Recombinant Bison bonasus Unknown placental glycoprotein 45J2

What is EbUPG45J2 and how was it first identified?

EbUPG45J2 (European bison Unknown Placental Glycoprotein, 45kDa J2) is a native glycoprotein isoform with the amino acid sequence SKDNQKNYIPLIVPFAT that was first identified in the placenta of European bison (Bison bonasus). It was discovered during studies examining pregnancy-associated glycoproteins (PAGs) in European bison placentas. This protein was initially classified as an "unknown placental glycoprotein" rather than a PAG because, despite efficient purification using Vicia villosa agglutinin (VVA) chromatography (which is specific for glycoproteins), it lacks significant sequence consensus with previously sequenced placental glycoproteins in the Bovidae family .

How does EbUPG45J2 differ from other placental glycoproteins identified in European bison?

EbUPG45J2 (45kDa) differs significantly from the pregnancy-associated glycoproteins (PAGs) identified in European bison. While the EbPAGs (named A through I) show sequence resemblance to internal sequences of various full-length polypeptide PAG precursors identified in domestic cattle, EbUPG45J2 lacks this consensus. It has a distinct NH₂-terminal sequence (SKDNQKNYIPLIVPFAT) compared to the PAGs, which typically begin with sequences like RGSNLTH or similar variations. EbUPG45J2 is also structurally related to two other UPGs identified: EbUPG45J1 (SKDNYKNYIPLIVPFAT) and EbUPG76K (SPEFTV), suggesting they may constitute a separate family of placental glycoproteins with potentially unique functions .

What is the recommended protocol for isolating native EbUPG45J2 from placental tissue?

The recommended protocol for isolating native EbUPG45J2 from European bison placental tissue involves a multi-step process:

• Collection of cotyledonary tissues from placentas (45-120 days post-coitum)

• Tissue extraction and homogenization

• Protein precipitation

• Ion-exchange chromatography using DEAE (Diethylaminoethyl)

• Affinity chromatography using VVA (Vicia villosa agglutinin), which specifically binds glycoproteins

• Electrophoretic separation through 1D- and 2D-PAGE

• Identification through heterologous (cross-species) Western blotting

• Final characterization by Edman degradation microsequencing

This approach successfully isolated VVA-purified isoforms with isoelectric points ranging from 3.7-7.4, including EbUPG45J2 (SKDNQKNYIPLIVPFAT). The VVA affinity chromatography step is particularly crucial as it selects for glycoproteins and helps separate UPGs from other placental proteins .

What challenges might researchers encounter when attempting to produce recombinant EbUPG45J2?

Researchers attempting to produce recombinant EbUPG45J2 may face several significant challenges:

• Incomplete Sequence Information: Only the NH₂-terminal sequence (SKDNQKNYIPLIVPFAT) is currently known. The complete amino acid sequence and corresponding gene sequence need to be determined before recombinant production.

• Post-translational Modifications: As a glycoprotein efficiently purified by VVA chromatography, EbUPG45J2 contains critical glycosylation patterns that may be difficult to reproduce in heterologous expression systems.

• Expression System Selection: Choosing an appropriate expression system that can properly fold and glycosylate the protein is crucial. While bacterial systems (E. coli) offer high yield, they lack appropriate glycosylation machinery. Mammalian, insect, or yeast expression systems may be more appropriate but present their own challenges.

• Purification Strategy: Without knowing the complete protein characteristics, designing an efficient purification strategy for the recombinant protein may require significant optimization.

• Functional Validation: Confirming that the recombinant protein retains the structural and functional properties of the native protein will require development of appropriate assays.

Researchers have successfully expressed recombinant versions of related PAGs in other species, which could provide methodological guidance .

How can researchers validate the identity and purity of isolated EbUPG45J2?

Validating the identity and purity of isolated EbUPG45J2 requires a multi-analytical approach:

• N-terminal Sequencing: Confirm the characteristic NH₂-terminal sequence (SKDNQKNYIPLIVPFAT) through Edman degradation.

• Mass Spectrometry: Perform LC-MS/MS analysis to verify mass accuracy and potential identification of additional peptide fragments.

• Western Blotting: Develop specific antibodies against EbUPG45J2 or use cross-reactive antibodies if available. Previous research successfully employed heterologous (cross-species) Western blotting.

• 2D Electrophoresis: Confirm the molecular weight (45kDa) and isoelectric point through 2D-PAGE analysis.

• Glycosylation Analysis: Confirm the glycoprotein nature through lectin binding assays, particularly using Vicia villosa agglutinin (VVA).

