Recombinant Human C-C motif chemokine 21 protein (CCL21)

What is the basic structure and molecular characteristics of CCL21?

CCL21 is a small cytokine belonging to the CC chemokine family, also known as 6Ckine (due to having six conserved cysteine residues instead of the typical four found in chemokines), exodus-2, and secondary lymphoid-tissue chemokine (SLC). The predicted molecular mass of recombinant human CCL21 is approximately 38.3 kDa, though its actual observed molecular weight on SDS-PAGE is significantly higher at 95-105 kDa, indicating potential post-translational modifications or structural characteristics affecting migration .

The protein structure features conserved cysteine residues that are crucial for its tertiary structure and biological function. When studying CCL21, researchers should consider that while the amino acid sequence suggests a certain molecular weight, observed experimental values may differ substantially due to glycosylation and other modifications.

What are the primary cellular receptors for CCL21 and signaling pathways involved?

CCL21 primarily exerts its effects by binding to the cell surface chemokine receptor CCR7. Research has shown that CCL21 binds locally to CCR7 at neuronal growth cones, activating the downstream MEK-ERK pathway, which subsequently activates N-WASP . This signaling cascade is important for various cellular processes including chemotaxis and cytoskeletal reorganization.

Interestingly, while CCL19 is another ligand for CCR7, experimental evidence indicates differential effects. CCL21 shows biased activation of CCR7, as demonstrated by studies where CCL21 administration increased neurite outgrowth while CCL19 did not produce the same effect . Western blot analysis has shown that CCL21 increases phosphorylated ERK (pERK) expression 50 minutes after administration, whereas CCL19 shows reduced pERK induction .

Methodologically, when studying CCL21 signaling pathways:

• Use MEK inhibitors like U0126 as experimental controls to confirm pathway involvement

• Monitor pERK levels as a readout of pathway activation

• Consider the timing of pathway activation (significant at 50 minutes post-treatment)

How can CCL21 be detected and quantified in biological samples?

CCL21 can be detected and quantified using several complementary approaches:

• Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative measurement of CCL21 in serum, tissue homogenates, or cell culture supernatants.

• Luminex-based multiplex assays: Allows simultaneous detection of CCL21 along with other cytokines/chemokines. Note that correlation between Luminex and ELISA measurements may be moderate (r=0.515, p<0.001), suggesting methodological considerations when comparing results from different platforms .

• Immunohistochemistry (IHC): Useful for localizing CCL21 expression in tissues. For instance, IHC has revealed that CCL21 is predominantly expressed in airway epithelial cells of systemic sclerosis patients with pulmonary arterial hypertension .

• Mass spectrometry (MS): Provides detailed information about protein structure and modifications. MS analysis has identified peptides located within amino acids 23-102 of CCL21, suggesting that it may circulate as a truncated protein without the C-terminal tail in certain conditions .

When quantifying CCL21, researchers should consider potential confounding factors such as anti-CCL21 antibodies present in some patient populations, though these appear to be present in only approximately 5% of systemic sclerosis patients .

How does CCL21 influence neuronal development and function?

CCL21 has been shown to play a significant role in neuronal development, particularly in dorsal root ganglia (DRG) neurons. Research demonstrates that CCL21 induces neurite outgrowth through a specific molecular mechanism:

• CCL21 binds to CCR7 receptors expressed on neurons, including parvalbumin-positive (PV+) proprioceptors, with relatively few CCR7+TRPV1+ nociceptors also expressing the receptor .

• This binding activates the MEK-ERK pathway, as demonstrated by inhibition studies. When CCL21 and U0126 (a MEK inhibitor) are co-administered, neurite outgrowth is significantly reduced compared to CCL21 administration alone .

• The specificity of this mechanism is evident from the following experimental findings:

  • CCR7-blocking antibodies inhibit CCL21-induced neurite outgrowth

  • CCL19, another CCR7 ligand, does not induce comparable neurite outgrowth

  • Western blot analysis confirms increased pERK expression 50 minutes after CCL21 administration

For researchers studying neuronal development, these findings suggest that CCL21 represents a nociception-dependent chemokine capable of inducing structural changes in sensory neurons through biased receptor activation and specific downstream pathways.

What role does CCL21 play in cancer immunotherapy and how can it be leveraged as a biomarker?

