Recombinant Bovine Ankyrin repeat domain-containing protein 46 (ANKRD46)

What is the basic structure of bovine ANKRD46?

ANKRD46 (Ankyrin repeat domain-containing protein 46) is a 228 amino acid protein in bovine species that contains multiple ankyrin repeat motifs, which are typically involved in protein-protein interactions. The bovine ANKRD46 protein contains these characteristic ankyrin repeat domains that form helix-turn-helix structures with a beta-hairpin/loop region projecting outward from the helices. The complete amino acid sequence includes MSYVFVNDSSQTNVPLLQACIDGDFNYSKRLLESGFDPNIRDSRGRTGLHLAAARGNVDICQLLHKFGADLLATDYQGNTALHLCGHVDTIQFLVSNGLKIDICNHQGATPLVLAKRRGVNKDVIRLLESLEEQEVKGFNRGTHSKLETMQTAESESAMESHSLLNPNLQQGEGVLSSFRTWQEFVEDLGFWRVLLLIFVIALLSLGIAYYVSGVLPFVENQPELVH . This structure suggests roles in cellular signaling and protein complex formation.

How is ANKRD46 genetically conserved across species?

ANKRD46 shows significant evolutionary conservation, indicating its biological importance. Comparative genomics reveal homologs across mammalian species including humans (chromosome 8q22.2), mice, and other vertebrates like Xenopus . This conservation suggests fundamental roles in cellular processes. Mouse Ankrd46 has been mapped and characterized with potential phenotypic impacts across multiple physiological systems including cardiovascular, nervous, and reproductive systems . The high degree of conservation provides researchers with opportunities to use model organisms for investigating ANKRD46 functions relevant to human and bovine biology.

What experimental approaches can verify ANKRD46's subcellular localization?

To determine ANKRD46's subcellular localization, researchers should employ multiple complementary techniques:

• Immunofluorescence microscopy: Using validated ANKRD46-specific antibodies with co-staining for organelle markers

• Cell fractionation followed by Western blotting: To quantitatively assess distribution across subcellular compartments

• Expression of fluorescently-tagged ANKRD46: Using constructs with GFP or other fluorescent proteins fused to either N- or C-terminus (verifying that tags don't disrupt targeting)

• Electron microscopy with immunogold labeling: For high-resolution localization studies

The transmembrane prediction in the protein sequence (positions 182-202) suggests potential membrane localization that should be experimentally verified . When designing these experiments, researchers should include appropriate controls and validate findings with multiple techniques to avoid artifacts from overexpression systems.

How does ANKRD46 function in embryo implantation?

ANKRD46 has been identified as a direct target of miR-451, which is specifically upregulated during the embryo implantation period. Research has demonstrated that ANKRD46 plays a crucial role in this process through its interaction with miR-451 . In mouse models, a dual-luciferase activity assay confirmed that Ankrd46 is directly targeted by miR-451. The functional significance of this interaction was demonstrated when loss-of-function experiments using LV-miR-451 sponge or miR-451 inhibitors resulted in a reduced number of embryo implantations without affecting fertilization . This suggests that the miR-451/ANKRD46 regulatory axis specifically influences the implantation process, potentially through modulating uterine receptivity or embryo-uterine interactions.

What methodological approaches are most effective for studying ANKRD46 in implantation?

For investigating ANKRD46's role in implantation, researchers should consider these methodological approaches:

• Laser-capture microdissection: To isolate luminal epithelium during specific implantation timepoints, as demonstrated in mouse models

• Model systems: Establish and utilize pseudopregnancy, delayed implantation, and artificial decidualization models for comparative analysis

• Expression analysis: Use qRT-PCR to validate miRNA and ANKRD46 expression patterns across implantation timepoints

• Functional validation:

  • Loss-of-function approaches: CRISPR/Cas9 knockout, miRNA inhibitors, or target protectors

  • Gain-of-function studies: Overexpression constructs or miRNA mimics

• Mechanistic determination: Luciferase reporter assays to confirm direct miRNA targeting of ANKRD46 3'UTR

These approaches can be combined with in vivo implantation models to assess embryo implantation rates under various experimental conditions, similar to the methodology reported for miR-451 functional studies .

