Recombinant Mouse C-C motif chemokine 21c protein (Ccl21c) (Active)

What is the molecular structure of Mouse CCL21C protein?

Recombinant Mouse CCL21C is a full-length protein belonging to the intercrine beta (chemokine CC) family. The mature protein spans amino acids 24-133 with a molecular weight of approximately 12.8 kDa. The protein sequence is: SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG . The protein maintains its native tertiary structure when properly stored and handled, with disulfide bonds critical for its biological function. CCL21C belongs to the C-C motif chemokine family, characterized by adjacent cysteine residues that form specific disulfide bonds essential for protein stability and receptor interaction.

How does CCL21C differ from other mouse CCL21 isoforms?

Mouse C-C motif chemokine 21 exists in multiple isoforms including CCL21a (Scya21a), CCL21b (Scya21b), and CCL21c (Scya21c). The primary differences between these isoforms lie in subtle amino acid variations. CCL21a and CCL21b share high sequence homology but differ in specific residues, with CCL21a containing serine at position 65, while CCL21b contains leucine at the same position . CCL21C (UniProtKB: P86793) is distinguished by its specific amino acid sequence that affects its biological activity profile, particularly in lymphoid tissue targeting. While all isoforms function primarily as chemotactic factors, their differential expression patterns and binding affinities for CCR7 receptors suggest tissue-specific roles in immune regulation.

What expression systems are commonly used for producing recombinant CCL21C?

Recombinant Mouse CCL21C is primarily produced using E. coli expression systems, which yield protein with greater than 85% purity as determined by SDS-PAGE . When expressing CCL21C in bacterial systems, researchers must address challenges including proper protein folding and disulfide bond formation. Alternative expression platforms include mammalian cell lines (which provide proper post-translational modifications) and plant-based systems as demonstrated in experimental studies using N. tabacum . Each expression system offers distinct advantages:

What are the primary biological functions of CCL21C in mouse immune system?

CCL21C functions primarily as a chemotactic agent that regulates immune cell trafficking and lymphoid organ development. It inhibits hemopoiesis while stimulating chemotaxis, particularly for thymocytes and activated T-cells . The protein demonstrates selective chemotactic activity for specific cell populations, showing preferential activity toward naïve T-cells while exhibiting minimal chemotactic effects on B-cells, macrophages, and neutrophils. CCL21C serves as a potent mesangial cell chemoattractant and plays a critical role in mediating lymphocyte homing to secondary lymphoid organs . This directed migration is essential for immune surveillance and the initiation of adaptive immune responses. The protein facilitates T-cell activation through recruitment and co-localization of naïve lymphocytes and antigen-presenting dendritic cells, creating microenvironments conducive to antigen presentation and immune response development .

How does CCL21C interact with its receptor, and what signaling pathways does it activate?

CCL21C primarily signals through the CCR7 receptor, a G protein-coupled receptor expressed on various immune cells and certain cancer cells . Upon binding to CCR7, CCL21C initiates a cascade of intracellular signaling events that regulate cell migration, survival, and proliferation. The key signaling pathways activated include:

• G protein-coupled signaling leading to calcium mobilization

• Activation of mitogen-activated protein kinases (MAPKs), particularly ERK1/2

• Phosphatidylinositol 3-kinase (PI3K)/Akt pathway activation

• Modulation of small GTPases (Rho, Rac, Cdc42) that control cytoskeletal rearrangements

The in silico analysis of CCL21 binding to CCR7 demonstrates specific structural interactions that determine signaling intensity and specificity . This receptor-ligand interaction is critical for CCL21C's role in directing cellular movement toward lymphoid tissues and within the lymphoid microenvironment.

What is the role of CCL21C in cancer research models?

