Recombinant Human ATP-binding cassette sub-family D member 2 (ABCD2)
Description
Fundamental Characteristics of ABCD2 Protein
ATP-binding cassette sub-family D member 2 (ABCD2) belongs to the superfamily of ATP-binding cassette transporters, a class of proteins that utilize ATP hydrolysis to transport various molecules across cellular membranes. ABCD2 is also known by alternative names including Adrenoleukodystrophy-like 1 and Adrenoleukodystrophy-related protein (hALDR) . This protein is encoded by the ABCD2 gene, which corresponds to Gene ID 225 in humans . The ABCD2 protein is primarily localized to the peroxisomal membrane where it plays a crucial role in the transport of very long-chain fatty acids (VLCFAs) into peroxisomes for subsequent β-oxidation. The protein's significance is particularly evident in the context of X-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder caused by mutations in the ABCD1 gene, where ABCD2 has been identified as the closest homolog of ABCD1 and a potential therapeutic target .
In human biological systems, ABCD2 functions as a transporter with broad substrate specificity that is dependent on ATP for its activity. The protein consists of 740 amino acids in its full-length form, with a molecular weight that positions it among medium-sized transport proteins . The sequence of ABCD2 is highly conserved across species, indicating its evolutionary importance in cellular metabolism, particularly in lipid processing and peroxisomal function. The preservation of key functional domains across species suggests a fundamental role in cellular homeostasis that has remained critical throughout evolutionary development.
Production Methods for Recombinant ABCD2
Recombinant Human ABCD2 protein is produced using various expression systems, with different approaches yielding proteins suitable for distinct research applications. Common expression systems include bacterial (E. coli), insect cell (Baculovirus), and mammalian cell platforms, each with inherent advantages for protein production. The choice of expression system impacts protein folding, post-translational modifications, and ultimately, the functionality of the recombinant protein.
The bacterial expression system using E. coli has been employed for the production of full-length Human ABCD2 protein with an N-terminal His tag . This system offers high protein yields and relative simplicity but may lack some post-translational modifications found in eukaryotic cells. Alternatively, the Baculovirus expression system has been utilized for the production of partial ABCD2 protein, offering a eukaryotic environment that can provide more authentic protein processing .
Post-production, recombinant ABCD2 undergoes purification procedures to remove contaminants and achieve high purity levels. For His-tagged proteins, affinity chromatography using nickel or cobalt resins is commonly employed to selectively isolate the tagged protein. The purified protein is then typically available in either lyophilized form or as a liquid with glycerol added for stability. Purity levels exceeding 85-90% as determined by SDS-PAGE are standard for commercial preparations .
Functional Significance of ABCD2
ABCD2 plays a crucial role in peroxisomal metabolism, particularly in the transport of very long-chain fatty acids (VLCFAs) across the peroxisomal membrane for β-oxidation. The protein's function is especially significant in the context of X-linked adrenoleukodystrophy (X-ALD), a disorder characterized by the accumulation of VLCFAs due to defective ABCD1 protein. Research has demonstrated that ABCD2 can functionally compensate for ABCD1 deficiency to some extent, highlighting its potential as a therapeutic target in X-ALD .
Studies have shown that the induction of ABCD2 gene expression can reduce the levels of VLCFAs in cells, including those from X-ALD patients. This compensatory effect suggests that pharmacological upregulation of ABCD2 could potentially mitigate the metabolic abnormalities associated with ABCD1 deficiency. Interestingly, ectopic expression of ABCD2-GFP, as well as the transcription factors β-catenin and TCF-4, has been observed to decrease VLCFA levels, further supporting the therapeutic potential of ABCD2 induction .
Beyond its role in VLCFA metabolism, ABCD2 is believed to have broader functions in lipid homeostasis, although these remain less well-characterized than its role in VLCFA transport. The protein's broad substrate specificity suggests it may transport multiple lipid species, potentially influencing various aspects of cellular lipid metabolism. Further research is needed to fully elucidate the complete substrate range and physiological functions of ABCD2 in different tissues and developmental stages.
