Recombinant Micromys minutus Phosphatidylcholine-sterol acyltransferase (LCAT)

What is Micromys minutus Phosphatidylcholine-sterol Acyltransferase and how does it function in lipid metabolism?

Micromys minutus Phosphatidylcholine-sterol Acyltransferase (LCAT) is an enzyme that catalyzes the transfer of fatty acids from phosphatidylcholine (lecithin) to free cholesterol, forming cholesteryl esters. This enzyme plays a crucial role in the maturation of high-density lipoproteins (HDL) and reverse cholesterol transport. In its native context, LCAT is primarily synthesized in the liver and secreted into the bloodstream, where it associates with lipoproteins. The enzyme has critical functions in lipogenesis, lipometabolism, and specifically cholesterol metabolism, as indicated by its protein function classification .

The catalytic mechanism involves the transfer of the sn-2 acyl group from phosphatidylcholine to the 3-β-hydroxyl group of cholesterol. This reaction is essential for maintaining proper plasma lipoprotein composition and function. Understanding this enzyme from Micromys minutus (harvest mouse) provides comparative insights into LCAT function across species.

What are the structural characteristics of Recombinant Micromys minutus LCAT?

Recombinant Micromys minutus LCAT belongs to the α/β-hydrolase fold enzyme family, characterized by a central β-sheet surrounded by α-helices. While the specific crystal structure of Micromys minutus LCAT has not been fully described in the provided search results, recombinant forms of this protein are available from both E. coli and yeast expression systems (CSB-EP012783MTV and CSB-YP012783MTV, respectively) .

The protein likely contains the canonical active site triad (serine, histidine, and aspartic/glutamic acid) that is characteristic of serine hydrolases. Post-translational modifications, particularly glycosylation, may differ between native LCAT and recombinant versions, especially those expressed in prokaryotic systems like E. coli, potentially affecting enzyme activity and stability.

How does Micromys minutus LCAT compare with LCAT from other species?

Micromys minutus (harvest mouse) LCAT is one of several mammalian LCAT proteins available as recombinant proteins for research. While specific sequence homology data is not provided in the search results, the availability of LCAT from multiple species including human, rat, mouse, rabbit, pig, and chicken suggests that this enzyme is highly conserved across various mammals and vertebrates .

The search results indicate that recombinant LCAT is available from multiple rodent species including Micromys minutus, Tatera kempi gambiana, Eliomys quercinus, and Myodes glareolus, suggesting these may serve as comparative models for researching LCAT function across rodent species . Researchers interested in evolutionary or comparative studies would benefit from analyzing the sequence homology and functional conservation between these different species-specific LCAT proteins.

What expression systems are optimal for producing Recombinant Micromys minutus LCAT?

Based on the available search results, Recombinant Micromys minutus LCAT can be produced in both E. coli (CSB-EP012783MTV) and yeast (CSB-YP012783MTV) expression systems . Each system offers distinct advantages:

E. coli Expression System:

• Advantages: Higher protein yield, faster growth, lower cost, and simpler cultivation requirements

• Limitations: Lacks eukaryotic post-translational modifications, potential for inclusion bodies formation, and possible endotoxin contamination

Yeast Expression System:

• Advantages: Provides eukaryotic post-translational modifications (particularly glycosylation), protein folding machinery similar to higher eukaryotes, and secretion of soluble protein

• Limitations: Lower yield compared to E. coli, longer cultivation time, and more complex media requirements

For functional studies requiring properly folded and post-translationally modified LCAT, the yeast-expressed version (CSB-YP012783MTV) may be preferable, particularly when enzymatic activity is critical. For structural studies or applications where post-translational modifications are less important, the E. coli-expressed version may be sufficient and more economical.

What are the recommended methods for assessing LCAT enzymatic activity?

LCAT activity can be assessed through several methodological approaches:

• Radiometric assays: Using radiolabeled substrates (typically [³H]cholesterol or [¹⁴C]phosphatidylcholine) to measure the formation of cholesteryl esters.

• Fluorescent substrate assays: Utilizing fluorescent cholesterol analogs that change emission properties upon esterification.

• ELISA-based methods: While primarily used for quantification rather than activity, sandwich ELISA techniques (as described in the search results) can be used to quantify LCAT protein levels . The assay principles described include:

  • Pre-coating microplates with LCAT-specific antibodies

  • Binding of LCAT from samples

  • Detection with biotin-conjugated antibodies

  • Signal amplification with avidin-HRP and TMB substrate development

  • Absorbance measurement at 450 nm

• Mass spectrometry-based assays: For detailed characterization of LCAT-generated cholesteryl ester species.