CCL21C and related isoforms have emerged as significant factors in cancer research due to their influence on immune cell trafficking and tumor microenvironments. The CCL21-CCR7 axis plays essential roles in lymphatic cancer metastasis, as CCR7 is expressed on many cancer tumor cells . In experimental models, CCL21 has demonstrated both pro-tumorigenic and anti-tumorigenic effects depending on the cancer type and microenvironmental context. The protein can attract CCR7-positive cancer cells to lymph nodes, potentially facilitating metastatic spread . Conversely, in certain experimental contexts, recombinant CCL21 has shown potential to reduce cancer cell proliferation. For instance, research indicates that purified CCL21/IL-1β fusion protein may decrease wound healing in MCF7CCR7+ cancer cell lines, suggesting potential anti-proliferative effects .

What are the recommended protocols for reconstitution and storage of CCL21C?

For optimal bioactivity retention, recombinant Mouse CCL21C requires specific handling protocols. Lyophilized CCL21C should be briefly centrifuged prior to opening to ensure all material is at the bottom of the vial . For reconstitution, dissolve the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . To enhance stability during storage, it is recommended to add glycerol to a final concentration of 5-50%, with 50% being the default recommendation for long-term storage . The reconstituted protein should be aliquoted to minimize freeze-thaw cycles and stored at -20°C to -80°C for maximum retention of biological activity. Quality control testing suggests that properly stored CCL21C maintains >90% of its activity for at least 12 months when stored as recommended. For short-term use (≤1 month), the reconstituted protein can be stored at 2-8°C.

How can researchers verify the biological activity of CCL21C in experimental settings?

Multiple complementary approaches can be employed to verify CCL21C's biological activity:

• Chemotaxis Assay: Using Boyden chamber or transwell migration assays with CCR7-expressing cells (such as naive T cells or specific dendritic cell populations). Active CCL21C induces dose-dependent migration with typical EC50 values in the 10-50 ng/mL range.

• Calcium Mobilization: Measuring intracellular calcium flux in CCR7-expressing cells following CCL21C stimulation using fluorescent calcium indicators.

• Receptor Binding Assays: Competitive binding assays using labeled CCL21C or displacement of known CCR7 ligands to confirm receptor specificity.

• Cell Proliferation Assays: MTT assays can be utilized to evaluate the effects of CCL21C on relevant cell populations .

• Wound Healing Assays: For cancer research applications, wound healing assays with CCR7-positive cancer cell lines can demonstrate CCL21C's impact on cell migration properties .

What considerations should researchers make when designing CCL21C-based experiments?

When designing experiments involving CCL21C, researchers should consider several critical factors:

• Receptor Expression: Verify CCR7 expression on target cells, as receptor density significantly impacts responsiveness to CCL21C. Flow cytometry quantification of CCR7 is recommended before experiments.

• Species Specificity: While mouse and human CCL21 share functional similarities, they exhibit species-specific differences in potency and receptor interaction profiles. Cross-species experiments should be interpreted with caution.

• Experimental Controls: Include appropriate positive controls (known CCR7 ligands) and negative controls (heat-inactivated protein) to validate assay specificity.

• Endotoxin Considerations: E. coli-expressed recombinant proteins may contain endotoxin, which can confound immunological experiments. High-quality preparations typically contain <1 EU/μg endotoxin .

• In vivo Considerations: CCL21C is an active protein and may elicit biological responses in vivo, requiring careful handling in animal studies .

How can CCL21C be utilized in lymphoid tissue engineering and immunotherapy?

CCL21C's ability to orchestrate immune cell migration makes it valuable for engineered lymphoid tissue construction and immunotherapeutic approaches. In tissue engineering applications, CCL21C can be incorporated into biomaterial scaffolds to create artificial lymphoid-like structures that facilitate immune cell interactions. Various delivery systems have been investigated, including:

• Controlled-release hydrogels: Incorporating CCL21C into hydrogels provides sustained release, creating chemokine gradients that direct immune cell migration and organization.

• Cell-based delivery: Genetically modified cells expressing CCL21C can establish localized chemokine production within specific tissue microenvironments.

• Fusion proteins: CCL21C-based fusion proteins, such as CCL21/IL1β constructs, represent an emerging approach for combining chemotactic activity with immunomodulatory functions .

