Recombinant Chicken Asparagine synthetase [glutamine-hydrolyzing] (ASNS), partial

What is the basic structure and function of chicken ASNS?

Chicken asparagine synthetase, like its human counterpart, is an aminotransferase responsible for biosynthesizing asparagine using aspartic acid and glutamine as substrates . Based on modeling of the human enzyme, ASNS likely functions as a homodimer where each monomer consists of two functional domains: an N-terminal domain containing the glutamine-binding pocket and a C-terminal domain housing the ATP-binding site . The reaction catalyzed by ASNS not only produces asparagine but also impacts glutamine, aspartate, and glutamate homeostasis .

The enzymatic reaction involves an ATP-dependent amidotransferase mechanism that transfers the amide nitrogen from glutamine to aspartate, producing asparagine and glutamate. This reaction can be represented as:

ATP + L-aspartate + L-glutamine → AMP + PPi + L-asparagine + L-glutamate

How does chicken ASNS expression vary across different chicken breeds and tissues?

Research indicates that ASNS expression in chicken muscles mirrors trends in muscle development parameters. The mRNA expression pattern of ASNS correlates with muscle fiber cross-sectional area, average daily weight gain, and muscle weight across different breeds . ASNS is highly expressed in fast-growing broilers compared to slower-growing breeds, suggesting its importance in regulating muscle development .

While tissue-specific expression data for chicken ASNS is limited in the provided search results, studies on human ASNS show that it is ubiquitously expressed at low levels in many organs but particularly high in the pancreas . A similar tissue distribution pattern might be expected in chickens, with potential enrichment in muscle tissues based on its role in muscle development.

What are the optimal conditions for expressing recombinant chicken ASNS?

While the search results don't provide specific conditions for chicken ASNS expression, insights can be drawn from human ASNS expression studies. Temperature is a critical factor for producing soluble and active recombinant ASNS. Expression at lower temperatures (21-30°C) favors the production of soluble enzyme, whereas higher temperatures (37°C) may yield insoluble protein .

For optimal expression of recombinant ASNS:

• Consider using a baculovirus-based expression system, which has been successful for human ASNS

• Maintain lower incubation temperatures (21-30°C) during expression

• Use C-terminal tagging strategies, which have been shown to preserve enzymatic activity

• Include appropriate cofactors such as ATP in purification and storage buffers to maintain enzyme stability

What assays can be used to measure chicken ASNS activity?

ASNS activity can be measured through several methodological approaches:

• Phosphate Release Assay: ASNS activity can be quantified by measuring the release of inorganic phosphate during the ATP-dependent reaction. The specific activity can be calculated using the formula:

$\text{Specific Activity (pmol/min/μg)} = \frac{\text{Phosphate released (nmol)} \times \text{1000 pmol/nmol}}{\text{Incubation time (min)} \times \text{amount of enzyme (μg)} \times \text{2}}$

• Glutamate Production Assay: Since the ASNS reaction produces glutamate, coupling with glutamate dehydrogenase can allow indirect measurement of ASNS activity through NAD(P)H consumption.

• Asparagine Production Measurement: Direct quantification of asparagine production using HPLC or LC-MS/MS methods.

Standard reaction conditions typically include:

• ASNS enzyme: 0.4 μg per reaction

• ATP: 1 mM

• L-Aspartic Acid: 4 mM

• L-Glutamine: 20 mM

How does ASNS affect chicken skeletal muscle satellite cell (SMSC) proliferation and differentiation?

ASNS plays a dual regulatory role in chicken muscle development by affecting both proliferation and differentiation of skeletal muscle satellite cells:

Effect on Proliferation:

• ASNS knockdown inhibits SMSC proliferation

• ASNS overexpression enhances SMSC proliferation

Effect on Differentiation:

• ASNS attenuates SMSC differentiation

• This inhibitory effect occurs through activation of the AMPK pathway

• 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) treatment suppresses cell differentiation induced by siRNA-ASNS

These findings suggest that ASNS functions as a molecular switch that promotes proliferation while inhibiting differentiation in chicken SMSCs, potentially regulating the balance between muscle growth and development.

What molecular pathways are affected by ASNS overexpression in chicken muscle cells?