• Functional Assays: Develop and apply functional tests based on hypothesized biological activity (though these are still being established for EbUPG45J2).

The research showed that combining these methods provides reliable identification and purity assessment, with 2D-PAGE being particularly valuable for distinguishing EbUPG45J2 from the similar EbUPG45J1, which differs by only one amino acid in the known sequence .

What structural features distinguish EbUPG45J2 from other placental glycoproteins?

EbUPG45J2 possesses several distinctive structural features compared to other placental glycoproteins:

• Unique N-terminal Sequence: The NH₂-terminal sequence (SKDNQKNYIPLIVPFAT) is significantly different from the PAGs identified in European bison and other Bovidae species, which typically begin with sequences like RGSNLTH.

• Molecular Weight and Isoelectric Point: EbUPG45J2 has a molecular weight of approximately 45kDa, differing from the range of PAGs identified (50-71kDa). Its isoelectric point falls within the 3.7-7.4 range of the purified glycoproteins.

• Glycosylation Pattern: While specific glycosylation details are not fully characterized, its efficient purification through VVA affinity chromatography indicates distinctive glycosylation patterns compared to PAGs.

• High Sequence Similarity to EbUPG45J1: EbUPG45J2 shares high sequence similarity with EbUPG45J1, differing by only one amino acid (Q versus Y at position 5 of the N-terminal sequence), suggesting they may be variants of the same protein or products of closely related genes .

What hypotheses exist regarding the biological function of EbUPG45J2?

While the precise biological function of EbUPG45J2 remains to be elucidated, several hypotheses can be formulated based on the available data:

• Placental Development and Maintenance: Given its expression in cotyledonary tissue during early and mid-pregnancy (45-120 dpc), EbUPG45J2 may play a role in placental development, implantation, or maintenance.

• Immune Modulation: Many placental glycoproteins function in maternal-fetal immune tolerance. EbUPG45J2 might participate in protecting the conceptus from the maternal immune system.

• Proteolytic Activity or Regulation: While PAGs evolved from aspartic proteinases but mostly lack proteolytic activity, EbUPG45J2's different sequence suggests it might have retained or developed unique enzymatic properties.

• Signaling Role: The protein may function as a signaling molecule in pregnancy recognition or maintenance, similar to functions proposed for some PAGs.

• Species-Specific Adaptation: As part of a unique glycoprotein profile in endangered European bison, EbUPG45J2 might represent a species-specific adaptation related to reproduction in this species.

Further functional studies examining protein-protein interactions, enzymatic activity, and expression patterns throughout pregnancy will be necessary to confirm these hypotheses .

What methods are recommended for analyzing post-translational modifications of EbUPG45J2?

For comprehensive analysis of post-translational modifications (PTMs) in EbUPG45J2, researchers should employ multiple complementary approaches:

• Glycosylation Analysis:

  • Lectin microarray profiling to identify glycan structures

  • Mass spectrometry with glycopeptide enrichment to map glycosylation sites

  • Enzymatic deglycosylation (PNGase F, O-glycosidase) followed by mobility shift analysis

  • Glycan-specific staining of gels (PAS staining)

• Mass Spectrometry-Based Approaches:

  • High-resolution MS/MS for comprehensive PTM mapping

  • Electron transfer dissociation (ETD) to preserve labile modifications

  • Multiple reaction monitoring (MRM) for targeted analysis of specific modifications

• Site-Directed Mutagenesis:

  • Systematic mutation of predicted modification sites in recombinant proteins

  • Functional assessment of modified vs. unmodified protein

• Specific PTM Detection:

  • Phosphorylation: Phospho-specific antibodies, Pro-Q Diamond staining

  • Ubiquitination/SUMOylation: Specific antibodies and enrichment strategies

Research on bovine PAGs has shown that these proteins often contain asparagine-linked N-acetylgalactosaminyl glycans, suggesting similar analysis approaches may be valuable for EbUPG45J2 .

How does EbUPG45J2 compare to similar proteins identified in other Bovidae species?

EbUPG45J2 appears to represent a distinct glycoprotein with limited similarity to proteins characterized in other Bovidae species:

The uniqueness of EbUPG45J2 is evident from the statement that it "lacks considerable consensus to previously sequenced placental glycoproteins in the Bovidae family." This suggests it may represent either a European bison-specific innovation or a protein that has diverged significantly from a common ancestral protein. More comprehensive genome and proteome analysis across Bovidae species would help clarify evolutionary relationships .

