Recombinant Mouse protein C-C motif chemokine (Ccl17) (Active)

What is mouse CCL17 and what are its primary biological functions?

Mouse CCL17 is a C-C motif chemokine that functions primarily as a chemoattractant for T lymphocytes, with preferential activity for Th2 cells, but not monocytes or granulocytes. It plays crucial roles in a wide range of inflammatory and immunological processes by binding to the CCR4 receptor at the T-cell surface . In addition to its immunomodulatory functions, CCL17 mediates GM-CSF/CSF2-driven pain and inflammation pathways .

Within the central nervous system, CCL17 is required to maintain the highly branched morphology of hippocampal microglia under homeostatic conditions and may be important for appropriate adaptation of microglial morphology and synaptic plasticity during neuroinflammation . It also plays a significant role in wound healing, primarily by inducing fibroblast migration into wounded areas .

Recent research has identified CCL17 as a contributor to age-related heart failure in both humans and mice through its regulation of T cell plasticity and differentiation . The expression of CCL17 exhibits an age-dependent increase, and its levels correlate with cardiac function status in heart failure patients .

What is the molecular structure of recombinant mouse CCL17 protein?

Recombinant mouse CCL17 protein is typically expressed as a full-length protein spanning amino acids 1-93 . The amino acid sequence of mouse CCL17 is:

MRSLQMLLLAALLGTFLQHARAARATNVGRECCLDYFKGAIPIRKLVSWYKTSVECSRDAIVFLTVQGKLIC ADPKDKHVKKAIRLVKNPRP

This chemokine shares 28% amino acid sequence identity and 57% similarity to CCL2, and 35% identity and 61% similarity to CCL11 . Within the chemokine family, CCL17 has several distinctive structural features that affect its binding properties, including:

• A glycine at position 17 instead of a positively charged residue found in other chemokines

• Absence of positively charged residues at positions 44 and/or 45 that are present in most other chemokines

• An arginine instead of the highly conserved tryptophan at position 57

These structural differences contribute to CCL17's unique binding properties, including its relatively weak affinity for viral CC chemokine inhibitor (vCCI) compared to other chemokines in the same family .

What expression systems are most effective for producing recombinant mouse CCL17?

For high-quality recombinant mouse CCL17 production, the baculovirus-infected insect cell expression system has proven particularly effective. This system can produce mouse CCL17 with >98% purity and endotoxin levels below 1 EU/μg, making it suitable for sensitive experimental applications .

When working with bacterial expression systems such as BL21(DE3) E. coli, researchers should incorporate the following methodology for optimal results:

• Express the protein with an appropriate tag (e.g., His-tag) for purification

• Solubilize the protein using 6 M guanidinium chloride

• Purify using Ni²⁺ affinity chromatography

• Perform protein refolding under controlled conditions

• Conduct final purification using reversed-phase chromatography

To verify proper folding of the recombinant protein, nuclear magnetic resonance (NMR) spectroscopy is recommended as demonstrated in previous studies . This methodology ensures that the recombinant protein maintains its native structural features essential for biological activity.

What analytical methods are recommended for assessing CCL17 binding affinities to its receptors or inhibitors?

Multiple complementary approaches are recommended for comprehensive analysis of CCL17 binding interactions:

• Size Exclusion Chromatography with Multiangle Light Scattering (SEC/MALS): This technique is effective for determining complex stoichiometry, as demonstrated in studies of vCCI:CCL17 complexes which form a 1:1 complex at high concentrations (10 mg/mL) .

• Competition Fluorescence Anisotropy: This method provides quantitative binding affinity measurements. The technique involves:

  • Forming a 1:1 complex of the binding partner with a tight-binding fluorescent ligand

  • Adding CCL17 at increasing concentrations

  • Measuring the decrease in anisotropy signal as CCL17 displaces the fluorescent ligand

  • Calculating the Kd value from the anisotropy versus concentration curve

• Molecular Dynamics (MD) Simulations: For structural analysis of CCL17 binding interfaces, molecular dynamics simulations offer valuable insights into persistent residue-residue interactions. Simulations of 1 microsecond or longer provide reliable data on binding conformations and interface surface areas .

The combination of these techniques provides a comprehensive assessment of binding properties, enabling researchers to characterize both wild-type and mutant CCL17 proteins with high precision.

How can researchers effectively measure and analyze CCL17 expression in clinical samples?

Based on established research protocols, several approaches are recommended for measuring CCL17 expression in clinical samples:

• Serum Protein Quantification: ELISA or similar immunoassay techniques can be used to measure circulating CCL17 levels in patient serum, as demonstrated in studies of heart failure patients . Serial measurements before and after treatment can provide valuable information on how CCL17 levels correlate with disease progression or response to therapy.

• Transcriptome Analysis: RNA sequencing or microarray analysis of tissue samples provides comprehensive information on CCL17 gene expression. This approach was successfully used to analyze left ventricular (LV) transcriptomes from heart failure patients, revealing significantly increased CCL17 expression compared to non-failing controls .

