Recombinant Ciona intestinalis Inosine triphosphate pyrophosphatase

What is Inosine triphosphate pyrophosphatase (ITPase) and what is its function in Ciona intestinalis?

Inosine triphosphate pyrophosphatase (ITPase) is a protective enzyme that hydrolyzes noncanonical purine nucleotides, specifically (deoxy)inosine and (deoxy)xanthosine triphosphate, into their corresponding monophosphates with the release of pyrophosphate . In Ciona intestinalis, like in other organisms, ITPase likely serves as a guardian of cellular nucleotide pools by preventing the accumulation of potentially mutagenic noncanonical nucleotides.

The enzyme functions to maintain genomic integrity by preventing the incorporation of these noncanonical nucleotides into DNA and RNA during replication and transcription. While human ITPase deficiency has been linked to various clinical manifestations including severe multisystem disorders, the specific role of ITPase in Ciona intestinalis may relate to developmental processes and tissue regeneration, given the organism's remarkable regenerative capabilities .

How conserved is ITPase across species from ascidians to humans?

ITPase is a highly conserved enzyme across diverse phylogenetic lineages. Comparative genomic analyses reveal significant structural and functional conservation from bacterial species through ascidians to mammals. The ITPA gene shows evolutionary conservation in key catalytic domains and substrate binding regions.

In humans, ITPase is encoded by the ITPA gene, and its homologs have been identified in various organisms including yeast (Ham1p) and bacteria (such as the Mj0226 protein from Methanococcus jannaschii) . While specific information about Ciona intestinalis ITPase conservation is limited in the provided search results, the Ciona genome has been well-annotated , allowing for comparative analysis with human ITPA.

The conservation pattern suggests that the fundamental mechanism of preventing noncanonical nucleotide incorporation into genetic material is evolutionarily ancient and critical for cellular function across species. Understanding these conserved elements can provide insights into the basic mechanisms of nucleotide pool sanitation across evolutionary time.

What is the genomic location and structure of the ITPA gene in Ciona intestinalis?

Based on the Ciona intestinalis genome annotation (NCBI Ciona intestinalis Annotation Release 102), the Ciona genome contains approximately 14,648 genes and pseudogenes, with 13,538 being protein-coding . While the specific genomic location of the ITPA gene is not directly provided in the search results, researchers can access this information through the NCBI genome browser using the reference genome assembly KH (GCF_000224145.2) .

The Ciona genome is organized into 15 assembled chromosomes plus unplaced scaffolds . To locate the ITPA gene precisely, researchers should:

• Access the NCBI Genome database for Ciona intestinalis

• Search for the ITPA gene or its annotated identifier

• Note the chromosomal position, exon-intron structure, and flanking genes

For detailed molecular phylogenetic analysis, techniques similar to those used for other Ciona genes (such as Fgf4/5/6, Foxa.a, Jade, Patched, and vAChTP) can be applied to confirm the identity and structural features of the ITPA gene .

What substrates does ITPase act upon in Ciona intestinalis?

While the specific substrate profile of Ciona intestinalis ITPase has not been directly characterized in the provided search results, based on the conserved function of ITPase across species, it likely hydrolyzes similar noncanonical purine nucleotides as its human counterpart.

The primary substrates for human ITPase include:

• Inosine triphosphate (ITP)

• Deoxyinosine triphosphate (dITP)

• Xanthosine triphosphate (XTP)

• Deoxy-xanthosine triphosphate (dXTP)

Human ITPase shows negligible activity toward other nucleoside triphosphates and no activity toward IDP or IMP . Given the evolutionary conservation of this enzyme, Ciona intestinalis ITPase likely possesses similar substrate specificity.

To experimentally determine the exact substrate profile of Ciona intestinalis ITPase, researchers would need to express and purify the recombinant enzyme and test its activity with various substrates under controlled conditions, measuring the release of pyrophosphate or the formation of nucleoside monophosphates.

What are the optimal biochemical conditions for Ciona intestinalis ITPase activity?

