Recombinant Pig Cytochrome c oxidase subunit 2 (MT-CO2)

What is the molecular structure and functional role of MT-CO2 in cellular metabolism?

MT-CO2 (also known as COX2, COII, or COXII) is one of the core subunits of mitochondrial cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain. The protein contains a dual core CuA active site that transfers electrons from cytochrome c via its binuclear copper A center to the bimetallic center of the catalytic subunit 1 . In the pig (Sus scrofa), the full-length MT-CO2 protein contains 228 amino acids (positions 1-228) according to the UniProt database (accession number P50667) .

As a component of Complex IV in the electron transport chain, MT-CO2 plays a significant role in energy metabolism, specifically in the process that catalyzes the reduction of oxygen to water, coupling this reaction with proton translocation across the inner mitochondrial membrane . This process is crucial for establishing the electrochemical gradient that drives ATP synthesis.

How does porcine MT-CO2 differ structurally and functionally from MT-CO2 in other species?

Multiple sequence alignment and phylogenetic analysis have shown that MT-CO2 proteins share high sequence identity across various mammalian species, indicating evolutionary conservation of this critical protein . The pig MT-CO2 protein has a molecular mass of approximately 26 kDa , similar to the molecular weight observed in other species such as the maize weevil (Sitophilus zeamais), which has been reported as 26.2 kDa .

What are the most effective expression systems for producing recombinant pig MT-CO2?

The most commonly used expression system for recombinant pig MT-CO2 is E. coli. According to available data, recombinant full-length pig MT-CO2 protein has been successfully expressed in E. coli systems with N-terminal His-tags . This approach allows for straightforward purification using affinity chromatography.

Similar to the approach used for Sitophilus zeamais COX2, the methodology typically involves:

• Subcloning the MT-CO2 gene into an appropriate expression vector (e.g., pET-32a)

• Transformation into a suitable E. coli strain (e.g., Transetta DE3)

• Induction of protein expression using IPTG (isopropyl β-d-thiogalactopyranoside)

• Purification of the recombinant protein using affinity chromatography with Ni²⁺-NTA agarose

While E. coli is the predominant expression system, researchers investigating complex functional studies might consider eukaryotic expression systems to ensure proper folding and post-translational modifications.

What purification strategies yield the highest purity and activity for recombinant MT-CO2?

For His-tagged recombinant MT-CO2, the following purification protocol typically yields high purity:

• Cell lysis under native or denaturing conditions depending on protein solubility

• Affinity chromatography using Ni²⁺-NTA agarose

• Washing with increasing concentrations of imidazole to remove non-specifically bound proteins

• Elution with high imidazole concentration buffer (typically 250-500 mM)

• Buffer exchange to remove imidazole and establish conditions suitable for downstream applications

The typical formulation for purified recombinant MT-CO2 includes buffering at physiological pH (around 7.4) with phosphate buffer, moderate salt concentration (0.5 M NaCl), and stabilizing agents . For recombinant proteins expressed in E. coli, purity levels exceeding 90% are typically achievable as determined by SDS-PAGE under reducing conditions and visualized by Coomassie blue staining .

How can researchers effectively measure MT-CO2 enzyme activity in vitro?

MT-CO2 enzyme activity can be measured through several methods:

• Spectrophotometric assays: The oxidation of reduced cytochrome c can be monitored at 550 nm, where a decrease in absorbance indicates enzyme activity. Based on protocols similar to those used for S. zeamais COX2, UV-spectrophotometer analysis can be employed to determine if recombinant MT-CO2 catalyzes the oxidation of substrate cytochrome c .

• Oxygen consumption assays: Using oxygen electrodes or fluorescence-based oxygen sensors to measure the rate of oxygen consumption during enzyme activity.

• Polarographic methods: These can be used to measure electron transfer rates.

A typical experimental setup based on related studies would include:

• Reaction buffer: 10-50 mM phosphate buffer (pH 7.0-7.4)

• Substrate: 10-100 μM reduced cytochrome c

• Enzyme: 0.1-10 μg purified recombinant MT-CO2

• Temperature: 25-37°C

• Monitoring: Continuous spectrophotometric reading at 550 nm for 3-5 minutes

The activity is typically expressed as μmol of cytochrome c oxidized per minute per mg of enzyme .

What are the recommended protocols for detecting MT-CO2 in tissue samples using immunological methods?

For detecting MT-CO2 in porcine tissue samples, sandwich enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) are the most commonly used methods.