• Immunohistochemistry: For validating CCL17 expression in tissue samples, immunohistochemistry offers visual confirmation of protein expression patterns. This technique has been applied to validate CCL17 expression in tumor and para-carcinoma tissue samples .

When analyzing expression data, bioinformatics tools can help categorize patients based on CCL17 expression levels. For example, the X-tile program has been used to categorize patients into high-expression and low-expression groups based on survival status, survival time, and CCL17 expression from RNA-seq data .

How does CCL17 contribute to age-related heart failure pathogenesis?

Recent research has identified CCL17 as a significant contributor to age-related heart failure through its immunomodulatory effects. Analysis of left ventricular transcriptomes from the Gene Expression Omnibus (GEO) database, comprising 366 samples (200 heart failure and 166 non-heart failure samples), revealed significantly increased CCL17 expression in heart failure patients compared to non-failing controls .

The pathogenic role of CCL17 in heart failure is supported by several key findings:

• Age-dependent expression increase: Serum CCL17 levels show a significant association with age, with expression in mice serum increasing with advancing age .

• Correlation with disease severity: Circulating CCL17 levels decrease with cardiac function recovery in patients with acute decompensated heart failure following standard treatment, suggesting its direct relationship with disease severity .

• Mechanism of action: CCL17 appears to contribute to heart failure through regulation of T cell plasticity and differentiation, affecting immune responses that influence cardiac remodeling and function .

• Differential expression pattern: Unlike CCL22, another CCR4-binding chemokine, CCL17 expression specifically increases with age, suggesting a unique role in age-related cardiac pathology .

These findings collectively suggest that CCL17 represents a novel therapeutic target for age-related heart failure, potentially through modulation of T cell-mediated immune responses affecting cardiac function.

What experimental models are most appropriate for studying CCL17 in cardiac pathology?

For investigating CCL17 in cardiac pathology, researchers should consider the following experimental approaches:

• Human cohort studies: Analysis of serum CCL17 levels in patients with heart failure compared to normal controls provides clinically relevant data. Particularly valuable are longitudinal studies of patients with acute decompensated heart failure before and after treatment, which can demonstrate how CCL17 levels correlate with cardiac function recovery .

• Murine models of age-related heart failure: Mice models allow for detailed mechanistic studies of CCL17's role in cardiac aging and dysfunction. Age-dependent increases in serum CCL17 levels in mice mirror human findings, making these models translationally relevant .

• Transcriptomic analysis: RNA sequencing of cardiac tissue provides comprehensive insights into CCL17 expression changes in heart failure. The GEO database (e.g., GSE141910) offers valuable resources for such analyses .

When designing experiments, researchers should consider incorporating the following parameters:

• Age as a critical variable in experimental design

• Correlation with established cardiac biomarkers (e.g., amino-terminal pro-brain natriuretic peptide)

• Assessment of T cell populations and differentiation states

• Longitudinal measurements to capture disease progression and treatment response

These approaches enable comprehensive investigation of CCL17's role in cardiac pathophysiology and potential therapeutic interventions.

How does CCL17 influence dendritic cell migration and immune cell trafficking?

CCL17 plays a critical role in dendritic cell (DC) migration through both direct and indirect mechanisms. Research using CCL17-deficient mice has revealed that this chemokine is required for CCR7- and CXCR4-dependent migration of cutaneous DCs .

Key findings on CCL17's role in dendritic cell migration include:

• Sensitization for chemokine responsiveness: CCL17 sensitizes DCs for CCR7- and CXCR4-dependent migration to lymph node-associated homeostatic chemokines under inflammatory conditions .

• Migration defects in CCL17 deficiency: CCL17-deficient Langerhans cells (LCs) showed impaired emigration from the skin after exposure to contact sensitizers. In an atopic dermatitis model, CCL17-deficient mice retained LCs in lesional skin after chronic allergen exposure, whereas most LCs emigrated from the epidermis of allergen-treated wild-type controls into draining lymph nodes .

• Mechanism of impaired migration: The major migratory defect of CCL17-deficient DCs is linked to:

  • Impaired mobility in response to CCL19/21 in 3D in vitro migration assays

  • Blockade of intracellular calcium release in response to CCR7 ligands

  • Impaired responsiveness to CXCL12

Interestingly, despite CCL17 being a ligand for CCR4, the absence of CCR4 had no effect on cutaneous DC migration and development of atopic dermatitis symptoms, suggesting CCL17 functions through different or additional mechanisms in this context .

What experimental approaches are recommended for studying CCL17 function in inflammatory disease models?

When investigating CCL17 function in inflammatory disease models, researchers should consider implementing these methodological approaches:

• Genetic modification models: CCL17-deficient mice provide valuable insights into the role of this chemokine in various inflammatory conditions. Comparing wild-type, CCL17-deficient, and CCR4-deficient mice can help distinguish receptor-dependent and independent functions of CCL17 .

