Recombinant Danio rerio Acyl-CoA-binding domain-containing protein 5A (acbd5a)

What is Acyl-CoA-binding domain-containing protein 5A (acbd5a) in zebrafish?

Acyl-CoA-binding domain-containing protein 5A (acbd5a) is a peroxisomal protein in zebrafish that contains an acyl-CoA binding domain (ACBD) at its N-terminal region. It is a tail-anchored membrane protein that exposes its ACBD to the cytosol. This protein plays a crucial role in lipid metabolism, particularly in the β-oxidation of very-long-chain fatty acids (VLCFAs) in peroxisomes. Zebrafish acbd5a is structurally and functionally similar to human ACBD5, making it valuable for studying human diseases related to peroxisomal dysfunction .

How does zebrafish acbd5a compare structurally to human ACBD5?

Zebrafish acbd5a shares significant sequence homology with human ACBD5, reflecting the evolutionary conservation of this protein. The zebrafish ortholog contains the characteristic N-terminal acyl-CoA binding domain that is critical for its function. Given that zebrafish share approximately 70% of their genes with humans and more specifically 84% of the genes associated with human genetic diseases, acbd5a likely retains key functional domains present in the human version . Both proteins are peroxisomal tail-anchored membrane proteins with similar topological organization, suggesting conserved mechanisms of action across species.

What is the significance of using zebrafish as a model for acbd5a research?

Zebrafish provide numerous advantages as a model organism for studying acbd5a:

• Rapid development - Most major organs form within 24 hours, allowing for efficient experimental timelines

• Transparent embryos - Enabling direct visualization of developmental processes and protein localization

• External fertilization - Facilitating genetic manipulation and experimental interventions

• High fecundity - Each female can produce up to 300 embryos every 2-3 days, providing sufficient samples for statistical power

• Genetic similarity to humans - Zebrafish possess orthologs of many human disease-causing genes, including those involved in peroxisomal disorders

• Amenability to drug administration - Novel methods allow for precise dosing of compounds in zebrafish studies

These characteristics make zebrafish an excellent model for investigating the physiological roles of acbd5a and its implications in human disease states.

What techniques are available for detecting acbd5a expression in zebrafish tissues?

Several methodologies can be employed to detect acbd5a expression in zebrafish tissues:

• RT-qPCR: For quantitative analysis of acbd5a mRNA expression levels in different tissues or developmental stages. This technique requires designing specific primers for the zebrafish acbd5a gene.

• In situ hybridization: For spatial mapping of acbd5a expression patterns during development and in adult tissues. This technique allows visualization of the specific cells expressing the gene.

• Immunohistochemistry/Immunofluorescence: Using specific antibodies against zebrafish acbd5a to detect protein localization in tissue sections. If antibodies against zebrafish acbd5a are not commercially available, recombinant acbd5a with epitope tags can be used to generate such antibodies.

• Western blotting: For quantitative analysis of acbd5a protein levels in tissue lysates. This technique can also reveal potential post-translational modifications.

• Transgenic reporter lines: Creating zebrafish lines expressing fluorescent proteins under the control of the acbd5a promoter to monitor gene expression in real-time, taking advantage of the transparency of zebrafish embryos .

How is acbd5a expression regulated during zebrafish development?

The regulation of acbd5a expression during zebrafish development involves:

• Temporal regulation: Expression patterns likely change throughout development, with specific peaks during organogenesis of tissues where peroxisomal metabolism is critical.

• Spatial regulation: Expression is likely highest in tissues with significant peroxisomal content, including liver, kidneys, and potentially the retina, given the association of human ACBD5 mutations with retinal dystrophy .

• Transcriptional regulation: The acbd5a promoter likely contains binding sites for transcription factors involved in peroxisomal biogenesis and lipid metabolism.

• Post-transcriptional regulation: mRNA stability and translation efficiency may be regulated by RNA-binding proteins and microRNAs specific to metabolic pathways.

Characterizing these regulatory mechanisms requires techniques such as chromatin immunoprecipitation (ChIP), reporter assays, and RNA stability measurements in zebrafish embryos and tissues.

What expression systems are optimal for producing recombinant zebrafish acbd5a?