How can contradictory data on ANKRD46 expression patterns during implantation be reconciled?

When facing contradictory data regarding ANKRD46 expression during implantation, researchers should:

• Examine temporal specificity: ANKRD46 expression may vary dramatically within narrow time windows during the implantation period. Ensure precise staging of samples (hours rather than days)

• Consider spatial heterogeneity: Expression may differ between luminal epithelium, stroma, and other uterine compartments. Use techniques like laser-capture microdissection to isolate specific cell populations

• Evaluate experimental model differences: Compare natural pregnancy versus pseudopregnancy or delayed implantation models

• Assess post-transcriptional regulation: Measure both mRNA and protein levels, as miR-451 regulation may affect translation without changing mRNA abundance

• Validate with multiple techniques: Combine qRT-PCR, Western blotting, and in situ hybridization/immunohistochemistry to confirm expression patterns

For rigorous validation, time-course experiments with at least 3-6 biological replicates at each timepoint should be performed, with appropriate statistical analysis to account for biological variation.

What strategies should be employed for ANKRD46 knockout using CRISPR/Cas9?

For effective ANKRD46 knockout using CRISPR/Cas9 technology, researchers should consider the following comprehensive strategy:

• Guide RNA design: Target 5' constitutive exons of ANKRD46 to ensure complete functional disruption. Commercial ANKRD46 CRISPR/Cas9 KO plasmids consist of a pool of 3 plasmids, each encoding Cas9 nuclease and target-specific 20 nt guide RNA designed for maximum knockout efficiency

• Delivery optimization:

  • For cell lines: Lipofection, electroporation, or viral delivery

  • For in vivo studies: Viral vectors (AAV, lentivirus) or direct embryonic manipulation

• Knockout verification strategy:

  • Genomic level: PCR amplification and sequencing of target region

  • Transcript level: RT-PCR and qRT-PCR

  • Protein level: Western blotting with validated antibodies

• Control design:

  • Negative controls: Non-targeting gRNA constructs

  • Rescue experiments: Re-expression of ANKRD46 to confirm phenotype specificity

• Clone isolation: Single-cell isolation and expansion to establish homogeneous knockout populations

This approach has been successfully employed in generating MDBK CD46-knockout cell lines and similar methodologies can be adapted for ANKRD46 .

How can off-target effects be minimized in ANKRD46 CRISPR experiments?

Minimizing off-target effects in ANKRD46 CRISPR experiments requires a multi-faceted approach:

• Improved guide RNA design:

  • Use algorithms that maximize specificity (e.g., CRISPOR, Cas-OFFinder)

  • Prioritize guides with minimal predicted off-target sites, especially those with fewer than 3 mismatches

  • Consider truncated guides (17-18nt) which can reduce off-target effects while maintaining on-target efficiency

• Enhanced Cas9 variants:

  • Use high-fidelity Cas9 variants (SpCas9-HF1, eSpCas9, HypaCas9)

  • Consider Cas9 nickase paired with dual guides for improved specificity

• Controlled delivery parameters:

  • Optimize Cas9:gRNA ratios

  • Use transient rather than constitutive Cas9 expression

  • Consider ribonucleoprotein (RNP) delivery for shorter Cas9 exposure

• Comprehensive validation:

  • Sequence verification of on-target modifications

  • Genome-wide off-target analysis (GUIDE-seq, CIRCLE-seq, or whole genome sequencing)

  • Use multiple independent guide RNAs and confirm consistent phenotypes

• Controls and rescue experiments:

  • Include validation with alternative knockout methods (e.g., RNAi)

  • Perform rescue experiments with wildtype ANKRD46 cDNA containing synonymous mutations at the guide RNA binding sites

These strategies collectively minimize the risk of misinterpreting ANKRD46 knockout phenotypes due to off-target effects.