CCL21 demonstrates significant potential as both a biomarker and therapeutic target in cancer immunotherapy:

Recent research has identified CCL21 as a predictive biomarker for immunotherapy response in hepatocellular carcinoma (HCC). Transcriptome analysis revealed that CCL21 levels were significantly higher in HCC patients who responded to immune checkpoint inhibitors (ICIs) . This predictive capacity has led to the development of a nomogram that can classify patients based on their likelihood of response.

The underlying mechanism appears to involve CCL21's ability to modulate the tumor microenvironment (TME):

• CCL21 inhibits N2 neutrophil polarization by suppressing the activation of the nuclear factor kappa B (NF-κB) pathway .

• This inhibition of immunosuppressive neutrophil phenotypes enhances the efficacy of immune checkpoint inhibitors.

• In experimental models, combining CCL21 with ICIs demonstrated enhanced therapeutic efficacy compared to ICI monotherapy .

For researchers studying cancer immunotherapy:

• Consider measuring CCL21 levels in tumor tissues to predict immunotherapy response

• Explore the relationship between CCL21 expression and neutrophil polarization states in the TME

• Investigate potential synergistic effects between recombinant CCL21 and various immunotherapeutic agents

What protein-protein interactions influence CCL21 function in biological systems?

Bioinformatic analysis reveals that CCL21 engages in multiple types of interactions with other proteins that significantly influence its biological functions:

CCL21 is co-expressed, co-localized, physically interacts with, and shares protein domains and pathways with several proteins, particularly CCL19, CCR7, and CCR6 . These interactions suggest functional relationships that extend beyond simple ligand-receptor binding.

Based on GeneMANIA network analysis, CCL21 and its interacting partners play crucial roles in:

• Regulation of leukocytes: This function has particular relevance to drug resistance, as studies have demonstrated a direct relationship between DNA damage in leukocytes and disease response to platinum-based treatments .

• Neutrophil chemotaxis: While indirectly linked to drug resistance, this function becomes relevant when considering compounds like celastrol (an inhibitor of neutrophil chemotaxis) that induce synergistic apoptosis when combined with conventional microtubule-targeting drugs .

• G-protein coupled receptor activity: These receptors regulate cellular processes fundamental to cancer pathology, including differentiation, proliferation, migration, tissue invasion, survival, and drug resistance .

• Calcium ion regulation: Calcium content increases in multidrug resistant cells, and resistance can be reversed by calcium channel blockers like verapamil, suggesting calcium's role in drug resistance mechanisms .

For researchers investigating CCL21's functional networks, these interaction data provide potential targets for modulating CCL21 activity in experimental and therapeutic contexts.

How do post-translational modifications affect CCL21 bioactivity?

Post-translational modifications significantly impact CCL21 bioactivity and should be carefully considered in experimental design:

The predicted molecular weight of recombinant human CCL21 is 38.3 kDa, but its observed migration on SDS-PAGE shows a significantly higher molecular weight of 95-105 kDa . This discrepancy suggests extensive post-translational modifications that affect protein size and potentially function.

Mass spectrometry analysis of circulating CCL21 in systemic sclerosis patients revealed that only peptides located within amino acids 23-102 were detected, indicating that CCL21 may circulate as a truncated protein without the C-terminal tail in certain pathological conditions . This finding has important implications:

• The C-terminal region of chemokines often contains critical functional domains that influence receptor binding affinity and specificity.

• Truncated forms may exhibit altered bioactivity compared to full-length protein.

• When designing experiments, researchers should consider which form of CCL21 (full-length vs. truncated) is most relevant to their biological question.

For accurate experimental results, researchers should:

• Specify which form of CCL21 is being used in experiments

• Consider using both forms to compare functional differences

• Develop assays that can distinguish between full-length and truncated forms in biological samples

What are the optimal conditions for using recombinant CCL21 in cell culture experiments?

When designing cell culture experiments with recombinant CCL21, consider the following evidence-based methodological guidelines:

• Concentration: Effective concentrations of CCL21 typically range from 10-100 nM, with 50 nM showing significant effects in neuronal outgrowth studies . Dose-response curves should be established for each cell type and experimental endpoint.

• Timing: Signaling effects occur relatively quickly, with phosphorylation of ERK detectable at 50 minutes post-treatment . For phenotypic changes like neurite outgrowth, longer timeframes (24 hours) may be necessary.

