider using FACS to isolate transfected cell populations

• Statistical approaches:

  • Increase biological replicates (minimum n=3)

  • Perform power calculations to determine adequate sample sizes

  • Use appropriate statistical tests that account for variability

  • Consider mixed-effects models for analyzing repeated experiments

Previous studies demonstrated that transfection of HUVECs with DGKE siRNAs at 10 nM or 20 nM led to a dose-dependent decrease in DGKE mRNA expression of approximately 50% and 60%, respectively . Researchers should aim for similar efficiency and explicitly report transfection parameters and knockdown levels to facilitate reproducibility.

What approaches help resolve contradictory results in DGKE functional studies across different model systems?

When encountering contradictory results in DGKE functional studies across different model systems, researchers should implement these systematic approaches:

• Comprehensive model characterization:

  • Document precise genotypes (knockout, knockdown, overexpression)

  • Quantify DGKE expression/activity levels across models

  • Assess compensatory changes in related DGK isoforms

  • Evaluate baseline differences in relevant signaling pathways

• Standardized experimental conditions:

  • Use consistent culture conditions for in vitro studies

  • Control for environmental variables in animal studies

  • Standardize stimulation protocols (dose, duration, reagent source)

  • Conduct experiments in parallel when possible

• Multi-parameter analysis:

  • Measure multiple readouts simultaneously

  • Perform time-course studies to capture dynamic responses

  • Assess cell-type specific responses

• Cross-validation strategies:

  • Replicate key findings using independent methodologies

  • Validate in vitro findings in ex vivo or in vivo systems

  • Corroborate findings using patient-derived samples

• Meta-analysis approach:

  • Systematically compare methodological differences across studies

  • Identify variables that correlate with divergent results

  • Develop unified models that account for context-dependent functions

The discrepancy between complement activation status in different DGKE-deficient models illustrates this challenge. While some patients with DGKE mutations show no evidence of complement activation, others do . Similarly, DGKE knockdown in endothelial cells decreases MCP expression, potentially increasing susceptibility to complement-mediated damage . These apparently contradictory findings may reflect genuine biological variability, differences in experimental conditions, or complex gene-environment interactions that require integrated analysis to fully understand.

How should researchers design experiments to investigate the relationship between DGKE and the complement system?

Designing experiments to investigate the relationship between DGKE and the complement system requires carefully structured approaches that can detect both direct and indirect interactions:

• Systematic genetic manipulation:

  • Generate models with:

    • DGKE deficiency alone

    • Complement component deficiencies

    • Combined deficiencies

  • Use inducible systems to control timing of gene inactivation

• Comprehensive complement assessment:

  • Measure expression of complement regulatory proteins (MCP/CD46, DAF/CD55, CD59) on relevant cell types

  • Quantify C3b deposition on cell surfaces following serum exposure

  • Assess terminal complement complex (C5b-9) formation

  • Measure fluid-phase complement activation markers

• Mechanistic dissection:

  • Perform pathway inhibition studies using:

    • PKC inhibitors

    • DAG analogues

    • Specific complement pathway blockers

  • Conduct rescue experiments with wild-type and mutant DGKE

• Temporal relationship analysis:

  • Establish time course of molecular events following DGKE inactivation

  • Determine whether complement activation precedes or follows other cellular changes

• Cell-specific effects:

  • Compare DGKE-complement interactions in:

    • Endothelial cells

    • Platelets

    • Podocytes

    • Leukocytes

Previous research has shown that DGKE knockdown in HUVECs and HMECs resulted in a marked decrease in MCP expression but had variable effects on other complement regulators (DAF expression slightly increased; CD59 expression unchanged) . This suggests cell-type specific regulatory mechanisms that should be systematically investigated. Researchers should also compare results in static versus flow conditions, as hemodynamic factors may influence complement-endothelial interactions in ways relevant to aHUS pathophysiology.

What novel applications of Recombinant Mouse Dgke are being explored beyond nephropathy research?

