Recombinant Bovine Acetolactate synthase-like protein (ILVBL)

What is Bovine Acetolactate Synthase-Like Protein (ILVBL) and what is its basic structure?

Bovine Acetolactate Synthase-Like Protein (ILVBL) is an important enzyme involved in the biosynthesis of branched-chain amino acids, playing a critical role in cellular metabolism and protein synthesis . The full-length bovine ILVBL protein consists of 632 amino acids and is classified as a membrane single-pass membrane protein . The protein is encoded by the ILVBL gene located on chromosome 19p13.1 in humans, while its bovine counterpart has been fully characterized and sequenced .

The protein shows significant homology with several thiamine pyrophosphate-binding proteins found in bacteria, yeast, and plants. Particularly, it shares highest homology with two bacterial enzymes: the B isozyme of the large catalytic subunit of Escherichia coli acetohydroxy-acid synthase (AHAS) and the oxalyl-CoA decarboxylase of Oxalobacter formigenes .

What is the primary cellular function of ILVBL?

ILVBL primarily functions in metabolic pathways related to branched-chain amino acid biosynthesis and pyruvate metabolism . As an enzyme that shares homology with thiamine pyrophosphate-binding proteins, it likely plays a role in catalyzing reactions that are essential for cellular energy production and protein synthesis . The protein's membrane localization suggests it may also be involved in signaling pathways or transport functions across cellular membranes .

Its involvement in branched-chain amino acid metabolism is particularly significant, as dysregulation of these pathways has been implicated in various metabolic disorders, including maple syrup urine disease . This makes ILVBL a valuable biomarker for studying these conditions and identifying potential therapeutic targets.

How does recombinant ILVBL differ from native ILVBL in terms of experimental applications?

Recombinant ILVBL, particularly those expressed with tags such as His-tags, offers several experimental advantages over native ILVBL. The recombinant version allows for easier purification through affinity chromatography using the attached tag, enabling researchers to obtain higher purity samples for structural and functional studies .

When expressed in systems like E. coli, recombinant ILVBL can be produced in larger quantities than would be possible through extraction from native bovine tissues . This abundance facilitates a wider range of experimental applications, including crystallography, enzymatic assays, and protein-protein interaction studies.

What are the optimal conditions for expressing and purifying recombinant bovine ILVBL protein?

For optimal expression of recombinant bovine ILVBL, E. coli has proven to be an effective host system . When designing expression constructs, researchers should consider using an N-terminal His-tag to facilitate purification while minimizing interference with the protein's C-terminal functional domains .

The expression should be carefully optimized for temperature, inducer concentration, and duration to maximize protein yield while avoiding inclusion body formation. Typically, lower temperatures (16-25°C) and longer induction times produce better results for complex proteins like ILVBL.

For purification, a multi-step approach is recommended:

• Initial capture using immobilized metal affinity chromatography (IMAC) leveraging the His-tag

• Intermediate purification using ion-exchange chromatography

• Final polishing step with size-exclusion chromatography to ensure homogeneity

Protein stability should be maintained throughout purification by including appropriate buffers containing stabilizing agents such as glycerol (10-15%) and potentially thiamine pyrophosphate, given ILVBL's predicted binding affinity for this cofactor .

What are the recommended approaches for quantifying ILVBL in biological samples?

Several approaches can be used for quantifying ILVBL in biological samples, with ELISA being one of the most sensitive and specific methods available. The Bovine Acetolactate Synthase-Like Protein ELISA Kit provides exceptional sensitivity and specificity for detecting ILVBL in bovine serum, plasma, and cell culture supernatants .

For gene expression analysis, reverse transcription quantitative PCR (RT-qPCR) offers an accurate and sensitive method for measuring ILVBL mRNA levels . When designing qPCR assays for ILVBL, researchers should:

• Carefully examine transcript variants and exon organization using databases like Ensembl

• Evaluate SNP positioning to avoid issues with primer and probe annealing

• Ensure primer and probe sequences are specific to ILVBL using BLAST analysis

• Include appropriate positive and negative controls and reference genes

• Develop a robust standard curve for accurate quantification

For protein-level detection in tissues or cells, immunohistochemistry or immunofluorescence using validated antibodies against ILVBL can provide spatial information about its expression patterns.