Multiple expression systems can be employed for recombinant zebrafish acbd5a production, each with specific advantages:

• E. coli-based expression:

  • Advantages: High yield, cost-effective, rapid production

  • Considerations: May require optimization of codon usage for zebrafish genes; the peroxisomal membrane-associated nature of acbd5a may cause inclusion body formation

  • Recommendation: Express only the soluble N-terminal ACBD domain for functional studies, as the full-length protein contains a transmembrane domain that may complicate purification

• Insect cell expression (Baculovirus system):

  • Advantages: Post-translational modifications closer to vertebrates, better folding of complex proteins

  • Considerations: Higher cost, longer production time

  • Recommendation: Preferable for full-length acbd5a expression with native folding

• Mammalian cell expression:

  • Advantages: Most authentic post-translational modifications, proper folding

  • Considerations: Highest cost, lower yield

  • Recommendation: Best for studies requiring physiologically relevant modifications

• Cell-free expression systems:

  • Advantages: Rapid production, direct incorporation of modified amino acids

  • Considerations: Lower yield, higher cost

  • Recommendation: Useful for preliminary functional studies or when rapid results are needed

The choice should be guided by the specific research questions and downstream applications. When precise binding studies with very-long-chain fatty acyl-CoAs (VLC-CoAs) are required, a system that ensures proper folding of the ACBD domain is essential .

What purification strategies work best for recombinant zebrafish acbd5a?

Effective purification strategies for recombinant zebrafish acbd5a include:

• Affinity chromatography:

  • His-tag purification: 6-8 histidine residues at N- or C-terminus

  • GST-fusion: Improves solubility but adds large tag

  • MBP-fusion: Enhances solubility for the full-length protein

• Ion exchange chromatography:

  • Useful as a secondary purification step

  • Selection of cation or anion exchange depends on the isoelectric point of acbd5a

• Size exclusion chromatography:

  • Final polishing step to remove aggregates and ensure monodispersity

  • Critical for structural studies and accurate binding assays

Purification Protocol Recommendation:

• Lyse cells in buffer containing mild detergents (for full-length protein)

• Perform initial purification via affinity chromatography

• Remove tags using specific proteases if necessary

• Further purify using ion exchange chromatography

• Perform final purification via size exclusion chromatography

• Verify purity by SDS-PAGE and Western blotting

• Confirm identity using mass spectrometry

For the N-terminal ACBD domain alone (without the transmembrane region), standard protein purification protocols can be followed with higher expected yields and simpler procedures.

How can I measure the binding affinity of recombinant zebrafish acbd5a to different acyl-CoA substrates?

Several methodologies can be employed to measure binding affinities:

• Isothermal Titration Calorimetry (ITC):

  • Directly measures thermodynamic parameters (ΔH, ΔS, ΔG) and binding constants (Kd)

  • Requires purified protein and ligands

  • Typical protocol: Titrate various concentrations of very-long-chain fatty acyl-CoAs into a solution of purified recombinant acbd5a while measuring heat changes

• Surface Plasmon Resonance (SPR):

  • Measures real-time binding kinetics (kon, koff)

  • Requires immobilization of either protein or ligand

  • Typical protocol: Immobilize recombinant acbd5a on a sensor chip and flow various acyl-CoA species at different concentrations

• Fluorescence-based assays:

  • Intrinsic tryptophan fluorescence quenching

  • Environmentally sensitive fluorescent probes

  • FRET-based approaches

  • Typical protocol: Monitor changes in fluorescence intensity as increasing concentrations of acyl-CoAs are added to a solution of recombinant acbd5a

• Ligand displacement assays:

  • Competitive binding assays using fluorescently labeled acyl-CoA probes

  • Typical protocol: Establish binding of a fluorescent acyl-CoA analog, then compete with unlabeled acyl-CoAs of various chain lengths

Based on research with human ACBD5, zebrafish acbd5a is expected to preferentially bind very-long-chain fatty acyl-CoAs (VLC-CoAs) . Create a comparative binding profile using acyl-CoAs of various chain lengths (C16 to C26) to determine the specificity of zebrafish acbd5a.

What functional assays can measure the impact of acbd5a on peroxisomal β-oxidation in zebrafish models?

To assess the impact of acbd5a on peroxisomal β-oxidation in zebrafish:

• VLCFA analysis by GC-MS or LC-MS/MS:

  • Quantify C22:0-C26:0 fatty acid levels in tissues from wild-type versus acbd5a-deficient zebrafish

  • Protocol: Extract total lipids from tissues, derive fatty acids, and analyze by mass spectrometry

  • Expected result: acbd5a-deficient fish would show elevated VLCFA levels if the protein functions similarly to human ACBD5

• Radioactive substrate-based β-oxidation assays:

  • Measure oxidation rates of [1-14C]-labeled VLCFAs in isolated peroxisomes or tissue homogenates

  • Protocol: Incubate samples with labeled substrates and measure radioactive CO2 production

  • Expected result: Reduced oxidation rates in acbd5a-deficient samples

• Phospholipid profiling:

  • Quantify phospholipid species containing VLCFAs

  • Protocol: Extract phospholipids and analyze by LC-MS/MS

  • Expected result: Elevated levels of VLCFA-containing phospholipids in acbd5a-deficient zebrafish

• Peroxisome morphology and abundance assessment:

  • Immunofluorescence microscopy using antibodies against peroxisomal markers

  • Protocol: Prepare tissue sections or culture cells, stain with appropriate antibodies, and analyze by confocal microscopy

  • Expected result: Normal peroxisome biogenesis but altered lipid content in acbd5a-deficient samples

• Zebrafish-specific behavioral tests:

  • Assess swimming patterns and responses to stimuli that might be affected by altered lipid metabolism

  • Protocol: Utilize standardized behavioral tests such as the light/dark preference test

  • Expected result: Potential behavioral abnormalities in acbd5a-deficient fish, particularly if VLCFA accumulation affects neural function

How can zebrafish acbd5a models inform our understanding of retinal dystrophy?