Beyond nephropathy research, Recombinant Mouse Dgke is being explored in several innovative research areas:

• Neuroscience applications:

  • Investigation of DGKE's role in neuronal signaling given the importance of DAG-PKC pathways in synaptic plasticity

  • Potential implications for neurodegenerative disorders where lipid metabolism is dysregulated

• Cardiovascular research:

  • Exploration of DGKE's role in regulating endothelial barrier function

  • Investigation of DGKE in atherosclerosis progression given its expression in endothelial cells and involvement in inflammatory pathways

  • Study of cardiac hypertrophy models, as PKC signaling is involved in cardiac remodeling

• Immunology applications:

  • Examination of DGKE's role in regulating immune cell activation and function

  • Investigation of potential involvement in autoimmune disorders

• Cancer biology:

  • Study of DGKE in tumor angiogenesis given its role in endothelial cell function

  • Exploration of DGKE-dependent signaling in cancer cell proliferation and survival

• Drug discovery platforms:

  • Development of high-throughput screening assays using Recombinant Mouse Dgke to identify novel modulators

  • Investigation of DGKE as a potential therapeutic target for diseases beyond nephropathy

These emerging applications leverage the fundamental understanding of DGKE's biochemical functions while exploring its roles in diverse physiological and pathological contexts. Researchers entering these fields should consider the technical challenges associated with measuring DGKE activity in different cellular contexts and develop tissue-specific assays.

How can researchers integrate multi-omics approaches to better understand DGKE function?

Integrating multi-omics approaches provides powerful strategies for comprehensively understanding DGKE function:

• Coordinated multi-omics experimental design:

  • Apply transcriptomics, proteomics, metabolomics, and lipidomics to the same experimental model

  • Include time-course sampling to capture dynamic responses to DGKE manipulation

  • Incorporate both basal and stimulated conditions (e.g., inflammatory challenges)

• Transcriptomics applications:

  • Compare gene expression profiles between wild-type and DGKE-deficient models

  • Identify transcriptional networks affected by DGKE deficiency

  • Analyze alternative splicing patterns of DGKE and related pathway components

• Proteomics strategies:

  • Quantify changes in protein abundance and post-translational modifications

  • Perform phosphoproteomics to map PKC-dependent phosphorylation events

  • Conduct protein-protein interaction studies using proximity labeling coupled with mass spectrometry

• Metabolomics and lipidomics:

  • Profile changes in DAG species with particular attention to arachidonate-containing DAGs

  • Analyze phosphatidic acid production as a direct measure of DGKE activity

  • Map alterations in downstream lipid metabolites and signaling molecules

• Computational integration frameworks:

  • Apply network analysis to identify pathway connections

  • Use machine learning approaches to predict functional relationships

  • Develop mathematical models of DGKE-dependent signaling

• Functional validation of multi-omics findings:

  • Prioritize targets for validation based on network centrality

  • Confirm key findings using orthogonal techniques

  • Test predictions using targeted genetic or pharmacological approaches

This integrated approach can help resolve seemingly contradictory findings in DGKE research by providing a systems-level view of its functions. For example, the variable complement activation observed in different DGKE-deficient models might be explained by identifying compensatory mechanisms or context-dependent regulatory networks through multi-omics analysis.

What are the prospects for developing targeted therapies based on understanding DGKE mechanisms?

The growing understanding of DGKE mechanisms offers several promising avenues for targeted therapeutic development:

• Enzyme replacement approaches:

  • Recombinant DGKE protein therapy, potentially with cell-targeting modifications

  • Development of stabilized DGKE variants with enhanced half-life

  • Engineering of delivery systems (liposomes, nanoparticles) to target affected tissues

• Gene therapy strategies:

  • AAV-mediated DGKE gene delivery to endothelial cells and podocytes

  • CRISPR-based correction of pathogenic DGKE mutations

  • RNA therapeutics to modulate DGKE expression or correct splicing defects

• Small molecule modulators:

  • PKC inhibitors to counteract downstream effects of DGKE deficiency

  • Development of DAG analogues that bypass the need for DGKE

  • Identification of compounds that enhance residual DGKE activity in hypomorphic mutations

• Pathway-targeted approaches:

  • Complement inhibitors for patients with evidence of complement activation

  • Anticoagulant strategies to prevent thrombotic complications

  • Anti-inflammatory agents to mitigate endothelial activation

• Biomarker-guided precision medicine:

  • Development of assays to measure DGKE activity in patient samples

  • Identification of biomarkers predictive of disease progression or treatment response

  • Patient stratification strategies based on molecular phenotyping

The therapeutic approach should be tailored to the specific disease mechanism. For instance, while complement inhibitors like eculizumab are standard therapy for many forms of aHUS, their efficacy in DGKE-associated disease may vary depending on whether complement activation is a primary or secondary phenomenon . Translational research should focus on identifying which patients with DGKE mutations might benefit from complement inhibition versus other targeted approaches.