• Cell types: Consider that CCL21 affects multiple cell types differently:

  • Neuronal cells: Induces neurite outgrowth in CCR7+ neurons

  • Immune cells: Attracts T cells and dendritic cells

  • Cancer cells: May attract CCR7-bearing cancer cells

  • Neutrophils: Inhibits N2 polarization

• Controls: Important experimental controls include:

  • CCL19 as a control CCR7 ligand with different bioactivity profile

  • CCR7-blocking antibodies (2-5 μg/ml has shown significant inhibition)

  • Pathway inhibitors (e.g., U0126 for MEK-ERK pathway)

• Readouts: When analyzing CCL21 effects, consider monitoring:

  • Phosphorylation of ERK via Western blot

  • Neurite outgrowth via Tuj-1 immunostaining

  • Neutrophil polarization markers

  • Cell migration and chemotaxis

How can researchers develop assays to study CCL21-CCR7 signaling specificity?

Developing assays to study the specificity of CCL21-CCR7 signaling requires careful consideration of the biased activation observed between different CCR7 ligands. The following methodological approaches may be useful:

• Comparative ligand studies: Include both CCL21 and CCL19 in parallel experiments to identify biased signaling effects. While both bind CCR7, they produce different downstream effects .

• Pathway-specific readouts: Monitor multiple downstream pathways simultaneously:

  • MEK/ERK pathway: Measure pERK levels via Western blot at 50 minutes post-treatment

  • G-protein vs. β-arrestin recruitment: Use BRET or FRET-based assays to distinguish pathway preferences

  • Cytoskeletal reorganization: Monitor N-WASP activation or actin polymerization

• Receptor blocking strategies:

  • Use CCR7-blocking antibodies at varying concentrations (2-5 μg/ml)

  • Compare effects of IgG controls to ensure specificity

  • Consider siRNA knockdown or CRISPR-based approaches for receptor depletion

• Functional readouts tailored to cell type:

  • Neurons: Measure neurite outgrowth and axon guidance

  • Immune cells: Assess chemotaxis and activation markers

  • Cancer cells: Evaluate invasion, migration, and therapy response

• In vitro vs. ex vivo approaches:

  • Cultured cell lines expressing CCR7

  • Primary cells from relevant tissues

  • Tissue explants (particularly useful for complex cellular interactions)

What are the methodological considerations for studying CCL21 in animal models of disease?

When designing animal studies involving CCL21, researchers should consider several methodological aspects that can influence experimental outcomes:

• Model selection: Different disease models may require specific approaches:

  • Cancer models: Subcutaneous tumor implantation has been used to study CCL21's role in immunotherapy response

  • Neurological models: Consider nociception-related models to study sensory neuronal responses

  • Autoimmune/inflammatory models: Relevant for studying systemic sclerosis and pulmonary arterial hypertension

• Administration routes and dosing:

  • Local vs. systemic administration will have different effects

  • For cancer studies, consider intra-tumoral vs. systemic delivery

  • Dosing should be determined through pilot studies with pharmacokinetic profiling

• Combinatorial approaches:

  • For cancer immunotherapy studies, CCL21 can be combined with immune checkpoint inhibitors to assess synergistic effects

  • Consider combinations with pathway inhibitors to verify mechanism

• Assessment techniques:

  • Immunohistochemistry to localize CCL21 expression in target tissues

  • Flow cytometry to characterize immune cell infiltration and phenotypes

  • Transcriptomic analysis to assess broader pathway effects

  • Functional assays specific to the disease model (tumor growth, neuronal function, etc.)

• Timing considerations:

  • Acute vs. chronic effects may differ substantially

  • Consider the temporal relationship between CCL21 administration and disease progression

  • Monitor outcomes at multiple timepoints to capture dynamic responses

Why might I observe discrepancies between different methods of CCL21 quantification?

Discrepancies between different methods of CCL21 quantification are common and may arise from several factors:

• Assay platform differences: Research has found only moderate correlation (r=0.515, p<0.001) between Luminex and ELISA measurements of CCL21 . This suggests inherent differences in assay sensitivity, dynamic range, or epitope recognition.

• Protein truncation and modification: Mass spectrometry studies have identified that CCL21 may circulate as a truncated protein without the C-terminal tail in certain conditions . Different assays may have varying abilities to detect these modified forms:

  • Antibodies targeting the C-terminal region will not detect truncated forms

  • Assays using antibodies recognizing central epitopes may detect both forms

• Anti-CCL21 antibodies in samples: Approximately 5% of systemic sclerosis patients have been found to have anti-CCL21 antibodies . These endogenous antibodies may interfere with assay performance by:

  • Blocking epitopes needed for detection

  • Creating false-negative results

  • Forming immune complexes that alter CCL21 detection

• Sample processing variations: Differences in:

  • Sample collection (timing, anticoagulants)

  • Storage conditions and freeze-thaw cycles

  • Pre-analytical processing steps

To minimize discrepancies, researchers should:

• Use multiple detection methods when possible

• Include appropriate controls

• Consider sample pre-treatment to address potential interfering factors

• Be consistent with assay platform choice when comparing across studies or timepoints

How can researchers address potential confounding factors when studying CCL21 in human samples?

