Recombinant Vulpes vulpes Calcium-binding tyrosine phosphorylation-regulated protein (CABYR)

What experimental approaches are most suitable for initial characterization of Vulpes vulpes CABYR?

For initial characterization of Vulpes vulpes CABYR, researchers should implement a sequential approach:

• Genomic and transcriptomic analysis:

  • Utilize the recent chromosome-level assembly of canid genomes to identify the CABYR gene in Vulpes vulpes

  • Perform RT-PCR to confirm expression patterns in reproductive tissues

• Protein isolation and identification:

  • Extract proteins from fox sperm samples

  • Perform 2D gel electrophoresis followed by Western blotting

  • Identify acidic isoforms with molecular weights similar to human CABYR (86-kDa)

• Phosphorylation and calcium binding assessment:

  • Analyze tyrosine phosphorylation patterns during in vitro capacitation

  • Perform calcium binding assays using 45Ca on 2D gels

  • Test whether dephosphorylation with alkaline phosphatase abolishes calcium binding

• Localization studies:

  • Develop antibodies against predicted conserved regions of Vulpes vulpes CABYR

  • Perform immunohistochemistry to determine subcellular localization in sperm cells

This methodological pipeline would establish fundamental characteristics of CABYR in Vulpes vulpes, creating a foundation for more advanced functional studies.

How does the structure of Vulpes vulpes CABYR likely compare to human CABYR?

Human CABYR contains two coding regions (CR-A and CR-B) with six identified variants containing alternative splice deletions . Key structural features include:

• A motif homologous to the RII dimerization domain of protein kinase A in the N-terminus of CR-A

• A single putative EF hand-like motif in CR-A at amino acids 197-209

• Seven potential tyrosine phosphorylation sites in CR-A and four in CR-B

• Pro-X-X-Pro (PXXP) modules in both N- and C-termini of CR-A and CR-B

For Vulpes vulpes CABYR, structural predictions should be based on comparative genomics with both humans and closer canid relatives. Researchers should focus particularly on:

• Conservation of the calcium-binding EF hand-like motif

• Presence and positioning of tyrosine phosphorylation sites

• Conservation of the RII dimerization domain, which in humans mediates interaction with AKAP3

• Potential canid-specific structural features that may have evolved due to differences in reproductive biology

The recently developed chromosome-level assembly for gray fox (Urocyon cinereoargenteus) and other canid genomes provides a valuable resource for such comparative structural analysis .

What are the optimal methods for expressing recombinant Vulpes vulpes CABYR?

Expressing recombinant Vulpes vulpes CABYR requires careful consideration of expression systems and conditions:

• Gene synthesis and cloning strategies:

  • Design the CABYR gene based on the Vulpes vulpes genome sequence

  • Consider codon optimization for the selected expression system

  • Include affinity tags (His, GST) for purification

  • Design constructs for both full-length protein and individual domains (CR-A, CR-B)

• Expression system selection:

  • Bacterial systems: E. coli BL21(DE3) for high yield, though proper folding may be compromised

  • Yeast systems: Pichia pastoris for improved folding and post-translational modifications

  • Mammalian systems: HEK293 or CHO cells for most native-like protein production, particularly important for studying phosphorylation-dependent calcium binding

• Expression optimization:

  • For bacterial systems: Test multiple temperatures (16°C, 25°C, 37°C), IPTG concentrations, and induction times

  • For mammalian systems: Compare transient versus stable expression

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation state

• Protein solubility enhancement:

  • Test fusion partners (MBP, SUMO) to improve solubility

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Consider mild detergents for extraction if membrane-associated

Careful attention to these methodological details will significantly impact the quality and yield of recombinant Vulpes vulpes CABYR.

How can researchers effectively study calcium binding properties of recombinant Vulpes vulpes CABYR?

