Recombinant Macaca mulatta Elongation of very long chain fatty acids protein 4 (ELOVL4)

What is Recombinant Macaca mulatta ELOVL4 protein and what is its role in cellular metabolism?

Elongation of very long chain fatty acids protein 4 (ELOVL4) is an essential enzyme that mediates tissue-specific biosynthesis of both very long chain polyunsaturated fatty acids (VLC-PUFA) and very long chain saturated fatty acids (VLC-SFA). In Macaca mulatta (Rhesus macaque), the full-length protein consists of 314 amino acids. The recombinant form is commonly expressed in E. coli with an N-terminal His tag to facilitate purification and detection .

ELOVL4 is primarily expressed in specific tissues including retina, brain, Meibomian glands, skin, testes, and sperm. The very long chain fatty acids produced by this enzyme play critical roles in:

• Maintaining retina and brain function

• Neuroprotection mechanisms

• Skin permeability barrier maintenance

• Sperm function and fertility

• Other important cellular processes

What are the optimal storage conditions for Recombinant Macaca mulatta ELOVL4 protein?

For optimal storage of Recombinant Macaca mulatta ELOVL4:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, add glycerol to a final concentration of 50% (or between 5-50% as needed)

• For long-term storage, aliquot and store at -20°C/-80°C

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

The protein is typically supplied in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, or alternatively in a Tris-based buffer with 50% glycerol, optimized specifically for this protein's stability .

What is the recommended reconstitution protocol for lyophilized ELOVL4 protein?

For optimal reconstitution of lyophilized Recombinant Macaca mulatta ELOVL4:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized, sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% (this can be adjusted between 5-50% based on experimental requirements)

• Prepare small aliquots to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week and long-term storage aliquots at -20°C/-80°C

This reconstitution protocol helps maintain protein stability and enzymatic activity for downstream applications.

How do mutations in ELOVL4 correlate with human diseases, and can the Macaca mulatta model provide insights?

Mutations in the human ELOVL4 gene cause several tissue-specific disorders that align with the protein's expression pattern. Studies of these mutations in various model systems, including non-human primates like Macaca mulatta, provide valuable insights into human disease mechanisms.

Key disease associations include:

• Stargardt-like macular dystrophy (STGD3): Caused by a five base pair deletion (797–801delAACT) in exon 6, resulting in truncation of the last 51 amino acids including the endoplasmic reticulum retention signal .

• Spinocerebellar ataxia-34 (SCA34): Associated with multiple mutations:

  • c.504G>C (p.L168F) mutation in French-Canadian families, often with erythrokeratodermia variabilis (EKV) skin disorder

  • c.736T>G (p.W246G) mutation in a Japanese family with selective degeneration of pontocerebellar tracts

  • c.539A>C (p.G180P) in exon 4 in a patient with progressive ataxia disorder

  • c.698C>T (p.T233M) in exon 6 in a patient with both EKV and SCA34

• More severe neurological disorders: Homozygous mutations lead to conditions characterized by seizures, intellectual disability, and childhood mortality .

The Macaca mulatta model is particularly valuable given the high sequence homology to human ELOVL4, allowing for more translatable insights than rodent models.

What is the relationship between ELOVL4 mutations and very long chain fatty acid metabolism in disease pathogenesis?

ELOVL4 is essential for the tissue-specific biosynthesis of very long chain fatty acids (VLC-FAs), including both VLC-PUFAs and VLC-SFAs, which have critical functions in their respective tissues. Mutations disrupt these functions through multiple mechanisms:

• Truncation mutations (as in STGD3) remove the ER retention signal, causing mislocalization of the protein and dominant-negative effects that impair VLC-PUFA synthesis in the retina, leading to photoreceptor degeneration .

• Missense mutations (as in SCA34) may affect specific enzymatic activities or substrate specificities, altering the balance of VLC-FAs in affected tissues like the cerebellum and skin .

