Recombinant Mouse C-C motif chemokine 20 protein (Ccl20) (Active)

What is the molecular structure of recombinant mouse CCL20 protein?

Recombinant mouse CCL20 is a low molecular weight chemokine (approximately 8.0 kDa) expressed as a tag-free protein spanning amino acids 28-97 of the native sequence. The protein contains the characteristic CC chemokine fold but exhibits unusual properties compared to other CC chemokines. Unlike most CC chemokines, crystal structures of CCL20 (PDB codes 2HCI and 1M8A) reveal that it can adopt a CXC-type dimeric arrangement, although NMR studies indicate this self-association is relatively weak and pH dependent . The mature recombinant protein produced in E. coli expression systems contains the full biological activity domain without additional tags that might interfere with receptor binding or structural studies .

What receptor does mouse CCL20 interact with and how does this affect its function?

Mouse CCL20 interacts specifically with the G-protein coupled receptor CCR6. This exclusive relationship between CCL20 and CCR6 is relatively unique in the chemokine family, as most chemokines can bind multiple receptors. The CCL20-CCR6 interaction mediates several critical biological processes including:

• Chemoattraction of dendritic cells and T lymphocytes (particularly Th17 cells)

• Recruitment of immune cells to mucosal surfaces

• Regulation of inflammatory responses

The binding of CCL20 to CCR6 triggers various signaling cascades that lead to cell migration, calcium mobilization, and gene expression changes. The specificity of this interaction makes it a potential target for therapeutic interventions in inflammatory conditions .

How can researchers differentiate recombinant mouse CCL20 from other chemokines in experimental systems?

Researchers can distinguish recombinant mouse CCL20 from other chemokines through several approaches:

• Receptor binding specificity: Using CCR6-expressing cells in competitive binding assays

• Antibody-based detection: Employing specific antibodies like the mouse CCL20/MIP-3 alpha Antibody (Clone #114908) in Western blotting, ELISA, or immunohistochemistry

• Functional assays: Measuring chemotaxis of CCR6+ cells (e.g., BaF3 cells transfected with human CCR6)

• Mass spectrometry: Confirming molecular weight (8.0 kDa for tag-free mouse CCL20)

• SDS-PAGE mobility: Monitoring migration patterns under reducing and non-reducing conditions

For optimal specificity, researchers should perform neutralization experiments using CCL20-specific antibodies to confirm that observed effects are truly CCL20-dependent. For example, chemotaxis elicited by recombinant mouse CCL20 (20 ng/mL) can be neutralized by increasing concentrations of anti-mouse CCL20 monoclonal antibody, with an ND50 typically between 3-15 μg/mL .

What are the optimal conditions for reconstitution and storage of recombinant mouse CCL20?

For optimal stability and biological activity, recombinant mouse CCL20 should be handled as follows:

Reconstitution Protocol:

• Briefly centrifuge the lyophilized protein vial to collect all material at the bottom

• Reconstitute in sterile PBS containing at least 0.1% carrier protein (BSA or HSA)

• Gently mix by pipetting or brief vortexing, avoiding foam formation

• Allow the protein to sit at room temperature for 10-15 minutes before use

Storage Conditions:

• Short-term (≤1 month): Store at 4°C in sterile conditions

• Long-term: Store at -20°C to -80°C in working aliquots to avoid repeated freeze-thaw cycles

• Add a preservative (e.g., 0.1% sodium azide) for longer storage periods when stored at 4°C

• Monitor activity periodically through functional assays to ensure protein viability

Researchers should validate protein activity after reconstitution using a chemotaxis assay with CCR6-expressing cells before employing the protein in critical experiments .

How can researchers accurately evaluate the biological activity of recombinant mouse CCL20?

