Recombinant Pongo abelii 1-acyl-sn-glycerol-3-phosphate acyltransferase delta (AGPAT4)
Description
Biological Function and Enzyme Activity
AGPAT4 belongs to the acyltransferase family and operates at the endoplasmic reticulum and mitochondrial membranes . Its primary enzymatic role involves:
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Substrate specificity: Transfers acyl groups to the sn-2 position of LPA to form PA .
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Metabolic pathways: Integral to the Kennedy pathway for glycerophospholipid synthesis and TAG biosynthesis .
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Tissue-specific roles: Studies in mice show AGPAT4 influences lipid storage in adipose tissue and phospholipid composition in the brain .
Key Functional Insights from Knockout Studies:
Recombinant Protein Production
Commercially available recombinant AGPAT4 is produced in bacterial expression systems:
Lipid Metabolism Studies
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Adipose tissue dynamics: AGPAT4 deficiency in mice increases TAG content in epididymal WAT, linked to reduced lipolysis and altered adipocyte size .
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Brain phospholipids: AGPAT4 supports phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis, critical for neuronal membrane integrity .
Regulatory Mechanisms
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Transcriptional control: Cardiac AGPAT4 activity is modulated by PPARα, influencing lipid oxidation pathways .
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Compensatory upregulation: Loss of AGPAT4 triggers isoform-specific redundancy (e.g., AGPAT3 in brain) .
Implications for Disease Models
AGPAT4’s role in lipid homeostasis positions it as a potential target for:
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement, and we will accommodate your needs.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. For the lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
Q&A
What is the fundamental role of AGPAT4 in phospholipid biosynthesis and how does the Pongo abelii variant compare to human AGPAT4?
AGPAT4 (1-acylglycerol-3-phosphate O-acyltransferase 4) catalyzes the acylation of lysophosphatidic acid (LPA) to produce phosphatidic acid (PA), representing the second step in de novo phospholipid biosynthesis . This conversion is critical for membrane glycerophospholipid production across species.
The Pongo abelii (Sumatran orangutan) AGPAT4 shares significant sequence homology with human AGPAT4, but contains species-specific amino acid variations that may affect substrate specificity and enzyme kinetics. Comparative analysis between recombinant human and Pongo abelii AGPAT4 reveals:
| Feature | Human AGPAT4 | Pongo abelii AGPAT4 |
|---|---|---|
| Amino acid sequence identity | Reference | ~97-98% homology to human |
| Substrate preference | Diverse acyl-CoA donors | Possible preference for specific polyunsaturated fatty acyl donors |
| Subcellular localization | Mitochondrial | Presumed mitochondrial (based on sequence conservation) |
| Tissue expression | Highest in brain | Predicted highest in brain (based on evolutionary conservation) |
Methodological approach: To investigate functional differences, researchers should perform side-by-side enzymatic assays using purified recombinant proteins from both species with various acyl-CoA donors and measure product formation via liquid chromatography-mass spectrometry (LC-MS) .
What expression patterns does AGPAT4 demonstrate during development, and what methodologies are recommended for studying these patterns?
Studies in murine models demonstrate that AGPAT4 exhibits significant temporal regulation during embryonic development . Research shows:
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AGPAT4 is upregulated 3.7-fold at embryonic day E14.5 compared to E10.5
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Expression dramatically decreases to only 4% of E14.5 levels by E18.5 (immediately prior to birth)
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AGPAT4 is immunodetectable in both cortical neurons and glial cells derived from developing mouse brain
This temporal expression pattern suggests AGPAT4 may play a critical role during specific developmental windows, particularly during mid-embryonic development when neural differentiation is prominent.
Recommended methodological approaches:
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RT-qPCR with appropriate reference genes (e.g., 18S rRNA) for temporal expression analysis across developmental timepoints
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Immunohistochemistry with cell-type specific markers (e.g., NESTIN for neurons, GFAP for glial cells) for cellular localization studies
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In situ hybridization to visualize tissue-specific expression patterns
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Conditional knockout models to investigate developmental stage-specific functions
For non-human primates like Pongo abelii, where direct embryonic studies face ethical limitations, researchers should consider post-mortem tissue analysis or induced pluripotent stem cell (iPSC) models that can recapitulate developmental trajectories.
What are the optimal conditions for solubilizing and purifying active recombinant AGPAT4 from Pongo abelii?
AGPAT4 presents significant purification challenges as an integral membrane protein. Standard purification protocols often result in loss of enzymatic activity, which has historically hampered biochemical characterization . Based on successful protocols with related enzymes:
Methodological considerations:
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Avoid repeated freeze-thaw cycles, which significantly reduce enzymatic activity
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Consider adding protease inhibitors during purification to prevent degradation
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Validate enzymatic activity immediately after purification using appropriate substrates
Advanced researchers should consider using nanodiscs or liposome reconstitution systems to maintain the protein in a more native membrane environment, which may better preserve activity for functional studies.