To determine the precise optimal conditions for the Ciona enzyme, researchers should conduct systematic biochemical characterization studies with the purified recombinant protein, testing activity across ranges of pH, temperature, and ionic conditions.

What are the key structural differences between human and Ciona intestinalis ITPase that might affect substrate specificity?

• Sequence alignment analysis: Comparing the amino acid sequences of human and Ciona ITPase to identify conserved catalytic residues and divergent regions that might influence substrate binding.

• Homology modeling: If crystal structures are unavailable for Ciona ITPase, researchers can create homology models based on known structures of human ITPase or bacterial homologs.

• Substrate binding pocket comparison: Focusing specifically on residues that line the substrate binding pocket to identify substitutions that might alter substrate preference or catalytic efficiency.

• Molecular dynamics simulations: Running simulations to predict how structural differences might affect protein dynamics and substrate interactions.

Key regions to examine include:

• The nucleotide binding domain

• The catalytic site for phosphohydrolase activity

• Regions involved in conformational changes during catalysis

• Surface residues that might influence protein stability under different environmental conditions

These structural differences could potentially explain any observed differences in substrate specificity, catalytic efficiency, or response to inhibitors between the human and Ciona enzymes.

How does ITPase activity contribute to nucleotide pool maintenance during rapid cell proliferation in Ciona intestinalis?

During rapid cell proliferation, such as during development or regeneration in Ciona intestinalis, maintaining nucleotide pool quality becomes particularly critical. While the specific role of ITPase in Ciona development isn't directly addressed in the search results, we can make informed hypotheses based on known ITPase functions:

• Prevention of genomic instability: During rapid cell division, increased DNA replication can lead to higher rates of cytosine deamination, producing inosine. ITPase likely helps prevent incorporation of these deaminated nucleotides into nascent DNA.

• Support for regenerative processes: Ciona intestinalis demonstrates remarkable regenerative capabilities, particularly in the basal body parts that can regenerate distal structures . This regeneration involves adult stem cells in the branchial sac that proliferate and produce migratory progenitor cells for tissue replacement .

• Interaction with apoptosis pathways: Regeneration in Ciona involves apoptosis at injury sites, which triggers Wnt signaling . ITPase may play a role in this process by preventing aberrant nucleotide incorporation during the cellular remodeling that accompanies regeneration.

To experimentally investigate this question, researchers could:

• Compare ITPase expression and activity levels in proliferating versus quiescent tissues

• Knock down ITPase expression and observe effects on development and regeneration

• Correlate nucleotide pool composition with ITPase activity during different developmental stages

What impact does temperature have on recombinant Ciona intestinalis ITPase stability and activity?

To thoroughly investigate this question, researchers should conduct:

• Thermal stability assays: Measure protein unfolding at different temperatures using techniques such as differential scanning fluorimetry or circular dichroism spectroscopy.

• Temperature-dependent activity assays: Determine enzymatic activity across a range of temperatures (5-40°C) to establish the temperature optimum and range.

• Arrhenius plot analysis: Calculate activation energy by measuring reaction rates at different temperatures and creating an Arrhenius plot.

• Long-term stability studies: Assess enzyme activity retention after storage at different temperatures over extended periods.

Expected findings might include:

• Lower temperature optima compared to mammalian enzymes, reflecting Ciona's marine habitat

• Potentially higher flexibility in cold environments compared to human ITPase

• Different temperature-dependent inactivation kinetics compared to terrestrial species' enzymes

These thermal characteristics could have important implications for experimental design, storage conditions, and understanding the evolutionary adaptations of nucleotide metabolism enzymes.

How does ITPase activity in Ciona intestinalis relate to developmental processes and regeneration?

While the specific connection between ITPase and regeneration in Ciona intestinalis has not been directly established in the search results, there are several potential relationships worth investigating:

• Regenerative capacity and nucleotide pool quality: Ciona intestinalis demonstrates unique regenerative abilities where basal body parts can regenerate distal structures, but distal parts cannot replace basal structures . This process involves adult stem cells in the branchial sac that proliferate to produce migratory progenitor cells .

