Recombinant Trachypithecus phayrei C-C chemokine receptor type 5 (CCR5)

How does Trachypithecus phayrei CCR5 compare to human CCR5 in terms of sequence homology?

Comparative analysis of CCR5 sequences across primates shows that nucleotide and amino acid sequences are highly homologous, with variations slightly concentrated at the amino and carboxyl termini . While specific homology percentages between human and Trachypithecus phayrei CCR5 aren't explicitly stated in the search results, evolutionary studies demonstrate that primate CCR5 genes have undergone negative or purifying selection, indicating functional conservation .

One notable difference is at position 13, where Trachypithecus phayrei and other nonhuman primates have an Asp (aspartic acid) residue that is critical for CD4-independent binding of SIV gp120 . This suggests that Trachypithecus phayrei CCR5 might bind SIV gp120 without CD4 co-receptor, unlike human CCR5 which typically requires CD4 for HIV binding.

The evolutionary rate calculations suggest a slowdown in CCR5 evolution in primates after they diverged from rodents, with a constant synonymous mutation rate in primates of approximately 1.1 × 10^-9 synonymous mutations per site per year .

What are the recommended storage and reconstitution procedures for recombinant Trachypithecus phayrei CCR5?

For optimal results with recombinant Trachypithecus phayrei CCR5, the following procedures are recommended:

• Storage: Store the lyophilized protein at -20°C/-80°C upon receipt. Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles .

• Reconstitution:

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

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

  • Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C

  • The default recommended final concentration of glycerol is 50%

• Working with the protein: Repeated freezing and thawing is not recommended. Working aliquots can be stored at 4°C for up to one week .

• Buffer information: The protein is supplied in Tris/PBS-based buffer with 6% Trehalose, pH 8.0 .

How do the functional properties of Trachypithecus phayrei CCR5 contribute to understanding viral resistance mechanisms in primates?

Trachypithecus phayrei belongs to the Colobinae subfamily (Old World monkeys), which provides important insights into CCR5 evolution and viral interaction. While New World monkeys (Platyrrhini) have no documented in natura lentivirus infections, Old World monkeys show various levels of susceptibility to SIV . The study of Trachypithecus phayrei CCR5 offers valuable perspective on intermediate evolutionary adaptations.

The presence of Asp13 in Trachypithecus phayrei CCR5, shared with other nonhuman primates including Macaca mulatta, suggests it might bind SIV gp120 independently of CD4 . This distinctive property could facilitate studies comparing CD4-dependent versus CD4-independent viral entry mechanisms.

Evolutionary analyses demonstrate that Ka/Ks ratios (nonsynonymous to synonymous substitution rates) from cercopithecines and colobines are significantly different, implying different selective pressures in these lineages . Trachypithecus phayrei, as a colobine, may have evolved unique properties in response to specific viral pressures or immunological challenges. Researchers can exploit these differences to identify critical residues involved in receptor-virus interactions and potentially develop novel antiviral strategies.

Comparative functional studies of Trachypithecus phayrei CCR5 with both human CCR5 and CCR5 from New World monkeys could illuminate evolutionary adaptations that confer resistance to lentiviral infection, potentially informing therapeutic approaches for HIV/AIDS in humans.

What are the implications of CCR5 structure-function relationship for drug development when using Trachypithecus phayrei as a model?

The structural insights from Trachypithecus phayrei CCR5 have significant implications for drug development targeting HIV and other lentiviruses. Current drugs like maraviroc act as negative allosteric modulators of human CCR5, blocking HIV protein gp120 from associating with the receptor and inhibiting viral cellular entry .

A critical consideration when using Trachypithecus phayrei CCR5 as a model is the role of the lipid environment in receptor function. Research with human CCR5 has demonstrated that cholesterol (Chol) considerably decreases maraviroc binding affinity to the CCR5 receptor, affecting receptor-conformational dynamics . Similar studies with Trachypithecus phayrei CCR5 would be essential to understand whether this lipid dependency is conserved across species.

When designing experimental approaches using Trachypithecus phayrei CCR5, researchers should consider:

• Expression system selection: Both Pichia pastoris and cell-free expression systems have been used successfully with human CCR5 . The choice may affect post-translational modifications and lipid interactions.

• Reconstitution in model lipid systems: Controlling lipid composition is crucial for accurate assessment of drug binding properties.