How does AGPAT4 contribute to phospholipid metabolism in neuronal tissue, and what experimental approaches best elucidate its function?
AGPAT4 demonstrates notably high expression in brain tissue compared to other organs, suggesting a specialized role in neural phospholipid metabolism . Research findings indicate:
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AGPAT4 is localized to mitochondria in neurons, with a distinct punctate staining pattern observable via immunofluorescence
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It is present in both neurons (co-localizing with NESTIN or Nissl stain) and glial cells (co-localizing with GFAP)
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Its temporal regulation during development suggests involvement in critical periods of neural membrane expansion and/or remodeling
Recommended experimental approaches:
| Approach | Application | Expected Outcome |
|---|---|---|
| Subcellular fractionation | Confirm mitochondrial localization | Enrichment in mitochondrial fraction |
| Lipidomics analysis | Identify altered phospholipid species in AGPAT4-deficient cells | Changes in phosphatidic acid species profile |
| Neurite outgrowth assays | Assess role in membrane expansion | Altered neurite length or branching patterns |
| Electrophysiology | Evaluate membrane functional properties | Changes in synaptic transmission parameters |
| CRISPR-Cas9 knockout | Determine essentiality in neurons | Viability and functional consequences |
For studies specifically focused on Pongo abelii AGPAT4, researchers should consider comparative analyses between human and orangutan iPSC-derived neural cultures to identify species-specific functions in neuronal development and maintenance.
What is the role of AGPAT4 in regulating immune responses in the tumor microenvironment?
Recent research has uncovered a surprising role for AGPAT4 in modulating immune responses within the tumor microenvironment, particularly in colorectal cancer (CRC) . Key findings include:
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AGPAT4 is aberrantly expressed in colorectal cancer tissues compared to normal tissues
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AGPAT4 expression levels correlate with poor survival in CRC patients
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While AGPAT4 manipulation doesn't affect CRC cell growth in vitro, silencing AGPAT4 suppresses tumor growth in xenograft models
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Mechanistically, AGPAT4 silencing induces LPA release from CRC cells, which polarizes macrophages toward an anti-tumor M1-like phenotype through LPA receptors 1 and 3
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This M1 activation is characterized by elevated p38/p65 signaling and increased proinflammatory cytokines
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The resulting inflammatory environment promotes CD4+ and CD8+ T cell infiltration and activation
Methodological approaches to investigate this pathway:
| Approach | Purpose | Key Parameters |
|---|---|---|
| AGPAT4 knockdown/overexpression | Modulate enzyme levels | siRNA, shRNA, or CRISPR-Cas9 systems |
| Co-culture systems | Study cancer cell-macrophage interactions | Transwell or direct co-culture conditions |
| Flow cytometry | Analyze immune cell polarization | M1 markers (CD45+F4/80+CD11c+) vs. M2 markers (CD45+F4/80+CD206+) |
| Cytokine profiling | Measure inflammatory mediators | IL-1β, IL-6, TNFα (M1) vs. IL-10, Arg-1 (M2) |
| Western blotting | Assess signaling pathway activation | Phosphorylated p38 and p65 levels |
| LPA measurements | Quantify LPA release | LC-MS based lipidomics |
While this function has been primarily characterized in human and mouse models, similar pathways likely exist in Pongo abelii. Comparative studies using recombinant Pongo abelii AGPAT4 could reveal evolutionary differences in immune modulation across primate species.
What evolutionary insights can be gained from studying Pongo abelii AGPAT4 in comparison to human and other primate AGPAT4 variants?
Evolutionary analysis of AGPAT4 across primate species offers valuable insights into the conservation and divergence of phospholipid metabolism pathways. Pongo abelii (Sumatran orangutan) represents an important branch in primate evolution, having diverged from the human lineage approximately 12-16 million years ago.
Key evolutionary considerations:
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Sequence conservation analysis reveals highly conserved catalytic domains across primates, suggesting functional importance
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Species-specific variations predominantly occur in non-catalytic regions, potentially affecting regulatory interactions
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Comparison of substrate preferences across species may reveal adaptive changes in membrane lipid composition
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Differences in expression patterns may reflect species-specific metabolic adaptations
Methodological approaches:
| Approach | Application | Expected Outcome |
|---|---|---|
| Phylogenetic analysis | Trace evolutionary history | Evolutionary rate and selective pressure metrics |
| Structural modeling | Predict functional consequences of variants | Identification of species-specific substrate binding determinants |
| Recombinant protein activity assays | Compare enzymatic parameters | Species-specific differences in substrate affinity and catalytic efficiency |
| Genomic sequence analysis | Identify regulatory element differences | Insights into expression pattern divergence |
The study of Pongo abelii AGPAT4 is particularly valuable for understanding primate-specific adaptations in phospholipid metabolism, potentially revealing how differences in membrane composition may have contributed to cognitive evolution and brain development across the primate lineage.
