Recombinant Rhinopithecus avunculus C-C chemokine receptor type 5 (CCR5)

What is Rhinopithecus avunculus CCR5 and why is it significant for research?

Rhinopithecus avunculus CCR5 is a seven-transmembrane G protein-coupled receptor expressed in T cells and macrophages of the critically endangered Tonkin snub-nosed monkey (Rhinopithecus avunculus). This receptor belongs to the beta chemokine receptor family and functions as a key component in immune cell migration and inflammatory responses. Its significance for research stems from Rhinopithecus avunculus being one of five endangered snub-nosed monkey species restricted to small areas in China, Vietnam, and Myanmar . CCR5 receptors are critical in primate immunology and serve as important co-receptors for viral entry, particularly for primate lentiviruses. Comparing CCR5 across primate species provides valuable insights into host-pathogen co-evolution and mechanisms of viral resistance or susceptibility.

How does Rhinopithecus avunculus CCR5 compare structurally to human CCR5?

While the exact sequence comparison data between Rhinopithecus avunculus CCR5 and human CCR5 is not directly reported in the search results, we can extrapolate from related primate data. CCR5 proteins among primates demonstrate high sequence conservation. For instance, African green monkey CCR5 proteins are 97.7-98.3% identical to human CCR5 . Based on evolutionary relationships, we would expect Rhinopithecus avunculus CCR5 to share significant structural homology with human CCR5, particularly in the seven-transmembrane domains and ligand-binding regions, though with potential species-specific polymorphisms in key extracellular regions that might impact viral interactions.

The core structure likely maintains the seven-transmembrane architecture characteristic of G protein-coupled receptors with three extracellular loops and an N-terminal domain crucial for ligand binding and viral interactions.

What are the primary ligands for Rhinopithecus avunculus CCR5?

Based on CCR5 conservation across primates, the primary ligands for Rhinopithecus avunculus CCR5 likely include chemokines similar to those that bind human CCR5, specifically:

• Macrophage inflammatory protein 1-alpha (MIP-1α/CCL3)

• Macrophage inflammatory protein 1-beta (MIP-1β/CCL4)

• RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted/CCL5)

• Monocyte chemoattractant protein 2 (MCP-2/CCL8)

These chemokines would interact with the receptor to mediate immune cell recruitment and inflammatory responses. The binding affinity and specificity may vary somewhat from human CCR5 due to species-specific amino acid variations, potentially impacting immune function in this endangered primate species.

What expression systems are recommended for producing recombinant Rhinopithecus avunculus CCR5?

Based on successful expression systems for other primate CCR5 proteins, researchers can consider the following expression systems for Rhinopithecus avunculus CCR5:

• Pichia pastoris expression system: This system has been successfully used for CCR5 expression and allows for proper protein folding and post-translational modifications critical for receptor functionality .

• Cell-free expression systems: These provide a controlled environment for synthesizing membrane proteins like CCR5 and can be advantageous for studying receptor-ligand interactions .

• Mammalian cell expression: HEK293 or CHO cell lines are suitable for expressing properly folded and functionally active CCR5 with appropriate post-translational modifications.

When choosing an expression system, researchers should consider that CCR5 is a membrane protein requiring appropriate lipid environments for proper folding and function. Expression yield, post-translational modifications (particularly tyrosine sulfation), and functional activity should be carefully evaluated when selecting the optimal system.

How do polymorphisms in Rhinopithecus avunculus CCR5 potentially affect viral susceptibility compared to other primates?

While specific polymorphisms in Rhinopithecus avunculus CCR5 are not detailed in the search results, insights can be drawn from studies of other primates. In African green monkeys, naturally occurring CCR5 polymorphisms significantly impact viral interactions. Notably, two distinct AGM CCR5 sequences have been identified with critical amino acid substitutions affecting viral susceptibility:

• The Y14N substitution in the amino-terminal extracellular region completely blocked HIV-1 infections while maintaining SIV susceptibility

• The Q93R substitution in extracellular loop 1 significantly reduced HIV-1 infection capability while preserving SIV susceptibility

This demonstrates how single amino acid polymorphisms in primate CCR5 can dramatically alter viral tropism. For Rhinopithecus avunculus, identifying natural polymorphisms would be valuable for understanding potential resistance to various lentiviruses and could provide evolutionary insights into host-pathogen adaptations in this endangered species.

