Recombinant Bison bonasus Pregnancy-associated glycoprotein 67A

What is Pregnancy-associated glycoprotein 67A from European bison and what is its significance?

Pregnancy-associated glycoprotein 67A (PAG 67A) is a member of the placental aspartic proteinase (AP) group found in European bison (Bison bonasus). This protein is encoded by distinct genes expressed in extra-embryonic cells (trophoblast) before implantation and subsequently in the chorionic epithelium of various ungulate species with different placental types . The significance of PAG 67A lies in its role as a pregnancy biomarker in bovine species and its potential utility in reproductive research, conservation breeding programs, and comparative placental biology studies. The protein has been assigned UniProt ID P85319 and is also known as EbPAG-A 67 kDa .

What is the amino acid sequence of the recombinant fragment of European bison PAG 67A?

The commercially available recombinant fragment of European bison PAG 67A corresponds to the N-terminal 1-20 amino acid region with the sequence: RGSNLTHPLR NIGDLFYVGN . This fragment is sufficient for various immunological applications and maintains biological activity as determined by its binding capability in functional ELISA tests . The sequence appears to be well-conserved and represents an important functional domain of the native protein. Researchers seeking to design antibodies or perform epitope mapping should note that this N-terminal region may contain important antigenic determinants.

How is recombinant European bison PAG 67A produced and what are its typical specifications?

Recombinant European bison PAG 67A (fragment 1-20 aa) is typically expressed in yeast expression systems, which allow for proper protein folding and post-translational modifications . The recombinant protein can be produced with a histidine tag (His-tag) for purification purposes or in a tag-free form depending on the experimental requirements . Standard specifications include:

Researchers should note that repeated freezing and thawing is not recommended, and working aliquots should be stored at 4°C for up to one week to maintain protein integrity .

What are the primary research applications for recombinant European bison PAG 67A?

Recombinant European bison PAG 67A serves multiple research applications in reproductive biology and conservation science. The protein can be used in various experimental techniques including:

• ELISA (Enzyme-Linked Immunosorbent Assay): For the development and calibration of pregnancy detection assays in European bison and related species .

• Western Blot (WB): For detection and characterization of native PAG proteins in biological samples, allowing for comparative analysis across different bovine species .

• Immunoprecipitation (IP): For isolation and enrichment of PAG complexes from biological samples to study protein-protein interactions .

• Antibody Production: As an immunogen for generating specific antibodies against European bison PAG 67A for research and diagnostic applications.

• Comparative Studies: For exploring evolutionary relationships and functional differences among PAGs across different ungulate species.

The high purity (>85-90%) ensures reliable results in these research applications, though optimization may be required depending on the specific experimental conditions .

How can researchers effectively detect PAG concentration changes throughout pregnancy in bovine species?

Detection of PAG concentration changes throughout pregnancy in bovine species requires appropriate timing and methodology. Based on studies in American Bison (a close relative of European bison), researchers should consider:

• Sampling Timeline: PAG concentrations show significant variations based on pregnancy stage. In American Bison, PAG levels remain low in the first trimester but increase significantly during the second and third trimesters .

• Assay Selection: Radio-Immunoassay (RIA) using specific antibodies (such as AS706) has been successfully employed to measure PAG concentrations in bovine species. Liquid Chromatography coupled with Mass Spectrometry (LC-MS) can also be used for more precise measurements .

• Control Samples: Include samples from non-pregnant females and males as negative controls. In American Bison studies, baseline PAG concentrations were approximately 0.21 ± 0.12 ng/mL in bulls and 0.25 ± 0.62 ng/mL in non-pregnant cows .

• Data Interpretation: PAG concentrations in American Bison during the second and third trimesters were found to be 16.00 ± 6.42 ng/mL and 20.14 ± 8.99 ng/mL respectively, with considerable individual variation . Researchers studying European bison should anticipate similar patterns while acknowledging potential species-specific differences.

• Correlation Analysis: Consider correlating PAG measurements with other pregnancy hormones. Studies in American Bison found a significant positive correlation between PAG and progesterone levels (r = 0.52) .

How does the N-glycodiversity of PAG proteins impact their functional characterization and assay development?

The N-glycodiversity of PAG proteins, including European bison PAG 67A, presents important considerations for functional characterization and assay development. Research has shown that:

• Structural Impact: N-glycosylation patterns can significantly affect protein folding, stability, and epitope presentation. Researchers must consider these post-translational modifications when designing experiments to study PAG function .

• Species-Specific Patterns: Wild pecoran Bovidae taxa, including European bison, exhibit unique PAG N-glycodiversity profiles that may differ from domesticated bovine species. This diversity must be accounted for when developing cross-reactive assays .