ELISA Protocol:
According to available commercial kit specifications, a sandwich ELISA method provides high sensitivity (approximately 0.054 ng/mL) with a detection range of 0.16-10 ng/mL . The test principle involves:

• Pre-coating microplates with an antibody specific to pig MT-CO2

• Adding standards or samples to appropriate wells

• Adding biotin-conjugated antibody specific to pig MT-CO2

• Adding avidin conjugated to horseradish peroxidase (HRP)

• Adding TMB substrate solution

• Measuring spectrophotometrically at 450 nm ± 10 nm

• Determining concentration by comparing OD values to a standard curve

Immunohistochemistry Protocol:
For formalin-fixed paraffin-embedded (FFPE) tissue samples, IHC can be performed using:

• Antigen retrieval buffer (typically citrate or EDTA-based)

• Blocking buffer to reduce non-specific binding

• Primary antibody (polyclonal anti-MT-CO2)

• Secondary antibody (typically polymer-HRP-goat anti-rabbit)

• Chromogen components for visualization

• Counterstaining reagent

• Mounting media for preservation

These methods provide high specificity and sensitivity for detecting MT-CO2 in various porcine tissue samples, including tissue homogenates, cell lysates, and biological fluids.

How can recombinant MT-CO2 be used to study mitochondrial dysfunction in metabolic disorders?

Recombinant MT-CO2 serves as a valuable tool for understanding mitochondrial dysfunction in metabolic disorders through several approaches:

• In vitro reconstitution studies: Purified recombinant MT-CO2 can be used to reconstitute cytochrome c oxidase activity in vitro, allowing researchers to study how specific mutations or post-translational modifications affect enzyme function.

• Structural studies: The recombinant protein can be used for crystallization trials to determine high-resolution structures, providing insights into the molecular mechanisms of disease-causing mutations.

• Interaction studies: Pull-down assays using His-tagged recombinant MT-CO2 can identify interacting partners and how these interactions are affected in disease states.

• Drug screening: The protein can be used to screen for compounds that might restore or enhance cytochrome c oxidase activity in conditions of mitochondrial dysfunction.

Research has shown that cytochrome c oxidase activity is altered in various metabolic conditions. For example, studies in pigs have demonstrated connections between MT-CO2 expression and fatness traits, suggesting its role in energy metabolism regulation . Understanding these mechanisms could lead to novel therapeutic approaches for metabolic disorders.

What computational modeling approaches best predict MT-CO2 interaction with other respiratory chain components?

Advanced computational modeling approaches for studying MT-CO2 interactions include:

• Molecular docking: This method can predict binding interactions between MT-CO2 and other proteins or small molecules. For example, molecular docking has been used to study the interaction between COX2 and allyl isothiocyanate (AITC), revealing that a sulfur atom of AITC can form a hydrogen bond with specific amino acid residues (e.g., Leu-31) .

• Molecular dynamics simulations: These provide insights into the dynamic behavior of MT-CO2 within the cytochrome c oxidase complex over time, revealing conformational changes during electron transfer.

• Quantum mechanical/molecular mechanical (QM/MM) approaches: These are particularly useful for studying the electron transfer process in the CuA center of MT-CO2.

• Network analysis: This can be used to identify co-expression patterns and regulatory relationships with other genes. Differential expression and co-expression gene network analysis has revealed that cytochrome c oxidase subunits, including MT-CO2, can be downregulated in certain conditions, such as in pigs with high backfat thickness .

These computational approaches can help predict how mutations or post-translational modifications might affect MT-CO2 function and interaction with other components of the respiratory chain.

What strategies can overcome insolubility issues when expressing recombinant MT-CO2?

Membrane proteins like MT-CO2 often face solubility challenges during recombinant expression. Based on established protocols for similar proteins, the following strategies can help overcome insolubility issues:

• Optimization of expression conditions:

  • Lowering induction temperature (16-25°C)

  • Reducing IPTG concentration (0.1-0.5 mM)

  • Using rich media formulations (e.g., Terrific Broth)

  • Extending expression time at lower temperatures

• Fusion tags:

  • N-terminal His-tags have been successfully used for pig MT-CO2

  • Solubility-enhancing fusion partners (MBP, SUMO, or TRX) can improve folding

• Solubilization methods:

  • Extraction using mild detergents (n-dodecyl-β-D-maltoside, digitonin)

  • Inclusion body solubilization with urea (6-8 M) followed by refolding

• Codon optimization:

  • Adjusting codons to match E. coli preference

  • Co-expression with rare tRNAs

• Alternative expression systems:

  • Eukaryotic systems for complex membrane proteins

  • Cell-free expression systems for toxic proteins

For specific cases where standard methods fail, newer approaches such as nanodiscs or amphipols may provide alternatives for maintaining MT-CO2 in a soluble, native-like environment for functional studies.

How can researchers address cross-reactivity concerns when using antibodies against MT-CO2 in immunological assays?

