Recombinant Xenopus laevis Carnitine O-palmitoyltransferase 2, mitochondrial (cpt2), partial

What is Xenopus laevis Carnitine O-palmitoyltransferase 2 and what are its primary functions?

Carnitine O-palmitoyltransferase 2, mitochondrial (cpt2) is an enzyme involved in fatty acid metabolism. In Xenopus laevis, as in other vertebrates, this enzyme plays a critical role in the transport of long-chain fatty acyl groups into mitochondria for beta-oxidation. The enzyme is located on the inner mitochondrial membrane and catalyzes the formation of acyl-CoA from carnitine derivatives. CPT2 functions as part of the carnitine shuttle system, which is essential for energy production from fatty acids. The Xenopus laevis variant has specific gene names including cpt2, cpt2.S, and cg2107, and is also known as carnitine palmitoyltransferase 2 S homeolog .

How is recombinant Xenopus laevis CPT2 typically expressed and purified for research applications?

Recombinant Xenopus laevis CPT2 can be expressed in multiple host systems including E. coli, yeast, baculovirus, or mammalian cells, each offering different advantages depending on research requirements. When expressed in prokaryotic systems like E. coli, the protein is typically tagged (often with a His-tag as seen with His6-N-hCPT2 in human studies) to facilitate purification . The purification process generally involves:

• Cell lysis under conditions that maintain protein stability

• Affinity chromatography (commonly using nickel columns for His-tagged proteins)

• Further purification through size exclusion or ion exchange chromatography

• Quality control assessment through SDS-PAGE with expected purity of ≥85%

The choice of expression system impacts post-translational modifications, with mammalian cells providing the most physiologically relevant modifications for vertebrate proteins. For kinetic studies and structural analysis, E. coli expression may be sufficient and more economical.

How do genomic and functional studies of Xenopus laevis CPT2 contribute to understanding evolutionary conservation of fatty acid metabolism?

Xenopus laevis presents a unique model for evolutionary studies due to its allotetraploid genome. The CPT2 gene in Xenopus laevis exists as homeologs (cpt2 and cpt2.S), offering insights into gene duplication and functional divergence . Comparative genomic analysis between Xenopus laevis and other species reveals patterns of conservation in metabolic pathways.

For conducting these analyses, researchers typically:

• Perform sequence alignments of CPT2 across multiple species

• Analyze conserved domains and catalytic sites

• Study gene expression patterns in different developmental stages

• Compare kinetic parameters of recombinant enzymes from different species

The advantage of using Xenopus laevis for these studies includes the ability to perform both genomic and functional analyses in the same experimental system, from embryonic development through adulthood . This comprehensive approach allows researchers to correlate genetic changes with functional consequences in a vertebrate model that bridges the evolutionary gap between aquatic and terrestrial species.

What are the advantages and limitations of using recombinant Xenopus laevis CPT2 as a model for human CPT2 deficiency research?

Advantages:

• Xenopus laevis produces large embryos excellent for gene overexpression analysis and biochemical studies

• The developmental biology of Xenopus is well-characterized, allowing for studies across life stages

• Genetic manipulation techniques are well-established in Xenopus

• Functional conservation of metabolic pathways between amphibians and mammals makes mechanistic insights potentially translatable

Limitations:

• The allotetraploid nature of Xenopus laevis can complicate genetic analyses compared to diploid models

• Differences in thermal regulation between amphibians and mammals may affect enzyme kinetics and stability studies

• Species-specific post-translational modifications may alter protein function

• Metabolic demands differ between Xenopus and humans, potentially affecting the phenotypic manifestation of enzyme deficiencies

Researchers working with recombinant Xenopus CPT2 to model human disease should consider:

• Complementing Xenopus studies with mammalian cell experiments

• Confirming key findings in human samples when possible

• Carefully controlling temperature conditions during enzyme assays to account for differences in thermal optima

• Analyzing conserved versus divergent regions when making cross-species inferences

Results from human CPT2 variant studies show that genetic mutations like p.Ser113Leu can impair enzyme kinetic stability , providing a framework for investigating similar mechanisms in Xenopus models.

How can site-directed mutagenesis of Xenopus laevis CPT2 be used to investigate structure-function relationships?