• Multiple binding assay approaches: Combining techniques like plasmon waveguide resonance and fluorescence anisotropy provides more robust data on ligand-receptor interactions .

• Molecular dynamics simulation: Coarse-grained molecular dynamics can help investigate the impact of lipid components on receptor conformational flexibility and dynamics .

These considerations are particularly relevant as small differences in receptor structure between species can significantly impact drug efficacy and binding profiles.

How can evolutionary patterns in CCR5 across primates inform research using Trachypithecus phayrei CCR5?

The evolutionary patterns of CCR5 across primates provide crucial context for research with Trachypithecus phayrei CCR5. Phylogenetic analyses reveal that Platyrrhini (New World monkeys) CCR5 genes show greater genetic diversity than their Catarrhini (Old World monkeys, apes, humans) counterparts . Trachypithecus phayrei, as a member of Catarrhini, falls within this evolutionary framework.

Notably, the topologies of CCR5 gene trees conflict with the traditional view that snub-nosed langurs form a monophyletic group, suggesting CCR5 may not be ideal for low-level phylogenetic analysis . This observation highlights the complex evolutionary history of this gene, possibly shaped by different selective pressures.

When designing comparative studies:

• Consider that selective pressures have played different roles in cercopithecines and colobines lineages, as evidenced by significantly different Ka/Ks ratios .

• Focus on amino acid sites that differ between Platyrrhini and Catarrhini, particularly those experimentally associated with lentiviral interaction, as they may be under strong selection for variation .

• Analyze the implications of purifying selection on CCR5 function, as comparisons of Ka and Ks suggest CCR5 genes have undergone negative or purifying selection .

• Investigate whether the constant synonymous mutation rate in primates (approximately 1.1 × 10^-9 synonymous mutations per site per year) has functional consequences for receptor properties .

This evolutionary context helps researchers interpret functional differences between Trachypithecus phayrei CCR5 and its human or other primate counterparts, particularly in viral interaction studies.

What are the recommended procedures for expressing and purifying recombinant Trachypithecus phayrei CCR5?

Based on established protocols for similar proteins, the following methodological approach is recommended for expressing and purifying recombinant Trachypithecus phayrei CCR5:

Expression Systems:

• E. coli expression system: The commercially available recombinant Trachypithecus phayrei CCR5 is expressed in E. coli with an N-terminal His tag . This system offers high yield but may lack post-translational modifications.

• Alternative systems for functional studies: For functional studies, consider:

  • Pichia pastoris expression system, which provides eukaryotic processing

  • Cell-free expression system, which allows controlled membrane protein synthesis

Purification Protocol:

• Harvest cells and disrupt using appropriate lysis buffer containing detergents suitable for membrane proteins

• Perform affinity chromatography using the His-tag (Ni-NTA or TALON resin)

• Further purify using size exclusion chromatography

• Confirm purity via SDS-PAGE (>90% purity expected)

• For functional studies, reconstitute in lipid systems of controlled composition

Quality Control:

• Verify protein identity using Western blot with anti-His antibodies

• Assess purity via Coomassie blue staining of SDS-PAGE

• Confirm functionality through binding assays with known ligands

• For membrane-reconstituted protein, verify proper folding using circular dichroism or thermal stability assays

Success with this procedure can be evaluated through activity assays, such as the functional ELISA binding assay used for human CCR5, where immobilized CCR5 binds anti-CCR5 recombinant antibody with an EC50 of approximately 1.1-1.3 ng/mL .

What experimental approaches can differentiate between CD4-dependent and CD4-independent viral interactions with Trachypithecus phayrei CCR5?

Given that Trachypithecus phayrei CCR5 contains Asp13, critical for CD4-independent binding of SIV gp120 , the following experimental approaches can differentiate between CD4-dependent and CD4-independent viral interactions:

Cell-Based Assays:

• Transfection studies: Express Trachypithecus phayrei CCR5 in cell lines lacking endogenous CD4 and CCR5, then challenge with viruses to assess CD4-independent entry.

• Co-expression studies: Create cell lines with variable expression levels of CD4 and Trachypithecus phayrei CCR5 to determine threshold requirements for viral entry.

• Mutagenesis approach: Generate Asp13 mutants (e.g., D13A, D13N) of Trachypithecus phayrei CCR5 to directly assess the role of this residue in CD4-independent viral binding.