Regulation of ABCD2 Expression
The transcriptional regulation of ABCD2 has emerged as an area of significant interest, particularly given its potential therapeutic implications for X-ALD. Recent research has identified specific transcription factors and regulatory elements that control ABCD2 gene expression. One notable finding is the direct regulation of ABCD2 by β-catenin and TCF-4, components of the Wnt signaling pathway .
Through in silico analysis, researchers have identified two putative TCF-4 binding elements in the promoter region of the ABCD2 gene, located between nucleotide positions -360 and -260. Experimental evidence has demonstrated that the transcriptional activity of the ABCD2 promoter is significantly enhanced by the ectopic expression of β-catenin and TCF-4. Site-directed mutagenesis of these TCF-4 binding elements results in decreased promoter activity, confirming their functional importance in regulating ABCD2 expression .
| Regulatory Element | Position in Promoter | Effect on Transcription |
|---|---|---|
| TCF-4 Binding Elements | Between -360 and -260 | Enhance transcriptional activity when bound by TCF-4 and β-catenin |
Chromatin immunoprecipitation assays have provided further evidence for this regulatory mechanism, demonstrating that β-catenin physically interacts with the ABCD2 promoter. Additionally, real-time PCR analysis has shown that β-catenin and TCF-4 increase ABCD2 mRNA levels in both hepatocellular carcinoma cell lines and primary fibroblasts from X-ALD patients . These findings establish a direct link between the Wnt signaling pathway and ABCD2 expression, opening new avenues for therapeutic intervention in conditions associated with ABCD2 dysfunction.
Applications in Research and Medicine
Recombinant Human ABCD2 protein has diverse applications in both basic research and potential therapeutic development. In research settings, the protein serves as a valuable tool for studying peroxisomal transport mechanisms, the pathophysiology of X-ALD, and broader aspects of cellular lipid metabolism. Recombinant ABCD2 is also utilized as an immunogen for antibody production, facilitating the development of detection tools for studying ABCD2 expression and localization in cells and tissues .
In the context of X-ALD, ABCD2 has attracted significant attention as a potential therapeutic target. The observation that ABCD2 can functionally compensate for ABCD1 deficiency has prompted research into strategies for upregulating ABCD2 expression as a treatment approach for X-ALD. The identification of regulatory mechanisms, such as the β-catenin/TCF-4 pathway, provides potential targets for pharmacological intervention aimed at enhancing ABCD2 expression .
The development of purified recombinant ABCD2 proteins has also facilitated structural studies of this important membrane protein, contributing to our understanding of ABC transporter architecture and function. These structural insights may ultimately inform the design of small molecules capable of modulating ABCD2 activity for therapeutic purposes. As research in this field progresses, recombinant ABCD2 is likely to continue playing a central role in advancing our understanding of peroxisomal biology and developing novel treatments for associated disorders.
Recent Research Developments
Recent scientific investigations have expanded our understanding of ABCD2's biological roles and regulatory mechanisms. The discovery that ABCD2 is a direct target of β-catenin and TCF-4 represents a significant advancement in understanding the transcriptional regulation of this important protein . This finding has implications not only for basic peroxisomal biology but also for potential therapeutic approaches in conditions such as X-ALD where ABCD2 upregulation could be beneficial.
Research has demonstrated that ectopic expression of β-catenin and TCF-4 increases ABCD2 mRNA levels in both hepatocellular carcinoma cells and primary fibroblasts from X-ALD patients. Furthermore, this upregulation of ABCD2 is associated with decreased levels of very long-chain fatty acids, suggesting a functional consequence of the increased expression . These findings provide a mechanistic link between the Wnt signaling pathway and peroxisomal lipid metabolism through ABCD2 regulation.
Technological advancements have also facilitated the development of improved recombinant ABCD2 protein preparations, including virus-like particles (VLPs) containing ABCD2, which may offer advantages for certain research applications . These innovations in protein production methods continue to expand the toolkit available for studying ABCD2 structure, function, and potential therapeutic applications.
