Recombinant Xenopus tropicalis Phosphoribosyltransferase domain-containing protein 1 (prtfdc1)

What is the functional role of PRTFDC1 in Xenopus tropicalis?

PRTFDC1 in Xenopus tropicalis, like its human ortholog, is a member of the phosphoribosyltransferase family, with structural similarity to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Based on human PRTFDC1 characterization, the X. tropicalis variant likely has low phosphoribosyltransferase activity toward hypoxanthine and guanine.

In humans, PRTFDC1 has been characterized as having only 0.26% and 0.09% of the catalytic efficiency of HPRT toward hypoxanthine and guanine, respectively . This suggests that X. tropicalis PRTFDC1 may not play a significant direct catalytic role in the purine salvage pathway. Instead, it may have alternative functions in development or metabolism that remain to be fully characterized.

The protein's expression during X. tropicalis metamorphosis has been documented , suggesting potential roles in tissue remodeling or developmental processes. Further research is needed to determine its precise function in amphibian systems.

How conserved is PRTFDC1 between Xenopus tropicalis and humans?

One key difference in human PRTFDC1 compared to HPRT is that PRTFDC1 has a glycine residue in the position of the proposed catalytic aspartate in HPRT . This substitution likely explains the dramatically reduced catalytic efficiency. In human PRTFDC1, a water molecule at the position of the aspartic acid side chain might act as a weak base, explaining the low activity observed.

Sequence alignment and structural comparison between X. tropicalis and human PRTFDC1 would reveal conservation of these key residues and help predict functional similarities and differences between the species.

What expression patterns does PRTFDC1 show during Xenopus tropicalis development?

PRTFDC1 expression in X. tropicalis has been documented in the context of metamorphosis, particularly in the remodeling intestine . The paper "Genome-wide identification of thyroid hormone receptor targets in the remodeling intestine during Xenopus tropicalis metamorphosis" by Fu et al. (2017) identifies PRTFDC1 as one of the genes of interest in this context.

To fully characterize expression patterns:

• Perform RT-PCR or RNA-seq analysis at different developmental stages

• Use in situ hybridization to visualize spatial expression patterns

• Generate transgenic reporter lines using the PRTFDC1 promoter region linked to fluorescent reporters, similar to techniques used for other X. tropicalis genes

Expression data could be compared with the known temporal regulation of metamorphosis and tissue remodeling to better understand PRTFDC1's developmental role.

How can I express and purify recombinant Xenopus tropicalis PRTFDC1?

For successful expression and purification of recombinant X. tropicalis PRTFDC1, consider the following methodology:

Expression System Selection:
Based on successful human PRTFDC1 expression, HEK293T cells provide an excellent mammalian expression system . For X. tropicalis PRTFDC1:

• Clone the full-length X. tropicalis PRTFDC1 coding sequence into an appropriate expression vector (e.g., with C-Myc/DDK tag)

• Transfect HEK293T cells for protein expression

• Incubate at 37°C for 48-72 hours for optimal expression

Purification Protocol:

• Harvest cells and lyse in buffer containing 25 mM Tris-HCl, pH 7.3, 100 mM glycine, 10% glycerol

• Purify using affinity chromatography based on the fusion tag

• Verify purity by SDS-PAGE with Coomassie blue staining (aim for >80% purity)

• Determine protein concentration using microplate BCA method

• Store at -80°C in aliquots to avoid freeze-thaw cycles

Expected Characteristics:
The predicted molecular weight should be approximately 25-26 kDa, similar to human PRTFDC1 . Verification of proper folding can be performed using thermal-melt assays with known ligands such as GMP, IMP, or PRPP.

What are optimal assay conditions for measuring PRTFDC1 enzymatic activity?

Based on human PRTFDC1 characterization, the following conditions are recommended for measuring X. tropicalis PRTFDC1 activity:

Assay Buffer Composition:

• 50 mM Tris-HCl (pH 7.4)

• 10 mM MgCl₂ (essential for activity as PRTFDC1 requires magnesium ion binding )

• 1 mM PRPP (α-D-5-phosphoribosyl 1-pyrophosphate)

• 1 mM DTT

Substrate Considerations:

• Test both hypoxanthine and guanine as substrates (typically at concentrations ranging from 10-500 μM)

• Include positive controls using human or X. tropicalis HPRT

Detection Methods:

• Spectrophotometric assay tracking the formation of IMP or GMP at 245-250 nm

• HPLC-based assay for direct quantification of nucleotide products

• Coupled enzyme assays that link PRTFDC1 activity to NAD+/NADH conversion

Important Considerations:

• Expect significantly lower activity compared to HPRT (~0.1-0.3% based on human studies )

• Include extended incubation times to detect the low expected activity

• Consider temperature optimization (25-30°C may be optimal for X. tropicalis proteins)

A comprehensive kinetic analysis should determine Km and kcat values for comparison with human PRTFDC1 and HPRT enzymes.