What is the connection between ANKRD46 and metabolic-cognitive disorders?

Research has identified ANKRD46 as a co-expressed gene in both mild cognitive impairment (MCI) and type 2 diabetes mellitus (T2DM), suggesting its potential role in the mechanistic link between these conditions . Quantitative RT-PCR validation confirmed that ANKRD46 expression patterns identified through bioinformatic analysis of GEO databases were consistent in clinical samples. This places ANKRD46 among several genes (including LNX2, BIRC6, TGFB1, PSEN1, and ALDH2) that may represent molecular connections between cognitive dysfunction and metabolic disorders .

The identification of ANKRD46 in this context provides potential new therapeutic targets for both diagnosis and treatment of these interconnected conditions. While the exact molecular mechanisms remain to be fully elucidated, the co-expression patterns suggest ANKRD46 may function in signaling pathways relevant to both glucose metabolism and neuronal function, possibly through protein-protein interactions mediated by its ankyrin repeat domains.

What experimental approaches can verify ANKRD46's role in disease mechanisms?

To verify ANKRD46's role in disease mechanisms, researchers should implement a multi-faceted experimental approach:

• Expression correlation studies:

  • qRT-PCR validation in patient cohorts with both MCI and T2DM compared to single-condition patients and healthy controls

  • Protein-level validation via Western blotting and immunohistochemistry

  • Single-cell RNA sequencing to identify cell type-specific expression patterns

• Functional genomics:

  • CRISPR/Cas9 knockout or knockdown in relevant cell types (neurons, pancreatic β-cells)

  • Phenotypic analysis of glucose metabolism and neuronal function in modified cells

  • Rescue experiments with wildtype or mutant ANKRD46

• Animal models:

  • Conditional and tissue-specific knockout mouse models evaluating both metabolic and cognitive parameters

  • Analyses across multiple timepoints to establish temporal relationships

• Mechanistic investigations:

  • Interactome analysis using co-immunoprecipitation followed by mass spectrometry

  • Phosphoproteomics to identify post-translational modifications

  • Pathway analysis integrating transcriptomic and proteomic data

• Translational validation:

  • Correlation of ANKRD46 expression/variants with clinical parameters in patient cohorts

  • Testing of potential therapeutic approaches targeting ANKRD46 or its pathways

This comprehensive approach would provide robust evidence for ANKRD46's functional roles in disease mechanisms linking cognitive and metabolic disorders .

What are the optimal expression systems for producing functional recombinant bovine ANKRD46?

For producing functional recombinant bovine ANKRD46, researchers should consider multiple expression systems, each with distinct advantages for different experimental applications:

Expression System Comparison for Recombinant Bovine ANKRD46 Production

How can the functional activity of recombinant ANKRD46 be verified?

Verifying the functional activity of recombinant ANKRD46 requires multiple complementary approaches:

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Size-exclusion chromatography to assess oligomerization state

  • Limited proteolysis to evaluate folding quality

• Protein-protein interaction assays:

  • Pull-down assays with known or predicted interaction partners

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Yeast two-hybrid or mammalian two-hybrid screening to identify novel interactors

• Functional complementation:

  • Rescue experiments in ANKRD46 knockout or knockdown models

  • Assays based on known functions (e.g., embryo implantation models if studying reproductive roles)

• Target binding validation:

  • Assessment of miR-451 targeting using luciferase reporter assays

  • Confirmation of protein complex formation via co-immunoprecipitation

• Cell-based functional assays:

  • Overexpression in relevant cell types followed by phenotypic assays

  • Subcellular localization studies with tagged recombinant protein to confirm proper targeting

When designing these validation experiments, researchers should include appropriate controls including inactive mutants (e.g., mutations in key ankyrin repeats) and comparative analysis with native ANKRD46 where possible.

What are the critical factors for successful purification of recombinant bovine ANKRD46?