Biochemical and Biophysical Methods:

• Surface Plasmon Resonance (SPR): Compare binding kinetics of viral envelope proteins to immobilized Trachypithecus phayrei CCR5 in the presence and absence of soluble CD4.

• Fluorescence-based binding assays: Utilize fluorescently labeled viral proteins to quantify binding to CCR5 with and without CD4.

• Pull-down assays: Use His-tagged Trachypithecus phayrei CCR5 in pull-down experiments with viral envelope proteins, with and without CD4.

Structural Studies:

• Cryo-electron microscopy: Visualize complexes of Trachypithecus phayrei CCR5 with viral envelope proteins in the presence and absence of CD4.

• X-ray crystallography: Attempt crystallization of CCR5-virus complexes under various conditions.

• Hydrogen-deuterium exchange mass spectrometry: Map interaction interfaces between CCR5 and viral proteins with and without CD4.

Functional Readouts:

• Calcium flux assays: Measure signaling responses upon virus binding in cells expressing different combinations of CD4 and CCR5.

• Viral entry assays: Use pseudotyped viruses carrying reporter genes to quantify entry efficiency under different CD4/CCR5 expression conditions.

These methodologies will help elucidate the molecular details of how Trachypithecus phayrei CCR5 interacts with viruses and the precise role of CD4 in these interactions.

How can researchers effectively reconstitute Trachypithecus phayrei CCR5 in membrane systems for functional studies?

Effective reconstitution of Trachypithecus phayrei CCR5 in membrane systems is crucial for functional studies, particularly when investigating receptor-ligand interactions and conformational dynamics. Based on established protocols for similar receptors, the following methodology is recommended:

Selection of Membrane Mimetics:

• Detergent micelles: Initial solubilization in mild detergents like DDM, LMNG, or digitonin

• Lipid nanodiscs: For a more native-like environment, incorporating specific lipid compositions

• Liposomes: For studies requiring a closed membrane system

• Reconstituted model lipid systems of controlled lipid composition: Essential for studying the influence of specific lipids on receptor function

Reconstitution Protocol:

• Solubilize purified CCR5 in appropriate detergent

• Prepare lipid mixture (consider including cholesterol given its demonstrated impact on CCR5 function)

• Mix protein and lipids at appropriate ratios

• Remove detergent via:

  • Dialysis (slow, gentle method)

  • Bio-beads adsorption (faster method)

  • Gel filtration (for more homogeneous preparations)

• Confirm successful reconstitution via:

  • Negative stain electron microscopy

  • Dynamic light scattering

  • Density gradient centrifugation with subsequent Coomassie blue staining

Functional Validation Methods:

• Ligand binding assays:

  • Fluorescence anisotropy for competition binding assays between fluorescent agonists and antagonists like maraviroc

  • Plasmon waveguide resonance for total binding assays

• Conformational analysis:

  • Coarse-grained molecular dynamics simulation to investigate lipid impact on receptor-conformational flexibility and dynamics

  • Fluorescence spectroscopy using strategically placed fluorophores to monitor conformational changes

• Comparison conditions:

  • Always include parallel reconstitutions with and without cholesterol

  • Consider testing different lipid headgroups and acyl chain compositions to identify optimal conditions

This methodology acknowledges the critical finding that cholesterol considerably decreases maraviroc binding affinity to CCR5 receptor and affects receptor-conformational dynamics , which may also apply to Trachypithecus phayrei CCR5.

How does Trachypithecus phayrei CCR5 compare to other nonhuman primate CCR5 receptors in virus binding studies?

Comparative analysis of Trachypithecus phayrei CCR5 with other nonhuman primate CCR5 receptors reveals important evolutionary and functional patterns relevant to virus binding:

Conservation and Variation Patterns:

The presence of Asp13 in Trachypithecus phayrei CCR5, shared with Macaca mulatta and other tested nonhuman primates, suggests a conserved mechanism for potential CD4-independent binding of SIV gp120 . This characteristic distinguishes nonhuman primate CCR5 from human CCR5 in virus interaction studies.

While nucleotide and amino acid sequences of CCR5 among primates are highly homologous, variations are slightly concentrated on the amino and carboxyl termini . These regions often interact with viral envelope proteins and represent potential determinants of species-specific virus susceptibility.

Evolutionary analyses have identified specific amino acid residues that differ between Platyrrhini and Catarrhini CCR5, some of which have been experimentally associated with lentiviral interaction . Some of these sites appear to be under strong selection for variation among Platyrrhini but not among Catarrhini species, suggesting their potential role in differential virus susceptibility .