How can researchers effectively design gene expression studies for ILVBL?

Effective gene expression studies for ILVBL require meticulous experimental design following the MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) to ensure quality and reproducibility . Key considerations include:

• Comprehensive knowledge of the ILVBL gene structure, including awareness of transcript variants, to design appropriate primers that capture all relevant isoforms or specifically target certain variants of interest

• Management of SNP positioning, particularly important for ILVBL given its polymorphic nature and association with conditions like AERD

• Ensuring primer and probe specificity through careful sequence analysis using BLAST or similar tools to prevent cross-reactivity with other genes

• Inclusion of appropriate controls:

  • Positive controls to establish baseline expression levels

  • Negative controls to identify potential contamination

  • No-reverse transcriptase controls to detect genomic DNA contamination

  • Proper reference genes selected based on expression stability across the experimental conditions

• Complete removal of RNA from cDNA samples, which is essential for obtaining accurate cDNA content used for data normalization

For more complex analyses such as examining ILVBL expression across different tissues or in response to various stimuli, researchers should consider whether bulk analysis or single-cell approaches are more appropriate for their specific research questions.

What is the relationship between ILVBL gene polymorphisms and aspirin-exacerbated respiratory disease (AERD)?

Studies have established a significant association between ILVBL gene polymorphisms and aspirin-exacerbated respiratory disease (AERD) . A comprehensive analysis of nine single nucleotide polymorphisms (SNPs) with minor allele frequencies above 0.05 in the region from 2 kb upstream to 0.5 kb downstream of ILVBL revealed that seven of these SNPs were significantly associated with the risk for AERD after correction for multiple comparisons .

In the codominant model, five SNPs comprising block2 (rs2240299, rs7507755, rs1468198, rs2074261, and rs13301) showed significant associations with the risk for AERD (corrected P = 0.001–0.004, OR = 0.59–0.64) . Particularly noteworthy, rs1468198 was significantly associated with the percent decline in FEV1 (forced expiratory volume in one second) during oral aspirin challenge tests after correction for multiple comparisons (corrected P = 0.033) .

The linkage disequilibrium analysis revealed that the ILVBL gene can be parsed into two LD blocks (BLs) with four major haplotypes (frequency > 0.01) for each block . This genetic association suggests potential functional implications of ILVBL in the pathophysiology of AERD, possibly through its role in metabolic pathways that intersect with aspirin sensitivity or arachidonic acid metabolism.

How might ILVBL contribute to metabolic disorders such as maple syrup urine disease?

ILVBL's involvement in branched-chain amino acid metabolism suggests a potential role in metabolic disorders like maple syrup urine disease (MSUD) . MSUD is characterized by defects in the catabolism of branched-chain amino acids (leucine, isoleucine, and valine), leading to their accumulation in body fluids along with their corresponding α-keto acids.

While direct evidence linking ILVBL dysregulation to MSUD is still emerging, several mechanistic hypotheses can be proposed based on ILVBL's predicted function:

• ILVBL may function in parallel or complementary pathways to the branched-chain α-keto acid dehydrogenase complex (BCKDC), which is typically defective in MSUD

• Alterations in ILVBL activity could potentially influence the flux through branched-chain amino acid metabolic pathways, either exacerbating or compensating for defects in BCKDC

• As ILVBL shares homology with acetohydroxy-acid synthase (AHAS), which catalyzes the first step in branched-chain amino acid biosynthesis in microorganisms, it may play a regulatory role in mammalian branched-chain amino acid homeostasis

Researchers investigating this connection should consider analyzing ILVBL expression and activity in MSUD models and patient samples, as well as exploring potential genetic associations between ILVBL variants and MSUD subtypes or severity.

What experimental approaches can elucidate the molecular interaction between ILVBL and thiamine pyrophosphate-dependent pathways?