Evaluating the biological activity of recombinant mouse CCL20 requires multiple complementary approaches:

Chemotaxis Assay:
The most direct functional assessment involves measuring CCL20-induced migration of CCR6-expressing cells. The BaF3 mouse pro-B cell line transfected with human CCR6 is commonly used for this purpose. In a dose-dependent chemotaxis assay, recombinant mouse CCL20 should induce cell migration at concentrations as low as 1-10 ng/mL, with optimal activity typically observed at 20-50 ng/mL. Cell migration can be quantified using Resazurin or similar viability dyes to measure the number of cells that migrate through a membrane .

Calcium Mobilization Assay:
Binding of CCL20 to CCR6 triggers intracellular calcium flux, which can be measured using calcium-sensitive fluorescent dyes like Fluo-4 AM. Researchers should observe a rapid and transient increase in fluorescence intensity following CCL20 stimulation.

Receptor Binding Assay:
Competitive binding assays using radiolabeled or fluorescently labeled CCL20 can determine binding affinity. The IC50 value for unlabeled CCL20 displacing labeled CCL20 provides an indirect measure of biological activity.

Cell Signaling Assays:
Western blotting for phosphorylated ERK1/2, Akt, or other downstream signaling molecules can confirm functional receptor activation. Researchers should observe time-dependent and dose-dependent changes in phosphorylation status following CCL20 stimulation.

To ensure specificity, control experiments should include:

• CCR6-negative cells (negative control)

• Anti-CCL20 neutralizing antibodies

• CCR6 receptor antagonists

• Heat-inactivated CCL20 (denatured control)

What approaches can be used to engineer modified forms of mouse CCL20 with altered functions?

Engineering modified forms of mouse CCL20 can yield variants with altered receptor binding, activation properties, or stability. Key approaches include:

Site-Directed Mutagenesis:
Strategic amino acid substitutions can create CCL20 variants with modified properties. For example, the S64C variant creates a constitutive dimer through introduction of a cysteine that forms an intermolecular disulfide bond. This engineered dimer binds and partially activates CCR6 but inhibits T cell chemotaxis, demonstrating potential therapeutic value .

The engineering process typically involves:

• Structure-based design using algorithms like Disulfide by Design

• Visual inspection of crystal structures (such as PDB ID 2HCI) to identify suitable modification sites

• Creating a panel of variants (e.g., V21C/T24C, G22C/T24C, F23C, V60C/V67C, S64C)

• Expression in E. coli and refolding

• Biophysical characterization to confirm proper folding and oligomeric state

• Functional testing to determine altered properties

Domain Swapping:
Chimeric proteins created by exchanging domains between CCL20 and other chemokines can yield insights into structure-function relationships and potentially create novel activities.

Fusion Proteins:
Linking CCL20 to other proteins can create bifunctional molecules with expanded capabilities, such as:

• CCL20-Fc fusion for extended half-life

• CCL20-toxin conjugates for targeted cell killing

• CCL20-reporter fusions for tracking receptor binding

Post-Translational Modifications:
Introduction of specific glycosylation sites or other modifications can alter stability, receptor binding, or immunogenicity.

Researchers should confirm proper folding of engineered variants using techniques such as circular dichroism, nuclear magnetic resonance (NMR) spectroscopy, or X-ray crystallography .

How does CCL20 contribute to inflammatory skin conditions like psoriasis?

CCL20 plays a crucial role in the pathogenesis of psoriasis through several mechanisms:

• T Cell Recruitment: CCL20 is upregulated in psoriatic lesions and attracts CCR6-expressing Th17 cells to the skin, which are key drivers of psoriatic inflammation

• Dendritic Cell Trafficking: CCL20 mediates recruitment of CCR6+ dendritic cells that present antigens and stimulate T cell activation

• Inflammatory Amplification Loop: In response to skin injury, keratinocytes produce CCL20, which recruits immune cells that release pro-inflammatory cytokines (IL-17, IL-22, IL-23), further stimulating CCL20 production

• Epithelial Barrier Disruption: CCL20-recruited inflammatory cells release cytokines that alter keratinocyte differentiation, contributing to the characteristic epidermal hyperplasia

In mouse models of psoriasis, such as the IL-23-dependent model, engineered CCL20 variants that bind CCR6 but inhibit T cell chemotaxis have been shown to reduce disease severity. The disulfide-linked CCL20 dimer (S64C variant) demonstrates therapeutic potential by blocking the recruitment of pathogenic T cells to the skin while partially activating the CCR6 receptor .