• Potential role in apoptosis-Wnt signaling axis: Regeneration in Ciona involves apoptosis at injury sites, which appears to trigger Wnt signaling required for successful regeneration . ITPase may potentially influence this process by regulating nucleotide pools during cellular remodeling.

• Developmental stage-specific requirements: The need for ITPase activity may vary across developmental stages, potentially correlating with periods of rapid cell division or differentiation.

Experimental approaches to explore this relationship could include:

• Analyzing ITPA expression patterns across developmental stages and regenerating tissues

• Using siRNA knockdown of ITPA (similar to methods used for other Ciona genes ) and observing effects on development and regeneration

• Investigating potential interactions between ITPase activity and Wnt signaling components

Understanding these relationships could provide insights into both the basic biology of nucleotide metabolism and the mechanisms underlying regenerative processes in chordates.

How does the amino acid sequence of Ciona intestinalis ITPase compare to known ITPA polymorphisms associated with drug responses in humans?

Human ITPA polymorphisms have been associated with altered drug responses, particularly to thiopurine drugs . While specific information about Ciona intestinalis ITPase polymorphisms is not provided in the search results, researchers investigating this question should:

• Sequence alignment analysis: Align Ciona and human ITPA sequences, focusing on regions containing known human polymorphisms (such as P32T, R139H, or 94C>A).

• Conservation assessment: Determine if the amino acid positions associated with clinically significant polymorphisms in humans are conserved in the Ciona sequence.

• Structural modeling: Model how any conserved or variable regions might affect enzyme function, stability, or substrate binding.

• Functional prediction: Predict how differences at these positions might affect enzyme function, potentially leading to different drug metabolism profiles.

A comparison table presenting key human polymorphic sites and corresponding Ciona residues would be valuable:

This comparative analysis could provide insights into the evolutionary conservation of functionally critical residues and the potential for modulating drug responses through engineered variants.

What expression systems are most effective for producing recombinant Ciona intestinalis ITPase?

While the search results don't specifically address expression systems for Ciona intestinalis ITPase, the following expression systems would likely be effective based on general recombinant protein methodology and the characteristics of ITPase:

• E. coli expression systems:

  • BL21(DE3) strain with pET vector systems for high-level expression

  • Arctic Express or Rosetta strains for improved folding at lower temperatures

  • Fusion tags: His6, GST, or MBP to improve solubility and facilitate purification

• Insect cell expression systems:

  • Baculovirus expression vector system (BEVS) with Sf9 or Hi5 cells

  • Advantages: Better post-translational modifications and folding for eukaryotic proteins

• Yeast expression systems:

  • Pichia pastoris for secreted expression

  • Saccharomyces cerevisiae for intracellular expression

• Mammalian expression systems:

  • HEK293 or CHO cells for highest authenticity of post-translational modifications

  • Useful if specific modifications are critical for function

Optimization strategies should include:

• Codon optimization for the chosen expression host

• Testing multiple fusion tags and cleavage sites

• Optimizing induction conditions (temperature, inducer concentration, time)

• Screening for soluble vs. insoluble expression

The ideal system will balance yield, activity, and ease of purification, with E. coli systems typically providing the highest yield for non-glycosylated proteins like ITPase.

What purification strategies yield the highest activity for recombinant Ciona intestinalis ITPase?