Given ILVBL's homology with thiamine pyrophosphate-binding proteins , several experimental approaches can be employed to investigate this molecular interaction:

• Binding affinity studies: Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can quantitatively measure the binding affinity and kinetics between purified recombinant ILVBL and thiamine pyrophosphate.

• Structural analysis: X-ray crystallography or cryo-electron microscopy of ILVBL in complex with thiamine pyrophosphate can reveal the precise binding site and conformational changes associated with cofactor binding.

• Functional enzymatic assays: Developing specific activity assays for ILVBL based on its predicted function in branched-chain amino acid or pyruvate metabolism, and testing how thiamine pyrophosphate concentration affects enzymatic activity.

• Site-directed mutagenesis: Identifying and mutating predicted thiamine pyrophosphate-binding residues in ILVBL to confirm their importance for cofactor binding and enzymatic activity.

• Cellular studies: Examining how thiamine deficiency or supplementation affects ILVBL expression, localization, and function in cell culture models.

• Metabolomic analysis: Comparing metabolite profiles in systems with normal versus altered ILVBL expression to identify specific metabolic pathways affected, particularly those known to involve thiamine pyrophosphate-dependent enzymes.

These approaches would provide comprehensive insights into how ILVBL functions within thiamine pyrophosphate-dependent metabolic networks and how disruptions might contribute to disease states.

How can ILVBL serve as a biomarker for respiratory and metabolic conditions?

ILVBL holds significant potential as a biomarker for various conditions, particularly for aspirin-exacerbated respiratory disease (AERD) and potentially for metabolic disorders like maple syrup urine disease .

For AERD, specific SNPs in the ILVBL gene have shown strong associations with both disease risk and phenotype severity, particularly regarding the decline in lung function following aspirin challenge . The consistent association of multiple SNPs within the gene suggests that genetic testing for these variants could help identify patients at risk for AERD before administering aspirin or other NSAIDs. This would be particularly valuable in asthmatic patients where aspirin sensitivity may not be apparent until adverse reactions occur.

A quantitative assessment approach using techniques like ELISA can precisely measure ILVBL protein levels in biological samples . Changes in ILVBL concentration might correlate with disease progression or treatment response, particularly in conditions where branched-chain amino acid metabolism is dysregulated.

For effective biomarker application, researchers should develop standardized protocols for sample collection, processing, and analysis, while establishing reference ranges for ILVBL levels in healthy individuals and those with specific conditions of interest.

What are the most effective experimental controls when studying ILVBL in different cellular contexts?

When studying ILVBL across different cellular contexts, implementing appropriate controls is crucial for data reliability and interpretation:

• Positive controls: Include samples known to express ILVBL at detectable levels, such as tissues or cell lines with confirmed high expression (e.g., certain metabolically active bovine tissues).

• Negative controls: Use samples where ILVBL expression is expected to be absent or use ILVBL knockout/knockdown systems generated through CRISPR-Cas9 or RNAi technology.

• Reference gene selection: For gene expression studies, carefully select reference genes that maintain stable expression across the experimental conditions being tested. Common references like GAPDH or β-actin may not be suitable in all contexts, particularly when studying metabolic genes like ILVBL .

• Isotype controls: For antibody-based detection methods, include appropriate isotype controls to distinguish specific from non-specific binding.

• Cellular fractionation controls: When studying the subcellular localization of ILVBL (a membrane protein), include markers for different cellular compartments to verify fractionation quality.

• Recombinant protein standards: Use purified recombinant ILVBL protein as a standard for absolute quantification and to validate detection methods .

• Wild-type vs. mutant comparisons: When studying specific ILVBL variants associated with disease (e.g., SNPs associated with AERD), include both wild-type and mutant versions in expression or functional studies to directly compare their properties .

How can researchers address contradictory findings in ILVBL association studies?

Addressing contradictory findings in ILVBL association studies requires a systematic approach to identify sources of variation and resolve discrepancies:

• Population stratification analysis: Carefully analyze the ethnic background of study populations, as SNP frequencies and linkage disequilibrium patterns can vary significantly between different ethnic groups . Restrict analysis to homogeneous populations or implement appropriate statistical corrections for population stratification.