What methodologies are optimal for assessing the binding affinity of ligands to recombinant Rhinopithecus avunculus CCR5?

For comprehensive binding affinity assessment, researchers should employ multiple complementary methodologies:

• Plasmon Waveguide Resonance: This technique provides direct measurement of total binding interactions between CCR5 and its ligands, offering high sensitivity for membrane protein studies .

• Fluorescence Anisotropy: Particularly useful for competition binding assays, this method can assess binding of non-fluorescent compounds like receptor antagonists by measuring their ability to displace fluorescent ligands .

• Radioligand Binding Assays: Using radiolabeled chemokines such as [125I]MIP-1β allows for quantitative determination of binding parameters (Kd, Bmax) through saturation and competition assays .

• Surface Plasmon Resonance (SPR): Provides real-time binding kinetics and allows determination of association/dissociation rate constants.

For optimal results, researchers should reconstitute the recombinant receptor in model membrane systems with controlled lipid composition, as lipid environment significantly impacts receptor conformation and ligand binding properties. For instance, cholesterol has been shown to considerably decrease maraviroc binding affinity to CCR5 .

How can chimeric constructs of Rhinopithecus avunculus CCR5 be designed to study species-specific determinants of viral entry?

To design effective chimeric constructs for studying species-specific viral entry determinants, researchers should:

• Target key functional domains: Focus on creating chimeras that swap the three main regions critical for viral interactions:

  • N-terminal extracellular domain

  • Extracellular loop 1 (ECL1)

  • Extracellular loop 2 (ECL2)

• Employ site-directed mutagenesis: Based on previous primate CCR5 studies, particular attention should be paid to residues identified as critical for viral interactions:

  • Position 14 in the N-terminus (Y14N substitution blocked HIV-1 in AGM CCR5)

  • Position 93 in ECL1 (Q93R reduced HIV-1 susceptibility)

  • Position 183 in ECL2 (P183L in mouse CCR5 prevented coreceptor activity)

• Validation methodology: After generating chimeric constructs, validate their expression and function through:

  • Surface expression analysis using specific antibodies or tagged constructs

  • Chemokine binding assays using [125I]MIP-1β or fluorescently labeled chemokines

  • Viral entry assays using pseudotyped viruses expressing various env proteins

This approach allows systematic identification of molecular determinants that confer species-specific viral susceptibility patterns.

What impact does the lipid environment have on recombinant Rhinopithecus avunculus CCR5 function and ligand binding?

The lipid environment critically influences CCR5 function and ligand binding properties, as demonstrated in reconstitution studies:

Molecular dynamics simulations have confirmed cholesterol's considerable impact on receptor conformational flexibility and dynamics . For Rhinopithecus avunculus CCR5, reconstitution in appropriate lipid environments is crucial for maintaining native-like properties. When designing binding studies, researchers should carefully consider:

• Cholesterol content in reconstitution systems

• Membrane fluidity parameters

• Lipid raft-mimicking components

• Lipid-to-protein ratios

These considerations will ensure that the recombinant receptor retains functional and structural properties representative of its native state in Rhinopithecus avunculus cells.

What purification strategy is most effective for obtaining high-purity recombinant Rhinopithecus avunculus CCR5?

A multi-step purification strategy is recommended for obtaining high-purity, functionally active recombinant Rhinopithecus avunculus CCR5:

• Solubilization: Use mild detergents (DDM, LMNG, or digitonin) for initial solubilization from expression system membranes to maintain protein integrity.

• Affinity Chromatography: Employ histidine-tag purification if the recombinant protein incorporates a 6xHis-tag. Alternatively, use immunoaffinity chromatography with CCR5-specific antibodies.

• Size Exclusion Chromatography: Further purify the receptor to remove aggregates and ensure monomeric state.

• Sucrose Gradient Centrifugation: As demonstrated effective for CCR5 purification, this step enables separation of the receptor from remaining contaminants .

Quality control should include:

• SDS-PAGE analysis to confirm purity (>95%)

• Western blot to confirm identity

• Ligand binding assays to verify functionality

• Circular dichroism to assess secondary structure

A successful purification should yield protein that appears as a single band around 40.5 kDa on SDS-PAGE, consistent with the expected molecular weight of CCR5 .