• Expression System Selection: When producing recombinant PAGs, the choice of expression system (e.g., yeast vs. mammalian cells) will impact glycosylation patterns. Yeast-expressed recombinant European bison PAG 67A may have different glycosylation compared to the native protein, potentially affecting certain functional studies .

• Assay Interference: Glycan structures may interfere with antibody binding in immunoassays, necessitating careful antibody selection and validation. Researchers should evaluate antibody recognition of both glycosylated and deglycosylated forms of the protein .

• Evolutionary Significance: Comparative analysis of N-glycodiversity across species can provide insights into the evolutionary adaptation of PAGs. Studies examining cotyledonary explants have revealed species-specific PAG protein profiles that reflect evolutionary relationships .

For comprehensive characterization, researchers should consider employing glycoproteomic approaches alongside traditional protein analysis methods when studying European bison PAG 67A.

What methodological challenges exist when comparing PAG expression patterns between wild bovine species like European bison and domesticated cattle?

Comparing PAG expression patterns between wild bovine species and domesticated cattle presents several methodological challenges that researchers must address:

• Sample Collection Limitations: Obtaining placental samples from wild species like European bison is logistically difficult and often restricted due to conservation concerns, limiting sample sizes and timepoints compared to domestic cattle studies .

• Transcript Diversity: European bison may express multiple PAG variants with different temporal and spatial patterns. In situ hybridization studies have revealed chorionic mRNA expression patterns that may differ from those in domestic cattle, requiring careful probe design and optimization .

• Cross-Reactivity Issues: Antibodies developed against cattle PAGs may show variable cross-reactivity with European bison PAGs due to sequence differences. Validation of antibody cross-reactivity is essential before conducting comparative studies .

• Reference Ranges: Establishing normal reference ranges for PAG concentrations in European bison requires larger population studies. Current data from American Bison suggest significant individual variation in PAG levels during pregnancy, complicating interpretation of results .

• Assay Standardization: Different assay methodologies (RIA, ELISA, LC-MS) may yield different absolute values, making direct comparisons between studies challenging. Researchers should consider using identical methodologies when comparing species .

• Environmental and Physiological Variables: Wild species experience different environmental conditions and stressors compared to domestic cattle, which may influence PAG expression patterns. Factors such as nutrition, season, and parity should be controlled for or documented .

To address these challenges, collaborative research leveraging both in vitro approaches (using cotyledonary explant cultures) and in vivo sampling (when ethically permitted) offers the most comprehensive approach to comparative PAG studies.

How can in vitro cotyledonary explant cultures advance our understanding of PAG secretion in European bison?

In vitro cotyledonary (CT) explant cultures represent a valuable methodology for studying PAG secretion in European bison, offering several advantages:

• Controlled Environment: CT explant cultures allow researchers to study PAG secretion under controlled conditions, enabling the manipulation of variables that might affect protein expression and secretion .

• Reduced Animal Use: This approach maximizes information obtained from limited sample availability, which is particularly important for protected species like European bison where invasive sampling is restricted .

• Temporal Profiling: CT explants can be maintained in culture for various durations, allowing researchers to study the kinetics of PAG secretion and responses to different stimuli over time.

• Protocol Optimization: For European bison studies, researchers should:

  • Collect placental tissues from ethically sourced specimens (veterinary cases, managed culls)

  • Prepare cotyledonary explants of consistent size (approximately 5-10 mg)

  • Culture in serum-free medium to avoid interference with PAG detection

  • Collect conditioned medium at regular intervals (24-72h)

  • Analyze secreted PAGs using techniques such as immunoprecipitation followed by mass spectrometry

• Comparative Applications: CT explant cultures from European bison can be directly compared with those from domestic cattle under identical conditions, providing insights into evolutionary adaptations of the PAG family .

• Glycosylation Studies: The N-glycodiversity of PAGs secreted by European bison CT explants can be characterized using lectins and glycosidases, providing insights into species-specific post-translational modifications .

This methodology has already revealed unique profiles of pecoran PAG protein families secreted by trophectoderm in wild bovine species, highlighting its utility for advancing our understanding of reproductive biology in European bison .

What are the optimal storage and handling conditions for recombinant European bison PAG 67A to maintain biological activity?

Maintaining the biological activity of recombinant European bison PAG 67A requires adherence to specific storage and handling protocols:

• Long-term Storage: Store at -20°C, or preferably at -80°C for extended preservation. The shelf life is approximately 6 months for liquid preparations and 12 months for lyophilized forms at these temperatures .

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (with 50% being optimal for longest preservation)

  • Prepare small working aliquots to minimize freeze-thaw cycles

• Working Conditions:

  • Store working aliquots at 4°C for a maximum of one week

  • Avoid repeated freeze-thaw cycles as they significantly reduce biological activity

  • When using in assays, maintain the protein at appropriate temperatures (typically 4°C or on ice) until use

• Quality Control:

  • Periodically verify protein activity using functional ELISA

  • Monitor purity by SDS-PAGE (should remain >85-90%)

  • Check for signs of degradation or aggregation before use in critical experiments

• Stability Considerations: The addition of stabilizing agents (beyond glycerol) may be necessary for certain applications. Consider including protease inhibitors when working with biological samples that might contain proteolytic enzymes.

