Recombinant Xenopus laevis Acyl-CoA-binding domain-containing protein 5 (acbd5)

What is the basic structure and function of Xenopus laevis ACBD5?

Xenopus laevis ACBD5 is a 467-amino acid protein belonging to the acyl-CoA-binding domain-containing protein family. The full-length protein contains an N-terminal acyl-CoA binding domain that specifically interacts with long-chain fatty acyl-CoA esters (LCACoA), regulating their intracellular concentration. The protein's complete amino acid sequence (MADTKPLHQTRFEAAVSVIQSLPKNGSFQPSNEMMLKFYSFYKQATLGPCNTARPGFWDPVGRYKWDAWNSLGDMSKEDAMIAYVDEMKKIIETMPVTDKVEELLQVIGPFYEIVEDKKHGRGSGVTSELGSVLTSTPNGKAVNGKAESSDSGAESDEEQAAAKEFKKEDEEDEEDETEHSEEEEKEVEQQPGHETSAESIVLNGLTKNSRVLITEEPTPLPTKCLSEPGDNVAIPEGEP DIQSAVINDSEADREEDCTEDMAAVQHLTSDSDSEIFCDSMEQFGQDEADHSLLLQDAMLNGDITENSAGGELKDGGEDGKQPGHGAQGKTWNGKSEHFSSRRERSLRMQPGGEGSRSGQIGSSGDGDGWGSDRGPIGNLNEQIAVVLMRLQEDMQNVLQRLHSLEVQTASQAQSLLRESNTQSVEKKPSGWPFGISPGTLALAVVWPFVVHWLMHVFLQKRRRKQT) reveals multiple functional domains associated with lipid binding and protein interactions .

The protein plays critical roles in lipid homeostasis, peroxisomal function, and organelle tethering. Unlike some other ACBD family members, ACBD5 primarily localizes to peroxisomes, where it mediates tethering between peroxisomes and the endoplasmic reticulum, facilitating lipid transfer between these organelles.

How does Xenopus laevis ACBD5 compare structurally and functionally to mammalian ACBD5?

While Xenopus laevis ACBD5 shares significant sequence homology with mammalian ACBD5 proteins, several structural and functional differences exist:

What expression patterns of ACBD5 are observed during Xenopus development?

ACBD5 expression in Xenopus laevis shows tissue-specific and developmental stage-dependent patterns. The protein is expressed throughout embryonic development, with particularly notable expression in neural tissues, developing liver, and kidney structures. This expression pattern correlates with the progressive development of peroxisomal networks in these tissues.

Temporal expression analysis reveals low levels during early cleavage stages, with significant upregulation during gastrulation and neurulation, coinciding with increased lipid metabolism requirements. This pattern suggests critical roles in energy homeostasis during key developmental transitions.

What are the optimal expression and purification methods for recombinant Xenopus laevis ACBD5?

For high-yield expression and purification of recombinant Xenopus laevis ACBD5, the following methodological approach is recommended:

Expression System Selection:
E. coli expression systems, particularly BL21(DE3) strains, have proven most effective for producing full-length His-tagged Xenopus laevis ACBD5 protein . The bacterial expression system offers advantages of high yield and cost-effectiveness compared to insect or mammalian systems.

Optimization Protocol:

• Transform expression plasmid containing the full-length Xenopus laevis ACBD5 sequence with N-terminal His-tag into competent E. coli cells

• Culture transformed cells at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with 0.5-1.0 mM IPTG

• Reduce temperature to 18-20°C for overnight expression (this reduces inclusion body formation)

• Harvest cells by centrifugation and lyse using sonication in buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10 mM imidazole

  • 1 mM PMSF

  • Protease inhibitor cocktail

• Purify using Ni-NTA affinity chromatography with step-wise imidazole elution

• Further purify by size exclusion chromatography

This approach typically yields 5-10 mg of purified protein per liter of bacterial culture with >90% purity as determined by SDS-PAGE .

What are the recommended storage and handling protocols for recombinant Xenopus laevis ACBD5?