How can post-translational modifications of Rhinopithecus avunculus CCR5 be characterized and their functional impact assessed?

Characterizing post-translational modifications (PTMs) of Rhinopithecus avunculus CCR5 requires a comprehensive analytical approach:

• Tyrosine Sulfation Analysis:

  • Mass spectrometry (MS) to identify sulfated peptides

  • Synthetic sulfopeptide ELISA assays to assess sulfation heterogeneity

  • Anti-sulfotyrosine antibody detection

• Glycosylation Analysis:

  • PNGase F digestion followed by mobility shift assays

  • Lectin binding assays

  • MS glycopeptide mapping

To assess functional impact:

• PTM-specific binding studies:

  • Compare binding properties of differentially modified receptor populations

  • Use competition binding assays with sulfated vs. non-sulfated receptor forms

• Site-directed mutagenesis:

  • Mutate PTM sites individually and in combination

  • Assess effects on ligand binding and signaling

Research has shown that CCR5 sulfation is heterogeneous, affecting binding properties of both native chemokines and antibodies . This heterogeneity likely extends to Rhinopithecus avunculus CCR5 and may contribute to species-specific differences in ligand and pathogen interactions.

What are the optimal reconstitution parameters for functional studies of recombinant Rhinopithecus avunculus CCR5?

Optimal reconstitution parameters for functional studies include:

When studying receptor-ligand interactions, it's essential to test both cholesterol-containing and cholesterol-free preparations, as cholesterol has been shown to considerably decrease binding affinity of ligands like maraviroc to CCR5 .

For functional verification post-reconstitution:

• Assess ligand binding using fluorescence anisotropy or plasmon waveguide resonance

• Verify correct orientation using antibodies targeting extracellular epitopes

• Confirm G-protein coupling capacity through GTPγS binding assays

These parameters will ensure that reconstituted Rhinopithecus avunculus CCR5 maintains native-like properties for reliable functional studies.

How should researchers interpret differences in ligand binding profiles between Rhinopithecus avunculus CCR5 and human CCR5?

When interpreting differences in ligand binding profiles, researchers should consider multiple factors:

If Rhinopithecus avunculus CCR5 shows altered binding to HIV-1 envelope proteins compared to human CCR5, this could reflect natural selection for resistance mechanisms, similar to the Y14N substitution found in some African green monkey CCR5 variants that blocks HIV-1 infection while maintaining SIV susceptibility .

What bioinformatic approaches are most valuable for analyzing Rhinopithecus avunculus CCR5 in evolutionary and functional contexts?

For comprehensive analysis of Rhinopithecus avunculus CCR5, researchers should employ multiple bioinformatic approaches:

• Phylogenetic analysis:

  • Maximum likelihood and Bayesian methods to position Rhinopithecus avunculus CCR5 in evolutionary context

  • Selection pressure analysis (dN/dS ratios) to identify sites under positive selection

  • Reconstruction of ancestral sequences to track evolutionary changes

• Structural prediction and analysis:

  • Homology modeling based on crystal structures of human CCR5

  • Molecular dynamics simulations to assess conformational dynamics

  • Binding site prediction and comparison across species

• Population genetics:

  • Analysis of polymorphisms within Rhinopithecus avunculus populations

  • Comparison with other endangered Rhinopithecus species to identify genus-specific adaptations

• Functional domain mapping:

  • Identification of conserved vs. variable regions

  • Correlation of sequence variations with functional differences in viral entry or signaling

These approaches can reveal how Rhinopithecus avunculus CCR5 evolved specific features that might relate to disease susceptibility or resistance, providing insights into both conservation genetics and potential therapeutic applications.

How can researchers differentiate between experimental artifacts and genuine species-specific properties when characterizing Rhinopithecus avunculus CCR5?