• Phenotype definition standardization: Ensure consistent definition of phenotypes across studies, particularly for complex conditions like AERD where diagnostic criteria might vary.

• Sample size considerations: Evaluate whether contradictory findings might result from underpowered studies with insufficient sample sizes. Consider performing meta-analyses combining data from multiple studies to increase statistical power.

• Genotyping methodology assessment: Compare genotyping methods used across studies to identify potential technical sources of variation. Different platforms or quality control thresholds could lead to discrepant results.

• Environmental factor analysis: Investigate whether environmental factors or gene-environment interactions could explain different associations observed across studies.

• Functional validation: Move beyond association studies to functional validation of identified variants, examining how they affect ILVBL expression, protein function, or relevant metabolic pathways.

• Replication studies: Design dedicated replication studies with appropriate power calculations based on effect sizes observed in initial studies, ensuring similar methodology and population characteristics.

By systematically addressing these factors, researchers can resolve contradictions and build a more coherent understanding of ILVBL's role in health and disease.

What novel technologies could advance the understanding of ILVBL's molecular function?

Several cutting-edge technologies could significantly advance our understanding of ILVBL's molecular function:

• CRISPR-Cas9 genome editing: Creating precise knockouts or introducing specific SNPs associated with AERD into cellular or animal models could help establish causal relationships between ILVBL variants and disease phenotypes .

• Single-cell transcriptomics: This technology can reveal cell-type-specific expression patterns of ILVBL and how they change in different physiological or pathological conditions, providing insights beyond what bulk RNA analysis can offer .

• Cryo-electron microscopy: High-resolution structural analysis of ILVBL alone or in complex with potential binding partners could reveal crucial insights into its functional mechanisms and how disease-associated mutations might disrupt these functions.

• Protein interactomics: Techniques like BioID, proximity labeling, or co-immunoprecipitation coupled with mass spectrometry can identify ILVBL's protein interaction network, revealing potential functional associates and signaling pathways.

• Metabolomics: Untargeted and targeted metabolomic approaches can identify specific metabolites affected by ILVBL activity or dysfunction, helping to define its precise role in cellular metabolism.

• Organoid models: Developing respiratory or metabolic tissue organoids with different ILVBL variants could provide physiologically relevant models to study its function in a three-dimensional tissue context.

• Proteomics with post-translational modification analysis: Investigating how post-translational modifications regulate ILVBL activity could reveal additional regulatory mechanisms and intervention points.

These technologies, especially when used in combination, could provide unprecedented insights into ILVBL biology and its role in disease.

What interdisciplinary approaches might yield new insights into ILVBL biology?

Interdisciplinary approaches combining diverse scientific fields could uncover novel aspects of ILVBL biology:

• Computational biology and machine learning: Applying sophisticated algorithms to predict ILVBL function, identify potential binding partners, or discover cryptic functional domains could generate new hypotheses for experimental validation.

• Systems biology: Integrating transcriptomic, proteomic, and metabolomic data to model how ILVBL functions within broader biological networks could reveal emergent properties not apparent from studying isolated components.

• Evolutionary biology: Comparative genomic analyses across species could illuminate the evolutionary history of ILVBL and how its function may have adapted in different organisms, potentially revealing conserved functional domains.

• Immunology and inflammation research: Given ILVBL's association with AERD , exploring its potential role in immune and inflammatory pathways could uncover unexpected functions beyond metabolism.

• Chemical biology: Developing small molecule modulators of ILVBL activity could provide valuable tools for studying its function and potential therapeutic leads for associated disorders.

• Microbiome research: Investigating potential interactions between host ILVBL and microbial metabolism, particularly in respiratory or gut microbiomes where aspirin sensitivity or metabolic disorders manifest.

• Clinical research: Correlating ILVBL genetic variants or expression levels with detailed clinical phenotypes in large patient cohorts could reveal subtle associations not captured in smaller studies.

These interdisciplinary approaches could break through conventional research silos and generate transformative insights into ILVBL biology.