Cross-reactivity is a significant concern in MT-CO2 immunoassays due to sequence conservation across species and similarity with other cytochrome c oxidase subunits. Strategies to address this include:

• Antibody validation:

  • Perform Western blotting using recombinant MT-CO2 and tissue lysates

  • Include appropriate positive and negative controls

  • Test antibodies on tissues from MT-CO2 knockout models if available

• Blocking optimization:

  • Extended blocking times (2-24 hours)

  • Use of alternative blocking agents (5% BSA, commercial blocking reagents)

  • Addition of 0.1-0.5% Triton X-100 or Tween-20 to reduce non-specific binding

• Absorption controls:

  • Pre-absorb antibodies with recombinant proteins of potential cross-reactive antigens

  • Use peptide competition assays to confirm specificity

• Optimization of antibody concentration:

  • Titrate primary antibodies (typical range: 1:200-1:2000)

  • Adjust secondary antibody dilutions accordingly (typical range: 1:400-1:5000)

• Enhanced washing protocols:

  • Increase washing steps (5-7 times)

  • Extended washing times (10-15 minutes per wash)

  • Addition of higher salt concentrations (150-500 mM NaCl)

When possible, using monoclonal antibodies targeting unique epitopes of pig MT-CO2 can significantly reduce cross-reactivity concerns. For ELISA applications, validation data has shown high specificity with inter-assay precision (CV% <10%) and good recovery in various sample matrices (serum: 99%, EDTA plasma: 93%, heparin plasma: 86%) .

How can genetic manipulation of MT-CO2 be used to study its role in cellular bioenergetics?

Genetic manipulation approaches for studying MT-CO2 function include:

• CRISPR/Cas9 gene editing:

  • Introduction of specific mutations to mimic disease states

  • Creation of conditional knockouts for temporal control

  • Gene tagging for visualization and interaction studies

• RNA interference (RNAi):

  • siRNA or shRNA targeting MT-CO2 mRNA

  • Inducible knockdown systems for controlled expression reduction

  • Tissue-specific knockdown using tissue-specific promoters

• Overexpression systems:

  • Viral vectors for stable or transient overexpression

  • Inducible expression systems for controlled upregulation

  • Expression of wild-type vs. mutant variants for comparative studies

• Reporter systems:

  • Fusion of MT-CO2 with fluorescent proteins to study localization

  • Luciferase reporters to monitor transcriptional regulation

  • FRET-based sensors to study protein-protein interactions

Studies have shown that alterations in MT-CO2 expression can significantly impact cellular bioenergetics. For instance, differential expression analysis has revealed that cytochrome c oxidase subunits can be regulated under specific physiological conditions, affecting energy metabolism and adipose deposition in pigs . These techniques allow researchers to investigate how MT-CO2 contributes to mitochondrial function and cellular energy production in both normal and pathological states.

What are the current methodologies for studying the role of MT-CO2 in oxidative stress and apoptosis?

Current methodologies for investigating MT-CO2's role in oxidative stress and apoptosis include:

• Oxidative stress assessment:

  • Measurement of reactive oxygen species (ROS) using fluorescent probes (DCF-DA, MitoSOX)

  • Assessment of lipid peroxidation (TBARS, 4-HNE, MDA levels)

  • Analysis of antioxidant enzyme activities (SOD, catalase, GPx)

  • Protein carbonylation and oxidative damage markers

• Apoptosis detection:

  • Annexin V/PI staining for flow cytometry

  • TUNEL assay for DNA fragmentation

  • Measurement of caspase activities

  • Western blotting for apoptotic markers (cleaved PARP, cytochrome c release)

• Functional assays:

  • Oxygen consumption rate (OCR) measurement using Seahorse XF analyzers

  • Membrane potential assessment using JC-1 or TMRM dyes

  • ATP production assays

  • Mitochondrial morphology assessment using electron microscopy or fluorescence imaging

• Protein interaction studies:

  • Co-immunoprecipitation to identify MT-CO2 interaction with apoptotic regulators

  • Proximity ligation assay for in situ detection of protein interactions

  • Molecular modeling of MT-CO2 interaction with anti-apoptotic proteins

Research indicates that cytochrome c oxidase function is directly linked to cellular responses to oxidative stress. Studies have shown interactions between cytochrome c oxidase subunits (such as COX5A) and anti-apoptotic proteins like Bcl-2, which can affect oxidative stress levels and cell viability . Understanding these interactions provides insights into how MT-CO2 might influence apoptotic pathways and cellular responses to oxidative damage.

How do sequence variations in MT-CO2 across species correlate with functional adaptations?

