Recombinant Pongo abelii Raftlin-2 (RFTN2)

What is Raftlin-2 (RFTN2) and what is its primary function in Pongo abelii?

Raftlin-2 (RFTN2) in Pongo abelii (Sumatran orangutan) is a protein involved in immune signaling pathways. Its primary function includes mediating clathrin-dependent internalization of TLR4 (Toll-like receptor 4) in dendritic cells, which leads to downstream immune activation . This protein plays an important role in the innate immune response, particularly in pathogen recognition and cellular signaling processes. Unlike its paralog RNFT2 (RING finger and transmembrane domain-containing protein 2), which has different structural properties and functions, RFTN2 is specifically involved in membrane organization and immune receptor trafficking.

How does the amino acid sequence of Pongo abelii RFTN2 compare to its human ortholog?

The amino acid sequence of Pongo abelii RFTN2 shares significant homology with its human ortholog, reflecting their evolutionary relationship. While complete sequence alignment data for RFTN2 specifically is not provided in the search results, orangutan proteins typically show 95-98% amino acid identity with their human counterparts. This high conservation suggests similar functional mechanisms, though species-specific variations may exist in certain domains that could affect protein-protein interactions or regulatory mechanisms. Researchers should perform detailed sequence analyses when designing experiments to account for these potential differences.

What is known about RFTN2 expression patterns in different tissues of Pongo abelii?

RFTN2 shows variable expression across different tissues in Pongo abelii. Based on available knowledge metrics, there is a high value (0.89 on a 0-1 scale) for cell type/tissue specificity information for this target . The protein appears to be expressed in immune-related tissues, particularly in dendritic cells where it functions in TLR4 internalization. Other tissues with notable expression likely include lymphoid organs and potentially epithelial barriers that serve as first-line immune defenses. Researchers should consider these expression patterns when designing experiments to study RFTN2 function in specific physiological contexts.

How do post-translational modifications affect RFTN2 function in orangutan immune cells?

Post-translational modifications (PTMs) likely play critical roles in regulating RFTN2 function in orangutan immune cells, though specific modification sites have not been fully characterized. Based on protein domain structure analysis, potential phosphorylation sites may regulate RFTN2's ability to mediate clathrin-dependent internalization of TLR4 . When investigating PTMs experimentally, researchers should consider:

• Phosphorylation analysis using phospho-specific antibodies or mass spectrometry

• Ubiquitination patterns that may regulate protein turnover

• Potential glycosylation sites that could affect membrane localization

• Comparative analysis with human RFTN2 PTMs as a starting reference

Understanding these modifications is crucial for deciphering the regulatory mechanisms controlling RFTN2's role in immune signaling and membrane organization in Pongo abelii.

What experimental approaches are optimal for studying RFTN2-TLR4 interactions in Pongo abelii dendritic cells?

To effectively study RFTN2-TLR4 interactions in Pongo abelii dendritic cells, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Using anti-RFTN2 antibodies to pull down protein complexes and probe for TLR4, or vice versa. This technique can verify direct protein-protein interactions.

• Proximity Ligation Assay (PLA): Providing spatial resolution of interactions within intact cells, revealing where in the dendritic cell RFTN2-TLR4 complexes form.

• CRISPR/Cas9-mediated knockout/knockin: Creating modified cell lines to study functional consequences of RFTN2 mutations on TLR4 trafficking.

• Live-cell imaging: Using fluorescently tagged proteins to track the dynamics of RFTN2-mediated TLR4 internalization in real-time.

• Electron microscopy: Visualizing clathrin-dependent endocytic vesicles containing both proteins at ultrastructural resolution.

These approaches should be adapted to primary orangutan cells or suitable cell models to maintain physiological relevance.

How might genetic variation in RFTN2 contribute to immune response differences between Sumatran and Bornean orangutan subspecies?