RNA-Seq analysis of chicken SMSCs overexpressing ASNS identified 1,968 differentially expressed genes (DEGs) during differentiation . These DEGs were involved in multiple important signaling pathways:

• JAK-STAT signaling pathway

• Tumor necrosis factor signaling pathway

• Toll-like receptor signaling pathway

• PI3K-Akt signaling pathway

Gene Ontology (GO) enrichment analysis revealed that these DEGs primarily participated in:

• 8 biological processes

• 8 cellular components

• 4 molecular functions

The AMPK pathway appears to be a key mediator of ASNS effects on muscle cell differentiation. Activation of this pathway by ASNS inhibits differentiation, while inhibition of AMPK can rescue the differentiation phenotype in ASNS-overexpressing cells .

How can one design CRISPR/Cas9 experiments to study chicken ASNS function?

CRISPR/Cas9 genome editing can be effectively applied to study chicken ASNS function using approaches similar to those described for RAG1 knockout in chickens:

• sgRNA Design: Design guide RNAs targeting exonic regions of the chicken ASNS gene. Multiple sgRNAs should be tested to identify those with highest editing efficiency.

• Delivery System: For in vitro studies, transfection of chicken primordial germ cells (PGCs) or cell lines with CRISPR/Cas9 and donor plasmids can be performed using Lipofectamine 2000 reagent (6 μl reagent with 2 μg each of CRISPR and donor plasmids) .

• Selection Strategy: Include selection markers such as neomycin resistance genes or fluorescent reporters (like tdTomato) in donor plasmids .

• Verification of Editing: Confirm editing through:

  • PCR amplification of the targeted region

  • Sequencing of the junction regions

  • Expression analysis via RT-PCR and Western blot

  • Functional assays measuring asparagine synthesis

• Phenotypic Analysis: For ASNS knockout or knockin chickens, analyze:

  • Muscle development parameters

  • Skeletal muscle satellite cell proliferation and differentiation

  • Amino acid metabolism in various tissues

How does chicken ASNS compare structurally and functionally to human and other mammalian ASNS?

While specific structural comparisons between chicken and human ASNS are not detailed in the search results, several functional and comparative aspects can be highlighted:

Structural Comparison:
Human ASNS is a cytosolic, 65 kDa ATP-dependent homodimer with an N-terminal domain containing the glutamine-binding pocket and a C-terminal domain housing the ATP-binding site . Chicken ASNS likely shares similar domain organization given the conserved function.

Functional Comparison:

• Both human and chicken ASNS catalyze the ATP-dependent synthesis of asparagine from aspartate using glutamine as an amide donor.

• Human ASNS is associated with asparaginase resistance in acute lymphoblastic leukemia (ALL) , while chicken ASNS has been linked to muscle development regulation .

• Chicken ASNS promotes SMSC proliferation while inhibiting differentiation through AMPK pathway activation . This tissue-specific regulatory role may differ in humans.

What are the challenges in producing sufficient quantities of active recombinant chicken ASNS for structural studies?

Production of active recombinant ASNS presents several challenges that researchers should address:

• Temperature Sensitivity: Expression of human ASNS is highly temperature-dependent, with no soluble enzyme produced at 37°C, while lower temperatures (21-30°C) favor soluble protein production . Similar temperature sensitivity likely applies to chicken ASNS.

• Protein Folding and Solubility: ASNS contains multiple domains that must fold correctly for enzyme activity. Expression systems must be optimized to ensure proper folding.

• Expression System Selection:

  • E. coli systems may produce improperly folded protein at higher temperatures

  • Baculovirus-based expression systems have been successful for human ASNS and may be appropriate for chicken ASNS

  • Mammalian expression systems might provide better post-translational processing

• Purification Strategy: C-terminal tagging has been successful for human ASNS , enabling purification without compromising activity. Similar strategies should be considered for chicken ASNS.

• Maintaining Enzyme Activity: ASNS requires ATP for activity, and storage conditions must preserve the active conformation of the enzyme. Including appropriate stabilizers and cofactors in storage buffers is essential.

A successful protocol for human ASNS involved a baculovirus-based expression system with C-terminal tagging, yielding multi-milligram quantities of correctly processed, catalytically active enzyme . This approach could be adapted for chicken ASNS.

How can one optimize RT-PCR protocols for studying chicken ASNS expression?