How can I generate CRISPR/Cas9-mediated PRTFDC1 knockout or knockin in Xenopus tropicalis?

For CRISPR/Cas9-mediated targeting of PRTFDC1 in X. tropicalis, follow these methodological guidelines:

Knockout Strategy:

• Design 2-3 sgRNAs targeting early exons of PRTFDC1

• Test sgRNA efficiency using in vitro cleavage assays

• Inject optimized sgRNA (300-500 pg) along with Cas9 protein (1500 pg) into fertilized X. tropicalis eggs at the one-cell stage

• Screen F0 embryos for mutations using T7 endonuclease assay or direct sequencing

• Raise mosaic F0 frogs to adulthood and outcross to wild-type to generate F1 heterozygotes

Knockin Strategy for Tagging PRTFDC1:
Apply homology-dependent or homology-independent strategies as described for X. tropicalis :

• Design a donor template with homology arms (~800 bp each) flanking the insertion site

• For C-terminal tagging, target the region just before the stop codon

• Include reporter genes (e.g., GFP) or epitope tags in frame with PRTFDC1

• Co-inject sgRNA, Cas9, and donor template

• Screen for successful integration using fluorescence (for fluorescent tags) or PCR

Phenotypic Analysis:
Examine PRTFDC1-modified animals for:

• Developmental abnormalities

• Metabolic deficiencies

• Changes during metamorphosis, particularly in intestinal remodeling

• Comparison with known HPRT-deficiency phenotypes

This approach has been successfully implemented for other genes in X. tropicalis with reported efficiencies of heritable modifications .

How does PRTFDC1 structure-function relationship compare between Xenopus tropicalis and humans?

The structure-function relationship of PRTFDC1 between X. tropicalis and humans requires detailed comparative analysis. Based on human PRTFDC1 data, the following approach is recommended:

Structural Comparison:
Human PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP . To compare with X. tropicalis PRTFDC1:

Functional Analysis:

The critical glycine residue in human PRTFDC1 (replacing the catalytic aspartate in HPRT) should be examined in X. tropicalis PRTFDC1 through sequence alignment and mutagenesis studies to determine if this key difference is conserved and contributes similarly to reduced catalytic activity.

What role does PRTFDC1 play in Xenopus tropicalis metamorphosis?

The role of PRTFDC1 in X. tropicalis metamorphosis requires investigation of its regulation and function during this critical developmental transition. Based on current knowledge:

PRTFDC1 has been identified among thyroid hormone receptor targets in the remodeling intestine during X. tropicalis metamorphosis , suggesting potential involvement in this process. To further characterize its role:

Expression Analysis During Metamorphosis:

• Perform stage-specific RT-qPCR to quantify PRTFDC1 expression throughout metamorphosis

• Use RNA-seq to identify co-regulated genes in the gene regulatory network

• Perform in situ hybridization to localize expression in remodeling tissues

Functional Studies:

• Generate PRTFDC1 knockouts or dominant-negative variants in X. tropicalis

• Analyze metamorphic phenotypes, particularly intestinal remodeling

• Perform rescue experiments with wild-type PRTFDC1

Thyroid Hormone Response:

• Identify thyroid hormone response elements in the PRTFDC1 promoter

• Perform ChIP assays to confirm direct thyroid hormone receptor binding

• Test PRTFDC1 expression response to T3/T4 treatment in tadpoles and in organ cultures

Understanding PRTFDC1's role in metamorphosis may reveal novel functions beyond the limited catalytic activity observed in purine metabolism, potentially identifying tissue-specific roles in cellular differentiation or programmed cell death during organ remodeling.

How can transgenic approaches be used to study PRTFDC1 function in Xenopus tropicalis?

Transgenic approaches offer powerful tools for studying PRTFDC1 function in X. tropicalis. Building on established transgenic methods :

Reporter Transgenic Lines:

• Create a PRTFDC1 promoter-fluorescent protein reporter construct

• Generate transgenic lines following the revised protocol for X. tropicalis transgenesis

• Use these lines to:

  • Monitor expression patterns throughout development

  • Perform live imaging during metamorphosis

  • Screen for factors affecting PRTFDC1 expression

Overexpression Studies:

• Generate transgenic lines expressing PRTFDC1 under tissue-specific or inducible promoters

• Create lines expressing mutant variants (e.g., with restored catalytic activity)

• Examine phenotypic consequences and molecular changes

Multi-reporter Systems:
Following established approaches for X. tropicalis , create multireporter lines combining:

• PRTFDC1 promoter driving one fluorescent protein

• HPRT promoter driving a different fluorescent protein

• Tissue-specific markers for co-localization studies

These approaches benefit from X. tropicalis' diploid genome, which simplifies genetic manipulation and analysis compared to the paleotetraploid X. laevis . The quicker maturation time of X. tropicalis (4-6 months vs. 12-18 months for X. laevis) also accelerates the establishment of stable transgenic lines .

Why might recombinant Xenopus tropicalis PRTFDC1 show different activity from expected?