Site-directed mutagenesis of Xenopus laevis CPT2 provides a powerful tool for understanding enzyme function. Based on research approaches with human CPT2 , a comprehensive mutagenesis strategy would include:

• Identification of target residues:

  • Conserved catalytic residues

  • Residues corresponding to human disease mutations (e.g., equivalents to S113L, Q46A, P50H, E174K, I502T, and R247W)

  • Substrate binding pocket residues

  • Membrane interaction domains

• Mutagenesis protocol:

  • PCR-based site-directed mutagenesis of cpt2 cDNA in expression vectors

  • Confirmation of mutations by sequencing

  • Expression in multiple systems (E. coli, yeast, mammalian cells) to assess system-dependent effects

• Functional characterization:

  • Enzymatic activity assays under varying conditions (temperature, pH, substrate concentrations)

  • Thermal stability studies comparing wild-type and mutant proteins

  • Substrate specificity assessments with various fatty acid chain lengths

• Structural analysis:

  • Circular dichroism to assess secondary structure changes

  • Limited proteolysis to identify conformational differences

  • Crystallization attempts for structural determination

This approach has successfully revealed that human CPT2 mutations like p.Ser113Leu significantly reduce thermal stability , suggesting that similar studies in Xenopus laevis CPT2 would provide valuable comparative data on enzyme evolution and the molecular basis of enzyme regulation.

What expression systems are optimal for producing functional recombinant Xenopus laevis CPT2, and how do they compare?

For most applications with recombinant Xenopus laevis CPT2, researchers report using E. coli or baculovirus systems, with protein purity reaching ≥85% as determined by SDS-PAGE . When selecting an expression system, researchers should consider:

• The specific research question (structural vs. functional studies)

• Required post-translational modifications

• Budget and timeline constraints

• Need for membrane association studies

For activity-based studies, it's essential to verify that the recombinant enzyme demonstrates appropriate catalytic activity regardless of the expression system used.

What are the most effective methods for assessing the enzymatic activity and kinetic parameters of recombinant Xenopus laevis CPT2?

Enzymatic activity and kinetic parameters of recombinant Xenopus laevis CPT2 can be assessed through several complementary methods:

• Spectrophotometric Assays:

  • Forward reaction: monitoring CoA-SH release using DTNB (5,5'-dithiobis-2-nitrobenzoic acid)

  • Reverse reaction: following acylcarnitine formation using radioisotope-labeled substrates

• Kinetic Parameter Determination:

  • Varying substrate concentrations to determine Km and Vmax

  • Temperature-dependent activity profiles (particularly important for comparing amphibian vs. mammalian enzymes)

  • pH optimization studies

  • Inhibitor sensitivity assays

• Thermal Stability Assessment:

  • Differential scanning fluorimetry (DSF) to determine melting temperatures

  • Activity retention after heat challenge at different temperatures

  • Time-course inactivation studies at elevated temperatures

Based on human CPT2 studies, temperature-dependent kinetic stability is a critical parameter, as mutations like p.Ser113Leu significantly impair stability at elevated temperatures . For Xenopus laevis CPT2, establishing baseline thermal stability profiles is essential due to the poikilothermic nature of amphibians compared to homeothermic mammals.

How can researchers effectively design experiments to compare Xenopus laevis CPT2 with human CPT2 for translational research?

Designing comparative experiments between Xenopus laevis and human CPT2 requires careful consideration of physiological differences while maintaining experimental consistency. A comprehensive approach includes:

• Sequence and Structure Comparison:

  • Align amino acid sequences to identify conserved and divergent regions

  • Construct homology models based on available crystal structures

  • Identify functionally important residues conserved across species

• Parallel Expression and Purification:

  • Express both proteins in identical systems (preferably multiple systems)

  • Use identical tags and purification protocols

  • Verify comparable purity (≥85% by SDS-PAGE)

• Comparative Functional Assays:

  • Conduct enzyme assays under identical conditions

  • Perform temperature-response curves relevant to both species (10-42°C)

  • Assess substrate specificity with panels of acyl-CoA substrates

  • Test inhibitor sensitivity profiles

• Mutation Analysis:

  • Create equivalent mutations in both proteins (particularly disease-associated variants)

  • Compare effects on stability and activity

  • Assess species-specific responses to perturbations

• Cellular Studies:

  • Express both proteins in identical cellular backgrounds

  • Assess subcellular localization

  • Measure cellular phenotypes (e.g., lipid metabolism, mitochondrial function)

When conducting these studies, it's crucial to consider the natural temperature ranges of each species and to interpret results in the appropriate physiological context.

How can recombinant Xenopus laevis CPT2 be utilized in high-throughput screening for metabolic disorder therapeutics?