For reliable quantification of chicken ASNS expression through RT-PCR:

• Primer Design:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Target conserved regions of the ASNS transcript

  • Verify primer specificity through BLAST analysis against the chicken genome

  • Optimal primer length: 18-25 nucleotides with GC content of 40-60%

• RNA Extraction and Quality Control:

  • Extract total RNA using commercial kits optimized for chicken tissues

  • Verify RNA integrity (RIN > 8) before cDNA synthesis

  • Treat samples with DNase to eliminate genomic DNA contamination

• RT-PCR Protocol Optimization:

  • Determine optimal annealing temperature through gradient PCR

  • Optimize primer concentrations (typically 0.1-0.5 μM)

  • Include appropriate reference genes for normalization (GAPDH, β-actin, 18S rRNA)

  • Validate PCR efficiency through standard curves (90-110% efficiency)

• Data Analysis:

  • Use the comparative Ct method (2^-ΔΔCt) for relative quantification

  • Perform statistical analysis to determine significance (student t-test or one-way ANOVA with P<0.05 considered significant)

What are the key considerations for studying ASNS-mediated regulation of muscle development in chickens?

When investigating ASNS's role in chicken muscle development, researchers should consider:

• Breed Selection:

  • Include both fast-growing (broiler) and slow-growing chicken breeds

  • Consider breeds with varying muscle development characteristics

  • Document growth parameters (average daily weight gain, muscle mass, etc.)

• Developmental Time Points:

  • Sample at multiple developmental stages (embryonic, post-hatching, juvenile, adult)

  • Track ASNS expression changes alongside muscle development parameters

• Cellular Models:

  • Skeletal muscle satellite cells (SMSCs) provide a valuable in vitro model

  • Establish primary SMSC cultures from different muscle types

  • Use standardized proliferation and differentiation protocols

• Molecular Approaches:

  • Gene expression modulation (overexpression, knockdown, knockout)

  • Pathway analysis targeting AMPK and other identified signaling pathways

  • Combine transcriptomic (RNA-Seq) with proteomic and metabolomic analyses

• Functional Assays:

  • EdU assay for proliferation assessment

  • Cell cycle analysis using flow cytometry

  • Immunofluorescence for differentiation markers

  • Metabolic profiling focusing on amino acid metabolism

What protein purification strategy is most effective for recombinant chicken ASNS?

Based on successful purification of human ASNS and general protein purification principles, the following strategy is recommended for recombinant chicken ASNS:

• Expression System:

  • Baculovirus-based expression in insect cells

  • Expression at lower temperatures (21-30°C) to maximize solubility

  • C-terminal His-tagging for purification

• Cell Lysis and Initial Clarification:

  • Gentle lysis in buffer containing protease inhibitors

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Clarification by centrifugation (30,000 × g, 30 min, 4°C)

• Purification Steps:

  • Immobilized Metal Affinity Chromatography (IMAC):

    • Ni-NTA or Co-NTA resin for His-tagged protein capture

    • Wash with increasing imidazole concentrations (10-30 mM)

    • Elution with higher imidazole (250 mM)

  • Size Exclusion Chromatography:

    • Separate dimeric active enzyme from aggregates and monomers

    • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

  • Optional Ion Exchange Chromatography:

    • Further purification if needed using anion exchange

• Quality Control:

  • SDS-PAGE to verify purity (expect a band at ~62-65 kDa)

  • Western blot using anti-ASNS antibodies

  • Activity assay measuring phosphate release or asparagine production

  • Thermal stability assessment using differential scanning fluorimetry

• Storage Conditions:

  • Store in buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

  • Flash-freeze in liquid nitrogen and store at -80°C in small aliquots

How has ASNS evolved across avian species and what are the functional implications?

While the search results don't provide direct evolutionary comparisons of ASNS across avian species, several inferences can be made:

• Evolutionary Conservation:
  The fundamental role of ASNS in asparagine biosynthesis suggests strong selection pressure for conservation of catalytic domains across species. The ATP-dependent amidotransferase mechanism is likely preserved in all avian ASNS proteins.

• Species-Specific Adaptations:
  Different avian species may show adaptations in ASNS regulation and tissue-specific expression patterns, particularly relating to:

  • Growth rate differences between wild and domesticated birds

  • Muscle development variations between flying and flightless birds

  • Metabolic adaptations to different diets and environments

• Domestication Effects:
  In chickens, ASNS expression correlates with muscle development parameters that have been strongly selected for during domestication . This suggests that ASNS regulation may have been indirectly affected by artificial selection for growth and meat production traits.

• Regulatory Evolution:
  While the protein sequence may be relatively conserved, regulatory elements controlling ASNS expression likely show greater variation between species, potentially accounting for differences in growth rates and tissue-specific expression patterns.

Comparative studies examining ASNS sequence, expression, and function across diverse avian species would provide valuable insights into how this enzyme has evolved in relation to different ecological niches and selective pressures.