Product Specs
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Note: The complete sequence including tag sequence, target protein sequence and linker sequence could be provided upon request.
Target Background
- The functional integrity of ABCD2 may play a significant role in OA pathogenesis through the accumulation of VLCFAs and the induction of apoptotic death by altering profiles of miRNAs targeting ACSL4. PMID: 30264402
- 13-cis-retinoic acid induces ABCD2 expression in human monocytes/macrophages. PMID: 25079382
- ABCD2 contributes, albeit not strongly, to the risk of early recurrent events after transient ischemic attack. PMID: 25604068
- Findings indicate that while patients with an ABCD2 score greater than 4 are more likely to develop recurrent TIA/CVA in the short term, those with lower scores still carry a considerable risk for TIA/CVA. PMID: 24338191
- The transcriptional activity of the ABCD2 promoter is significantly enhanced by ectopic expression of beta-catenin and TCF-4. PMID: 23437103
- LXRalpha acts as a negative modulator of Abcd2, employing a novel regulatory mechanism involving overlapping SREBP and LXRalpha binding sites. PMID: 16249184
- Testosterone metabolites enhance the expression of ABCD2 mRNA in fibroblasts derived from X-linked adrenoleukodystrophy patients. PMID: 17602313
- These findings hold particular importance for exploring pharmacological induction of ABCD2 as a potential therapeutic approach in X-linked adrenoleukodystrophy. PMID: 18834860
- LDRP (ABCD2) interacts with both farnesylated wild-type and farnesylation-deficient mutant PEX19. This interaction is mediated by amino acids 1-218 of ALDRP. PMID: 10777694
- ALDRP (ABCD2) forms homodimers via the C terminal half. This interaction is modeled on the demonstrated homodimerization of murine ALDRP (ABCD2). PMID: 10551832
- ALDRP interacts with PMP70. This interaction occurs through the ALDRP C-terminus [374-740] and the PMP70 C-terminus [338-659]. This interaction was demonstrated using human PMP70 and mouse ALDRP. PMID: 10551832
- ALDRP interacts with PEX19 splice variants PEX19-delta-E2 and PEX19-delta-E8. PMID: 11883941
Q&A
What is the molecular structure of ABCD2?
ABCD2 is a member of the D subfamily of ATP-binding cassette transporters. The single protein has a molecular mass of approximately 82 kDa, while its EGFP-tagged version (ABCD2-EGFP) has a molecular mass of approximately 111 kDa. In its native state in the peroxisomal membrane, ABCD2 primarily exists as part of higher molecular weight complexes, predominantly tetramers with apparent molecular masses of ~480 kDa for ABCD2-EGFP .
Where is ABCD2 localized in cells?
ABCD2 is primarily localized in the peroxisomal membrane, where it functions as a transporter. Research has shown that ABCD2 can be extracted from peroxisome-enriched fractions, confirming its peroxisomal localization .
How does ABCD2 relate to other ABCD transporters?
ABCD2 belongs to the D subfamily of ATP-binding cassette transporters, which includes other members like ABCD1. Both ABCD1 and ABCD2 show similar oligomeric organization in the peroxisomal membrane, predominantly forming tetramers, although they appear to assemble in distinct complexes with minimal overlap in native PAGE analysis .
What are effective techniques for solubilizing ABCD2 from peroxisomal membranes?
The extraction of ABCD2 from peroxisomal membranes is significantly enhanced when peroxisomes are preincubated with ATP before detergent treatment. Using the milder detergent α-DDM (n-dodecyl-α-D-maltoside), ABCD2-EGFP solubility increases from approximately 7.9% to 43.6% when peroxisomes are pretreated with ATP. This significant improvement in solubilization efficiency suggests an ATP-dependent conformational change that impacts the membrane association or protein-protein interactions of ABCD2 .
What electrophoretic methods are suitable for analyzing native ABCD2 complexes?