Successful purification of recombinant bovine ANKRD46 requires careful optimization of several critical factors:

• Solubility enhancement strategies:

  • Optimize expression temperature (typically 16-25°C for improved folding)

  • Co-expression with chaperones (GroEL/ES, DnaK, etc.)

  • Fusion tags beyond purification tags (e.g., SUMO, MBP, or Trx)

  • Specialized lysis buffers with mild detergents if transmembrane regions are present

• Purification protocol optimization:

  • For His-tagged ANKRD46: IMAC purification with optimized imidazole gradients

  • Secondary purification step (ion exchange or size exclusion chromatography)

  • Consider on-column refolding protocols if inclusion bodies form

  • Detergent screening if membrane association occurs

• Stability considerations:

  • Buffer optimization through thermal shift assays

  • Addition of stabilizing agents (glycerol, specific ions, reducing agents)

  • Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • Aliquoting to avoid freeze-thaw cycles

• Quality control assessments:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Mass spectrometry for accurate mass determination

  • Dynamic light scattering to assess aggregation state

  • Endotoxin testing if intended for cell-based assays

• Tag removal considerations:

  • If tag cleavage is performed, optimize protease digestion conditions

  • Perform secondary purification after tag removal

  • Verify activity retention following tag removal

For optimal results with recombinant bovine ANKRD46, it's recommended to store the final product at -20°C/-80°C, with lyophilized preparations showing longer shelf life (12 months) compared to liquid formulations (6 months) .

How can ANKRD46 be effectively studied in viral receptor interactions?

While ANKRD46 itself has not been directly implicated in viral entry, methodologies similar to those used for studying CD46 (a confirmed receptor for Bovine Viral Diarrhea Virus) can be adapted for investigating potential ANKRD46 involvement in viral interactions:

• Knockout cell line generation:

  • Develop stable ANKRD46 knockout cell lines using CRISPR/Cas9 technology similar to the MDBK CD46-knockout model

  • Create isogenic control lines to minimize experimental variation

  • Validate knockout at genomic, transcript, and protein levels

• Viral challenge experiments:

  • Perform comparative infection assays between wildtype and knockout cells

  • Analyze infection rates, viral replication kinetics, and cytopathic effects

  • Conduct sequential passaging to identify potential adaptive mutations

• Binding studies:

  • Direct binding assays between recombinant ANKRD46 and viral proteins

  • Competition assays with known receptor molecules

  • Surface plasmon resonance or ELISA-based quantification

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography of ANKRD46-viral protein complexes

  • Molecular modeling and docking simulations

  • Mutagenesis of key interaction residues based on structural data

• Receptor dynamics:

  • Live-cell imaging to track ANKRD46 during viral entry process

  • Analysis of ANKRD46 redistribution upon viral exposure

  • Co-localization studies with viral proteins during entry

These approaches can determine whether ANKRD46 functions as a viral receptor, co-receptor, or has indirect roles in viral infection processes .

What approaches should be used to investigate tissue-specific functions of ANKRD46?

Investigating tissue-specific functions of ANKRD46 requires a comprehensive strategy that combines genomic, transcriptomic, and functional approaches:

• Expression profiling across tissues:

  • Analyze existing databases (e.g., MGI for mouse orthologs) for expression patterns across tissues

  • Perform qRT-PCR on diverse tissue panels for both mRNA and potential isoforms

  • Conduct immunohistochemistry to determine cellular and subcellular localization

• Tissue-specific knockout models:

  • Generate conditional knockouts using Cre-loxP systems under tissue-specific promoters

  • Analyze phenotypes across multiple physiological systems (cardiovascular, nervous, reproductive, etc.)