When designing activity assays, researchers should consider physiologically relevant substrates, buffer conditions (pH 7.4, presence of appropriate cofactors), and potential interfering factors in sample matrices.

What protocols are recommended for purifying Recombinant Micromys minutus LCAT?

While specific purification protocols for Micromys minutus LCAT are not detailed in the search results, standard approaches for recombinant protein purification would apply:

For E. coli-expressed LCAT:

• Cell lysis by sonication or mechanical disruption in appropriate buffer (typically PBS with protease inhibitors)

• Clarification of lysate by centrifugation

• Initial capture using affinity chromatography (if His-tagged, use Ni-NTA; if GST-tagged, use glutathione sepharose)

• Intermediate purification using ion exchange chromatography (based on LCAT's isoelectric point)

• Polishing step using size exclusion chromatography

• Buffer exchange to storage buffer (typically PBS with 10-20% glycerol)

For yeast-expressed LCAT:

• Harvesting of culture supernatant (if secreted) or cell lysis

• Ammonium sulfate precipitation to concentrate protein

• Affinity chromatography using anti-LCAT antibodies or tagged purification

• Deglycosylation (if required for specific applications)

• Polishing with size exclusion chromatography

Maintaining enzyme activity during purification is critical, so all steps should be performed at 4°C with appropriate protease inhibitors, and exposure to air-water interfaces should be minimized to prevent protein denaturation.

How can Recombinant Micromys minutus LCAT be used in lipoprotein metabolism studies?

Recombinant Micromys minutus LCAT can be instrumental in studying various aspects of lipoprotein metabolism through the following approaches:

• Reconstituted HDL particle studies: The recombinant enzyme can be used to convert discoidal pre-β HDL to spherical α-HDL in vitro, allowing researchers to study this maturation process.

• Species-comparative lipoprotein metabolism: Using LCAT from different species including Micromys minutus allows researchers to investigate evolutionary adaptations in lipoprotein metabolism across rodents and other mammals.

• Structure-function relationship studies: Site-directed mutagenesis of recombinant LCAT can help identify critical residues involved in substrate binding, catalysis, and interaction with lipoprotein particles.

• Drug discovery platforms: Recombinant LCAT can be used in high-throughput screening assays to identify compounds that modulate its activity, potentially leading to therapeutic interventions for dyslipidemias.

For these applications, researchers should consider using the yeast-expressed version (CSB-YP012783MTV) which likely retains more native-like post-translational modifications important for interactions with lipoprotein components.

What are the critical factors affecting LCAT stability and activity in experimental settings?

Several factors critically affect LCAT stability and activity that researchers should consider:

• Temperature sensitivity: LCAT activity typically decreases significantly above 37°C, and the enzyme should be stored at -80°C for long-term storage or at 4°C for short-term use during experiments.

• Oxidative inactivation: LCAT contains free sulfhydryl groups that are susceptible to oxidation, leading to inactivation. Addition of reducing agents like DTT or β-mercaptoethanol (typically 1-5 mM) can help maintain activity.

• Interfacial denaturation: LCAT is susceptible to denaturation at air-water interfaces. Inclusion of non-ionic detergents (0.01-0.05% Triton X-100) or carrier proteins (0.1-1% BSA) can help stabilize the enzyme.

• Proteolytic degradation: LCAT is sensitive to proteolytic degradation, necessitating the use of protease inhibitor cocktails during isolation and storage.

• Freeze-thaw cycles: Repeated freeze-thaw cycles significantly reduce LCAT activity. As noted in the sample storage guidance for LCAT ELISA kits, repeated freeze-thaw cycles should be avoided .

• Metal ion dependence: LCAT activity can be influenced by certain metal ions, with some reports suggesting that calcium may enhance activity.

These factors should be carefully controlled in experimental designs to ensure reproducible results when working with Recombinant Micromys minutus LCAT.

How can researchers design experiments to compare LCAT function across different species?

To effectively compare LCAT function across species including Micromys minutus, researchers should consider the following experimental design approaches:

• Standardized activity assays: Use identical substrates, concentrations, and reaction conditions when testing LCAT from different species. The enzymatic principles described for LCAT assays can be applied consistently across species-specific variants .

• Substrate preference profiling: Compare the kinetic parameters (Km, Vmax, kcat) of LCAT from different species using various phospholipid and sterol substrates to identify species-specific preferences.

• Lipoprotein interaction studies: Evaluate how LCAT from different species interacts with human, mouse, or rat lipoprotein particles using techniques such as surface plasmon resonance or pull-down assays.

• Cross-species complementation: Test whether Micromys minutus LCAT can functionally replace LCAT in cell models derived from other species.