Comparative analysis of MT-CO2 sequences across species reveals important insights into evolutionary adaptations:

• Sequence conservation and variation:

  • Core functional domains, particularly those involved in electron transfer, show high conservation

  • Species-specific variations often occur in regions interacting with nuclear-encoded subunits

  • Analysis of sequence alignments has shown high sequence identity between pig MT-CO2 and MT-CO2 from other mammalian species

• Adaptive evolution markers:

  • Ratio of non-synonymous to synonymous substitutions (dN/dS) indicates selection pressure

  • Positively selected sites often correlate with environmental adaptations

  • Conserved sites typically represent functionally critical residues

• Structure-function relationships:

  • Mutations in copper-binding regions can alter electron transfer efficiency

  • Variations in protein-protein interaction domains may affect assembly with other subunits

  • Changes in transmembrane regions may influence membrane integration and stability

The giant panda provides an interesting case study, where MT-CO2 has adapted to support the species' unique bamboo-based diet despite its low energy content . Similar comparative studies can reveal how MT-CO2 variations contribute to metabolic adaptations across species with different energy demands, environmental niches, or dietary specializations.

What bioinformatic approaches best identify regulatory elements affecting MT-CO2 expression?

Bioinformatic approaches for identifying regulatory elements affecting MT-CO2 expression include:

• Promoter analysis:

  • Identification of transcription factor binding sites using tools like JASPAR, TRANSFAC

  • Comparative genomics to identify conserved regulatory regions

  • Analysis of CpG islands and methylation patterns

• Epigenetic profiling:

  • ChIP-seq data analysis for histone modifications

  • ATAC-seq for chromatin accessibility

  • DNA methylation patterns from bisulfite sequencing

• RNA regulation analysis:

  • miRNA binding site prediction

  • RNA-binding protein motif identification

  • mRNA stability and secondary structure prediction

• Co-expression network analysis:

  • Weighted Gene Co-expression Network Analysis (WGCNA) to identify genes with similar expression patterns

  • Identification of transcriptional regulators through enrichment analysis

  • Studies have successfully applied WGCNA to identify important DE genes in gene expression profiles associated with MT-CO2 and other cytochrome c oxidase subunits

• Pathway analysis:

  • Gene Ontology (GO) enrichment

  • KEGG pathway mapping

  • Ingenuity Pathway Analysis (IPA) for regulatory networks

These approaches can help researchers understand the complex regulatory mechanisms controlling MT-CO2 expression under different physiological and pathological conditions, providing insights into mitochondrial biogenesis and energy metabolism regulation.

How can recombinant MT-CO2 be utilized in developing diagnostic tools for mitochondrial disorders?

Recombinant MT-CO2 has significant potential for developing diagnostic tools for mitochondrial disorders:

• Antibody production and validation:

  • Generating high-specificity antibodies using purified recombinant MT-CO2

  • Validating antibodies across different sample types and assay conditions

  • Developing standardized immunoassays for clinical use

• Reference standards for quantitative assays:

  • Creating calibration curves for absolute quantification

  • Developing quality control materials for clinical laboratories

  • Establishing reference ranges for MT-CO2 levels in different tissues

• Functional assays:

  • Developing high-throughput screening methods to measure cytochrome c oxidase activity

  • Creating reporter systems to detect mutations or functional defects

  • Establishing patient-derived cell-based assays for personalized diagnostics

• Point-of-care testing development:

  • Integrating recombinant MT-CO2-based detection systems into portable devices

  • Developing rapid diagnostic tests for mitochondrial dysfunction

  • Creating biosensors for continuous monitoring applications

Studies have shown that defects in COX2 can cause mitochondrial complex IV deficiency (MT-C4D) , highlighting the importance of accurate diagnostic tools. By utilizing recombinant MT-CO2 as a standard, researchers can develop more precise and reliable methods for detecting abnormalities in cytochrome c oxidase function, potentially leading to earlier diagnosis and intervention for patients with mitochondrial disorders.

What is the potential of MT-CO2 as a therapeutic target for metabolic and mitochondrial diseases?

MT-CO2 presents several opportunities as a therapeutic target:

• Small molecule modulators:

  • Development of compounds that enhance cytochrome c oxidase activity

  • Identification of molecules that stabilize mutant MT-CO2 proteins

  • Design of allosteric regulators based on structural insights

• Protein replacement strategies:

  • Delivery of recombinant MT-CO2 using nanoparticle or liposomal systems

  • Development of cell-penetrating peptide conjugates for mitochondrial targeting

  • Using modular assembly mechanisms that naturally occur for COX maintenance

• Gene therapy approaches:

  • Development of mitochondrially-targeted gene delivery systems

  • Correction of MT-CO2 mutations using CRISPR/Cas9 or base editing

  • Allotopic expression of engineered MT-CO2 genes from the nucleus

• Metabolic bypass strategies:

  • Identification of alternative electron acceptors

  • Development of synthetic electron transport chains

  • Enhancement of compensatory metabolic pathways