To characterize calcium binding properties of recombinant Vulpes vulpes CABYR, researchers should employ multiple complementary techniques:

• 45Ca overlay assays:

  • Separate proteins on 2D gels

  • Overlay with 45Ca

  • Detect binding through autoradiography

  • This method successfully demonstrated calcium binding in human CABYR

• Isothermal titration calorimetry (ITC):

  • Quantitatively determine binding constants and thermodynamic parameters

  • Compare binding parameters between phosphorylated and dephosphorylated forms

  • Measure calcium binding at different pH values and ionic strengths

• Circular dichroism (CD) spectroscopy:

  • Monitor conformational changes upon calcium binding

  • Compare structural changes in phosphorylated versus non-phosphorylated protein

• Phosphorylation-dependent calcium binding:

  • Perform in vitro tyrosine phosphorylation using appropriate kinases

  • Compare calcium binding before and after phosphorylation

  • Treat with alkaline phosphatase to test if calcium binding is abolished, as observed in human CABYR

This experimental framework would establish the relationship between phosphorylation state and calcium binding in Vulpes vulpes CABYR, providing critical insights into its functional regulation.

What approaches can be used to study protein-protein interactions involving Vulpes vulpes CABYR?

Based on human CABYR studies, investigating protein-protein interactions involving Vulpes vulpes CABYR should focus on its interactions with fibrous sheath proteins. The following methodological approaches are recommended:

• Yeast two-hybrid screening:

  • Use different domains of CABYR (CR-A, CR-B, and truncated CR-A lacking the RII domain) as bait

  • Screen against a Vulpes vulpes testis cDNA library

  • Perform targeted screening with candidate partners like AKAP3 and Ropporin

  • Quantify interaction strength using α-galactosidase assays

• Co-immunoprecipitation:

  • Generate antibodies against Vulpes vulpes CABYR

  • Perform co-IP from fox sperm lysates

  • Identify binding partners through mass spectrometry

  • Validate interactions with reverse co-IP using antibodies against identified partners

• Pull-down assays:

  • Express recombinant GST-tagged CABYR domains

  • Incubate with fox sperm lysates

  • Identify binding proteins by mass spectrometry

  • Confirm direct interactions with purified recombinant partners

• Domain mapping:

  • Create truncation mutants to identify specific binding interfaces

  • Pay particular attention to the RII-like domain, which in humans mediates interaction with AKAP3

  • Test if deletion of the RII domain abolishes interaction with AKAP3 but not entirely with Ropporin, as seen in human studies

Human CABYR studies demonstrated that:

• CABYR-A binds strongly to AKAP3 via its RII domain

• CABYR-B binds weakly to AKAP3

• CABYR-A binds weakly to Ropporin, partially through the RII domain and partially through other domains

• CABYR-B does not interact with Ropporin

Testing whether these interaction patterns are conserved in Vulpes vulpes would provide valuable insights into the evolutionary conservation of the sperm fibrous sheath interactome.

How does tyrosine phosphorylation regulate CABYR function in Vulpes vulpes, and what experimental approaches can address this question?

The relationship between tyrosine phosphorylation and CABYR function represents a critical research question. Human studies have demonstrated that tyrosine phosphorylation regulates calcium binding of CABYR during capacitation . To investigate this relationship in Vulpes vulpes CABYR:

• Identification of phosphorylation sites:

  • Perform phosphoproteomic analysis of native CABYR isolated from fox sperm

  • Create a phosphorylation map using mass spectrometry

  • Compare with predicted sites based on sequence analysis

  • Generate phosphospecific antibodies against key sites

• Site-directed mutagenesis studies:

  • Create tyrosine-to-phenylalanine mutants at predicted phosphorylation sites

  • Generate phosphomimetic mutants (tyrosine-to-glutamate)

  • Assess impact on calcium binding and protein-protein interactions

  • Create combination mutants to identify critical sites

• Phosphorylation kinetics during capacitation:

  • Develop an in vitro capacitation system for fox sperm

  • Monitor phosphorylation of CABYR at different time points

  • Correlate phosphorylation with calcium binding and sperm functional parameters

  • Compare with capacitation timeline established for other species

• Kinase identification:

  • Use specific tyrosine kinase inhibitors during capacitation

  • Perform in vitro phosphorylation with candidate kinases

  • Identify kinases co-immunoprecipitating with CABYR

This comprehensive approach would elucidate the critical regulatory role of tyrosine phosphorylation in Vulpes vulpes CABYR function during sperm capacitation.

What are the considerations for studying CABYR in the context of comparative reproductive biology across canid species?