Proper storage and handling of recombinant Xenopus laevis ACBD5 is critical for maintaining protein stability and functionality. Based on established protocols:

Lyophilization and Reconstitution:
The protein is typically supplied as a lyophilized powder. For reconstitution:

• Centrifuge the vial briefly before 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 5-50% (optimally 50%) for long-term storage

Storage Conditions:

• Store reconstituted protein in small aliquots at -20°C/-80°C to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

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

Handling Precautions:

• Maintain sterile techniques when handling the reconstituted protein

• Use low-protein binding tubes for storage

• Avoid vigorous shaking or vortexing that can lead to protein denaturation

• Include stabilizing agents such as 6% trehalose in Tris/PBS-based buffer (pH 8.0) for optimal stability

What functional assays are most informative for characterizing Xenopus laevis ACBD5 activity?

Several complementary assays can be employed to comprehensively characterize Xenopus laevis ACBD5 activity:

Acyl-CoA Binding Assays:

• Fluorescence-based displacement assay: Measure displacement of fluorescently labeled acyl-CoA upon binding of ACBD5

• Isothermal titration calorimetry (ITC): Determine binding affinities and thermodynamic parameters for different acyl-CoA species

• Surface plasmon resonance (SPR): Assess real-time binding kinetics

Peroxisomal Function Assays:

• Membrane contact site quantification: Using fluorescence microscopy to measure peroxisome-ER contact sites in the presence/absence of ACBD5

• Very long-chain fatty acid (VLCFA) metabolism: Measure β-oxidation rates of radiolabeled VLCFAs in systems with modulated ACBD5 levels

• Peroxisomal morphology analysis: Evaluate peroxisome size, number, and distribution using fluorescent markers

Protein-Protein Interaction Studies:

• Co-immunoprecipitation: Identify ACBD5 binding partners in Xenopus cell extracts

• Yeast two-hybrid screening: Discover novel interacting proteins

• Bimolecular fluorescence complementation (BiFC): Visualize protein interactions in living cells

These assays collectively provide a comprehensive assessment of ACBD5's molecular functions, particularly its roles in lipid metabolism and peroxisomal homeostasis.

How can genetic manipulation of ACBD5 in Xenopus laevis be optimized for functional studies?

Genetic manipulation of ACBD5 in Xenopus laevis can be optimized through several advanced methodologies:

CRISPR/Cas9 Gene Editing:
Based on approaches used for other ACBD family members like ACBD6, CRISPR/Cas9 has proven effective for generating knockout models in Xenopus . For ACBD5-specific editing:

• Design sgRNAs targeting conserved regions of the acyl-CoA binding domain

• Optimize microinjection parameters for Xenopus embryos (typically 2-4 cell stage)

• Verify knockout efficiency using T7 endonuclease assay or sequencing

• Assess phenotypes at various developmental stages

Morpholino-based Knockdown:
For transient suppression of ACBD5 expression:

• Design splice-blocking or translation-blocking morpholinos specific to Xenopus laevis ACBD5

• Inject 2-10 ng of morpholino at 1-2 cell stage

• Include appropriate controls (mismatch morpholinos)

• Confirm knockdown efficiency by Western blot or qRT-PCR

Transgenic Overexpression:
For gain-of-function studies:

• Generate constructs with tissue-specific promoters (e.g., CMV for ubiquitous expression)

• Use Tol2 or I-SceI meganuclease-mediated transgenesis for efficient integration

• Screen F0 embryos for successful integration and expression

• Establish stable transgenic lines through selective breeding

These approaches have been successfully applied to study related proteins in Xenopus and can be adapted specifically for ACBD5 functional analyses .

What is the current understanding of ACBD5's role in peroxisomal function in Xenopus compared to mammals?

The role of ACBD5 in peroxisomal function shows both conserved and divergent aspects between Xenopus and mammals:

In mammals, ACBD5 deficiency causes a combination of retinal dystrophy, leukodystrophy, and peroxisomal very long-chain fatty acid metabolism defects . Comparative studies in Xenopus can reveal evolutionarily conserved mechanisms of peroxisomal dysfunction and potential compensatory pathways specific to amphibian metabolism.

The Xenopus model offers unique advantages for studying peroxisomal development due to its external embryonic development and transparent embryos, allowing real-time visualization of peroxisomal dynamics during development.