For analyzing native ABCD2 complexes, the Deriphat (N-lauryl-β-iminodipropionate)-PAGE system has been successfully employed. This system, adapted from Peter and Thornber's work, has proven effective for analyzing full-length ABC transporters. When combined with α-DDM solubilization and ATP pretreatment, this technique reveals ABCD2-EGFP in high molecular weight complexes with apparent masses of ~480 kDa, consistent with tetrameric assemblies .
How can protein-protein interactions of ABCD2 be effectively studied?
Co-immunoprecipitation (co-IP) coupled with quantitative mass spectrometry provides a powerful approach for identifying ABCD2 interaction partners. In studies using ABCD2-EGFP expressing cells, anti-GFP co-IP assays followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) have successfully identified ABCD2 with high sequence coverage (51.7%). This approach allows for differential analysis between samples expressing or not expressing ABCD2-EGFP to eliminate false-positive interactions .
What is the predominant oligomeric state of ABCD2 in the peroxisomal membrane?
Research evidence strongly indicates that ABCD2 predominantly exists as tetramers in the peroxisomal membrane. Native PAGE analysis of solubilized ABCD2-EGFP reveals a prominent band with an apparent molecular mass of ~480 kDa, which is approximately four times the molecular mass of the single ABCD2-EGFP protein (111 kDa). This tetrameric organization appears to be a common feature shared with other ABCD transporters like ABCD1 .
Does ATP binding affect the quaternary structure of ABCD2?
ATP appears to significantly influence the membrane association or structural organization of ABCD2. Preincubation of peroxisomes with ATP before detergent solubilization dramatically increases the extraction efficiency of ABCD2 from peroxisomal membranes. This suggests that ATP binding induces conformational changes in ABCD2 that affect its interaction with the membrane or other proteins, potentially as part of its normal catalytic cycle .
Do ABCD1 and ABCD2 form heterooligomeric complexes?
While both ABCD1 and ABCD2 form tetrameric complexes in the peroxisomal membrane, evidence suggests they primarily assemble in distinct complexes rather than heterooligomers. Native PAGE analysis shows that ABCD1 and ABCD2-EGFP bands have minimal overlap, as demonstrated by densitometric tracing of their electrophoretic patterns. This suggests functional specialization between these related transporters despite their structural similarities .
What methodological approaches can differentiate between homo- and heterooligomeric ABCD2 complexes?
To distinguish between homo- and heterooligomeric ABCD2 complexes, researchers should consider a multi-technique approach:
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Native PAGE with densitometric analysis: Comparing the migration patterns of ABCD2 and other potential partners can reveal distinct or overlapping complexes.
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Two-color co-localization: Using differently tagged versions of ABCD transporters to assess co-localization in the peroxisomal membrane.
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Sequential co-immunoprecipitation: Using antibodies against different potential partners in sequence to isolate specific complex compositions.
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Cross-linking studies: Employing chemical cross-linkers of defined lengths to capture protein-protein interactions within oligomeric assemblies .
How can researchers accurately assess the molecular mass of ABCD2 complexes?
Accurately determining the molecular mass of membrane protein complexes presents several challenges. For ABCD2 complexes, a comprehensive approach should include:
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Native PAGE with appropriate markers: Using well-characterized membrane protein standards.
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Size exclusion chromatography: Providing solution-based estimates of complex size.
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Analytical ultracentrifugation: Offering high-resolution mass determination.
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Mass photometry: A newer technique allowing single-molecule analysis of complex sizes.
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Accounting for detergent contributions: Considering that detergent micelles can contribute significantly to apparent mass measurements of membrane protein complexes .
What are the key considerations when designing co-IP/MS experiments for identifying ABCD2 interaction partners?
When designing co-IP/MS experiments to identify ABCD2 interaction partners, researchers should consider:
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Appropriate controls: Include samples not expressing ABCD2 (e.g., −dox/+dox inducible systems) to identify false-positive interactions.
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Detergent selection: Choose detergents that maintain native interactions while effectively solubilizing ABCD2 (α-DDM has proven effective).
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ATP pretreatment: Consider the dramatic impact of ATP preincubation on ABCD2 solubilization.
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Quantitative MS approach: Employ quantitative LC-ESI-MS/MS to reliably identify specific interaction partners.