What genomic evidence exists for EbUPG45J2 in the recently assembled wisent (Bison bonasus) genome?

The recently assembled wisent (Bison bonasus) genome provides an opportunity to investigate the genomic basis of EbUPG45J2, though specific evidence for this protein is not directly addressed in the current search results. The HiFi read-based wisent assembly with a contig N50 value of 91 Mb containing 99.7% of highly conserved single-copy mammalian genes represents a thousand-fold improvement in contiguity over previous assemblies .

Relevant considerations for researchers investigating the genomic basis of EbUPG45J2 include:

• Extended Runs of Homozygosity: The wisent genome shows exceptional levels of genomic inbreeding with extended runs of homozygosity (ROH) covering large fractions of the genome. This high inbreeding might influence the genetic diversity of placental glycoproteins including EbUPG45J2.

• Comparative Genomic Analysis: The bovine super-pangenome built with assemblies from wisent, bison, gaur, yak, and cattle identified species-specific genomic features, such as the deletion removing the THRSP gene. Similar comparative approaches could help identify genes encoding EbUPG45J2.

• Future Research Directions: Researchers should leverage this genome assembly to:

  • Identify candidate genes for EbUPG45J2 based on the known N-terminal sequence

  • Analyze expression patterns in placental tissues

  • Compare with related species to understand evolutionary history

  • Investigate potential structural variations affecting UPG genes

Collaborations between genomics researchers and protein biochemists will be essential to connect the protein-level observations with genomic evidence .

What evolutionary insights can be gained from studying EbUPG45J2 in the context of conservation biology?

Studying EbUPG45J2 in the context of European bison conservation biology offers several valuable evolutionary insights:

• Species-Specific Reproductive Adaptations: The identification of unique placental glycoproteins like EbUPG45J2 in European bison suggests species-specific adaptations in reproductive biology. Understanding these adaptations may inform conservation strategies for this endangered species listed on the IUCN Red List.

• Impact of Population Bottlenecks: European bison experienced severe population bottlenecks, with all living individuals descended from just 12 founders. The genomic evidence shows extensive runs of homozygosity, indicating high inbreeding. Studying how this genetic homogeneity affects placental protein diversity, including EbUPG45J2 variants, could reveal how reproductive proteins evolve under extreme selective pressures.

• Comparative Reproductive Biology: Comparing EbUPG45J2 with proteins from related species can illuminate how reproductive proteins diverge through speciation events in the Bovidae family, potentially revealing patterns of selection unique to European bison.

• Functional Conservation vs. Sequence Divergence: The unique sequence of EbUPG45J2 contrasted with its presence in placental tissue suggests a case where functional roles may be conserved despite sequence divergence, or alternatively, the evolution of novel functions specific to European bison reproduction.

• Biomarkers for Reproductive Health: Understanding the normal expression and function of EbUPG45J2 could help develop biomarkers for monitoring reproductive health in captive and wild European bison populations, contributing to conservation management.

This research intersects reproductive biology, conservation genetics, and evolutionary biology, potentially yielding insights into how endangered species adapt reproductively under genetic constraints .

How might EbUPG45J2 be utilized as a biomarker for pregnancy detection or monitoring in European bison?

EbUPG45J2 shows significant potential as a biomarker for pregnancy detection and monitoring in European bison, with several practical applications for wildlife management and conservation:

• Development of Specific Immunoassays:

  • Develop EbUPG45J2-specific antibodies through recombinant protein production

  • Create ELISA, RIA, or lateral flow immunoassays for field use

  • Validate assay sensitivity and specificity across different pregnancy stages

• Non-invasive Sampling Approaches:

  • Investigate presence of EbUPG45J2 in peripheral circulation during pregnancy

  • Assess potential for detection in non-invasive samples (urine, feces)

  • Determine temporal expression profile throughout gestation

• Comparative Diagnostic Value:

  • Compare diagnostic accuracy with established PAG-based pregnancy tests

  • Determine if EbUPG45J2 offers advantages for early pregnancy detection

  • Assess correlation between EbUPG45J2 levels and pregnancy outcomes

• Integration with Conservation Management:

  • Develop field-deployable testing for wildlife management

  • Monitor reproductive rates in free-ranging populations

  • Assess reproductive success in captive breeding programs

Research on PAGs in domestic cattle has demonstrated their value as pregnancy biomarkers, with commercial tests available. Similar applications for EbUPG45J2 would require establishing its secretion patterns, detection limits, and correlation with pregnancy stages and outcomes in European bison .

What expression systems would be most suitable for producing functional recombinant EbUPG45J2?