Genetic variation in RFTN2 may contribute significantly to immune response differences between orangutan subspecies, particularly given the evolutionary divergence between Sumatran (Pongo abelii) and Bornean orangutans. Short Tandem Repeats (STRs) analysis has proven valuable for capturing recent evolutionary changes in orangutans , and may reveal subspecies-specific variations in RFTN2. These variations could affect:

• RFTN2 expression levels in response to pathogens

• Protein-protein interaction affinities in immune signaling complexes

• Trafficking efficiency of TLR4 receptors

• Downstream signaling outcomes and cytokine production profiles

Researchers investigating these differences should consider examining:

These investigations could reveal adaptations to the distinct pathogen pressures faced by orangutan populations in different island environments.

What are the optimal conditions for expressing and purifying recombinant Pongo abelii RFTN2?

For optimal expression and purification of recombinant Pongo abelii RFTN2, researchers should consider the following protocol guidelines:

• Expression System Selection:

  • Mammalian expression systems (HEK293 or CHO cells) are preferable for maintaining proper folding and post-translational modifications

  • Insect cell systems can be used for higher yield but may have different glycosylation patterns

  • E. coli systems should be avoided for full-length protein due to potential transmembrane domains

• Construct Design:

  • Include a cleavable tag (His6 or GST) for purification

  • Consider codon optimization for the expression system

  • Include appropriate signal sequences if secretion is desired

• Purification Strategy:

  • Initial capture via affinity chromatography (Ni-NTA for His-tagged proteins)

  • Size exclusion chromatography for removing aggregates

  • Ion exchange chromatography for final polishing

• Storage Conditions:

  • Store in Tris-based buffer with 50% glycerol at -20°C for routine use

  • For extended storage, maintain at -80°C

  • Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

Following these guidelines will help ensure the production of functional protein suitable for downstream applications including structural studies and functional assays.

How can researchers accurately assess RFTN2 protein quality and functional activity after recombinant expression?

Assessing the quality and functional activity of recombinant Pongo abelii RFTN2 requires multiple analytical approaches:

• Purity Assessment:

  • SDS-PAGE with Coomassie staining (>95% purity desired)

  • Western blotting with RFTN2-specific antibodies

  • Mass spectrometry for precise molecular weight confirmation

• Structural Integrity:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Limited proteolysis to assess proper folding

  • Dynamic light scattering (DLS) to detect aggregation

• Functional Activity Assays:

  • TLR4 binding assays using surface plasmon resonance (SPR) or bio-layer interferometry

  • Clathrin recruitment assays in vitro

  • Cell-based internalization assays measuring TLR4 endocytosis

  • Downstream signaling activation measuring NF-κB translocation or cytokine production

• Comparative Analysis:

  • Side-by-side comparison with human RFTN2

  • Activity benchmarking against known standards

  • Dose-response relationships to determine EC50 values

These comprehensive quality control steps are essential to ensure that experimental findings accurately reflect genuine RFTN2 properties rather than artifacts of improper protein preparation.

What considerations are important when designing antibodies against Pongo abelii RFTN2 for research applications?

When designing antibodies against Pongo abelii RFTN2 for research applications, several important considerations must be addressed:

• Epitope Selection:

  • Target unique, surface-exposed regions that distinguish RFTN2 from related proteins

  • Avoid highly conserved domains if species specificity is required

  • Consider using multiple epitopes spanning different protein regions for comprehensive detection

• Antibody Format:

  • Monoclonal antibodies for consistent reproducibility in quantitative applications

  • Polyclonal antibodies for robust detection across multiple epitopes

  • Recombinant antibody fragments (Fab, scFv) for applications with space constraints

• Validation Strategy:

  • Western blotting against recombinant protein and native tissue lysates

  • Immunoprecipitation efficiency testing

  • Immunofluorescence localization compared to known RFTN2 distribution

  • Testing on RFTN2 knockout cells as negative controls

• Application-Specific Considerations:

  • For live cell imaging, test for non-interference with protein function

  • For co-IP studies, validate epitope accessibility in protein complexes

  • For flow cytometry, ensure antibodies recognize native conformations

While commercial antibodies are available (with 111 antibodies reported in the knowledge database ), researchers should thoroughly validate these reagents in their specific experimental systems before conducting critical experiments.

How can researchers distinguish between direct and indirect effects when studying RFTN2 in immune signaling pathways?

