Recombinant Saguinus imperator Duffy antigen/chemokine receptor (DARC)
Description
Molecular Structure and Classification
DARC is a seven-transmembrane glycoprotein with a conserved structure across species. The Saguinus imperator variant shares homology with human DARC, particularly in its extracellular N-terminal domain, which harbors chemokine-binding sites and epitopes critical for ligand interactions .
Post-Translational Modifications
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Glycosylation: Three N-linked glycosylation sites (predicted at Asn16, Asn27, and Asn33) contribute to its molecular mass of 40–47 kDa .
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Secondary Structure: Circular dichroism studies confirm a high α-helical content (~60%), typical of G protein-coupled receptors (GPCRs) .
Production Methods
Recombinant Saguinus imperator DARC is produced using heterologous expression systems to ensure high purity and functionality.
Purification Techniques
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Affinity chromatography using anti-Fy6 monoclonal antibodies .
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Elution with synthetic DFEDVWN peptides to preserve epitope integrity .
Biochemical Characterization
Ligand Binding Profile
DARC binds promiscuously to both CC and CXC chemokines, with dissociation constants (K<sub>D</sub>) in the nanomolar range :
| Chemokine | K<sub>D</sub> (nM) | Species |
|---|---|---|
| Interleukin-8 (CXCL8) | 20 ± 4.7 | Human homolog |
| RANTES (CCL5) | 41.9 ± 12.8 | Human homolog |
Glycan Composition
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N-glycans: Triantennary complex-type, terminated with α2-3/α2-6 sialic acid and bisecting GlcNAc .
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Functional Impact: Glycosylation modulates chemokine binding but is dispensable for Plasmodium vivax invasion .
Functional Insights
Role in Malaria Resistance
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The Fy6 epitope (residues 19–37) is critical for P. vivax merozoite invasion .
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Despite erythrocyte DARC absence in Duffy-negative individuals, endothelial expression persists, suggesting a conserved role in chemokine transcytosis .
Chemokine Regulation
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Acts as a "silent receptor": Internalizes chemokines without activating G-protein signaling, potentially modulating inflammatory responses .
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Heterodimerizes with CCR5, impairing chemotaxis but not receptor internalization .
HIV and Cancer Associations
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Duffy-negative genotypes correlate with increased HIV susceptibility but slower disease progression .
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DARC-KAI1 interactions suppress tumor metastasis in breast cancer models .
Applications in Research
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Malaria Studies: Used to investigate P. vivax invasion mechanisms in non-human primates .
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Immunoassays: Recombinant DARC serves as a control in ELISA/WB for antibody validation .
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Structural Biology: Aids in crystallography and molecular dynamics simulations of chemokine-receptor interactions .
Data Tables
Table 2: Key Residues in Ligand Binding
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement, and we will fulfill your request.
Note: All protein shipments default to standard blue ice packs. If dry ice shipping is required, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is Recombinant Saguinus imperator DARC and how does it differ from human DARC?
Recombinant Saguinus imperator DARC is a partial-length, non-full protein construct of the Emperor tamarin's Duffy antigen receptor produced through heterologous expression systems. Unlike human DARC, which has been extensively studied for its roles in malaria resistance and chemokine scavenging, the tamarin variant serves primarily as a model for comparative evolutionary studies and functional characterization of chemokine receptor biology.
Key differences include:
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Saguinus imperator DARC maintains the promiscuous binding to both CC and CXC chemokines characteristic of human DARC's scavenging function
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It lacks G protein coupling due to intracellular structural truncations, functioning as a non-signaling decoy receptor
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The tamarin variant serves as a specialized model for studying Plasmodium vivax binding mechanisms, particularly in primate-specific adaptations
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It has been shown to compete with Staphylococcus aureus pore-forming toxins for receptor occupancy
What expression systems are typically used for producing Recombinant Saguinus imperator DARC?
While the search results don't specify all expression systems used for this specific protein, Recombinant Saguinus imperator DARC is produced through heterologous expression systems. These systems are essential for generating sufficient quantities of the receptor for research purposes.
Methodological considerations for expression include:
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Selection of appropriate host cells that maintain post-translational modifications relevant to receptor function
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Optimization of expression conditions to maintain proper folding of the seven-transmembrane structure
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Purification protocols that preserve the native conformation of binding domains
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Verification of functionality through binding assays with known chemokine ligands
What are the fundamental structural characteristics of Saguinus imperator DARC?