Based on general enzyme purification principles and what is known about ITPase, the following purification strategy would likely be effective for Ciona intestinalis ITPase:

Multi-step purification protocol:

• Initial capture: Affinity chromatography

  • His-tag purification using Ni-NTA or TALON resin

  • Buffer conditions: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5-20 mM imidazole (wash), 250 mM imidazole (elution)

  • Include 5-10% glycerol and 1 mM DTT to maintain stability

• Intermediate purification: Ion exchange chromatography

  • Anion exchange (Q Sepharose) for removing contaminants

  • pH selection based on the theoretical pI of Ciona ITPase

• Polishing step: Size exclusion chromatography

  • Superdex 75/200 to remove aggregates and ensure monodispersity

  • Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

Activity preservation strategies:

• Maintain divalent metal ions (Mg²⁺ or Mn²⁺) at 1-5 mM throughout purification

• Keep temperature at 4°C during all steps

• Monitor activity after each purification step

• Consider adding stabilizing agents (glycerol, reducing agents, specific substrates)

Quality control assessment:

• SDS-PAGE for purity evaluation (target >95%)

• Western blot for identity confirmation

• Dynamic light scattering for homogeneity assessment

• Initial activity tests using colorimetric pyrophosphate detection

This strategy should yield highly pure, active enzyme suitable for biochemical and structural characterization.

How can I optimize conditions for measuring the enzymatic activity of recombinant Ciona intestinalis ITPase?

To optimize conditions for measuring recombinant Ciona intestinalis ITPase activity, researchers should establish a robust assay system and systematically test key parameters:

Recommended assay systems:

• Colorimetric pyrophosphate detection:

  • Malachite green assay for phosphate released after pyrophosphate hydrolysis

  • Enzymatic coupling with pyrophosphatase to release inorganic phosphate

  • Monitoring at 620-640 nm

• HPLC-based substrate depletion/product formation:

  • Direct measurement of ITP/XTP decrease or IMP/XMP formation

  • Reverse-phase HPLC with UV detection at 245-260 nm

• Coupled enzyme assays:

  • Link pyrophosphate release to NADH oxidation through enzymatic coupling

  • Monitor absorbance decrease at 340 nm

Optimization parameters table:

Assay validation steps:

• Determine linear range with respect to time and enzyme concentration

• Confirm substrate saturation behavior

• Verify that product inhibition is not affecting measurements

• Include appropriate positive controls (known ITPase enzymes)

• Include negative controls (heat-inactivated enzyme)

These optimized conditions will provide reliable kinetic data for characterizing the enzymatic properties of Ciona intestinalis ITPase.

What are effective methods for studying ITPase localization in Ciona intestinalis tissues?

For studying ITPase localization in Ciona intestinalis tissues, researchers can employ several complementary approaches:

1. Immunohistochemistry (IHC)/Immunofluorescence (IF):

• Generate specific antibodies against Ciona intestinalis ITPase

• If commercial antibodies are unavailable, express recombinant protein and develop custom antibodies

• Process tissue sections through:

  • Fixation with 4% paraformaldehyde

  • Permeabilization with 0.1-0.5% Triton X-100

  • Blocking with 5% BSA or normal serum

  • Primary antibody incubation (anti-ITPase)

  • Secondary antibody with fluorescent tag (for IF) or HRP/AP (for IHC)

  • Counterstaining with DAPI for nuclei

2. In situ hybridization:

• Design RNA probes targeting ITPA mRNA

• Use whole-mount in situ hybridization for embryos and larvae

• For adult tissues, employ either whole-mount or sectioned tissue approaches

• Follow protocols similar to those used for other Ciona genes

3. Fluorescent protein tagging:

• Create GFP or mCherry fusion constructs with Ciona ITPA

• Employ electroporation techniques for embryo transformation

• Use tissue-specific promoters to examine expression patterns

4. Subcellular fractionation:

• Isolate different cellular compartments (cytosol, nucleus, mitochondria)

• Perform Western blotting to detect ITPase in different fractions

• Confirm findings with organelle-specific markers

These methods can be particularly useful for examining ITPase expression during development and regeneration processes, which are well-studied in Ciona intestinalis . When analyzing branchial sac tissues, which contain stem cells involved in regeneration , special attention should be paid to ITPase localization in relation to proliferating cells and apoptotic regions.

How can I design siRNA experiments to knock down ITPA expression in Ciona intestinalis?