For cancer immunotherapy, CCL21C-based approaches aim to enhance anti-tumor immune responses by promoting interactions between tumor antigens, dendritic cells, and T lymphocytes. Experimental evidence suggests that local delivery of CCL21C may enhance tumor infiltration by immune cells, potentially converting immunologically "cold" tumors to "hot" tumors more responsive to checkpoint inhibitor therapies.

What experimental approaches can distinguish between the functional roles of different mouse CCL21 isoforms?

Distinguishing the specific roles of mouse CCL21 isoforms (CCL21a, CCL21b, CCL21c) requires sophisticated experimental strategies due to their high sequence homology. Recommended approaches include:

• Isoform-specific gene editing: CRISPR-Cas9 technology targeting unique regions of each isoform gene allows selective knockout of individual isoforms while preserving others.

• Selective neutralization: Developing highly specific antibodies that recognize unique epitopes in each isoform permits selective neutralization in complex biological systems.

• Differential expression analysis: RNAseq and quantitative PCR with isoform-specific primers can reveal tissue-specific and condition-dependent expression patterns of each isoform.

• Receptor binding kinetics: Surface plasmon resonance (SPR) analysis comparing binding kinetics (kon, koff, KD) of each isoform to CCR7 can reveal subtle functional differences.

• Domain swapping experiments: Creating chimeric proteins by swapping domains between isoforms helps identify regions responsible for specific functional properties.

Experiments combining these approaches have revealed that despite their similarity, CCL21 isoforms may exhibit tissue-specific expression patterns and potentially different efficacies in immune cell recruitment and activation.

How does glycosylation affect CCL21C structure and function?

While E. coli-expressed recombinant CCL21C lacks glycosylation, native mouse CCL21C may undergo post-translational modifications including glycosylation, which can significantly impact protein properties. Studies comparing glycosylated versus non-glycosylated forms reveal several important differences:

• Receptor binding: Glycosylation can modulate CCR7 binding affinity and specificity, with glycosylated forms typically showing altered receptor interaction kinetics.

• Proteolytic resistance: Glycosylated CCL21C demonstrates enhanced resistance to proteolytic degradation, potentially extending its half-life in biological systems.

• Solubility and diffusion: Glycosylation affects the protein's biophysical properties, influencing solubility, diffusion rates, and gradient formation in tissues.

• Immunogenicity: Non-glycosylated recombinant CCL21C may exhibit different immunogenic properties compared to native glycosylated forms.

For research applications requiring glycosylated CCL21C, mammalian expression systems are preferable to E. coli production. Alternatively, enzymatic glycosylation of bacterially expressed protein can be employed to study specific glycoform effects.

How does mouse CCL21C compare structurally and functionally with human CCL21?

Mouse CCL21C and human CCL21 share approximately 70% sequence identity, with conserved cysteine residues critical for their tertiary structure. Key differences and similarities include:

These differences have significant implications for translational research, particularly when extrapolating findings from mouse models to human applications. Cross-species activity exists but with reduced potency, necessitating species-matched ligands for optimal experimental outcomes.

What challenges exist in studying CCL21C in complex disease models?

Researchers face several significant challenges when investigating CCL21C in disease models:

• Redundancy in the chemokine system: Multiple chemokines may signal through CCR7, creating functional redundancy that complicates interpretation of CCL21C-specific effects.

• Context-dependent activity: CCL21C function varies considerably depending on the tissue microenvironment, with different outcomes observed in lymphoid versus non-lymphoid tissues.

• Gradient formation complexity: CCL21C's biological activity depends on precise concentration gradients that are difficult to recreate experimentally, particularly in three-dimensional tissue models.

• Receptor regulation dynamics: CCR7 expression and signaling capacity are dynamically regulated during immune responses, creating temporal windows of sensitivity to CCL21C that may be missed in static experimental models.

• Isoform-specific tools limitation: Limited availability of tools that can distinguish between mouse CCL21 isoforms with high specificity restricts mechanistic studies.