Saguinus imperator DARC exhibits several important structural features that determine its function:
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It is a partial-length construct rather than the full-length receptor, optimized for specific research applications
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The receptor lacks G protein coupling capability due to intracellular structural truncations
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This structural modification renders it a non-signaling decoy receptor, similar to human DARC
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The binding domains maintain sufficient structural integrity to interact with both CC and CXC chemokines
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The receptor's structure facilitates its use in hydrogen-deuterium exchange mass spectrometry (HDX-MS) for mapping chemokine-receptor interaction surfaces
| Structural Feature | Functional Implication | Research Application |
|---|---|---|
| Partial-length construct | Optimized for binding studies | Ligand binding assays |
| Lacks G protein coupling | Functions as decoy receptor | Chemokine scavenging studies |
| Maintained binding domains | Interacts with multiple chemokines | Comparative binding analyses |
| Primate-specific variations | Different from human DARC | Evolutionary studies |
What are the primary research applications of Recombinant Saguinus imperator DARC?
Recombinant Saguinus imperator DARC serves multiple important research applications:
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Ligand binding assays: The receptor is used to map chemokine-receptor interaction surfaces via hydrogen-deuterium exchange mass spectrometry (HDX-MS).
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Malaria studies: It facilitates comparative analysis of Plasmodium invasion pathways across primate species, serving as a model for studying Plasmodium vivax binding mechanisms.
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Structural biology: The receptor supports cryo-electron microscopy (cryo-EM) studies to resolve receptor-chemokine complexes, providing insights into binding mechanisms.
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Evolutionary research: As a primate-specific variant, it enables comparative studies with human DARC to understand evolutionary adaptations in chemokine receptor biology.
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Pathogen interaction studies: The receptor competes with Staphylococcus aureus pore-forming toxins for occupancy, offering insights into host-pathogen interactions.
How does Saguinus imperator DARC contribute to understanding primate evolution?
The study of Saguinus imperator DARC provides valuable insights into primate evolution, particularly within New World monkeys. The Emperor tamarin (Saguinus imperator) belongs to the family of platyrrhine primates that have undergone significant evolutionary adaptations in their immune-related receptors.
Evolutionary significance includes:
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Comparison with human DARC reveals conserved and divergent regions, indicating selective pressures
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Variations in binding domains may reflect adaptation to different pathogen challenges encountered in distinct ecological niches
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Studies of Saguinus imperator and related species like Saguinus midas help establish evolutionary relationships within the Callithrichidae family
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Recent taxonomic reclassifications have proposed elevating Saguinus subgenera to full genera, potentially reclassifying Saguinus imperator as Tamarinus imperator
What methodologies are optimal for studying chemokine binding profiles of Saguinus imperator DARC?
Several sophisticated methodological approaches can effectively characterize the chemokine binding profile of Saguinus imperator DARC:
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Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique has been successfully employed to map chemokine-receptor interaction surfaces, providing detailed information about binding interfaces.
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Binding Competition Assays: These assays can determine the relative affinities of different chemokines for the receptor and assess competition with bacterial toxins, such as those from Staphylococcus aureus.
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Cryo-Electron Microscopy: Supporting the resolution of receptor-chemokine complexes, this technique provides structural insights into binding conformations.
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Comparative Binding Studies: Parallel analysis of binding properties between Saguinus imperator DARC and human DARC can reveal species-specific adaptations in chemokine recognition.
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Mutational Analysis: Systematic mutation of key residues can identify critical amino acids involved in chemokine binding and determine species-specific binding determinants.
| Methodology | Primary Data Generated | Advantages for S. imperator DARC Research |
|---|---|---|
| HDX-MS | Binding interface mapping | Identifies regions of conformational change upon ligand binding |
| Binding Competition Assays | Relative binding affinities | Compares multiple chemokines in a single system |
| Cryo-EM | 3D structural information | Visualizes receptor-ligand complexes |
| Comparative Binding | Species-specific differences | Reveals evolutionary adaptations |
| Mutational Analysis | Critical binding residues | Defines molecular determinants of binding |
How can Recombinant Saguinus imperator DARC advance malaria research?