The Ka/Ks ratios from cercopithecines and colobines (including Trachypithecus phayrei) are significantly different, indicating varying selective pressures in these lineages . This difference may translate to functional variations in virus binding and entry mechanisms that researchers should consider when designing comparative studies.

What research applications benefit most from using Trachypithecus phayrei CCR5 versus human CCR5?

Several research applications gain unique advantages from using Trachypithecus phayrei CCR5 instead of or alongside human CCR5:

Evolutionary Virology Studies:
Trachypithecus phayrei CCR5 represents an evolutionary intermediate that can help trace the co-evolution of primates and lentiviruses. The colobine lineage (which includes Trachypithecus phayrei) has experienced different selective pressures than cercopithecines, as evidenced by different Ka/Ks ratios . This makes it valuable for understanding how virus-host interactions have shaped receptor evolution.

Comparative Receptor Pharmacology:
Studies of drug interactions with Trachypithecus phayrei CCR5 can reveal:

• Species-specific binding pockets that might inform more selective drug design

• Conserved binding sites that represent robust targets for broad-spectrum antivirals

• Variations in allosteric modulation mechanisms, as seen with drugs like maraviroc

CD4-Independent Viral Entry Models:
The presence of Asp13 in Trachypithecus phayrei CCR5 makes it particularly valuable for studying CD4-independent viral entry mechanisms . Researchers can:

• Compare entry efficiency of the same virus via CD4-dependent (human) versus potentially CD4-independent (Trachypithecus phayrei) pathways

• Identify structural elements required for CD4-independence

• Develop novel intervention strategies targeting this alternate entry pathway

Structure-Function Analysis:
The subtle variations between human and Trachypithecus phayrei CCR5, particularly at the termini , provide natural "mutants" for structure-function studies:

• Chimeric receptors combining domains from both species can identify functionally important regions

• Comparative analysis of ligand binding profiles can reveal species-specific signaling biases

• Variations in post-translational modifications between species can elucidate their functional significance

Drug Resistance Studies:
Comparing drug effects on human versus Trachypithecus phayrei CCR5 can:

• Identify resistance pathways that might emerge in human patients

• Develop more robust therapeutics that target conserved receptor elements

• Understand the evolutionary constraints on drug binding sites

By leveraging these comparative advantages, researchers can gain insights that would not be possible with human CCR5 studies alone.

What are the key considerations when interpreting data from lipid interaction studies with Trachypithecus phayrei CCR5?

When interpreting data from lipid interaction studies with Trachypithecus phayrei CCR5, researchers should consider several critical factors that influence receptor behavior and experimental outcomes:

Lipid Composition Effects:
Research with human CCR5 has demonstrated that cholesterol considerably decreases maraviroc binding affinity and affects receptor-conformational dynamics . When working with Trachypithecus phayrei CCR5:

• Cholesterol dependency: Always compare receptor function in the presence and absence of cholesterol, as this lipid may similarly modulate ligand binding and signaling properties.

• Native membrane mimicry: Consider that the natural membrane environment of Trachypithecus phayrei immune cells may differ from human cells, potentially affecting receptor behavior in reconstituted systems.

• Lipid raft association: G protein-coupled receptors often localize to lipid rafts, so domain-forming lipids should be included in reconstitution studies to capture native behavior.

Experimental System Considerations:

Data Interpretation Guidelines:

• Binding assay selection: Different techniques (fluorescence anisotropy, plasmon waveguide resonance ) may yield slightly different results due to assay-specific constraints. Cross-validate findings using multiple methods.

• Conformational dynamics: Coarse-grained molecular dynamics simulations can provide valuable insights into how lipids affect receptor flexibility , but models should be validated against experimental data.

• Species comparison: Always run parallel experiments with human CCR5 to distinguish species-specific effects from general lipid-receptor interactions.

• Evolutionary context: Consider that lipid sensitivity may reflect evolutionary adaptations to specific viral challenges or immune functions in the species' ecological niche.

• Physiological relevance: Correlate in vitro findings with cellular assays when possible to establish biological significance of observed lipid effects.

By carefully considering these factors, researchers can generate more robust and interpretable data on how lipids modulate Trachypithecus phayrei CCR5 function, potentially revealing conserved and divergent mechanisms of receptor regulation across primate species.