• Structural comparison: Perform homology modeling based on available crystal structures to identify structural differences that might explain functional divergence between species.

• Environmental sensitivity: Compare how LCAT from different species responds to variables such as pH, temperature, and ionic strength to identify evolutionary adaptations.

When designing these experiments, researchers should account for differences in post-translational modifications between recombinant proteins expressed in E. coli versus yeast systems, as these can significantly impact function and stability.

What are common issues in LCAT activity assays and how can they be resolved?

Researchers frequently encounter several challenges when performing LCAT activity assays:

• Low or no detectable activity:

  • Possible causes: Enzyme denaturation, improper storage, oxidation of critical residues

  • Solutions: Add reducing agents (1-5 mM DTT), use freshly prepared enzyme, optimize buffer conditions (pH 7.2-7.4 is typically optimal)

• High background in radiometric or fluorescent assays:

  • Possible causes: Non-enzymatic hydrolysis of substrates, contaminating esterases

  • Solutions: Include appropriate controls (heat-inactivated enzyme), use enzyme-specific inhibitors to confirm LCAT-specific activity

• Poor reproducibility:

  • Possible causes: Variations in substrate preparation, inconsistent reaction termination

  • Solutions: Standardize substrate preparation methods, use precise timing and consistent stopping methods

• Interfering factors in complex samples:

  • Possible causes: Endogenous inhibitors, competing enzymes, lipoproteins binding the substrate

  • Solutions: Pre-extract or dilute samples, use immunoprecipitation to isolate LCAT before activity measurement

• Substrate solubility issues:

  • Possible causes: Poor dispersion of lipid substrates

  • Solutions: Optimize substrate preparation methods (sonication, extrusion), include appropriate detergents at concentrations below their critical micelle concentration

Following the detailed procedural steps outlined for ELISA-based detection systems, with attention to temperature control and incubation timing, may help address some of these issues .

What quality control measures should be implemented when working with Recombinant Micromys minutus LCAT?

Comprehensive quality control for Recombinant Micromys minutus LCAT should include:

• Purity assessment:

  • SDS-PAGE analysis (expect >90% purity for most applications)

  • Mass spectrometry confirmation of protein identity

  • Endotoxin testing for E. coli-expressed proteins (should be <0.1 EU/μg protein for cell culture applications)

• Activity verification:

  • Specific activity determination (nmol substrate converted/min/mg protein)

  • Comparison to reference standards or previous lots

  • Substrate specificity confirmation

• Stability monitoring:

  • Accelerated stability studies at different temperatures

  • Activity retention after freeze-thaw cycles

  • Long-term storage stability assessment

• Structural integrity:

  • Circular dichroism spectroscopy to assess secondary structure

  • Dynamic light scattering to detect aggregation

  • Thermal shift assays to determine stability

• Batch consistency:

  • Lot-to-lot comparison of activity and purity

  • Standardization against reference materials

  • Detailed certificate of analysis documentation

Similar quality control principles are applied to LCAT ELISA kits, which demonstrate intra-assay precision with CV% <8% and inter-assay precision with CV% <10%, indicating the importance of consistency in LCAT-related assays .

How can researchers verify the specificity of their LCAT activity measurements?

Verifying the specificity of LCAT activity measurements is crucial for accurate research outcomes. Researchers should implement the following approaches:

• Inhibitor studies:

  • Use known LCAT-specific inhibitors to confirm that observed activity is indeed from LCAT

  • Compare inhibition profiles against other lipid-metabolizing enzymes

• Substrate specificity analysis:

  • Test activity with LCAT-specific substrates versus substrates for related enzymes

  • Analyze product formation using mass spectrometry to confirm expected cholesteryl ester species

• Immunodepletion experiments:

  • Remove LCAT from samples using specific antibodies and confirm loss of activity

  • The high specificity noted in the ELISA kit (no significant cross-reactivity or interference between Rat LCAT and analogues) suggests species-specific antibodies are available

• Control experiments:

  • Use heat-inactivated enzyme as a negative control

  • Include purified LCAT from a reference source as a positive control

  • Test activity in the presence of EDTA (which may inhibit LCAT by chelating necessary metal cofactors)

• Genetic validation:

  • Use LCAT-knockout cell lines or tissues as negative controls

  • Perform rescue experiments with recombinant LCAT to restore activity

• Product verification:

  • Use chromatographic methods (HPLC, TLC) to separate and identify specific cholesteryl ester products

  • Employ mass spectrometry to confirm the molecular species of products formed

By implementing these specificity controls, researchers can ensure that their observations truly reflect LCAT activity rather than that of other esterases or non-enzymatic reactions.