What technical considerations are important when designing activity assays for recombinant Pongo abelii AGPAT4?
Designing robust enzymatic assays for AGPAT4 requires careful consideration of multiple factors to ensure physiologically relevant results. Based on successful approaches with related enzymes:
| Parameter | Recommendation | Rationale |
|---|---|---|
| Substrate preparation | Use freshly prepared or carefully stored lysophosphatidic acid | LPA is prone to degradation |
| Acyl-CoA donors | Test multiple chain lengths and saturation levels | AGPAT4 may have species-specific substrate preferences |
| Detergent concentration | Optimize to maintain enzyme activity without disrupting substrate presentation | Critical for membrane protein assays |
| pH optimization | Test range from 6.5-8.0 | pH affects both enzyme activity and substrate stability |
| Divalent cations | Include Mg²⁺ and test Mn²⁺ | Essential cofactors for acyltransferase activity |
| Reaction time | Establish linear range (typically 5-30 minutes) | Ensures accurate initial velocity measurements |
| Product detection | LC-MS or radioactive assays | Most sensitive and specific detection methods |
Common technical challenges:
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Background acyltransferase activity in expression systems requires careful negative controls
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Substrate solubility issues may necessitate optimization of delivery methods (liposomes, mixed micelles)
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Product extraction efficiency can significantly impact quantitative results
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Protein stability during the assay period must be verified
For Pongo abelii AGPAT4 specifically, researchers should consider comparative assays with human AGPAT4 under identical conditions to identify any species-specific kinetic parameters or substrate preferences.
How can researchers study AGPAT4 function in the context of neurodevelopmental disorders?
The high expression of AGPAT4 in neural tissues and its dynamic regulation during development suggest potential roles in neurodevelopmental processes . This presents opportunities for studying its function in relation to neurodevelopmental disorders:
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Temporal expression patterns indicate AGPAT4 is significantly upregulated during mid-embryonic development (E14.5) when critical neural differentiation and migration occur
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AGPAT4 is present in both neurons and glial cells, suggesting roles in multiple neural cell types
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As a phospholipid biosynthesis enzyme, AGPAT4 may influence membrane composition affecting neurite outgrowth, synaptogenesis, and myelination
Recommended research approaches:
| Approach | Application | Potential Insights |
|---|---|---|
| iPSC-derived neural models | Study species-specific neural development | Differences in phospholipid composition during neurogenesis |
| CRISPR-engineered mutations | Introduce disorder-associated variants | Functional consequences on neural development |
| Organoid cultures | Examine 3D developmental contexts | Effects on cortical organization and cell migration |
| Comparative developmental expression | Map expression across species | Evolutionary differences in neurodevelopmental timing |
| Lipidomics of neurodevelopmental stages | Profile phospholipid changes | Critical periods of membrane remodeling |
For studies using Pongo abelii AGPAT4, researchers could develop comparative models between human and orangutan neural development, potentially revealing how species-specific differences in phospholipid metabolism contribute to unique aspects of primate brain development and function.
What are the current limitations in AGPAT4 research and what methodological advances could address these challenges?
Despite increasing interest in AGPAT4 biology, several significant research challenges remain:
For Pongo abelii AGPAT4 specifically, additional challenges include limited sample availability and ethical considerations for primate research. Researchers can address these limitations through:
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Development of cell-based models using genetic engineering of accessible cell lines
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Computational approaches to predict species-specific functions based on sequence variations
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Non-invasive sampling methods when appropriate for endangered species
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Advanced recombinant expression systems that better replicate post-translational modifications
How does AGPAT4 interact with the broader lipid metabolic network in Pongo abelii compared to humans?
AGPAT4 functions within a complex network of lipid metabolic enzymes. Understanding its position within this network in Pongo abelii compared to humans provides insights into potential species-specific metabolic adaptations:
| Metabolic Pathway | AGPAT4 Involvement | Species Comparison Considerations |
|---|---|---|
| Glycerophospholipid metabolism | Direct role converting LPA to PA | Compare substrate preferences and product profiles |
| Glycerolipid metabolism | Indirect influence through PA production | Assess downstream metabolite differences |
| General metabolic pathways | Integration with broader cellular metabolism | Examine metabolic flux differences between species |
Key interaction partners to investigate:
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Other AGPAT family members (AGPAT1-5) - potential compensatory or cooperative mechanisms
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Phospholipases - may regulate substrate availability
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PA phosphatases - influence product utilization
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Membrane transport proteins - affect substrate accessibility
Methodological approaches for network studies:
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Metabolic flux analysis using stable isotope labeling
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Protein-protein interaction studies (co-immunoprecipitation, proximity labeling)
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Competitive enzyme assays to determine relative activities
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Systems biology modeling of lipid metabolic networks
For comparative Pongo abelii studies, researchers should consider how evolutionary adaptations in phospholipid metabolism might relate to species-specific traits such as brain development, cognitive function, and metabolic adaptations to different ecological niches.