This suggests that targeting the CCL20-CCR6 axis represents a promising approach for treating psoriasis and potentially other inflammatory skin conditions.

What is the role of CCL20 in cancer progression and how can it be targeted therapeutically?

CCL20 contributes to cancer progression through multiple mechanisms:

Cancer-Promoting Mechanisms of CCL20:

• Immunosuppression: CCL20 can recruit regulatory T cells (Tregs) to the tumor microenvironment, suppressing anti-tumor immune responses

• Angiogenesis: CCL20 promotes formation of new blood vessels to support tumor growth

• Epithelial-to-Mesenchymal Transition (EMT): CCL20 signaling induces EMT, enhancing cancer cell invasiveness

• Migration/Invasion: Activation of CCR6 by CCL20 stimulates cancer cell motility through various signaling pathways

• Chemoresistance: CCL20 can induce resistance to chemotherapeutic agents through activation of survival pathways

CCL20 is particularly implicated in breast cancer progression, where it has emerged as a potential therapeutic target .

Therapeutic Targeting Approaches:

• Monoclonal Antibodies: Neutralizing antibodies against CCL20 can block its interaction with CCR6

• Receptor Antagonists: Small molecule inhibitors of CCR6 can prevent CCL20-mediated signaling

• Engineered CCL20 Variants: Modified versions of CCL20 that bind CCR6 without activating downstream signaling can serve as competitive inhibitors

• Signaling Pathway Inhibitors: Targeting the downstream pathways activated by CCL20/CCR6 interaction

• Combination Therapies: Blocking CCL20 in combination with immune checkpoint inhibitors may enhance anti-tumor immunity

For effective therapeutic development, researchers must consider the complex cytokine network within the tumor microenvironment, as multiple factors collectively regulate tumor progression. Understanding the signaling pathways that regulate CCL20 function provides additional targets for intervention .

What experimental models are most appropriate for studying CCL20 function in immune regulation?

Several experimental models are valuable for investigating CCL20 function in immune regulation:

In Vitro Models:

• Chemotaxis Assays: Using transwell migration systems with CCR6-expressing cells (e.g., BaF3 cells transfected with CCR6, Th17 cells, or dendritic cells) to assess CCL20-mediated cell recruitment

• 3D Organoid Cultures: Intestinal or skin organoids can recapitulate tissue architecture and demonstrate CCL20's role in immune cell recruitment to epithelial surfaces

• Co-culture Systems: Combining epithelial cells with immune cells to study CCL20-mediated interactions

In Vivo Models:

• IL-23-Induced Psoriasis Model: Intradermal injection of IL-23 induces psoriasiform skin inflammation dependent on CCL20-CCR6 interaction, allowing assessment of therapeutic interventions targeting this axis

• Imiquimod-Induced Psoriasis Model: Topical application of imiquimod cream induces psoriasis-like inflammation with upregulation of CCL20

• DSS-Induced Colitis: Dextran sodium sulfate administration causes intestinal inflammation where CCL20 plays a role in immune cell recruitment

• CCR6 and CCL20 Knockout Mice: Genetic models lacking CCR6 or CCL20 help elucidate their roles in homeostasis and inflammation

• Humanized Mouse Models: Mice reconstituted with human immune cells allow study of human CCL20-CCR6 interactions

Experimental Readouts:

• Flow cytometry for immune cell recruitment and phenotyping

• Histological assessment of tissue inflammation

• Cytokine profiling by ELISA or multiplex assays

• Gene expression analysis by qPCR or RNA-seq

• In vivo imaging of fluorescently labeled immune cells

The choice of model should be guided by the specific research question, with consideration of species differences in CCL20 function between mice and humans .