  • Compare with global knockout phenotypes to identify tissue-autonomous effects

• Single-cell resolution approaches:

  • Perform single-cell RNA sequencing of tissues with ANKRD46 expression

  • Identify cell populations with enriched expression

  • Correlate with cell type-specific markers and functions

• In vitro modeling:

  • Derive primary cells or use tissue-specific cell lines for ANKRD46 modulation

  • Analyze phenotypic changes in tissue-relevant functional assays

  • Perform rescue experiments with tissue-specific isoforms

• Proteomics analysis:

  • Conduct tissue-specific interactome studies using proximity labeling

  • Compare ANKRD46 interaction partners across different tissues

  • Identify tissue-specific post-translational modifications

These approaches can uncover how ANKRD46 functions may vary across adipose tissue, nervous system, cardiovascular system, and other tissues, as suggested by phenotypic data from mouse models .

How do post-translational modifications affect ANKRD46 function?

While specific post-translational modifications (PTMs) of ANKRD46 have not been extensively characterized in the provided literature, researchers investigating PTMs should employ these approaches:

• Computational prediction and mass spectrometry verification:

  • In silico analysis using PTM prediction algorithms to identify potential sites

  • Targeted mass spectrometry to verify predicted modifications

  • Phosphoproteomics, ubiquitylome, and other PTM-specific enrichment approaches

• Site-directed mutagenesis studies:

  • Generate point mutations at predicted PTM sites

  • Compare functional outcomes between wildtype and mutant proteins

  • Create phosphomimetic mutants (e.g., S/T to D/E) to mimic constitutive phosphorylation

• Regulation analysis:

  • Investigate PTM changes in response to relevant stimuli (e.g., hormonal changes during implantation period)

  • Identify kinases, phosphatases, or other modifying enzymes using inhibitor studies

  • Determine half-life and stability differences between modified forms

• Structural implications:

  • Model structural changes induced by PTMs using molecular dynamics simulations

  • Analyze effects on protein-protein interactions

  • Determine if PTMs affect subcellular localization, particularly regarding potential membrane association

• Functional consequences:

  • Compare activity of differentially modified ANKRD46 in relevant assays

  • Assess impact on miR-451 targeting efficiency in embryo implantation models

  • Evaluate effects on protein complex formation and signaling pathway activity

Understanding PTMs will provide critical insights into how ANKRD46 function is dynamically regulated in different physiological contexts, particularly during processes like embryo implantation where timing of protein activity is crucial.

How can inconsistent antibody specificity for ANKRD46 detection be addressed?

Researchers facing challenges with ANKRD46 antibody specificity should implement a systematic validation strategy:

• Multi-antibody validation approach:

  • Test multiple antibodies targeting different epitopes of ANKRD46

  • Validate with positive controls (overexpression systems)

  • Confirm specificity with negative controls (CRISPR knockout cells)

• Optimization of detection conditions:

  • Systematic titration of antibody concentrations

  • Test multiple blocking agents and buffer compositions

  • Optimize antigen retrieval methods for tissue samples

  • Compare fixation protocols for immunofluorescence

• Cross-validation with alternative detection methods:

  • Complement antibody detection with tagged recombinant proteins

  • Correlate protein detection with mRNA expression (qRT-PCR)

  • Use mass spectrometry-based approaches for protein confirmation

• Custom antibody development considerations:

  • Design immunogens based on unique, accessible epitopes

  • Consider species-specific sequences for bovine-specific detection

  • Validate new antibodies against recombinant protein standards

• Reporting standards:

  • Document all validation methods used

  • Report antibody catalog numbers, dilutions, and conditions

  • Include all controls in publications

Following these approaches will increase confidence in ANKRD46 detection and improve experimental reproducibility across studies.

What are the best approaches for analyzing ANKRD46 expression in heterogeneous tissue samples?