Zebrafish acbd5a models can provide valuable insights into retinal dystrophy mechanisms through:

• Generation of mutant lines:

  • Create acbd5a knockout or knockdown zebrafish using CRISPR/Cas9 or morpholinos

  • Introduce specific human patient mutations to recapitulate disease phenotypes

• Retinal phenotype characterization:

  • Histological analysis of retinal layers and cell types

  • Immunohistochemistry for photoreceptor markers

  • Electron microscopy to examine subcellular structures, particularly photoreceptor outer segments

  • Functional assessment using electroretinography (ERG)

• Mechanistic investigations:

  • Analysis of lipid composition in retinal tissues

  • Assessment of peroxisomal function in retinal cells

  • Evaluation of photoreceptor-specific gene expression

• Intervention studies:

  • Test potential therapeutic approaches using the novel drug administration method for zebrafish

  • Assess compounds that modulate lipid metabolism or enhance peroxisomal function

  • Evaluate gene therapy approaches

Human ACBD5 mutations have been associated with syndromic retinal dystrophy , making zebrafish models particularly valuable for understanding pathogenic mechanisms. The transparency of zebrafish embryos allows for real-time visualization of retinal development and degeneration processes , providing unique advantages for studying the progression of retinal pathology.

What are the expected phenotypic consequences of acbd5a deficiency in zebrafish?

Based on knowledge of human ACBD5 and peroxisomal disorders, acbd5a-deficient zebrafish would likely exhibit:

• Biochemical alterations:

  • Elevated levels of very-long-chain fatty acids (VLCFAs) in tissues

  • Increased VLCFA-containing phospholipids

  • Moderately impaired peroxisomal β-oxidation

• Tissue-specific manifestations:

  • Retinal abnormalities, potentially including photoreceptor degeneration

  • Liver phenotypes due to altered lipid metabolism

  • Potential brain involvement, given the neurological aspects of human peroxisomal disorders

• Developmental effects:

  • Possible delays in specific developmental processes

  • Potentially normal gross morphology but with subtle organ-specific defects

• Behavioral phenotypes:

  • Altered swimming patterns

  • Potential vision-dependent behavioral changes

  • Possible abnormalities in light/dark preference tests

• Stress response:

  • Potentially enhanced sensitivity to oxidative stress

  • Possible altered response to lipid-modulating compounds

These phenotypes would likely be more subtle than those observed in zebrafish models of severe peroxisomal biogenesis disorders, as acbd5a deficiency affects specific aspects of peroxisomal function rather than global peroxisome formation .

What CRISPR/Cas9 strategies are most effective for acbd5a modification in zebrafish?

Optimal CRISPR/Cas9 strategies for acbd5a modification in zebrafish include:

• Target selection:

  • Target early exons to ensure complete loss-of-function

  • Focus on the ACBD domain-encoding region for functional studies

  • Design multiple gRNAs to increase targeting efficiency

  • Avoid regions with potential off-target sites in the zebrafish genome

• Delivery methods:

  • Microinjection of Cas9 protein:gRNA ribonucleoprotein complexes into one-cell stage embryos

  • Co-injection with a donor template for precise mutations or insertions

  • Potential use of tissue-specific Cas9 expression for conditional knockout

• Verification strategies:

  • T7 endonuclease assay for initial screening

  • Direct sequencing of targeted regions

  • Western blotting to confirm protein loss

  • RT-qPCR to assess mRNA levels and potential compensatory mechanisms

• Specific applications:

  • Complete knockout: Target coding sequences to create frameshift mutations

  • Domain-specific studies: Create in-frame deletions of specific functional domains

  • Patient mutations: Introduce specific point mutations using homology-directed repair

  • Reporter tagging: Insert fluorescent protein sequences in-frame with acbd5a

• Addressing potential limitations:

  • Screen for genetic compensation by related genes (potentially acbd5b if it exists in zebrafish)

  • Generate multiple independent lines to control for off-target effects

  • Consider generating tissue-specific conditional knockouts if complete knockout is lethal

What experimental approaches can determine the in vivo interaction partners of zebrafish acbd5a?