Designing effective siRNA experiments to knock down ITPA expression in Ciona intestinalis requires careful consideration of several key factors:

siRNA design and delivery:

• Target sequence selection:

  • Identify 3-4 regions in the Ciona ITPA mRNA sequence for targeting

  • Select 19-25 nucleotide regions with 40-60% GC content

  • Avoid regions with secondary structure

  • Perform BLAST analysis against the Ciona genome to ensure specificity

  • Target conserved regions across ITPA transcript variants

• Control design:

  • Include negative control siRNAs (non-targeting)

  • Include positive control siRNAs targeting a housekeeping gene

  • Consider including a fluorescently labeled siRNA to monitor uptake

• Delivery methods:

  • Microinjection for embryos

  • Electroporation for larvae and tissue explants

  • Lipofection for cell cultures derived from Ciona tissues

Experimental protocol:

• Preparation:

  • Synthesize or purchase custom siRNAs targeting Ciona ITPA

  • Prepare siRNA at 10-50 μM concentration in appropriate buffer

• Delivery:

  • For microinjection: 0.5-1.0 pmol siRNA per embryo

  • For electroporation: Similar to methods used in regeneration studies

  • For lipofection: Follow manufacturer's protocol with optimization

• Validation of knockdown:

  • RT-qPCR to measure ITPA mRNA levels (primary validation)

  • Western blot to confirm protein reduction

  • Enzymatic activity assay to verify functional knockdown

• Phenotypic analysis:

  • Monitor development and regeneration capabilities

  • Assess nucleotide pool composition

  • Observe cellular effects (proliferation, apoptosis)

  • Relate to Wnt signaling pathways known to be involved in regeneration

This approach allows for specific modulation of ITPA expression and function to study its role in Ciona intestinalis development, regeneration, and nucleotide metabolism.

What statistical approaches are appropriate for comparing ITPase activity across different experimental conditions?

Recommended statistical methods:

• For comparing two conditions:

  • Student's t-test (parametric) if data are normally distributed

  • Mann-Whitney U test (non-parametric) if normality cannot be assumed

  • Paired versions of above tests if samples are matched

• For multiple conditions:

  • One-way ANOVA followed by post-hoc tests (Tukey's HSD, Bonferroni) for normally distributed data

  • Kruskal-Wallis test followed by Dunn's test for non-parametric analysis

  • Two-way ANOVA when testing two factors (e.g., temperature and pH effects)

• For time-course or concentration-response data:

  • Repeated measures ANOVA

  • Mixed-effects models for handling missing data points

  • Non-linear regression for determining EC50 or IC50 values

Experimental design considerations:

Data quality assessment:

• Test for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests

• Check for homogeneity of variance using Levene's test

• Identify outliers using Grubbs' test or box plots

• Consider data transformations (log, square root) if appropriate

How can I resolve contradictory results regarding ITPase activity in different Ciona intestinalis tissues?

When faced with contradictory results regarding ITPase activity across different Ciona intestinalis tissues, researchers should implement a systematic troubleshooting and validation approach:

Systematic investigation process:

• Methodological validation and standardization:

  • Verify enzyme assay specificity using recombinant Ciona ITPase as positive control

  • Standardize tissue collection, storage, and processing protocols

  • Ensure consistent protein extraction methods across all tissues

  • Normalize activity measurements (per mg protein, per cell, per tissue weight)

• Biological factors to consider:

  • Developmental stage variation: Compare specimens at identical developmental points

  • Sexual maturity: Separate analyses for mature and immature specimens

  • Regenerative state: Note if tissues are actively regenerating

  • Circadian rhythms: Standardize collection times

• Technical considerations:

  • Tissue-specific inhibitors: Test for presence of endogenous inhibitors

  • Isoform expression: Investigate potential tissue-specific isoforms

  • Post-translational modifications: Examine by Western blot with phospho-specific antibodies

  • Sample degradation: Measure activity immediately after collection vs. after storage

Reconciliation strategies:

• Multi-method verification:

  • Combine enzymatic activity assays with protein quantification (Western blot)

  • Correlate with mRNA expression (RT-qPCR)

  • Verify localization (immunohistochemistry)

• Independent laboratory validation:

  • Have key experiments replicated by independent researchers

  • Compare results using different assay methodologies

• Statistical meta-analysis:

  • Pool data across experiments with appropriate statistical corrections

  • Identify patterns that may explain apparent contradictions

By systematically investigating these factors, researchers can determine whether contradictory results represent true biological variation (e.g., tissue-specific regulation of ITPase) or stem from methodological inconsistencies, leading to a more accurate understanding of ITPase distribution and function in Ciona intestinalis.

What approaches can be used to correlate ITPase activity with developmental stage in Ciona intestinalis?

To effectively correlate ITPase activity with developmental stages in Ciona intestinalis, researchers should employ a multi-faceted approach combining biochemical, molecular, and imaging techniques:

Experimental approach framework:

• Developmental timeline sampling:

  • Collect samples at defined developmental stages (fertilized egg, cleavage, gastrula, neurula, tailbud, larva, juvenile, adult)

  • Maintain consistent staging criteria across experiments

  • Consider synchronized development under controlled temperature conditions

• ITPase activity profiling:

  • Measure enzymatic activity using standardized assays

  • Normalize to total protein, DNA content, or cell number

  • Track activity patterns across development

  • Compare with known developmental milestones

• Expression analysis:

  • Perform RT-qPCR for ITPA mRNA quantification at each stage

  • Conduct Western blotting for protein expression patterns

  • Use whole-mount in situ hybridization to visualize spatial expression patterns

  • Consider RNA-seq for transcriptome-wide context

• Correlation with developmental processes:

  • Compare activity peaks with periods of rapid cell division

  • Analyze relationship to apoptotic events during development

  • Investigate potential connections to Wnt signaling, which is known to be important in Ciona regeneration

Visualization and quantification methods:

Functional validation approaches:

• Perturb ITPase expression at specific developmental stages using siRNA knockdown

• Overexpress ITPase and assess developmental consequences

• Inhibit ITPase activity using chemical inhibitors at defined developmental points

These approaches will provide comprehensive insights into how ITPase activity changes throughout Ciona intestinalis development and how these changes may relate to specific developmental processes, particularly those involving nucleotide metabolism and quality control.

How can differential gene expression data be used to understand the regulation of ITPase in Ciona intestinalis?

Differential gene expression data can provide valuable insights into the regulation of ITPase in Ciona intestinalis across various conditions. Here's a comprehensive approach to utilizing such data:

Analysis framework for ITPA regulation:

• Experimental design for differential expression studies:

  • Compare expression across developmental stages

  • Analyze expression in regenerating versus non-regenerating tissues

  • Examine expression under stress conditions (temperature, pH, oxidative stress)

  • Investigate tissue-specific expression patterns

• Data generation and processing:

  • RNA-seq for genome-wide expression profiling

  • Normalize data appropriately (TPM, FPKM, or DESeq2 normalization)

  • Implement strict quality control measures

  • Validate key findings using RT-qPCR

• Regulatory network analysis:

  • Identify genes co-expressed with ITPA

  • Perform pathway enrichment analysis for co-expressed genes

  • Conduct promoter analysis to identify potential transcription factor binding sites

  • Investigate correlation with Wnt signaling components, given their role in Ciona regeneration

Integrative analysis approaches:

Functional validation strategies:

• Test predicted transcription factor binding using reporter assays

• Verify protein-DNA interactions through ChIP assays

• Manipulate predicted regulatory pathways and observe effects on ITPA expression

• Correlate expression changes with functional ITPase activity measurements

This integrative approach to analyzing differential gene expression data will help elucidate the regulatory mechanisms controlling ITPA expression in Ciona intestinalis across different conditions, providing insights into how this important enzyme is regulated in response to developmental, environmental, and physiological changes.