• Complete loss of function (homozygous mutations or deletions) prevents synthesis of essential VLC-FAs in multiple tissues, explaining the neonatal lethality observed in mouse models .

The tissue-specific expression of ELOVL4 and its specialized role in producing tissue-specific VLC-FAs explains why different mutations manifest with different organ-specific pathologies, from retina-specific disease to skin disorders to neurological conditions.

How can recombinant ELOVL4 be used to study the enzymatic elongation of very long chain fatty acids in vitro?

Recombinant ELOVL4 provides a powerful tool for studying the enzymatic mechanisms of very long chain fatty acid elongation. Advanced in vitro applications include:

• Enzymatic activity assays:

  • Using purified recombinant ELOVL4 with appropriate fatty acid substrates to measure elongation activity

  • Analyzing substrate specificity by testing different chain length fatty acids

  • Determining kinetic parameters (Km, Vmax) for different substrates

• Structure-function studies:

  • Site-directed mutagenesis to create disease-associated mutations and assess their impact on enzyme activity

  • Domain swapping experiments to identify regions critical for substrate recognition

  • Protein interaction studies to identify cofactors or regulatory proteins

• High-throughput drug screening:

  • Using recombinant ELOVL4 to screen for small molecules that modulate enzyme activity

  • Developing therapeutic approaches for ELOVL4-associated diseases

For such applications, it's important to ensure the recombinant protein maintains its native conformation and enzymatic activity. The availability of His-tagged recombinant Macaca mulatta ELOVL4 facilitates purification and immobilization for these advanced applications .

What experimental approaches can be used to study the tissue-specific functions of ELOVL4 in different cellular contexts?

Understanding the tissue-specific functions of ELOVL4 requires specialized experimental approaches:

• Tissue-specific expression systems:

  • Primary cell cultures from tissues where ELOVL4 is naturally expressed (retina, brain, skin, etc.)

  • Development of organoid models to recapitulate tissue-specific cellular environments

  • Conditional expression systems in relevant cell lines

• Lipidomic profiling:

  • Comprehensive analysis of VLC-FA profiles in different tissues expressing ELOVL4

  • Comparison of lipid profiles between wild-type and mutant ELOVL4 expression

  • Analysis of downstream lipid species that incorporate VLC-FAs

• Functional assays:

  • Membrane fluidity measurements to assess the impact of ELOVL4-derived VLC-FAs

  • Electrophysiological studies in neuronal cells expressing ELOVL4

  • Skin barrier function tests in models expressing ELOVL4 variants

• Integration with disease models:

  • Transgenic expression of Macaca mulatta ELOVL4 in relevant disease models

  • Rescue experiments using wild-type ELOVL4 in systems with mutant expression

  • Comparative studies between human and Macaca mulatta ELOVL4 functions

What are the critical quality control parameters for validating recombinant ELOVL4 protein preparations?

To ensure experimental reproducibility and valid results when working with recombinant ELOVL4, researchers should assess the following quality control parameters:

• Purity assessment:

  • SDS-PAGE analysis (should show >90% purity)

  • Mass spectrometry confirmation of intact protein mass

  • Absence of significant degradation products or contaminants

• Protein identity confirmation:

  • Western blot with ELOVL4-specific antibodies

  • N-terminal sequencing to confirm correct starting sequence

  • Peptide mass fingerprinting after digestion

• Functional validation:

  • Enzymatic activity assays using appropriate fatty acid substrates

  • Proper subcellular localization in expression systems

  • Thermal stability assessments

• Storage stability:

  • Activity retention after storage under recommended conditions

  • Assessment of freeze-thaw stability

  • Monitoring for aggregation or precipitation

These quality control measures help ensure that experimental results are attributable to properly folded, functional ELOVL4 protein rather than artifacts from degraded or misfolded protein preparations.

How should researchers evaluate and interpret genetic association studies involving ELOVL4?