Recombinant Xenopus laevis CPT2 provides a valuable tool for high-throughput screening (HTS) of potential therapeutics for CPT2 deficiency and related metabolic disorders. An effective HTS platform would include:

• Assay Development:

  • Miniaturized spectrophotometric assays adaptable to 384-well format

  • Fluorescence-based activity assays for improved sensitivity

  • Thermal shift assays to identify stabilizing compounds

  • Counter-screens to eliminate false positives

• Compound Library Selection:

  • Natural product libraries relevant to metabolic pathways

  • FDA-approved drug libraries for repurposing potential

  • Targeted libraries of lipid metabolism modulators

  • Fragment-based approaches for novel scaffold identification

• Screening Strategy:

  • Primary screens against wild-type enzyme for activity modulators

  • Secondary screens against mutant forms corresponding to disease variants

  • Dose-response confirmation of hits

  • Orthogonal assays to confirm mechanism of action

• Validation Pipeline:

  • Parallel testing with human CPT2

  • Cell-based assays in relevant metabolic models

  • Xenopus embryo studies for developmental toxicity assessment

  • Evaluation in mammalian disease models

The advantage of using Xenopus laevis CPT2 in initial screens includes cost-effectiveness and the ability to easily transition positive hits to in vivo testing in Xenopus embryos before advancing to mammalian models.

What are the best approaches for using recombinant Xenopus laevis CPT2 to study mitochondrial fatty acid metabolism in developmental contexts?

Xenopus laevis provides an exceptional model for studying developmental regulation of mitochondrial metabolism due to its well-characterized embryonic development and the large size of its embryos . Research approaches include:

• Developmental Expression Analysis:

  • Temporal profiling of cpt2 expression during embryogenesis

  • Spatial mapping using in situ hybridization

  • Correlation with mitochondrial biogenesis markers

  • Comparison between cpt2 and cpt2.S homeologs expression patterns

• Functional Metabolic Studies:

  • Microinjection of wild-type or mutant recombinant CPT2 into embryos

  • Metabolic flux analysis using isotope-labeled fatty acids

  • Mitochondrial respiration measurements at different developmental stages

  • Correlation of CPT2 activity with developmental energy demands

• Genetic Manipulation Approaches:

  • CRISPR/Cas9 genome editing of endogenous cpt2 genes

  • Morpholino knockdown for transient loss-of-function

  • Rescue experiments with recombinant wild-type or mutant proteins

  • Overexpression studies to assess metabolic pathway regulation

• Imaging-Based Analyses:

  • Live imaging of mitochondrial dynamics in embryos following CPT2 manipulation

  • Confocal microscopy of tissue-specific mitochondrial changes

  • Correlation of mitochondrial morphology with metabolic activity

  • Super-resolution imaging of CPT2 localization during development

These approaches can leverage the established methods for Xenopus husbandry, tissue preparation, and microscopy described in the literature , allowing for comprehensive analysis of mitochondrial fatty acid metabolism across developmental stages.

How can protein engineering of Xenopus laevis CPT2 contribute to creating improved biocatalysts for biotechnological applications?

Protein engineering of Xenopus laevis CPT2 presents opportunities for developing specialized biocatalysts with applications in biotechnology and synthetic biology. Strategic approaches include:

• Stability Engineering:

  • Introducing disulfide bridges to enhance thermal stability

  • Consensus-based design incorporating thermostable features from related enzymes

  • Directed evolution for increased solvent tolerance

  • Computational design of stabilizing interactions

• Substrate Specificity Modification:

  • Active site mutagenesis to accommodate non-natural substrates

  • Directed evolution for altered chain-length preferences

  • Creation of chimeric enzymes with features from related transferases

  • Rational design based on molecular dynamics simulations

• Immobilization Strategies:

  • Addition of specific tags for oriented immobilization

  • Engineering surface residues for enhanced stability on solid supports

  • Creation of self-assembling enzyme arrays

  • Development of enzyme-nanomaterial conjugates

• Performance Optimization:

  • Activity enhancement through ancestral sequence reconstruction

  • Modification of regulatory sites to reduce product inhibition

  • pH-tolerance engineering for industrial conditions

  • Creation of fusion proteins with complementary enzymatic activities

These engineering approaches can build upon the knowledge gained from structure-function studies of natural variants, such as those observed in human CPT2 deficiency research , while taking advantage of the unique properties of the amphibian enzyme that may offer advantages for specific biotechnological applications.