How can researchers accurately quantify CCL20 expression in different experimental contexts?

Accurate quantification of CCL20 expression requires selecting appropriate methods based on the experimental context:

Protein Level Quantification:

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • Sandwich ELISA using capture and detection antibodies specific for mouse CCL20

  • Typical detection range: 15-1000 pg/mL

  • Sample types: Cell culture supernatants, tissue lysates, serum, plasma

  • Note: For optimal sensitivity, use antibody pairs validated for mouse CCL20 detection

• Western Blotting:

  • Use of specific anti-CCL20 antibodies (e.g., Clone #114908)

  • Sample preparation: Reduce samples with DTT or β-mercaptoethanol

  • Expected molecular weight: ~8.0 kDa

  • Controls: Recombinant mouse CCL20 as positive control

• Luminex/Multiplex Assays:

  • Allow simultaneous detection of CCL20 alongside other cytokines

  • Useful for comprehensive analysis of inflammatory profiles

  • Higher throughput than traditional ELISA

• Mass Spectrometry:

  • For unbiased detection and absolute quantification

  • Particularly useful in complex biological samples

  • Requires appropriate sample preparation and internal standards

mRNA Level Quantification:

• Quantitative Real-Time PCR (qRT-PCR):

  • Design primers specific for mouse Ccl20 gene

  • Normalize to appropriate housekeeping genes (e.g., GAPDH, β-actin)

  • Include no-template and no-RT controls

• RNA-Sequencing:

  • Provides comprehensive transcriptome analysis

  • Allows detection of alternative splicing variants

  • Requires bioinformatic analysis pipelines

In Situ Detection:

• Immunohistochemistry/Immunofluorescence:

  • Use of validated anti-CCL20 antibodies on tissue sections

  • Include appropriate positive and negative controls

  • Consider co-staining for cell type markers to identify CCL20-producing cells

• RNA in situ Hybridization:

  • Detection of Ccl20 mRNA in tissue sections

  • Useful for localizing expression at cellular level

  • RNAscope or similar technologies provide single-molecule sensitivity

When comparing CCL20 levels across different experimental conditions, researchers should maintain consistent sample collection, processing, and analysis methods to minimize technical variability .

What are the challenges and solutions in producing high-quality recombinant mouse CCL20 for research applications?

Producing high-quality recombinant mouse CCL20 presents several challenges, each with specific solutions:

Challenges and Solutions in Production:

Expression Systems Comparison:

• E. coli Expression System:

  • Advantages: High yield, cost-effective, tag-free expression possible

  • Disadvantages: Lacks post-translational modifications, inclusion body formation common

  • Best practices: Expression as inclusion bodies followed by denaturation and refolding

• Mammalian Expression System:

  • Advantages: Proper post-translational modifications, direct secretion into medium

  • Disadvantages: Lower yield, higher cost, potential for heterogeneous glycosylation

  • Best practices: Use of optimized signal sequences, serum-free adaptation

• Insect Cell Expression System:

  • Advantages: Higher yield than mammalian cells, some post-translational modifications

  • Disadvantages: Different glycosylation patterns than mammalian systems

  • Best practices: Optimization of multiplicity of infection and harvest time

The most commonly used system for recombinant mouse CCL20 production is E. coli, as evidenced by commercially available products. For optimal quality, purified protein should be subjected to rigorous quality control testing, including mass spectrometry, SDS-PAGE under reducing and non-reducing conditions, and functional assays to confirm biological activity .

How can structural analysis of CCL20 inform the development of therapeutic antagonists or agonists?

Structural analysis of CCL20 provides critical insights for rational drug design of therapeutic antagonists or agonists:

Key Structural Features Informing Drug Design:

• Dimerization Interface: The unusual CXC-type dimeric arrangement observed in CCL20 crystal structures (PDB ID codes 2HCI and 1M8A) presents opportunities for designing molecules that can stabilize or disrupt dimerization. The successful engineering of the S64C variant demonstrates how introducing an intermolecular disulfide bond can create a stable dimer with altered function, inhibiting T cell chemotaxis while maintaining receptor binding .