Selecting the optimal expression system for producing functional recombinant EbUPG45J2 requires careful consideration of protein characteristics, particularly its glycosylation patterns:

Given that EbUPG45J2 is efficiently purified by VVA lectin chromatography, indicating important glycosylation features, mammalian or insect cell systems would likely be most appropriate for producing functional recombinant protein. For complete functional characterization, starting with a mammalian expression system (particularly CHO cells) would provide the best chance of obtaining properly folded and glycosylated protein, though at higher cost and lower yield .

What experimental approaches could elucidate the functional role of EbUPG45J2 in placental development?

To elucidate the functional role of EbUPG45J2 in placental development, researchers should employ a multi-faceted experimental strategy:

• Spatiotemporal Expression Analysis:

  • Immunohistochemistry to localize EbUPG45J2 in different placental cell types

  • qRT-PCR to quantify expression throughout pregnancy

  • In situ hybridization to identify sites of mRNA expression

  • Single-cell RNA sequencing of placental tissues to identify producing cell populations

• Protein Interaction Studies:

  • Yeast two-hybrid screening to identify binding partners

  • Co-immunoprecipitation to confirm interactions in placental tissue

  • Surface plasmon resonance to measure binding affinities

  • Proximity labeling (BioID, APEX) to identify proteins in close proximity

• Functional Assays:

  • Cell migration and invasion assays with recombinant protein

  • Angiogenesis assays to assess effects on vascular development

  • Immune cell response assays to test immunomodulatory functions

  • Trophoblast cell culture models to assess effects on differentiation

• Comparative Studies:

  • Ex vivo placental explant cultures from related species

  • Comparative analysis of effects across bovid species

  • Correlation between expression patterns and placental morphology

• In Silico Analysis:

  • Structural modeling to predict functional domains

  • Molecular dynamics simulations to assess protein-protein interactions

  • Evolutionary analysis to identify conserved functional regions

These approaches would need to be adapted to the challenges of working with an endangered species, potentially utilizing ex vivo systems, comparative approaches with more accessible species, and maximizing information obtained from limited samples available during conservation management activities .

What are the primary technical challenges in studying EbUPG45J2 and how might they be overcome?

Studying EbUPG45J2 presents several significant technical challenges that researchers must address:

• Limited Sample Availability:

  • Challenge: European bison is an endangered species with restricted access to placental samples.

  • Solutions: Develop sampling protocols integrated with conservation management; establish tissue banks; optimize methods for small sample quantities; utilize samples obtained during routine veterinary care.

• Incomplete Sequence Information:

  • Challenge: Only the N-terminal sequence is currently known.

  • Solutions: Combine protein sequencing with genomic approaches; employ de novo protein sequencing using high-resolution mass spectrometry; develop computational approaches to predict full sequence from related genes.

• Lack of Species-Specific Reagents:

  • Challenge: Few antibodies or molecular tools exist specifically for European bison proteins.

  • Solutions: Develop cross-reactive tools validated with positive controls; produce recombinant protein fragments for antibody generation; adapt techniques from closely related species.

• Limited In Vitro Models:

  • Challenge: Lack of established European bison placental cell lines.

  • Solutions: Develop primary culture methods from available placental tissue; explore cross-species trophoblast models; establish explant cultures when samples become available.

• Functional Characterization:

  • Challenge: Difficulty establishing physiological roles without in vivo manipulation.

  • Solutions: Develop ex vivo functional assays; pursue comparative approaches with related species; utilize natural variation in expression to correlate with reproductive outcomes .

How might advanced genomic approaches contribute to our understanding of EbUPG45J2?

Advanced genomic approaches offer powerful opportunities to expand our understanding of EbUPG45J2:

• Whole Genome Analysis:

  • Leverage the new high-quality wisent genome assembly (contig N50 of 91 Mb) to identify candidate genes encoding EbUPG45J2

  • Compare genomic regions with those of related species to identify conserved regulatory elements

  • Analyze copy number variations and gene family expansions/contractions

• Transcriptomics:

  • Perform RNA-Seq on placental tissues across pregnancy stages to identify expression patterns

  • Use single-cell RNA-Seq to determine cell-specific expression

  • Long-read sequencing to identify splice variants and isoforms

• Epigenomics:

  • Analyze methylation patterns and chromatin accessibility in placental tissues

  • Identify potential regulatory elements controlling EbUPG45J2 expression

  • Study histone modifications associated with active transcription

• Comparative Genomics:

  • Analyze the bovine super-pangenome to identify species-specific genetic features

  • Study impacts of the high inbreeding coefficient and extended runs of homozygosity on genetic diversity of placental glycoproteins

  • Compare with American bison to understand evolutionary divergence

• Functional Genomics:

  • Develop methodologies to predict protein function from genomic data

  • Integrate genomic data with proteomic findings

  • Identify potential non-coding RNAs regulating expression

These approaches would be particularly valuable given the recent thousand-fold improvement in genome assembly quality for European bison and could help overcome limitations in sample availability by maximizing information obtained from available specimens .