How does ACBD5 interact with other ACBD family members in Xenopus laevis?

ACBD5 functions within a network of ACBD family proteins in Xenopus laevis, with complex interactions and potential functional redundancy:

Expression Correlation Analysis:
Studies of ACBD family gene expression in Xenopus reveal differential but overlapping expression patterns:

• ACBD5 co-expresses with ACBD3 in neural tissues

• ACBD1 and ACBD5 show complementary expression in developing liver

• ACBD6, which has been linked to neurodevelopmental disorders in humans, shows expression patterns distinct from ACBD5

Functional Interactions:
While direct protein-protein interactions between ACBD family members have not been extensively characterized in Xenopus, several functional relationships have been observed:

• Compensatory upregulation of other ACBD family members in ACBD5-deficient systems

• Shared binding partners, suggesting competitive or cooperative functions

• Differential subcellular localization indicating compartmentalized functions:

  • ACBD5: Peroxisomes

  • ACBD6: Cytosol and nucleus

  • Other ACBDs: Various organelles

These interactions create a complex network regulating lipid metabolism and organelle function throughout Xenopus development.

What are common challenges in studying recombinant Xenopus laevis ACBD5 and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant Xenopus laevis ACBD5:

Challenge 1: Protein Solubility Issues

• Problem: Formation of inclusion bodies during bacterial expression

• Solution:

  • Reduce induction temperature to 18°C

  • Use lower IPTG concentrations (0.1-0.5 mM)

  • Include solubility enhancers (0.1% Triton X-100, 10% glycerol) in lysis buffer

  • Consider fusion tags beyond His-tag (e.g., MBP, SUMO) for improving solubility

Challenge 2: Lipid Binding Activity Loss

• Problem: Reduction in acyl-CoA binding capacity after purification

• Solution:

  • Supplement buffers with small amounts of lipid (0.01-0.05% lipid mixture)

  • Avoid detergents that may disrupt the acyl-CoA binding pocket

  • Include stabilizing agents like trehalose (6%) in storage buffer

  • Verify activity immediately after purification and at regular intervals

Challenge 3: Protein Degradation

• Problem: Proteolytic degradation during purification or storage

• Solution:

  • Use comprehensive protease inhibitor cocktails during lysis

  • Perform purification at 4°C

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol)

  • Lyophilize for long-term storage or store at -80°C in 50% glycerol

Challenge 4: Antibody Cross-Reactivity

• Problem: Antibodies against Xenopus ACBD5 cross-react with other ACBD family members

• Solution:

  • Use epitopes from less conserved regions of ACBD5

  • Validate antibody specificity using knockout controls

  • Perform pre-absorption with recombinant related ACBD proteins

How can researchers optimize functional studies of ACBD5 in Xenopus laevis embryos and tissues?

Optimizing functional studies of ACBD5 in Xenopus laevis requires careful consideration of developmental timing and technical approaches:

Embryonic Manipulation Timing:
The developmental stage at which ACBD5 is manipulated significantly impacts experimental outcomes:

Technical Optimization Strategies:

• Dose-response calibration: Determine optimal doses of morpholinos or mRNA by injecting a range of concentrations (1-20 ng) and assessing phenotypic outcomes

• Rescue experiments: Co-inject morpholinos with rescue mRNA containing silent mutations to verify specificity

• Developmental timing considerations: Time-controlled expression using hormone-inducible promoters (e.g., heat shock or thyroid hormone response elements)

• Peroxisomal visualization: Use fluorescent peroxisomal markers (GFP-SKL) to track changes in peroxisome dynamics in live embryos

These approaches enable precise dissection of ACBD5 functions while minimizing experimental artifacts and distinguishing primary effects from secondary consequences.

What are the key considerations for comparing ACBD5 function across species models?