Following these guidelines will ensure optimal performance in research applications and extend the usable lifetime of this valuable reagent.

What are the key considerations for designing antibodies against European bison PAG 67A for immunological research?

Designing effective antibodies against European bison PAG 67A requires careful consideration of several factors:

Studies in American Bison have shown that antibody selection significantly impacts PAG detection sensitivity, with some antibodies detecting PAGs in early pregnancy (0-45 days) while others are more effective in later stages .

How do PAG expression patterns in European bison compare with other bovine species throughout pregnancy?

PAG expression patterns show both similarities and notable differences between European bison and other bovine species throughout pregnancy:

Understanding these comparative patterns provides insights into the evolution of pregnancy signaling mechanisms across bovine species and can inform the development of species-specific pregnancy detection methods.

What insights can PAG protein diversity provide about the evolutionary relationships among Bovidae species?

PAG protein diversity offers valuable insights into evolutionary relationships among Bovidae species, serving as molecular markers for understanding placental evolution:

• Multigene Family Evolution: The PAG family represents a multigene family that has undergone significant expansion during the evolution of ruminants. European bison PAG 67A is part of this diverse family, with its sequence and structure reflecting evolutionary adaptations specific to the Bison lineage .

• Phylogenetic Relationships: Comparative analysis of PAG sequences, including European bison PAG 67A, can help reconstruct phylogenetic relationships among Bovidae species. The degree of sequence conservation or divergence correlates with evolutionary distance and can support or challenge existing taxonomic classifications .

• Functional Adaptations: N-glycodiversity patterns in PAGs secreted by cotyledonary explants differ among wild pecoran Bovidae taxa, suggesting species-specific adaptations in placental function . These differences may reflect:

  • Adaptations to different reproductive strategies (seasonal vs. year-round breeding)

  • Placental structure variations

  • Maternal-fetal immunological interactions

• Selection Pressures: The diversity and conservation patterns in PAG proteins provide evidence of different selection pressures acting on placental proteins. Some regions show high conservation (suggesting functional importance), while others show rapid evolution (possibly reflecting immunological adaptations) .

• Divergence Timing: Molecular clock analyses based on PAG sequence differences can help estimate the timing of divergence between European bison and other bovine species, contributing to our understanding of speciation events in the Bovidae family.

• Conservation Implications: Understanding the unique aspects of European bison PAG diversity has implications for conservation biology, particularly for managing genetic diversity in small populations and developing species-specific reproductive monitoring tools .

This molecular evidence complements morphological, behavioral, and ecological data in reconstructing the evolutionary history of bovine species, with European bison PAG 67A serving as one piece of this complex evolutionary puzzle.

What novel research applications might emerge from further characterization of European bison PAG 67A?

Further characterization of European bison PAG 67A could lead to several novel research applications with implications for conservation biology, reproductive science, and comparative physiology:

• Non-invasive Pregnancy Monitoring: Development of highly sensitive assays specific for European bison PAG 67A could enable non-invasive pregnancy detection using fecal or urinary samples, reducing handling stress in this endangered species . This would be particularly valuable for conservation breeding programs.

• Placental Health Biomarkers: PAG 67A variants or abnormal expression patterns might serve as indicators of placental dysfunction or pregnancy complications in European bison. Establishing normal reference ranges and identifying deviation patterns could improve reproductive management .

• Immunomodulatory Functions: Investigation of potential immunomodulatory roles of PAG 67A at the maternal-fetal interface could reveal novel aspects of pregnancy maintenance in wild bovine species. The aspartic proteinase activity suggests possible roles in protein processing or signaling pathways .

• Evolutionary Adaptation Markers: Comparative analysis of PAG 67A across European bison populations from different habitats could reveal potential adaptive variations linked to environmental conditions, providing insights into local adaptation mechanisms .

• Synthetic Biology Applications: The unique structural and functional properties of European bison PAG 67A might inspire biomimetic approaches for developing novel enzymatic tools or diagnostic platforms based on its binding or catalytic properties.

• Structure-Function Relationship Studies: Advanced structural characterization through techniques such as cryo-EM or X-ray crystallography could reveal specific structural features that determine the unique functional properties of European bison PAG 67A compared to related proteins.

• In vitro Models of Placentation: Using recombinant PAG 67A in trophoblast cell culture models could help develop in vitro systems for studying placental development and function in endangered species where direct tissue access is limited .

These future directions highlight the potential broader impacts of basic research on European bison PAG 67A beyond its current applications in reproductive biology.