To differentiate between artifacts and genuine species-specific properties, researchers should implement rigorous controls and validation approaches:

• Expression system controls:

  • Express human CCR5 in parallel using identical systems

  • Compare multiple expression systems (e.g., Pichia pastoris vs. cell-free) to identify system-dependent artifacts

  • Verify that expression tags don't interfere with function

• Post-translational modification validation:

  • Assess sulfation heterogeneity using synthetic sulfopeptide ELISA assays

  • Compare glycosylation patterns between recombinant and native receptor (if available)

• Lipid environment standardization:

  • Test multiple reconstitution conditions with defined lipid compositions

  • Include cholesterol-dependent binding studies to identify lipid-sensitive properties

• Multiple methodological approaches:

  • Verify key findings using orthogonal techniques (e.g., both fluorescence anisotropy and plasmon waveguide resonance for binding studies)

  • Employ both binding and functional readouts to confirm observations

• Chimeric receptor studies:

  • Create domain-swapped chimeras between human and Rhinopithecus avunculus CCR5

  • Map species-specific properties to defined receptor regions

By implementing these approaches, researchers can confidently attribute observed differences to genuine species-specific properties rather than methodological artifacts.

How can recombinant Rhinopithecus avunculus CCR5 contribute to conservation genomics and population studies of this endangered species?

Recombinant Rhinopithecus avunculus CCR5 research can make significant contributions to conservation efforts:

• Genetic diversity assessment:

  • CCR5 polymorphism studies can reveal population-level genetic diversity

  • CCR5 variants may serve as markers for population structure analysis in this endangered species

• Disease susceptibility profiling:

  • Functional characterization of CCR5 variants can predict vulnerability to emerging pathogens

  • Insight into potential viral susceptibilities can inform management strategies

• Evolutionary history reconstruction:

  • CCR5 sequence comparisons across the five Rhinopithecus species can illuminate evolutionary relationships

  • Analysis of selection pressures on CCR5 can reveal historical pathogen challenges

• Conservation management applications:

  • Identifying individuals with protective or vulnerable CCR5 genotypes to inform breeding programs

  • Using CCR5 as part of genetic monitoring programs to track population health

As noted in the research literature, genomic information can significantly contribute to conservation strategies for snub-nosed monkeys, with CCR5 being an important immunogenetic marker that can provide insights into both evolutionary history and future disease risks .

What insights can comparative studies of Rhinopithecus avunculus CCR5 provide for developing novel antiviral strategies?

Comparative studies of Rhinopithecus avunculus CCR5 offer valuable insights for antiviral development:

• Natural resistance mechanisms:

  • Identification of species-specific amino acid variations that confer viral resistance

  • Similar to how Y14N and Q93R substitutions in African green monkey CCR5 affect HIV-1 susceptibility

• Novel binding site targets:

  • Comparison of binding domains across species may reveal previously unexplored inhibitor binding sites

  • Species-specific differences in extracellular loops can inform structure-based drug design

• Broad-spectrum inhibitor development:

  • Targeting conserved CCR5 regions found in both human and non-human primates

  • Designing inhibitors that function across species barriers for zoonotic virus protection

• Resistance prediction:

  • Understanding natural CCR5 polymorphisms can help predict potential resistance pathways

  • Informing the design of combination therapies that address multiple entry pathways

The documented differential effects of CCR5 amino acid variations on HIV-1 versus SIV entry highlight how comparative primate studies can reveal virus-specific receptor interactions that might be exploited for targeted antiviral development.

What considerations are important when developing antibodies or other detection reagents specific for Rhinopithecus avunculus CCR5?

Developing specific detection reagents requires careful consideration of several factors:

• Epitope selection strategy:

  • Target species-specific regions to ensure specificity for Rhinopithecus avunculus CCR5

  • Alternatively, target conserved regions for cross-reactive reagents

  • Consider accessibility of epitopes in native conformation

• Post-translational modification awareness:

  • Account for tyrosine sulfation heterogeneity, which affects antibody binding

  • Consider glycosylation patterns that may mask epitopes

• Validation requirements:

  • Test against CCR5 from closely related species to confirm specificity

  • Validate in multiple formats (Western blot, flow cytometry, immunohistochemistry)

  • Verify recognition of both denatured and native conformations

• Application-specific considerations:

  • For functional blocking antibodies, target ligand-binding regions

  • For detection antibodies, select epitopes unlikely to be affected by ligand binding

  • For conformation-sensitive applications, develop antibodies against structurally intact receptor

Research has shown that CCR5 sulfation heterogeneity affects the binding properties of antibodies , highlighting the importance of characterizing post-translational modifications when developing detection reagents for Rhinopithecus avunculus CCR5.