How can research on Trachypithecus phayrei CCR5 contribute to broader understanding of primate immunology and viral resistance?

Research on Trachypithecus phayrei CCR5 offers unique opportunities to advance our understanding of primate immunology and viral resistance mechanisms through several key contributions:

First, as a member of the Colobinae subfamily, Trachypithecus phayrei occupies an important evolutionary position that helps complete our understanding of CCR5 evolution across primates. The comparative analysis of CCR5 from diverse primates, including Trachypithecus phayrei, reveals patterns of conservation and variation that highlight functionally critical regions of the receptor . These evolutionary insights can identify novel targets for therapeutic intervention in viral diseases.

Second, the presence of Asp13 in Trachypithecus phayrei CCR5, shared with other nonhuman primates, suggests potential CD4-independent binding of SIV gp120 . This characteristic provides a valuable model for studying alternative viral entry mechanisms that may inform new antiviral strategies targeting entry processes. The distinct binding properties of Trachypithecus phayrei CCR5 could explain differences in viral susceptibility between primate species and potentially inspire biomimetic approaches to viral resistance.

Third, studies of Trachypithecus phayrei CCR5 contribute to our understanding of how the lipid environment modulates receptor function. Research with human CCR5 has demonstrated that cholesterol significantly affects binding affinity and receptor dynamics . Similar investigations with Trachypithecus phayrei CCR5 can reveal whether this lipid dependency is conserved across species and represents a fundamental aspect of chemokine receptor biology.

Finally, the significant differences in selective pressures between cercopithecines and colobines (including Trachypithecus phayrei) as indicated by Ka/Ks ratios suggest distinct evolutionary trajectories that may correlate with differences in immune function and viral susceptibility. By studying these patterns, researchers can better understand how host-pathogen co-evolution shapes receptor properties and immune defenses across the primate lineage.

Together, these contributions enhance our fundamental understanding of primate immunology and may ultimately inform novel approaches to addressing viral infections in humans.

What are the most promising future research directions involving Trachypithecus phayrei CCR5?

Several promising future research directions emerge from our current understanding of Trachypithecus phayrei CCR5, each with potential to yield significant insights into receptor biology, viral interactions, and therapeutic development:

Comparative Viral Entry Mechanisms

Investigating how diverse lentiviruses interact with Trachypithecus phayrei CCR5 compared to human CCR5 could reveal critical determinants of viral tropism and host range. The Asp13 residue in Trachypithecus phayrei CCR5, implicated in CD4-independent SIV binding , presents a particularly interesting target for mutagenesis studies to elucidate the molecular basis of this interaction. Such research might identify novel viral vulnerability points for therapeutic targeting.

Structural Biology Approaches

Determining the high-resolution structure of Trachypithecus phayrei CCR5, particularly in complex with various ligands, would advance our understanding of species-specific receptor properties. Cryo-electron microscopy or X-ray crystallography of the receptor in nanodiscs with controlled lipid compositions could reveal how membrane environment affects receptor conformation . Structural comparisons with human CCR5 might identify subtle differences that explain functional variations between species.

Lipid-Receptor Interaction Studies

Building on findings that cholesterol significantly impacts human CCR5 function , systematic investigation of how various lipids modulate Trachypithecus phayrei CCR5 dynamics and signaling could reveal conserved mechanisms of receptor regulation. Molecular dynamics simulations paired with experimental validation would be particularly valuable for understanding these complex interactions at the molecular level.

Evolutionary Immunology

Further exploration of the selective pressures that have shaped CCR5 evolution in colobines versus cercopithecines could illuminate how primates have adapted to historical viral challenges. Reconstruction of ancestral CCR5 sequences and functional testing of these reconstructed receptors might reveal evolutionary transitions in receptor function relevant to viral resistance.

Development of Novel Research Tools

Creating research tools based on Trachypithecus phayrei CCR5—such as transgenic cell lines expressing the receptor, monoclonal antibodies specific to its unique epitopes, or fluorescent biosensors based on its structure—could facilitate broader investigation of its properties and applications in virology and immunology research.

Therapeutic Applications

Exploring how drugs developed for human CCR5, such as maraviroc, interact with Trachypithecus phayrei CCR5 could identify structural determinants of drug efficacy and resistance. The distinct pharmacological properties of Trachypithecus phayrei CCR5 might inspire novel therapeutic approaches targeting conserved receptor elements less prone to resistance mutations.