When evaluating genetic association studies for ELOVL4 variants, researchers should apply rigorous criteria to assess the credibility of findings:

• Amount of evidence:

  • Category A (strong): Sample size >1000 in the least common genetic group

  • Category B (moderate): Sample size 100-1000 in this group

  • Category C (weak): Sample size <100 in this group

• Replication quality:

  • Category A: Extensive replication including well-conducted meta-analysis with little between-study inconsistency

  • Category B: Well-conducted meta-analysis with some methodological limitations or moderate inconsistency

  • Category C: No independent replication, failed replication, or inconsistent results

• Protection from bias:

  • Category A: Minimal bias affecting magnitude but not presence of association

  • Category B: No obvious bias but missing information on evidence generation

  • Category C: Considerable potential bias affecting presence/absence of association

• Specific considerations for ELOVL4 studies:

  • Heterogeneity in phenotyping methods across tissue-specific disorders

  • Variation in mutation detection techniques and coverage

  • Potential confounding from population stratification

  • Small effect sizes requiring larger samples for detection

This framework helps researchers critically evaluate evidence about ELOVL4 genetic associations and appropriately interpret their significance in research contexts.

How does Macaca mulatta ELOVL4 compare with ELOVL4 proteins from other species, and what are the implications for translational research?

Comparing Macaca mulatta ELOVL4 with homologs from other species provides valuable insights for translational research. While the search results don't provide direct comparative data, we can infer from the high conservation of this protein that:

• Primate models:

  • Macaca mulatta ELOVL4 likely shares high sequence homology with human ELOVL4

  • Functional domains are likely to be highly conserved

  • Rhesus macaque models may provide more translatable results than rodent models for human disease studies

• Evolutionary conservation:

  • The essential function of ELOVL4 in VLC-FA synthesis has likely maintained structural conservation

  • Species-specific differences may reflect adaptations to different tissue requirements

  • Comparison across species can identify absolutely conserved residues critical for function

• Translational implications:

  • Drug development targeting conserved regions might have cross-species efficacy

  • Species-specific differences must be considered when translating findings to human applications

  • Recombinant Macaca mulatta ELOVL4 may be a suitable surrogate for human protein in certain assays

Understanding these comparative aspects helps researchers make informed decisions about model systems and experimental design when studying ELOVL4-related diseases.

What are common challenges in working with recombinant membrane proteins like ELOVL4, and how can they be addressed?

ELOVL4 is a membrane-bound enzyme that functions in the endoplasmic reticulum, posing several technical challenges for researchers:

• Solubility and purification:

  • Challenge: Membrane proteins tend to aggregate when removed from lipid environments

  • Solution: Use appropriate detergents during extraction and purification

  • Recommendation: Consider using mild non-ionic detergents and optimize detergent-to-protein ratios

• Maintaining native conformation:

  • Challenge: Detergents may alter protein folding and activity

  • Solution: Consider reconstitution into lipid nanodiscs or liposomes after purification

  • Recommendation: Validate activity after purification with functional assays

• Expression systems:

  • Challenge: E. coli lacks appropriate post-translational modifications and membrane environment

  • Solution: Consider mammalian or insect cell expression systems for more complex studies

  • Note: The available recombinant Macaca mulatta ELOVL4 is expressed in E. coli, which may be sufficient for many applications but could limit others

• Storage stability:

  • Challenge: Membrane proteins are prone to aggregation during freeze-thaw cycles

  • Solution: Add stabilizers like glycerol (recommended at 50%) and store in small aliquots

  • Recommendation: Avoid repeated freeze-thaw cycles and store working aliquots at 4°C

• Activity assays:

  • Challenge: Creating appropriate conditions for measuring elongase activity in vitro

  • Solution: Design assays that include necessary cofactors and maintain an appropriate membrane environment

  • Recommendation: Include positive controls with established activity profiles

Addressing these challenges requires careful experimental design and optimization based on the specific research questions being addressed.