How do mammalian and avian ASNS differ in their roles in disease and development?

Mammalian and avian ASNS show both similarities and differences in their physiological roles:

Mammalian ASNS:

• Disease Associations:

  • Up-regulation of ASNS in human T-cells is associated with asparaginase-resistant forms of acute lymphoblastic leukemia (ALL)

  • Mutations in human ASNS cause Asparagine Synthetase Deficiency (ASD), a rare neurological disorder

• Developmental Roles:

  • Ubiquitously expressed but particularly high in the pancreas

  • Critical for normal brain development, as evidenced by neurological abnormalities in ASD patients

Avian (Chicken) ASNS:

• Developmental Roles:

  • Highly expressed in fast-growing broilers

  • Regulates muscle development by promoting proliferation while inhibiting differentiation of skeletal muscle satellite cells

  • Expression pattern mirrors trends in muscle fiber cross-sectional area and muscle weight

• Signaling Pathway Integration:

  • Activates the AMPK pathway to regulate muscle cell differentiation

  • Affects multiple signaling pathways including JAK-STAT, TNF, Toll-like receptor, and PI3K-Akt

These differences likely reflect evolutionary divergence in tissue-specific regulation and developmental requirements between mammals and birds, particularly relating to muscle development strategies that may be especially important for avian species.

What potential inhibitors of chicken ASNS could be developed for research applications?

Development of specific inhibitors for chicken ASNS would provide valuable research tools for studying its function. Potential approaches include:

• Structure-Based Design:
  Using homology models based on human ASNS structures, rational design of inhibitors targeting:

  • The ATP-binding pocket in the C-terminal domain

  • The glutamine-binding pocket in the N-terminal domain

  • The aspartate-binding site

  • The interface between monomers in the functional dimer

• Classes of Potential Inhibitors:

  • ATP analogs: Non-hydrolyzable ATP analogs that compete for the ATP-binding site

  • Transition state analogs: Compounds mimicking the transition state of the amidotransferase reaction

  • Glutamine analogs: Modified glutamine structures that bind but cannot undergo amide transfer

  • Allosteric inhibitors: Compounds binding to regulatory sites affecting enzyme conformation

• Screening Approaches:

  • High-throughput screening of compound libraries against purified recombinant chicken ASNS

  • Fragment-based screening to identify building blocks for inhibitor development

  • In silico screening using molecular docking and virtual library screening

• Validation Methods:

  • Enzyme activity assays measuring phosphate release or asparagine production

  • Cellular assays examining effects on SMSC proliferation and differentiation

  • Studies of amino acid metabolism in chicken cell cultures

Human ASNS inhibitors might serve as starting points, though modifications may be needed to achieve specificity for the chicken enzyme.

How can transcriptomic and proteomic approaches be integrated to study chicken ASNS regulatory networks?

A comprehensive multi-omics approach to studying chicken ASNS regulatory networks would involve:

• Integrated Experimental Design:

  • Parallel sampling for RNA-Seq, proteomics, and metabolomics from the same biological specimens

  • Time-course experiments capturing dynamic changes during muscle development

  • Comparison of normal conditions with ASNS overexpression, knockdown, and inhibition

• Transcriptomic Approaches:

  • RNA-Seq to identify differentially expressed genes (DEGs) as already demonstrated with 1,968 DEGs identified in ASNS-overexpressing cells

  • Small RNA sequencing to identify miRNAs potentially regulating ASNS or its downstream targets

  • Targeted RT-PCR validation of key genes in the network

• Proteomic Methods:

  • Mass spectrometry-based proteomics to identify changes in protein abundance

  • Phosphoproteomics to map signaling pathway activation (especially AMPK pathway)

  • Protein-protein interaction studies to identify ASNS binding partners

• Metabolomic Analysis:

  • Targeted metabolomics focusing on amino acids (especially asparagine, aspartate, glutamine, glutamate)

  • Broader metabolomic profiling to identify metabolic pathway alterations

• Data Integration Strategies:

  • Correlation analysis between transcript and protein levels

  • Pathway enrichment analysis across multiple omics datasets

  • Network analysis to identify key nodes and regulatory hubs

  • Causal network modeling to infer regulatory relationships

• Validation Approaches:

  • Functional studies targeting key nodes identified through multi-omics

  • CRISPR/Cas9-mediated genome editing of regulatory elements

  • Specific inhibitor studies to validate network predictions

This integrated approach would provide a comprehensive view of how ASNS regulates and is regulated within the context of chicken muscle development.