What statistical approaches are most appropriate for analyzing LCAT activity data?

When analyzing LCAT activity data, researchers should consider the following statistical approaches:

• For dose-response studies:

  • Nonlinear regression analysis to determine EC50 or IC50 values

  • Four-parameter logistic (4-PL) curve fitting, as recommended for ELISA standard curves

  • Analysis of curve parameters (Hill slope, maximum response) for mechanistic insights

• For comparative studies:

  • ANOVA with appropriate post-hoc tests for comparing multiple groups

  • t-tests (paired or unpaired) for two-group comparisons

  • Consider log-transformation of data if variance increases with mean values

• For kinetic analyses:

  • Michaelis-Menten or allosteric models fitted using nonlinear regression

  • Lineweaver-Burk, Eadie-Hofstee, or Hanes-Woolf plots for visualization of kinetic parameters

  • Global fitting approaches for complex kinetic mechanisms

• For assay validation:

  • Calculation of Z' factor to assess assay quality

  • Determination of intra-assay (CV% <8%) and inter-assay (CV% <10%) precision as noted in the ELISA kit specifications

  • Assessment of limits of detection and quantification

• For time-course experiments:

  • Area under the curve (AUC) analysis

  • First-order or more complex kinetic modeling

  • Repeated measures ANOVA for statistical comparison

• For processing raw data:

  • Subtraction of background readings (as recommended in the ELISA protocol)

  • Normalization to controls or reference standards

  • Correction for dilution factors when necessary

The appropriate statistical approach should be selected based on the specific experimental design and research question, with attention to assumptions of each statistical test.

How should researchers interpret species differences in LCAT activity and structure?

When interpreting species differences in LCAT activity and structure between Micromys minutus and other species, researchers should consider:

• Evolutionary context:

  • Phylogenetic relationships between species (e.g., Micromys minutus versus other rodents)

  • Selective pressures that might drive functional divergence

  • Conservation of catalytic residues versus variation in regulatory domains

• Physiological relevance:

  • Correlation with species differences in lipoprotein profiles

  • Relationship to dietary patterns (herbivore, omnivore, carnivore)

  • Potential adaptation to environmental factors

• Structural basis for functional differences:

  • Amino acid substitutions in substrate binding regions

  • Alterations in surface properties affecting lipoprotein interaction

  • Differences in post-translational modifications between species

• Catalytic parameters:

  • Differences in substrate specificity may reflect adaptation to species-specific lipid compositions

  • Variations in catalytic efficiency (kcat/Km) may indicate evolutionary optimization

  • Temperature optima may correlate with body temperature differences

• Translational implications:

  • Relevance of Micromys minutus as a model for human LCAT function

  • Potential impact on cross-species extrapolation of LCAT-targeting therapies

  • Insights into structure-function relationships that could inform drug design

Researchers should be cautious about over-interpreting modest species differences, as these may reflect neutral evolution rather than functional adaptation. Convergent studies using multiple approaches (structural, biochemical, genetic) provide the strongest evidence for functionally significant species differences.

What are the key considerations when comparing recombinant LCAT data with endogenous LCAT studies?

When comparing results obtained with Recombinant Micromys minutus LCAT to studies of endogenous LCAT, researchers should consider several critical factors:

• Expression system effects:

  • E. coli-expressed LCAT (CSB-EP012783MTV) lacks mammalian post-translational modifications

  • Yeast-expressed LCAT (CSB-YP012783MTV) has different glycosylation patterns than mammalian cells

  • These differences may affect activity, stability, and interactions with other proteins

• Structural considerations:

  • Presence or absence of purification tags (His, GST, etc.)

  • Potential differences in folding between recombinant and native proteins

  • Oligomerization state may differ between recombinant and endogenous LCAT

• Experimental context:

  • Endogenous LCAT functions in a complex environment with natural cofactors and activators

  • Recombinant systems may lack physiological regulators of LCAT activity

  • Buffer conditions for recombinant protein assays may not perfectly mimic physiological conditions

• Sample processing effects:

  • Methods used to collect and process samples for endogenous LCAT measurement (as described for serum/plasma collection)

  • Storage conditions may affect both recombinant and endogenous LCAT differently

  • Freeze-thaw cycles can impact activity measurements for both systems

• Quantification methods:

  • Absolute amounts of recombinant versus endogenous LCAT may be measured differently

  • ELISA measurements may detect total LCAT rather than active enzyme

  • Activity assays may be more relevant than concentration measurements for functional comparisons

To bridge the gap between recombinant and endogenous systems, researchers should consider parallel experiments, careful standardization, and validation of findings across multiple experimental approaches.