• Receptor Binding Domains: Structural studies identifying the precise regions of CCL20 that interact with CCR6 can inform the design of:

  • Peptide mimetics that compete for receptor binding

  • Small molecules that disrupt the protein-protein interaction

  • Antibodies targeting specific epitopes involved in receptor recognition

• Conformational Dynamics: NMR studies indicating that CCL20 self-association is weak and pH-dependent suggest that pH-sensitive variants could be developed for targeted activity in specific tissue microenvironments.

Structure-Based Drug Design Approaches:

• Computational Modeling:

  • Molecular dynamics simulations to identify stable binding conformations

  • Virtual screening of compound libraries against CCL20 or CCR6 binding pockets

  • In silico prediction of binding affinities for candidate molecules

• Biophysical Characterization:

  • X-ray crystallography of CCL20 complexed with receptor fragments

  • NMR studies to map binding interfaces

  • Surface plasmon resonance to determine binding kinetics

• Rational Protein Engineering:

  • Structure-guided mutations to create variants with enhanced receptor affinity but reduced signaling capacity (antagonists)

  • Engineering of constitutive dimers like CCL20 S64C that demonstrate partial receptor activation

  • Creation of fusion proteins that combine CCL20 with other functional domains

Therapeutic Applications Based on Structure:

The engineered CCL20 S64C dimer has already demonstrated therapeutic potential in a mouse model of psoriasis, suggesting broader applications for engineered CCL20 variants in:

• Treatment of autoimmune diseases (rheumatoid arthritis, multiple sclerosis)

• Cancer immunotherapy

• Modulation of mucosal immunity

• Treatment of inflammatory bowel diseases

By combining structural insights with functional studies, researchers can develop more potent and selective CCL20 inhibitors or modulators with improved pharmacokinetic properties and reduced off-target effects .

What signaling pathways are activated by CCL20-CCR6 interaction and how do they contribute to different cellular responses?

The CCL20-CCR6 interaction activates multiple signaling pathways that orchestrate diverse cellular responses:

Primary Signaling Cascades:

• G Protein-Coupled Signaling:

  • Gαi protein activation leads to inhibition of adenylyl cyclase and reduction in cAMP levels

  • Gβγ subunits activate phospholipase C (PLC), leading to formation of IP3 and DAG

  • IP3 triggers calcium release from intracellular stores, important for cellular motility

  • DAG activates protein kinase C (PKC), regulating cytoskeletal reorganization

• MAPK Pathways:

  • ERK1/2 activation promotes cell proliferation and survival

  • p38 MAPK regulates inflammatory cytokine production

  • JNK pathway activation influences cell migration and stress responses

• PI3K/Akt Pathway:

  • Phosphatidylinositol 3-kinase (PI3K) activation leads to Akt phosphorylation

  • Promotes cell survival, metabolism, and cytoskeletal rearrangements

  • Regulates mammalian target of rapamycin (mTOR) signaling

• Small GTPases:

  • Activation of Rho family GTPases (RhoA, Rac1, Cdc42)

  • Critical for actin polymerization, cell polarization, and directional migration

  • Controls formation of lamellipodia and filopodia during chemotaxis

Cell Type-Specific Responses:

Regulatory Mechanisms:

• Receptor Desensitization:

  • GRK-mediated phosphorylation of CCR6

  • β-arrestin recruitment leading to receptor internalization

  • Temporal limitation of signaling

• Crosstalk with Other Pathways:

  • Integration with TLR signaling in dendritic cells

  • Synergy with cytokine receptor signaling (e.g., IL-17R)

  • Modulation by microenvironmental factors

Understanding these signaling pathways provides opportunities for therapeutic targeting, either at the level of CCL20-CCR6 interaction or at downstream signaling nodes. Pathway-specific inhibitors could potentially modulate specific aspects of CCL20 function while preserving others, allowing for more precise therapeutic interventions in diseases where CCL20 plays a pathogenic role .