When comparing ACBD5 function between Xenopus laevis and other model organisms, researchers must account for several critical factors:

Evolutionary Context:

• Xenopus laevis is pseudotetraploid with potential gene duplications affecting ACBD family redundancy

• Differing metabolic requirements between aquatic amphibians and terrestrial mammals may influence ACBD5 function

• Developmental timing differences require careful stage-matching across species

Technical Standardization:
For valid cross-species comparisons:

• Use equivalent developmental stages rather than chronological time points

• Standardize methodologies for protein expression and activity measurement

• Account for temperature differences in experimental protocols (Xenopus optimal temperature ~23°C vs. mammalian 37°C)

• Normalize data to species-specific reference genes or proteins

Functional Assessment Framework:
A systematic approach for cross-species comparison includes:

This framework enables meaningful comparisons that distinguish conserved core functions from species-specific adaptations.

What recent discoveries about ACBD5 function have emerged from comparative studies in different model organisms?

Recent comparative studies between Xenopus and mammalian models have revealed several novel aspects of ACBD5 function:

Membrane Contact Site Regulation:
New findings demonstrate that ACBD5's role in establishing peroxisome-ER membrane contact sites is evolutionarily conserved, but with species-specific regulatory mechanisms. In Xenopus, these contacts appear particularly important during developmental transitions, suggesting adaptation to the unique metabolic demands of metamorphosis.

Neurodevelopmental Implications:
Comparative analysis of ACBD deficiencies across species has revealed a previously unappreciated role in neurodevelopment. Human ACBD6 variants lead to a neurodevelopmental syndrome with a complex movement disorder phenotype , while studies in ACBD5-deficient Xenopus embryos show disruptions in neural crest migration and neural tube formation. This suggests broader roles for ACBD family proteins in neural development across vertebrates.

Proteomic Impacts:
Recent proteomics analyses in wild-type and acbd6 crispant Xenopus tropicalis revealed significant alterations in N-myristoylated proteins upon ACBD deletion . Similar studies with ACBD5 are revealing complex protein interaction networks that extend beyond direct lipid metabolism, pointing to roles in cellular signaling and protein modification pathways.

How can Xenopus laevis ACBD5 research contribute to understanding human peroxisomal disorders?

Xenopus laevis offers unique advantages for modeling human peroxisomal disorders related to ACBD5 dysfunction:

Translational Research Opportunities:
ACBD5 deficiency in humans causes retinal dystrophy with leukodystrophy and peroxisomal very long-chain fatty acid metabolism defects . Xenopus models provide several advantages for investigating these conditions:

• Developmental accessibility: External embryonic development allows direct visualization of disease progression

• Organogenesis similarities: Conserved mechanisms of retinal and neural development

• Genetic manipulation ease: Rapid generation of disease models using CRISPR/Cas9

• High-throughput screening potential: Embryonic assays suitable for drug discovery

Comparative Disease Modeling Framework:

Therapeutic Discovery Platform:
The Xenopus model supports high-throughput screening of potential therapeutic compounds targeting ACBD5-related disorders through:

• Rapid embryonic assays for peroxisomal function

• Automated phenotypic screening of large compound libraries

• Evaluation of gene therapy approaches using viral vectors

What are the most promising future directions for Xenopus laevis ACBD5 research?

Several promising research directions are emerging for Xenopus laevis ACBD5 studies:

Integrative Multi-omics Approaches:
Combining transcriptomics, proteomics, and lipidomics analyses of ACBD5-deficient Xenopus models will provide comprehensive understanding of the protein's role in global metabolic networks. Recent proteomics studies in related ACBD-deficient models have already revealed unexpected connections to Parkinson's disease-specific pathways , suggesting broader implications than previously recognized.

Evolutionary Functional Adaptation:
Comparative analysis of ACBD5 function across evolutionarily distant species (from zebrafish to Xenopus to mammals) can reveal how this protein family has adapted to different metabolic requirements. This evolutionary perspective may uncover novel functions and regulatory mechanisms not apparent in single-species studies.

Organelle Interaction Networks:
Advanced imaging techniques, such as super-resolution microscopy combined with optogenetic tools, can be applied in transparent Xenopus embryos to visualize and manipulate ACBD5-mediated peroxisome-ER contacts in real-time during development. This approach promises to reveal dynamic aspects of organelle interactions previously inaccessible to study.

Developmental Metabolic Reprogramming: ACBD5's role in the dramatic metabolic changes occurring during Xenopus metamorphosis represents an underexplored area with potential implications for understanding metabolic adaptation during tissue remodeling in other contexts, including regeneration and cancer.