Studying CABYR across canid species offers valuable insights into reproductive protein evolution. Researchers should consider:

• Phylogenetic analysis and selection pressure:

  • Compare CABYR sequences across canids, including gray fox, red fox, domestic dog, and wolf

  • Analyze nucleotide substitution patterns to identify regions under purifying or positive selection

  • Focus on functional domains like the calcium-binding EF hand and phosphorylation sites

  • Consider the unique position of Urocyon (gray fox) as a basal genus within Canidae

• Reproductive strategy correlation:

  • Compare CABYR structure in species with different mating systems and reproductive seasonality

  • Analyze CABYR expression patterns in relation to breeding seasons

  • Consider environmental adaptations that might influence sperm function

• Technical considerations:

  • Develop universal primers and antibodies targeting conserved regions

  • Create species-specific reagents for divergent regions

  • Consider tissue collection timing in seasonally breeding species

• Genomic context analysis:

  • Examine chromosomal location and synteny across canid genomes

  • Identify species-specific splice variants

  • Analyze repetitive elements and simple sequence repeats (SSRs) near the CABYR gene

Recent genomic resources, including chromosome-level assemblies for gray fox and genetic characterization of red fox populations , provide excellent foundations for such comparative studies. Understanding CABYR evolution might contribute to broader questions about canid speciation and reproductive isolation.

How should researchers design experiments to analyze the potential role of CABYR in fertility issues in red fox populations?

To investigate CABYR's potential role in fox fertility, researchers should design a multifaceted approach:

• Population sampling and genetic analysis:

  • Collect samples from fox populations with documented fertility differences

  • Target populations with geographic separation, such as those identified in genetic studies between Eastern and Dinaric Alps

  • Sequence the CABYR gene to identify polymorphisms

  • Correlate genetic variants with fertility parameters

• Functional validation of variants:

  • Express recombinant versions of identified CABYR variants

  • Compare calcium binding properties and phosphorylation patterns

  • Assess protein-protein interactions with key partners

  • Test function in heterologous expression systems

• Sperm function analysis:

  • Develop parameters for fox sperm quality assessment

  • Compare CABYR expression, localization, and phosphorylation in samples with different fertility outcomes

  • Correlate CABYR characteristics with hyperactivated motility and acrosome reaction capacity

  • Develop functional blocking assays using anti-CABYR antibodies

• Environmental and toxicological considerations:

  • Assess whether environmental contaminants affect CABYR phosphorylation or function

  • Consider seasonal variations in CABYR expression and function

  • Investigate potential links between habitat quality and CABYR integrity

This experimental framework would provide insights into whether CABYR variants contribute to fertility differences in wild fox populations, potentially informing conservation strategies for threatened fox populations.

What controls and validation steps are essential when working with recombinant Vulpes vulpes CABYR?

Rigorous validation is critical when studying recombinant Vulpes vulpes CABYR. Essential controls and validation steps include:

• Expression and purification validation:

  • Confirm protein identity via mass spectrometry

  • Verify size and purity by SDS-PAGE

  • Validate immunoreactivity with anti-CABYR antibodies

  • Test for post-translational modifications, especially phosphorylation

• Functional validation:

  • Verify calcium binding using multiple independent methods

  • Compare properties with native CABYR from fox sperm when possible

  • Ensure proper folding through circular dichroism or other structural analyses

  • Validate expected protein-protein interactions

• Critical experimental controls:

  • Include non-phosphorylatable mutants (Y→F substitutions)

  • Use calcium-binding deficient mutants (EF-hand domain mutations)

  • Include heat-denatured protein as negative control

  • Test calcium binding in the presence of EGTA as chelation control

• Antibody validation:

  • Perform pre-absorption controls to confirm specificity

  • Validate antibody recognition of both recombinant and native protein

  • Test cross-reactivity with CABYR from related species

  • Include secondary antibody-only controls in immunostaining

• Physiological relevance controls:

  • Compare recombinant protein behavior with native CABYR during capacitation

  • Validate key findings in primary tissue samples or cells

How can qualitative research approaches contribute to CABYR studies, and what methodological frameworks are appropriate?