How do researchers troubleshoot common challenges in CCL20-related experiments?

Researchers frequently encounter several challenges when working with CCL20 in experimental settings. Here are common issues and troubleshooting approaches:

Challenge 1: Inconsistent Chemotaxis Results

Challenge 2: Protein Stability and Activity Loss

Challenge 3: Antibody Cross-Reactivity

Challenge 4: Gene Expression Analysis Issues

Challenge 5: Engineered Variant Production

When troubleshooting, researchers should systematically test each variable while maintaining appropriate positive and negative controls. For example, when evaluating chemotaxis, include a known chemoattractant as positive control and buffer-only conditions as negative control .

How does the tumor microenvironment regulate CCL20 expression and function in cancer progression?

The tumor microenvironment (TME) profoundly influences CCL20 expression and function through multiple regulatory mechanisms:

Regulatory Factors in the Tumor Microenvironment:

• Hypoxia:

  • Hypoxic conditions activate HIF-1α, which can upregulate CCL20 expression

  • Creates a feedback loop enhancing angiogenesis and immune cell recruitment

  • Cancer cells in hypoxic regions produce more CCL20, promoting invasion and metastasis

• Inflammatory Cytokines:

  • TNF-α, IL-1β, and IL-17 strongly induce CCL20 expression in tumor and stromal cells

  • NF-κB pathway activation serves as a major mediator of cytokine-induced CCL20 expression

  • Cytokine-rich environments amplify CCL20 production, creating chemotactic gradients

• Tumor-Associated Macrophages (TAMs):

  • M2-polarized TAMs can produce CCL20, recruiting CCR6+ regulatory T cells

  • This creates an immunosuppressive microenvironment favoring tumor growth

  • TAM-derived factors may also enhance tumor cell expression of CCL20

• Microbial Products:

  • In certain cancers (e.g., colorectal cancer), microbial components like LPS stimulate CCL20 production

  • Pattern recognition receptor activation links microbiome alterations to CCL20-mediated inflammation

Signaling Pathways Regulating CCL20 in Cancer:

• NF-κB Pathway:

  • Primary transcriptional regulator of CCL20 expression

  • Activated by inflammatory cytokines, growth factors, and cellular stress

  • Inhibition of NF-κB can substantially reduce CCL20 production

• STAT3 Signaling:

  • Activated by IL-6 family cytokines abundant in the TME

  • Promotes CCL20 expression and CCR6 upregulation on cancer cells

  • Creates autocrine/paracrine signaling loops enhancing tumor progression

• Wnt/β-catenin Pathway:

  • Dysregulated in many cancers and can promote CCL20 expression

  • Links developmental pathways to chemokine production

  • β-catenin accumulation correlates with increased CCL20 levels in some tumors

• MAPK Pathways:

  • ERK1/2 activation enhances CCL20 production in response to growth factors

  • p38 MAPK mediates stress-induced CCL20 expression

  • JNK pathway regulates CCL20 in response to inflammatory stimuli

Functional Consequences in Cancer Progression:

CCL20 produced in the TME contributes to cancer progression through:

• Immunosuppression: Recruitment of CCR6+ regulatory T cells that suppress anti-tumor immunity

• Angiogenesis: Stimulation of endothelial cell migration and tube formation

• EMT Promotion: Induction of epithelial-to-mesenchymal transition in cancer cells

• Invasion/Metastasis: Enhanced migration and matrix degradation capacity

• Chemoresistance: Activation of survival pathways protecting against apoptosis

Understanding these complex regulatory networks provides opportunities for therapeutic targeting of CCL20 in cancer, potentially through:

• Inhibition of upstream inflammatory cytokines

• Blockade of key signaling pathways (NF-κB, STAT3)

• Neutralization of CCL20 or antagonism of CCR6

• Combination approaches targeting multiple aspects of the CCL20-CCR6 axis