• Atopic dermatitis models: Chronic allergen exposure models in mice allow for studying how CCL17 influences:

  • Dermal inflammation

  • T helper 2-type cytokine production

  • Allergen-specific humoral immune responses

  • Langerhans cell migration patterns

• Contact hypersensitivity assays: These models help evaluate how CCL17 affects dendritic cell emigration from the skin after exposure to contact sensitizers .

• 3D in vitro migration assays: These provide controlled environments to assess the intrinsic migratory capacity of CCL17-deficient versus wild-type DCs in response to various chemokines .

• Calcium flux measurements: Assessing intracellular calcium release in response to chemokine stimulation helps elucidate the molecular mechanisms underlying migration defects in CCL17-deficient cells .

These approaches, used in combination, provide comprehensive insights into the multifaceted roles of CCL17 in inflammatory disease pathogenesis and potential therapeutic interventions.

How can protein engineering improve CCL17 binding characteristics and potential therapeutic applications?

Protein engineering offers powerful approaches for modifying CCL17 binding properties for research and therapeutic applications. A successful example of this approach is the rational design of CCL17 mutants with enhanced binding to viral CC chemokine inhibitor (vCCI) .

The following methodology was effective in engineering CCL17 with improved binding:

• Sequence alignment analysis: Comparing CCL17 with five other chemokines with high affinity for vCCI identified key residues that differ in CCL17, including:

  • Position 17: Glycine in CCL17 vs. positively charged residue in other chemokines

  • Positions 44/45: Absence of positively charged residues in CCL17

  • Position 57: Arginine in CCL17 vs. highly conserved tryptophan in other chemokines

• Molecular dynamics simulations: 1 microsecond simulations of wild-type CCL17 and designed mutants bound to vCCI provided insights into persistent residue-residue interactions and binding interface characteristics .

• Targeted mutations: Based on computational predictions, mutations G17R, V44K, and Q45R were introduced to improve binding .

• Experimental validation: Binding affinity was assessed using competition fluorescence anisotropy, revealing:

The triple mutant G17R/V44K/Q45R showed a remarkable 68-fold increase in affinity compared to wild-type CCL17 .

This rational design approach demonstrates the potential for engineering CCL17 variants with modified binding properties for research applications and potential therapeutic development.

What is the role of CCL17 in tumor immunology and how might it be targeted in cancer research?

Recent research has revealed complex roles for CCL17 in tumor immunology, particularly in thyroid carcinoma (THCA). CCL17's functions in the tumor microenvironment present both challenges and opportunities for cancer research and potential therapeutic development .

Key findings regarding CCL17 in tumor immunology include:

For cancer researchers, these findings suggest several approaches for investigating CCL17 in tumor immunology:

• Bioinformatic analysis of publicly available datasets (TCGA, GEO) to correlate CCL17 expression with clinical outcomes and immune cell infiltration

• Immunohistochemical validation of CCL17 expression in tumor and para-carcinoma tissue samples

• Functional studies to elucidate the mechanisms by which CCL17 influences immune cell recruitment and function in the tumor microenvironment

• Exploration of CCL17 as a potential biomarker or therapeutic target in cancer immunotherapy strategies

Understanding the dual role of CCL17 in promoting immune cell infiltration while potentially worsening clinical outcomes requires further investigation into the specific immune cell populations recruited and their functional states within the tumor microenvironment.

How does CCL17 interact with other chemokines and cytokines in complex inflammatory networks?

CCL17 operates within intricate inflammatory networks, interacting with multiple chemokines, cytokines, and their receptors. Understanding these interactions is crucial for comprehending its role in various pathological conditions .

Key aspects of CCL17's interactions within inflammatory networks include:

• Receptor interactions: CCL17 primarily acts through binding to CCR4 on T-cell surfaces, but its effects on dendritic cell migration suggest involvement in additional signaling pathways .

• Functional overlap with CCL22: CCL17 and CCL22 are both CCR4-binding chemokines, but they show distinct expression patterns. Unlike CCL22, CCL17 expression increases with age, suggesting differential regulation and function in age-related inflammatory conditions .

• Influence on CCR7 and CXCR4 signaling: CCL17 sensitizes dendritic cells for CCR7- and CXCR4-dependent migration to lymph node-associated homeostatic chemokines under inflammatory conditions. This indicates a role in modulating cellular responsiveness to other chemokine signals .

• T helper cell subset recruitment: CCL17 displays preferential chemotactic activity for Th2 cells, potentially influencing the balance between different T helper cell subsets in inflammatory conditions .

• Cross-talk with inflammatory mediators: In atopic dermatitis models, CCL17 deficiency leads to decreased T helper 2-type cytokine production and altered allergen-specific humoral immune responses, indicating bidirectional interactions with other inflammatory mediators .

When studying these complex interactions, researchers should consider multi-parameter approaches:

• Co-expression analysis of multiple chemokines and cytokines

• Perturbation studies examining how modulation of one component affects others

• Systems biology approaches to model interaction networks

• In vivo studies comparing single and multiple knockout/inhibition models

Understanding these complex interactions provides deeper insights into how CCL17 contributes to diverse pathological conditions and may reveal novel therapeutic opportunities through selective modulation of inflammatory networks.