To identify in vivo interaction partners of zebrafish acbd5a:

• Proximity-dependent biotinylation (BioID or TurboID):

  • Generate transgenic zebrafish expressing acbd5a fused to a biotin ligase

  • Harvest tissues at different developmental stages

  • Purify biotinylated proteins and identify by mass spectrometry

  • Advantages: Captures transient interactions and works in native cellular environment

• Co-immunoprecipitation coupled with mass spectrometry:

  • Express tagged versions of acbd5a in zebrafish

  • Perform immunoprecipitation from tissue lysates

  • Identify co-precipitated proteins by mass spectrometry

  • Advantages: Direct evidence for physical interactions

• Yeast two-hybrid screening:

  • Use zebrafish acbd5a as bait against a zebrafish cDNA library

  • Verify positive interactions in zebrafish cells

  • Advantages: Systematic screening approach

• Fluorescence resonance energy transfer (FRET) in vivo:

  • Generate transgenic zebrafish expressing acbd5a fused to a fluorescent protein

  • Co-express candidate interactors with complementary fluorescent tags

  • Analyze interaction by FRET microscopy in live embryos

  • Advantages: Direct visualization of interactions in real-time in living organisms

• Split-GFP complementation:

  • Express acbd5a and candidate partners fused to complementary GFP fragments

  • Visualize interactions through reconstituted GFP fluorescence

  • Advantages: Direct visualization with potentially less interference than full fluorescent proteins

Based on human ACBD5 studies, potential interaction partners to investigate include peroxisomal membrane proteins, components of the fatty acid transport machinery, and proteins involved in phospholipid metabolism .

How can I address issues with recombinant zebrafish acbd5a solubility and stability?

Recombinant acbd5a solubility and stability challenges can be addressed through:

• Expression strategy optimization:

  • Express only the soluble N-terminal ACBD domain without the transmembrane region

  • Use solubility-enhancing fusion partners (MBP, SUMO, or TrxA)

  • Lower induction temperature (16-18°C) for slower expression and better folding

  • Reduce inducer concentration to slow expression rate

• Buffer optimization:

  • Screen different pH ranges (typically 7.0-8.0)

  • Test various salt concentrations (150-500 mM NaCl)

  • Include stabilizing agents: glycerol (5-10%), mild detergents (for full-length protein)

  • Add reducing agents (DTT or β-mercaptoethanol) to prevent disulfide-mediated aggregation

• Storage conditions:

  • Determine optimal protein concentration (typically 1-5 mg/ml)

  • Test stability at different temperatures (4°C, -20°C, -80°C)

  • Evaluate freeze-thaw stability and consider flash-freezing aliquots

  • Test lyophilization with appropriate protectants

• Stability assessment methods:

  • Differential scanning fluorimetry to identify stabilizing conditions

  • Size exclusion chromatography to monitor aggregation state

  • Dynamic light scattering to assess homogeneity

  • Activity assays to confirm functional integrity over time

For full-length acbd5a containing the transmembrane domain, consider using mild detergents such as CHAPS, DDM, or Triton X-100 at concentrations just above their critical micelle concentration to maintain the native structure without causing aggregation.

What are the best approaches for resolving inconsistent phenotypes in acbd5a-modified zebrafish?

To address inconsistent phenotypes in acbd5a-modified zebrafish:

• Genetic background considerations:

  • Maintain consistent genetic background across experiments

  • Backcross mutant lines to wild-type for multiple generations

  • Use siblings as controls whenever possible

  • Consider generating mutations on multiple background strains to assess robustness

• Molecular verification:

  • Thoroughly verify mutations at DNA, RNA, and protein levels

  • Check for potential genetic compensation by related genes

  • Assess potential alternative splicing around the mutation site

  • Quantify residual protein levels using sensitive detection methods

• Experimental standardization:

  • Implement rigorous environmental control (temperature, light cycles, water quality)

  • Standardize feeding regimes and housing density

  • Use consistent time points for analysis

  • Employ the novel dosing method described for drug administration to ensure consistent exposure

• Phenotyping strategies:

  • Increase sample sizes to account for biological variability

  • Use quantitative rather than qualitative assessments

  • Implement blinded analysis to prevent observer bias

  • Employ automated systems for behavioral analysis

• Data analysis approaches:

  • Use appropriate statistical methods for the data type

  • Account for potential covariates

  • Consider employing multiple analytical approaches

  • Report effect sizes along with p-values

When using behavioral assays such as the light/dark preference test, maintain consistent environmental conditions and use standardized protocols as described in the research methodology literature to ensure replicability across experiments .

Table 1: Predicted Physiochemical Properties of Zebrafish acbd5a

Table 2: Recommended Expression Systems for Different Research Applications

Table 3: Expected Phenotypic Effects of acbd5a Manipulation in Zebrafish