Analyzing ANKRD46 expression in heterogeneous tissues requires specialized techniques to overcome cellular complexity:

• Spatial resolution techniques:

  • Laser capture microdissection to isolate specific cell populations, as demonstrated in uterine luminal epithelium studies

  • Spatial transcriptomics to map expression while preserving tissue architecture

  • In situ hybridization combined with immunohistochemistry for co-localization studies

• Single-cell approaches:

  • Single-cell RNA sequencing to profile expression across all cell types

  • Flow cytometry or FACS with validated antibodies for protein-level analysis

  • Single-cell western blotting for protein analysis with limited cell numbers

• Deconvolution methods:

  • Computational deconvolution of bulk RNA-seq data using cell type-specific signatures

  • Reference-based approaches using purified cell type transcriptomes

  • Marker gene analysis to identify cell type-specific contributions

• Quantitative considerations:

  • Normalize to cell type-specific markers rather than housekeeping genes

  • Use multiple reference genes for qRT-PCR normalization

  • Employ absolute quantification with standard curves when possible

• Visualization approaches:

  • Multiplex immunofluorescence imaging with cell type markers

  • Tissue clearing techniques for thick section 3D analysis

  • High-resolution imaging with digital quantification

These approaches will help researchers distinguish cell type-specific ANKRD46 expression patterns within complex tissues such as the uterus during implantation or in analyzing expression across multiple physiological systems .

What are the most promising research avenues for understanding ANKRD46's role in reproductive biology?

Based on current evidence, particularly regarding ANKRD46's interaction with miR-451 in embryo implantation , the most promising research avenues include:

• Mechanistic elucidation of the miR-451/ANKRD46 axis:

  • Characterize downstream signaling pathways affected by this regulation

  • Identify additional targets of miR-451 that may work in concert with ANKRD46

  • Determine how ANKRD46 levels affect uterine receptivity markers

• Translational research in reproductive medicine:

  • Explore ANKRD46 expression in patients with implantation failure

  • Develop diagnostic biomarkers based on miR-451/ANKRD46 expression ratios

  • Investigate potential therapeutic approaches targeting this pathway

• Comparative studies across species:

  • Analyze conservation of the miR-451/ANKRD46 mechanism across mammalian species

  • Compare roles in various reproductive strategies (e.g., delayed implantation)

  • Evaluate evolutionary conservation of regulatory mechanisms

• Integration within the implantation regulatory network:

  • Map interactions between ANKRD46 and known implantation factors

  • Determine temporal coordination with hormone-responsive pathways

  • Identify cell type-specific functions in the embryo-maternal interface

• Development of advanced in vitro models:

  • Create organoid or microfluidic systems to model the implantation environment

  • Use these systems to manipulate ANKRD46 levels and observe effects on implantation

  • Implement gene editing in these models to create reporter systems

These research directions would significantly advance understanding of ANKRD46's role in reproductive biology and potentially lead to clinical applications for addressing implantation-related infertility.

How might ANKRD46 research intersect with emerging technologies in gene therapy?

The intersection of ANKRD46 research with gene therapy technologies presents several innovative possibilities:

• CRISPR-based therapeutic approaches:

  • Utilization of CRISPR/Cas9 KO plasmids targeting ANKRD46 could be adapted for therapeutic applications

  • Development of base editing or prime editing approaches for precise modification of ANKRD46 regulatory regions

  • Design of RNA-targeting CRISPR systems to modulate ANKRD46 expression temporarily

• miRNA therapeutics targeting the miR-451/ANKRD46 axis:

  • Design of miRNA mimics or inhibitors to modulate this pathway in reproductive disorders

  • Development of targeted delivery systems for uterine-specific intervention

  • Creation of modified miRNAs with enhanced stability and specificity

• AAV-mediated gene therapy:

  • Engineering tissue-specific expression systems for ANKRD46 modulation

  • Development of regulatable expression systems for temporal control

  • Optimization of delivery methods for reproductive tract targeting

• Nanomedicine approaches:

  • Design of nanoparticles for targeted delivery of ANKRD46 modulators

  • Creation of bioresponsive systems that respond to the implantation environment

  • Development of theranostic platforms combining imaging and therapeutic capabilities

• Cell therapy applications:

  • Engineering of endometrial cells with modified ANKRD46 expression

  • Development of stem cell-based approaches for endometrial regeneration

  • Creation of biosensor cells to monitor ANKRD46-related pathway activation