While CABYR research is predominantly laboratory-based, qualitative research methodologies can provide valuable contextual information for more comprehensive understanding:

• Research question formulation:

  • Use reflective and interrogative processes to develop effective questions

  • Consider using the PEO (Population, Exposure, Outcome) framework for questions about CABYR in wild fox populations

  • Apply SPIDER (Sample, Phenomenon of Interest, Design, Evaluation, Research type) for mixed-methods studies integrating laboratory and field data

• Observational studies of reproductive behavior:

  • Document mating patterns and reproductive success in wild or captive foxes

  • Correlate behavioral observations with laboratory findings on CABYR function

  • Create a framework for integrating molecular data with behavioral observations

• Experimental design considerations:

  • Address both the "why" (establishes need for study) and "how" (selection of measures) in experimental design

  • Consider whether quasi-experimental designs might be appropriate for field studies where randomization is challenging

  • Ensure appropriate sample selection to avoid introduction of bias

• Data collection approaches:

  • Employ mixed-methods approaches combining molecular analysis with field observations

  • Develop standardized protocols for sample collection from wild populations

  • Consider seasonal timing for sample collection given reproductive cyclicity

As noted by researchers in qualitative methodology: "Good research questions do not necessarily produce good research, but poorly conceived or constructed questions will likely create problems that affect all subsequent stages of a study" . This principle applies equally to molecular studies of CABYR, highlighting the importance of thoughtful experimental design.

How do CABYR properties likely differ between human and Vulpes vulpes, and what are the implications for research?

Understanding the differences between human and Vulpes vulpes CABYR is crucial for research design and interpretation:

• Sequence and structural differences:

  • Human CABYR contains two coding regions (CR-A and CR-B) with multiple splice variants

  • Fox CABYR likely has similar domain organization but may exhibit canid-specific features

  • The calcium-binding EF-hand motif is likely conserved but may show species-specific adaptations

  • Phosphorylation sites may differ in number and position

• Functional differences:

  • In humans, CABYR was originally identified as testis-specific but later found in lung and brain tumors

  • CABYR expression patterns in foxes may differ from humans

  • Calcium binding properties may be adapted to fox-specific sperm function requirements

  • Phosphorylation kinetics during capacitation may reflect species-specific fertilization timing

• Research implications:

  • Antibodies developed against human CABYR may have unpredictable cross-reactivity with fox CABYR

  • Capacitation media and conditions optimized for human sperm may require significant modification for fox studies

  • Protein interaction networks may differ between species

  • The relationship between CABYR and fertility may have species-specific characteristics

• Evolutionary context:

  • Consider reproductive adaptations specific to canids such as seasonal breeding

  • Investigate whether CABYR shows evidence of adaptive evolution related to mating systems or sperm competition

This comparative perspective provides context for interpreting results from fox studies and highlights the importance of species-specific validation of methods developed for human CABYR research.

What is known about CABYR as a potential cancer antigen, and how might this inform research in Vulpes vulpes?

Human CABYR has been identified as a cancer-testis antigen with potential implications for cancer immunotherapy. This knowledge may inform Vulpes vulpes research:

• CABYR as a cancer-testis antigen:

  • Initially reported as testis-specific, CABYR has been observed in lung and brain tumors in humans

  • Classified as a novel cancer-testis antigen with potential immunogenic properties

  • Has been proposed as a candidate antigen for lung cancer immunotherapy

• Expression pattern analysis in Vulpes vulpes:

  • Researchers should investigate whether CABYR expression in foxes is strictly testis-specific or more broadly distributed

  • Examine CABYR expression in fox tumor samples if available

  • Compare tissue expression patterns across healthy fox tissues using RT-PCR and immunohistochemistry

• Research applications:

  • The dual roles of CABYR in reproduction and potentially in cancer make it an interesting model for studying protein repurposing during evolution

  • If CABYR shows similar cancer-associated expression in foxes, it could serve as a model for comparative oncology

  • Understanding the regulation of CABYR expression across tissues may provide insights into gene silencing mechanisms

• Methodological considerations:

  • When analyzing CABYR expression, include a wide range of tissues beyond reproductive organs

  • Use both mRNA and protein detection methods to confirm expression patterns

  • Consider epigenetic analyses to understand tissue-specific expression regulation

While cancer studies may not be the primary focus of Vulpes vulpes CABYR research, awareness of its potential dual role can inform comprehensive experimental design and potentially open new research directions.