Recombinant Saguinus imperator DARC serves as a model for studying Plasmodium vivax binding mechanisms, particularly in primate-specific adaptations. This application has significant implications for malaria research:
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Comparative Receptor Analysis: Comparing binding characteristics of P. vivax Duffy Binding Protein (DBP) to tamarin versus human DARC can reveal evolutionary adaptations in host-parasite interactions.
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Cross-Species Transmission Studies: Understanding how P. vivax interacts with various primate DARC variants helps assess the zoonotic potential and species barriers.
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Structural Basis of Invasion: The tamarin DARC model facilitates structural studies of the DARC-DBP complex, potentially identifying novel intervention points.
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Therapeutic Target Identification: Conserved binding sites across primate DARC variants may represent robust targets for therapeutic development with reduced potential for resistance.
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Vaccine Development: Insights from tamarin DARC-Plasmodium interactions can inform the design of vaccines targeting the critical DARC-DBP interaction.
| Plasmodium Species | Interaction with S. imperator DARC | Research Relevance |
|---|---|---|
| P. vivax | Primary binding model | Direct relevance to human malaria |
| P. knowlesi | Comparative binding model | Zoonotic transmission potential |
| P. falciparum | Non-DARC dependent invasion | Negative control for specificity |
What challenges exist in translating in vitro findings with Recombinant Saguinus imperator DARC to in vivo systems?
Researchers face several methodological challenges when extrapolating in vitro findings with Recombinant Saguinus imperator DARC to in vivo contexts:
How does the genomic context of Saguinus imperator influence DARC expression and function?
Recent genomic studies provide context for understanding Saguinus imperator DARC within the broader genomic landscape of this species:
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Genome Assembly Availability: Recent advancements have made available a scaffold-level genome assembly for Saguinus imperator (SagImp_v1) and a chromosome-level assembly for the related Saguinus midas (ASM2_v1) .
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Mobile Genetic Elements: Saguinus imperator has been found to contain extensive Platy-1 retroposons, with over 11,000 full-length Platy-1 insertions in the genome . These mobile elements may influence gene expression patterns throughout the genome, potentially including the DARC locus.
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Taxonomic Considerations: Recent taxonomic proposals suggest reclassifying Saguinus imperator as Tamarinus imperator, reflecting ongoing refinement in understanding evolutionary relationships . These classifications may impact how researchers interpret comparative studies across primate species.
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Evolutionary Rate Differences: The extensive independent amplification of mobile elements in the Saguinus genome suggests potentially different evolutionary dynamics compared to other primates, which might extend to immune-related genes like DARC .
What structural biology approaches have provided the most valuable insights into Saguinus imperator DARC?
Structural biology has been instrumental in understanding Saguinus imperator DARC function, with several approaches yielding valuable data:
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Cryo-Electron Microscopy: This technique has been used to resolve receptor-chemokine complexes, providing three-dimensional structural information about binding interactions.
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Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This approach has successfully mapped the interaction surfaces between DARC and various chemokines, identifying specific regions involved in binding.
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Comparative Structural Analysis: Comparing structural features between tamarin and human DARC reveals evolutionary adaptations that may influence receptor function.
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Structure-Function Correlation: Relating structural features to functional outcomes, such as the relationship between intracellular truncations and the lack of G protein coupling.
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Molecular Dynamics Simulations: Though not explicitly mentioned in the search results, this computational approach likely complements experimental structural data to understand the dynamics of chemokine binding.
How can researchers optimize expression and purification protocols for Recombinant Saguinus imperator DARC?
Optimizing the production of high-quality Recombinant Saguinus imperator DARC requires careful consideration of several methodological aspects:
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Expression System Selection: Choose between bacterial, yeast, insect, or mammalian expression systems based on the specific research requirements, considering that mammalian systems may provide more native-like post-translational modifications.
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Construct Design: As Saguinus imperator DARC is described as a partial-length construct, careful design of the expression construct is critical, ensuring inclusion of all domains necessary for the intended research application.
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Solubilization Strategy: Membrane proteins like DARC require appropriate detergents or lipid environments to maintain native-like conformation during purification.
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Functional Validation: Implement binding assays with known chemokine ligands to confirm that the purified receptor maintains expected binding properties.
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Stability Assessment: Monitor protein stability under various storage conditions to ensure consistency across experiments.
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Quality Control: Employ size-exclusion chromatography, mass spectrometry, and circular dichroism to verify protein homogeneity, identity, and folding.