How can Recombinant Micromys minutus LCAT be used in drug discovery research?

Recombinant Micromys minutus LCAT offers several valuable applications in drug discovery:

• High-throughput screening platforms:

  • Development of miniaturized LCAT activity assays for compound library screening

  • Identification of species-selective inhibitors or activators

  • Counter-screening using LCAT from multiple species to assess selectivity

• Structure-based drug design:

  • Using purified recombinant LCAT for crystallization and structure determination

  • Molecular docking studies to identify potential binding pockets

  • Fragment-based screening approaches using biophysical methods

• Target validation studies:

  • Comparison of drug effects on LCAT from different species to validate translational relevance

  • In vitro assessment of compound effects on LCAT before moving to in vivo studies

  • Evaluation of structure-activity relationships for lead optimization

• Biomarker development:

  • Using recombinant LCAT as a standard in assays measuring LCAT concentration or activity

  • Development of competitive assays for measuring auto-antibodies against LCAT

  • Establishing reference ranges based on standardized recombinant protein

• Therapeutic protein development:

  • Engineering recombinant LCAT with enhanced stability or activity

  • Developing enzyme replacement therapies for LCAT deficiency

  • Creating fusion proteins to improve pharmacokinetic properties

The availability of both E. coli and yeast expression systems provides flexibility for different drug discovery applications depending on whether native-like post-translational modifications are critical for the specific screening approach .

What emerging techniques are advancing LCAT research beyond traditional methodologies?

Several cutting-edge techniques are advancing LCAT research beyond traditional biochemical assays:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize LCAT interactions with lipoprotein particles

  • Single-molecule tracking to observe LCAT dynamics on lipoprotein surfaces

  • Cryo-electron microscopy for high-resolution structural studies of LCAT-lipoprotein complexes

• 'Omics' approaches:

  • Lipidomics to comprehensively profile LCAT-generated cholesteryl ester species

  • Proteomics to identify LCAT-interacting proteins in different physiological contexts

  • Transcriptomics to understand regulatory networks controlling LCAT expression

• Computational methods:

  • Molecular dynamics simulations to study LCAT conformational changes during catalysis

  • Systems biology modeling of LCAT's role in lipoprotein metabolism

  • Machine learning approaches to predict LCAT substrates and inhibitors

• Genetic engineering:

  • CRISPR/Cas9-mediated generation of species-specific LCAT mutations

  • Creation of humanized LCAT mice for improved translational research

  • Directed evolution to develop LCAT variants with enhanced properties

• Microfluidic technologies:

  • Organ-on-a-chip models incorporating LCAT function

  • Droplet-based high-throughput enzyme assays

  • Artificial lipoprotein particles for studying LCAT in defined contexts

• Biosensor development:

  • FRET-based sensors to monitor LCAT activity in real-time

  • Surface plasmon resonance for studying LCAT-substrate interactions

  • Electrochemical detection of LCAT activity for point-of-care diagnostics

These emerging techniques complement traditional methodologies like the ELISA-based detection systems described in the search results , enabling more detailed mechanistic studies and expanding potential applications.

What are the potential applications of comparative LCAT studies across rodent species including Micromys minutus?

Comparative studies of LCAT across rodent species including Micromys minutus offer several valuable research opportunities:

• Evolutionary insights:

  • Understanding the selective pressures that have shaped LCAT function across rodent lineages

  • Identifying conserved functional domains versus regions that have undergone adaptive evolution

  • Correlating LCAT properties with ecological niches and dietary adaptations

• Biomedical model development:

  • Evaluating the suitability of different rodent species as models for human LCAT function

  • Identifying species with LCAT properties most similar to human for translational research

  • Developing specialized rodent models for specific aspects of lipid metabolism

• Structure-function relationships:

  • Using natural variation across species to identify residues critical for LCAT function

  • Naturalistic mutagenesis studies comparing functional consequences of species-specific residues

  • Understanding the molecular basis for differences in substrate preference or catalytic efficiency

• Physiological adaptation studies:

  • Correlating LCAT properties with plasma lipoprotein profiles across species

  • Investigating relationships between LCAT function and hibernation in species like Eliomys quercinus

  • Examining LCAT's role in adaptation to different environmental temperatures

• Biotechnological applications:

  • Developing chimeric LCAT proteins with optimized properties

  • Identifying naturally evolved LCAT variants with enhanced stability or activity

  • Creating species-selective inhibitors for research tools

With recombinant LCAT available from multiple rodent species including Micromys minutus, Tatera kempi gambiana, Eliomys quercinus, and Myodes glareolus , researchers can conduct systematic comparative studies to address these questions.