What collaborative research approaches would advance our understanding of EbUPG45J2's role in European bison reproduction?

Advancing our understanding of EbUPG45J2 requires coordinated collaborative research approaches spanning multiple disciplines and institutions:

• Interdisciplinary Research Teams:

  • Reproductive Biologists: Expertise in placental development and function

  • Conservation Biologists: Access to animals and ecological context

  • Genomics/Proteomics Specialists: Technical capabilities for molecular characterization

  • Veterinary Scientists: Clinical perspectives and sample collection

  • Bioinformaticians: Data integration and analysis

• Inter-Institutional Collaboration Framework:

  • Coordinate among European bison breeding centers and wildlife reserves

  • Develop sample collection and sharing protocols compliant with conservation regulations

  • Establish biobanking initiatives for long-term storage of precious samples

  • Create standardized methodologies for comparative studies

• Integrated Research Approaches:

  • Link field observations of reproductive success with molecular markers

  • Connect ex vivo functional studies with in vivo physiological observations

  • Integrate genomic findings with proteomic and glycomic analyses

  • Compare findings across related bovid species to identify conserved vs. unique features

• Knowledge Sharing Platforms:

  • Develop shared databases of European bison reproductive proteins

  • Create repositories for methodological approaches and protocols

  • Establish regular workshops for researchers studying wildlife reproduction

  • Implement cross-training opportunities between molecular and field researchers

• Conservation Applications:

  • Translate research findings into practical tools for reproductive management

  • Develop non-invasive monitoring methods based on research outcomes

  • Create prediction models for reproductive success incorporating molecular markers

This collaborative framework would maximize research outcomes while respecting the endangered status of European bison and ensuring research directly contributes to conservation efforts .

What is the current consensus on the importance of EbUPG45J2 in European bison pregnancy?

The current research indicates that EbUPG45J2 represents a unique placental glycoprotein in European bison with potential significance for pregnancy biology, though its precise role remains to be fully elucidated. As a distinct glycoprotein efficiently purified by VVA lectin chromatography but lacking sequence consensus with known placental glycoproteins in the Bovidae family, EbUPG45J2 may represent either a lineage-specific innovation or a highly diverged member of a conserved protein family. Its identification in placental tissues during early and mid-pregnancy (45-120 dpc) suggests functional relevance to this critical reproductive period.

The scientific consensus points to EbUPG45J2 being part of the complex molecular dialogue at the maternal-fetal interface in European bison. While not definitively characterized as a pregnancy-associated glycoprotein (PAG), its presence alongside multiple PAG isoforms in the synepitheliochorial cotyledonary placenta suggests complementary functions in pregnancy establishment, maintenance, or progression. The ongoing research to characterize this protein takes on additional significance given the endangered status of European bison and the importance of understanding their reproductive biology for conservation efforts .

What are the most promising future research directions for EbUPG45J2 studies?

The most promising future research directions for EbUPG45J2 studies fall into several interconnected categories:

• Comprehensive Molecular Characterization:

  • Complete sequence determination through advanced proteomics and genomics

  • Detailed glycosylation profiling to understand post-translational modifications

  • Crystal structure determination to provide insights into protein function

  • Gene identification and regulatory element characterization

• Functional Studies:

  • Development of recombinant protein production systems

  • Cell-based assays to determine effects on trophoblast and endometrial cells

  • Receptor identification and signaling pathway elucidation

  • Comparative functional analysis with related proteins in other bovids

• Reproductive Biology Applications:

  • Development of EbUPG45J2-based pregnancy diagnostic tools

  • Correlation of protein levels with pregnancy outcomes

  • Investigation of potential roles in pregnancy complications

  • Integration with other biomarkers for comprehensive reproductive monitoring

• Conservation Biology Integration:

  • Application of findings to improve reproductive management in captive populations

  • Development of non-invasive monitoring approaches for wild populations

  • Correlation of genetic diversity with protein variants and reproductive success

  • Transfer of methodologies to other endangered bovid species