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Validation studies: Confirm key interactions through orthogonal methods like reciprocal co-IP or proximity labeling approaches .
What approaches can be used to study the transport function of ABCD2?
Investigating the transport function of ABCD2 requires specialized approaches for membrane transporters:
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Proteoliposome reconstitution: Purified ABCD2 can be reconstituted into liposomes to study transport of specific substrates.
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Cellular uptake assays: Comparing substrate uptake in cells overexpressing or lacking ABCD2.
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ATPase activity measurements: Assessing ATP hydrolysis rates in response to potential transported substrates.
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Transport inhibition studies: Using specific inhibitors to validate transport mechanisms.
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Site-directed mutagenesis: Creating variants with altered transport properties to identify critical residues .
How does the tetrameric structure of ABCD2 relate to its transport mechanism?
The tetrameric structure of ABCD2 raises important questions about its transport mechanism:
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Functional unit determination: Whether each monomer functions independently or cooperatively within the tetramer.
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Conformational coupling: How ATP binding and hydrolysis in one monomer influences others.
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Substrate binding sites: Whether each monomer has its own substrate binding site or if they form a common translocation pathway.
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Regulatory mechanisms: How the tetrameric structure might enable regulation not possible in monomeric transporters.
These aspects can be investigated using combinations of biochemical, structural, and functional approaches to correlate oligomeric state with transport activity .
What strategies can overcome poor solubilization of ABCD2 from peroxisomal membranes?
Based on research findings, the following approaches can improve ABCD2 solubilization:
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ATP pretreatment: Incubating peroxisomes with ATP before detergent extraction significantly increases ABCD2 solubility (from 7.9% to 43.6%).
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Detergent selection: α-DDM has proven effective for ABCD2 solubilization while maintaining native interactions.
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Buffer optimization: Adjusting ionic strength, pH, and glycerol content can improve extraction efficiency.
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Temperature considerations: Performing extractions at specific temperatures may help maintain protein stability.
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Protease inhibitors: Including appropriate protease inhibitors prevents degradation during extraction .
How can researchers validate the physiological relevance of observed ABCD2 tetramers?
To confirm that ABCD2 tetramers represent physiologically relevant structures rather than extraction artifacts:
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Cross-linking studies in intact cells: Applying membrane-permeable cross-linkers before extraction.
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Comparison across multiple detergents: Consistent results with different detergents suggest native structures.
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Functional correlation: Demonstrating that tetrameric forms correlate with transport activity.
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In situ structural approaches: Using techniques like FRET or proximity labeling in intact cells.
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Correlation with disease-causing mutations: Examining how mutations that affect function impact oligomerization .
What controls are essential when analyzing ABCD2 oligomeric structures?
Proper controls for analyzing ABCD2 oligomeric structures include:
| Control Type | Purpose | Implementation |
|---|---|---|
| Negative controls | Identify false-positive signals | Samples not expressing ABCD2 |
| Detergent controls | Assess detergent effects on oligomerization | Multiple detergent types and concentrations |
| ATP controls | Evaluate nucleotide effects | ±ATP, ±non-hydrolyzable ATP analogs |
| Denaturation controls | Confirm specificity of native interactions | Samples subjected to denaturing conditions |
| Related protein controls | Establish specificity | Analysis of related ABCD transporters |
These controls help ensure that observed oligomeric structures represent physiologically relevant ABCD2 complexes rather than experimental artifacts .
How should researchers interpret differences in ABCD2 complex sizes across different studies?
When evaluating variations in reported ABCD2 complex sizes:
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Consider methodological differences: Detergent type, concentration, and extraction protocols can significantly impact observed complex sizes.
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Evaluate calibration approaches: Different molecular weight standards and calibration methods affect size estimates.
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Account for detergent contributions: Detergent micelles contribute to apparent size, varying by detergent type.
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Assess protein tags: Tags like EGFP can influence both actual and apparent complex size.
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Examine experimental conditions: ATP presence, salt concentration, and pH can all affect oligomeric state.