Advanced techniques including intravital imaging, single-cell RNA sequencing, and computational modeling of chemokine gradients are emerging as valuable approaches to address these challenges.

What are the current contradictions in CCL21C research literature?

The scientific literature contains several apparent contradictions regarding CCL21C function that merit careful consideration:

• Pro-tumorigenic versus anti-tumorigenic effects: Some studies report CCL21C promotes tumor metastasis through CCR7-dependent mechanisms, while others demonstrate anti-tumor effects through enhanced immune cell recruitment .

• Inflammatory versus homeostatic functions: While often classified as a homeostatic chemokine lacking inflammatory responses , CCL21C expression can be upregulated in certain inflammatory conditions, suggesting context-dependent roles.

• Target cell specificity: Reports vary regarding CCL21C's ability to attract specific immune cell subsets, with some studies showing broader target cell ranges than others.

• Therapeutic potential discrepancies: Therapeutic applications of CCL21C show variable efficacy across different disease models, with beneficial effects in some contexts but negligible or detrimental effects in others.

These contradictions likely reflect the complex biology of CCL21C and the diversity of experimental systems used to study it. Resolving these discrepancies requires standardized experimental approaches and careful consideration of context-dependent factors.

What emerging applications exist for CCL21C in immunoengineering?

Cutting-edge immunoengineering applications for CCL21C include:

• Engineered organoids: CCL21C-containing organoids that recreate lymphoid tissue microenvironments for studying immune cell interactions and drug screening.

• Biomaterial-based vaccination: CCL21C-functionalized biomaterials that enhance dendritic cell recruitment and antigen presentation at vaccination sites.

• CAR-T cell enhancement: CCL21C-secreting CAR-T cells designed to recruit endogenous immune cells to tumor sites, potentially amplifying anti-tumor responses.

• Tissue-specific immune modulation: Targeted delivery systems that create localized CCL21C gradients to modify regional immune responses without systemic effects.

These approaches leverage CCL21C's natural role in coordinating immune cell positioning to create novel therapeutic strategies with potentially enhanced specificity and reduced side effects compared to conventional immunomodulatory approaches.

How might single-cell analysis technologies advance our understanding of CCL21C biology?

Single-cell technologies offer unprecedented opportunities to elucidate CCL21C function at cellular resolution:

• Single-cell RNA sequencing (scRNA-seq): Reveals cell-specific responses to CCL21C stimulation, identifying previously unrecognized responsive cell populations and heterogeneity within known target cells.

• CyTOF and spectral flow cytometry: Enable comprehensive phenotyping of CCL21C-responsive cells based on surface receptor expression and signaling pathway activation.

• Spatial transcriptomics: Maps CCL21C expression and responding cells within intact tissues, providing critical contextual information about chemokine gradient formation and cellular responses.

• Live-cell imaging with reporter systems: Visualizes real-time cellular responses to CCL21C at single-cell resolution, capturing dynamic behaviors including directional migration, velocity changes, and cell-cell interactions.

These technologies are revealing that cellular responses to CCL21C are more heterogeneous than previously recognized, with significant implications for understanding its role in normal physiology and disease states.

What potential exists for developing CCL21C-based precision medicine approaches?

The specific immunomodulatory properties of CCL21C offer several promising avenues for precision medicine:

• Biomarker development: CCL21C levels or associated signaling patterns may serve as predictive biomarkers for immunotherapy response or lymphoid malignancy progression.

• Personalized cancer vaccines: CCL21C incorporation into cancer vaccines could be tailored based on individual patients' immune profiles and tumor CCR7 expression patterns.

• Engineered therapeutic cells: Patient-derived cells engineered to express CCL21C could provide personalized immunomodulatory therapies for autoimmune conditions or cancer.

• Targeted delivery systems: Nanoparticle formulations designed to deliver CCL21C to specific tissue microenvironments based on individual disease characteristics.

Preliminary research suggests that CCL21C-based approaches may be particularly valuable in precision medicine contexts where modulating specific immune cell populations is desired without broad immunosuppression or activation.