When working with human samples to study CCL21, several confounding factors may impact results and interpretation:

• Patient heterogeneity: Individual variations in:

  • Disease state and severity

  • Concurrent medications

  • Comorbidities

  • Age and sex differences

• Sample-specific factors:

  • Presence of anti-CCL21 antibodies in approximately 5% of systemic sclerosis patients

  • Protein truncation (loss of C-terminal tail) in circulating CCL21

  • Varying levels of binding proteins or soluble receptors

• Methodological considerations:

  • Timing of sample collection relative to disease activity or treatment

  • Sample processing and storage conditions

  • Assay selection (considering moderate correlation between platforms)

To address these confounding factors, researchers should:

• Implement rigorous inclusion/exclusion criteria and document patient characteristics thoroughly

• Include adequate sample sizes to account for biological variability

• Use multiple detection methods when possible (e.g., both ELISA and Luminex)

• Consider testing for the presence of anti-CCL21 antibodies

• Use assays capable of distinguishing between full-length and truncated forms

• Include appropriate healthy controls matched for demographic variables

• Analyze data stratified by potential confounding variables

• Consider longitudinal sampling to account for temporal variations

What emerging technologies might advance CCL21 research?

Several cutting-edge technologies show promise for advancing CCL21 research:

• Single-cell technologies:

  • Single-cell RNA sequencing to identify cell populations responding to CCL21

  • Single-cell proteomics to characterize signaling at the individual cell level

  • Spatial transcriptomics to map CCL21 and CCR7 expression in complex tissues

• Advanced imaging approaches:

  • Intravital microscopy to visualize CCL21-mediated cell trafficking in vivo

  • Super-resolution microscopy to study CCL21-CCR7 interactions at the molecular level

  • Multi-parameter imaging mass cytometry to characterize cellular responses in tissue context

• Protein engineering and analysis:

  • CRISPR-based approaches to modify CCL21 or CCR7 in cellular models

  • Engineered variants of CCL21 to study structure-function relationships

  • Mass spectrometry imaging to localize and characterize CCL21 modifications in tissues

• Computational approaches:

  • Systems biology modeling of CCL21-dependent signaling networks

  • Machine learning to identify patterns in CCL21-associated gene expression

  • Prediction of CCL21 structure-function relationships through molecular dynamics simulations

• Translational platforms:

  • Organoid and microphysiological systems to study CCL21 in tissue-like environments

  • Patient-derived xenografts to evaluate CCL21-based therapeutic approaches

  • Combination therapy screening platforms incorporating CCL21 modulators

How might understanding CCL21 biology lead to novel therapeutic approaches?

The multifaceted roles of CCL21 suggest several promising therapeutic directions:

• Cancer immunotherapy enhancement:

  • CCL21 level assessment as a biomarker for immunotherapy response prediction

  • Combination therapy approaches using CCL21 with immune checkpoint inhibitors

  • Targeted delivery of CCL21 to tumors to enhance T cell and dendritic cell recruitment

• Neurological applications:

  • Modulation of CCL21-CCR7 signaling to promote neuronal regeneration after injury

  • Targeting MEK-ERK pathway activation in sensory neurons for pain management

  • Exploiting biased signaling to selectively activate beneficial neuronal responses

• Inflammatory and autoimmune conditions:

  • CCL21 monitoring as a biomarker for pulmonary arterial hypertension in systemic sclerosis

  • Development of agents that modify CCL21 structure or function

  • Targeting CCL21-mediated neutrophil polarization in inflammatory environments

• Drug resistance mechanisms:

  • Exploiting CCL21's relationships with leukocyte regulation and neutrophil chemotaxis

  • Combining CCL21-targeting approaches with conventional therapies to overcome resistance

  • Modulating calcium signaling pathways associated with CCL21 to address multidrug resistance

• Delivery strategies:

  • Localized delivery systems for tissue-specific CCL21 effects

  • Modified versions of CCL21 with enhanced stability or selective receptor activation

  • Biomaterial approaches for sustained release of CCL21 in target tissues