How does recombinant Xenopus laevis CPT2 research intersect with studies on mitochondrial dysfunction in neurodevelopmental disorders?

Recombinant Xenopus laevis CPT2 research provides a valuable platform for investigating mitochondrial dysfunction in neurodevelopmental contexts. Integration approaches include:

• Xenopus as a Neurological Model:

  • Xenopus laevis embryos serve as excellent models for neural development studies, allowing for the examination of how CPT2 dysfunction affects neural tissue specifically

  • The well-characterized developmental stages enable temporal analysis of when metabolic deficiencies impact neural development

• Translation to Human Disease:

  • Parallels between Xenopus and human CPT2 function can illuminate mechanisms underlying neurological manifestations in CPT2 deficiency

  • Common variants in human CPT2 (such as S113L, P50H, and others) can be recreated in Xenopus CPT2 to study neural-specific effects

• Methodological Approaches:

  • Neural stem progenitor cells from Xenopus exhibit specific mitochondrial responses that can be characterized using electron and confocal microscopy

  • Spinal cord injury models in Xenopus provide insights into how metabolic enzymes like CPT2 influence regenerative capacity in neural tissue

• Research Workflow:

  • Begin with in vitro studies using recombinant Xenopus CPT2

  • Progress to cell-based assays using Xenopus neural progenitors

  • Advance to embryonic manipulations and neural development assessment

  • Correlate findings with human patient data on neurological manifestations

By leveraging the established techniques for Xenopus neural tissue studies and combining them with recombinant protein approaches, researchers can create comprehensive models of how fatty acid metabolism influences neurodevelopment and neurological function.

What computational approaches are most effective for modeling structure-function relationships in Xenopus laevis CPT2?

Computational modeling of Xenopus laevis CPT2 requires specialized approaches to account for the unique aspects of this enzyme while leveraging established computational techniques:

• Homology Modeling Approaches:

  • Template selection ideally using crystal structures of related CPT enzymes

  • Multiple template modeling to improve accuracy in variable regions

  • Refinement focusing on mitochondrial membrane interaction domains

  • Validation through integration with experimental data

• Molecular Dynamics Simulations:

  • Membrane-embedded simulations to accurately represent the native environment

  • Temperature-varied simulations to capture amphibian-specific dynamics

  • Substrate binding and product release pathway analysis

  • Conformational change modeling during the catalytic cycle

• Virtual Screening Workflows:

  • Pharmacophore development based on conserved substrate binding features

  • Docking studies to identify potential modulators

  • Machine learning integration for improved hit prediction

  • Comparative analysis with human CPT2 for translational insights

• Network Analysis:

  • Integration of CPT2 into metabolic network models

  • Systems biology approaches to predict effects of CPT2 modulation

  • Developmental stage-specific network models

  • Comparative network analysis across species

These computational approaches should incorporate data from experimental studies on human CPT2 variants that affect enzyme stability and function , while accounting for the specific evolutionary and physiological context of Xenopus laevis.

What are the most promising future directions for research involving recombinant Xenopus laevis CPT2?

The study of recombinant Xenopus laevis Carnitine O-palmitoyltransferase 2 offers multiple promising research avenues:

• Comparative Metabolic Studies:

  • Cross-species analysis of CPT2 function between amphibians and mammals

  • Evolutionary adaptation of fatty acid metabolism across vertebrates

  • Temperature adaptation mechanisms in poikilothermic versus homeothermic species

• Developmental Metabolism:

  • Characterization of metabolic shifts during Xenopus embryogenesis

  • Role of fatty acid metabolism in tissue differentiation and organogenesis

  • Homeolog-specific functions of cpt2 and cpt2.S in development

• Therapeutic Applications:

  • Development of protein-based therapies for CPT2 deficiency

  • Screening platforms for small molecule modulators

  • Enzyme replacement strategies leveraging recombinant production

• Biotechnology Development:

  • Engineered variants with enhanced catalytic properties

  • Development of biosensors incorporating CPT2 for fatty acid detection

  • Biocatalytic applications in lipid modification

• Methodological Advancements:

  • Improved recombinant production systems for membrane-associated enzymes

  • Structural biology approaches for mitochondrial proteins

  • Integration of multi-omics data in metabolic research

These directions leverage the advantages of Xenopus laevis as an experimental system, including its well-characterized developmental biology, the ability to produce large embryos excellent for biochemical studies , and established methods for genetic manipulation and imaging .