Several factors can contribute to unexpected activity levels in recombinant X. tropicalis PRTFDC1:

Protein Structure and Folding Issues:

• Improper folding during expression (verify by circular dichroism spectroscopy)

• Missing post-translational modifications present in vivo

• Incorrect disulfide bond formation

Buffer and Assay Conditions:

• Suboptimal magnesium concentration (PRTFDC1 requires magnesium ion binding )

• Inappropriate pH (test range 6.8-8.0)

• Missing or inhibitory buffer components

• Temperature sensitivity (X. tropicalis proteins may have different temperature optima)

Expression System Considerations:

• Codon usage differences between expression system and X. tropicalis

• Presence of inhibitory contaminants from purification

• Tag interference with enzyme activity (consider tag removal or alternative tag positions)

Comparative Activity Table:

Remember that based on human PRTFDC1 data, the expected activity would be only 0.1-0.3% of HPRT activity , which may require sensitive detection methods and extended reaction times.

How can I determine if phenotypes in PRTFDC1-modified Xenopus tropicalis are specific to the protein's function?

To confirm that observed phenotypes in PRTFDC1-modified X. tropicalis are specifically related to the protein's function, implement the following methodological approaches:

Rescue Experiments:

• Reintroduce wild-type PRTFDC1 into knockout animals through mRNA injection or transgenic expression

• Test whether this rescues the observed phenotypes

• Use structure-based mutants (e.g., binding-deficient variants) to determine which functions are essential

Specificity Controls:

• Generate multiple independent PRTFDC1 knockout or knockdown lines using different targeting strategies

• Confirm consistent phenotypes across these different lines

• Use CRISPR off-target prediction tools to identify and sequence potential off-target sites

Molecular Pathway Validation:

• Perform RNA-seq on normal and PRTFDC1-deficient tissues to identify dysregulated pathways

• Use metabolomics to identify changes in purine metabolism or other affected pathways

• Conduct ChIP-seq if PRTFDC1 is suspected to have nuclear functions

Comparative Analysis:

• Compare phenotypes with HPRT-deficient X. tropicalis (if available)

• Correlate phenotype severity with the degree of PRTFDC1 depletion/mutation

• Examine tissue-specific effects in relation to normal PRTFDC1 expression patterns

For developmental phenotypes, carefully document timing, tissue specificity, and penetrance. This systematic approach will help distinguish specific PRTFDC1-related effects from background variability or off-target effects of genetic manipulation.

What is the evolutionary significance of PRTFDC1 conservation across vertebrates?

The conservation of PRTFDC1 across vertebrates, including humans and X. tropicalis, raises intriguing questions about its evolutionary significance, especially given its low catalytic activity compared to HPRT.

Comparative Genomic Analysis:

• Perform phylogenetic analysis of PRTFDC1 across vertebrate species

• Calculate selection pressure (dN/dS ratios) to identify conserved domains

• Correlate PRTFDC1 sequence conservation with species-specific metabolic or developmental adaptations

Functional Evolution Hypotheses:

• PRTFDC1 may have evolved from HPRT duplication with subsequent functional divergence

• The consistently low catalytic activity suggests evolution toward regulatory rather than enzymatic functions

• Conservation may indicate important protein-protein interaction capabilities distinct from catalytic activity

Experimental Approaches:

• Generate chimeric proteins between X. tropicalis PRTFDC1 and HPRT to map functional domains

• Perform interactome studies to identify binding partners that may be evolutionarily conserved

• Conduct cross-species complementation experiments to test functional conservation

The continued maintenance of PRTFDC1 in vertebrate genomes despite its low catalytic efficiency suggests important non-canonical functions that warrant further investigation in model systems like X. tropicalis.

How can proteomics approaches enhance our understanding of PRTFDC1 interaction networks?

Proteomics approaches offer powerful methods to decipher PRTFDC1 interaction networks and post-translational modifications, providing deeper insight into its biological functions:

Interaction Network Mapping:

• Perform immunoprecipitation coupled with mass spectrometry (IP-MS) using tagged PRTFDC1

• Use proximity labeling methods (BioID or APEX) to identify transient interactors

• Compare interaction networks between different developmental stages or tissues

• Validate key interactions using techniques such as co-immunoprecipitation or FRET

Post-translational Modification Analysis:

• Use mass spectrometry to identify phosphorylation, acetylation, or other modifications on PRTFDC1

• Map modification sites to structural features of the protein

• Determine how modifications affect activity, localization, or protein interactions

• Identify enzymes responsible for these modifications

Quantitative Proteomics:

• Compare proteome changes in PRTFDC1-knockout versus wild-type X. tropicalis tissues

• Use SILAC or TMT labeling for precise quantification

• Perform temporal proteomic analysis during metamorphosis to correlate with PRTFDC1 expression changes

Systems Analysis:
Integrate proteomic data with other -omics approaches to build comprehensive models of PRTFDC1 function in cellular networks, potentially revealing unexpected roles beyond purine metabolism.