Distinguishing between direct and indirect effects of RFTN2 in immune signaling pathways requires a multi-faceted experimental approach:

• Temporal Resolution Studies:

  • Use high-resolution time course experiments to determine the sequence of molecular events

  • Implement rapid induction systems (e.g., optogenetics) to precisely control RFTN2 activity

  • Compare kinetics of RFTN2 recruitment with downstream signaling events

• Protein Interaction Network Analysis:

  • Perform systematic protein-protein interaction mapping using proximity labeling methods (BioID, APEX)

  • Identify direct binding partners through crosslinking mass spectrometry (XL-MS)

  • Validate direct interactions with purified components in vitro

• Domain Mutation Approach:

  • Create domain-specific mutations that selectively disrupt particular interaction interfaces

  • Assess which downstream pathways are affected by specific mutations

  • Use these mutants to create interaction-deficient but expression-normal controls

• Systems Biology Integration:

  • Develop computational models incorporating known signaling components

  • Use perturbation data to refine model predictions

  • Identify nodes where RFTN2 directly impinges on the signaling network

These strategies collectively provide the evidence needed to discriminate between direct effects of RFTN2 on TLR4 trafficking and secondary consequences on downstream immune activation pathways.

What genomic analysis approaches are most effective for studying RFTN2 evolution in the context of orangutan speciation?

For studying RFTN2 evolution in the context of orangutan speciation, researchers should employ several genomic analysis approaches:

• Comparative Sequence Analysis:

  • Whole genome alignment of Sumatran (Pongo abelii) and Bornean (Pongo pygmaeus) orangutan RFTN2 loci

  • Identification of single nucleotide variants, insertions/deletions, and structural variants

  • Analysis of selection signatures using dN/dS ratios and McDonald-Kreitman tests

• Short Tandem Repeat (STR) Analysis:

  • STRs serve as high-resolution markers for recent evolutionary changes

  • Genotype STRs in and around the RFTN2 locus across orangutan populations

  • Use STR variation patterns to infer recent selective events, particularly those occurring since the Sumatran-Bornean divergence approximately 10,000 years ago

• Demographic History Integration:

  • Incorporate known orangutan population histories into evolutionary models

  • Account for bottlenecks, expansions, and geographic isolation events

  • Use coalescent-based approaches to estimate divergence times for RFTN2 variants

• Functional Genomics Correlation:

  • Integrate expression data from different tissues across subspecies

  • Correlate sequence changes with expression differences

  • Identify potential regulatory elements showing signs of rapid evolution

This comprehensive approach can reveal how RFTN2 has evolved in response to distinct pathogen pressures and environmental conditions on Sumatra versus Borneo, providing insights into adaptive immune evolution.

What are the most promising future research directions for Pongo abelii RFTN2 studies?

The most promising future research directions for Pongo abelii RFTN2 studies span several interconnected areas:

• Comparative Immunology:

  • Systematic comparison of RFTN2 function across great ape species

  • Investigation of RFTN2's role in species-specific immune responses to pathogens

  • Examination of RFTN2 evolution as part of broader innate immunity adaptations

• Structural Biology:

  • Determination of RFTN2 protein structure through cryo-EM or X-ray crystallography

  • Characterization of conformational changes during TLR4 binding

  • Structure-guided development of tools to modulate RFTN2 function

• Systems Immunology:

  • Integration of RFTN2 into comprehensive models of orangutan immune signaling

  • Network analysis of RFTN2-dependent pathways across immune cell types

  • Comparison with human systems to identify conserved and divergent mechanisms

• Conservation Applications:

  • Assessment of RFTN2 variants as potential markers for population health

  • Investigation of RFTN2 polymorphisms in relation to disease susceptibility

  • Development of non-invasive methods to monitor immune function in wild populations

• Emerging Technology Applications:

  • Application of single-cell sequencing to map RFTN2 expression across immune cell subtypes

  • Development of orangutan-specific organoid systems to study RFTN2 in relevant tissue contexts

  • Implementation of gene editing approaches to study RFTN2 function in appropriate cell models