Recombinant Mouse C-C motif chemokine 27 protein (Ccl27), partial (Active)

What is the primary function of Recombinant Mouse C-C motif chemokine 27 protein (Ccl27)?

Recombinant Mouse C-C motif chemokine 27 protein functions primarily as a chemotactic factor that selectively attracts skin-associated memory T-lymphocytes. It plays a crucial role in mediating homing of lymphocytes to cutaneous sites and may participate in cell migration during embryogenesis . The protein belongs to the intercrine beta (chemokine CC) family and specifically binds to the chemokine receptor CCR10, making it highly selective in its cellular targeting mechanisms . For experimental verification of biological activity, researchers can use dose-dependent measurements of beta-arrestin recruitment on CCR10 using overexpression systems such as U2OS cells with human CCR10/beta-arrestin/beta-galactosidase complementation .

How does Ccl27 differ structurally from other chemokines in the CC family?

While Ccl27 maintains the standard chemokine motif in its monomeric form, it demonstrates unique oligomerization patterns that contribute to its functional diversity. Unlike many other chemokines that maintain stable oligomeric states, Ccl27 transitions between monomeric, dimeric, and tetrameric states over a relatively narrow concentration range . Structural analysis through NMR spectroscopy reveals that this dynamic equilibrium involves different interfaces simultaneously at work, suggesting a complex quaternary structure regulation . The protein's distinctive C-terminal region plays a significant role in receptor activation, which differs from the conventional mechanism of other chemokines where the N-terminal region typically dominates receptor interactions . These structural peculiarities make Ccl27 an interesting subject for comparative chemokine studies.

What expression systems are optimal for producing functional Recombinant Mouse Ccl27?

For optimal expression of functional Recombinant Mouse Ccl27, HEK 293 cells have proven effective in producing the biologically active protein . When designing expression protocols, researchers should focus on the partial active fragment spanning amino acids 26-120, as this region contains the essential functional domains . The expression process requires careful quality control to ensure endotoxin levels remain below 0.005 EU/μg, which is critical for immunological applications . Following expression, the protein can be effectively characterized using SDS-PAGE, mass spectrometry (MS), and high-performance liquid chromatography (HPLC) . For NMR studies requiring isotopically labeled protein, specialized expression and purification methods have been developed involving milligram-scale production of both unlabeled and isotopically labeled functional protein .

How can researchers effectively analyze the oligomerization states of Ccl27 for structure-function studies?

To effectively analyze Ccl27 oligomerization states, researchers should implement a multi-technique approach centered on NMR spectroscopy. Pulsed field gradient (PFG) NMR diffusion experiments provide crucial insights into the transition between monomeric, dimeric, and tetrameric species across concentration gradients . This methodology should be complemented with 15N-1H HSQC chemical shift perturbation analysis to identify interface residues involved in oligomerization .

For researchers examining the relationship between oligomerization and function, the following methodological workflow is recommended:

• Concentration-dependent PFG-NMR diffusion studies (50-1000 μM range)

• Filtered (HC)NH-NOEs experiments to identify intermonomer contacts

• Site-directed mutagenesis of putative interface residues

• Validation using chemical cross-linking assays with concentration gradients

When analyzing glycosaminoglycan (GAG)-induced oligomerization, heparin binding assays provide valuable complementary data. The experimental χ² value should be compared to theoretical models to assess agreement of the diffusion tensor model with experimental data . This comprehensive approach allows researchers to establish correlations between oligomeric state and specific biological functions.

What are the optimal assays for evaluating Ccl27-CCR10 receptor activation mechanisms?

Evaluating Ccl27-CCR10 receptor activation requires a systematic approach combining cellular and molecular techniques. The gold standard assay involves measuring beta-arrestin recruitment on CCR10 using U2OS cells overexpressing human CCR10/beta-arrestin/beta-galactosidase complementation systems in a dose-dependent manner . For comprehensive mechanistic studies, researchers should employ:

• Cellular migration assays with varying Ccl27 concentrations (10-1000 ng/ml)

• Calcium influx measurements following receptor binding

• Receptor desensitization assays with sequential stimulations

• Targeted mutagenesis of both N-terminal and C-terminal regions

Particular attention should be given to modifications of the N-terminal phenylalanine residue, as it is critical for optimal function. Introduction of a second phenylalanine at the N-terminus creates a "super-agonist" with approximately 10-fold increased activity . Researchers should also examine C-terminal modifications, which unusually play a significant role in receptor activation for this chemokine . These methodological approaches allow researchers to identify partial agonists and antagonists for potential therapeutic applications.

How does Ccl27 contribute to cutaneous immune surveillance and what models best demonstrate this function?

Ccl27 plays a critical role in cutaneous immune surveillance by regulating T cell homing under both homeostatic and inflammatory conditions . To effectively study this process, researchers should utilize murine models with fluorescently labeled T cells to track migration patterns following Ccl27 administration or neutralization .

The following experimental approaches yield robust data on Ccl27's role in skin immunity:

• DNFB (2,4-dinitrofluorobenzene) application to trigger inflammation and monitoring Ccl27 accumulation in skin-draining lymph nodes

• Quantitative RT-PCR measurement of CCR10 mRNA in skin-draining lymph nodes, normalized to CD2 expression

• Temporal association analysis between Ccl27 transport and increased numbers of CCR10-expressing T cells

These models reveal that skin-derived Ccl27 is transported to draining lymph nodes following inflammatory stimuli, resulting in a transient ~5-fold increase in normalized CCR10 mRNA expression, suggesting influx of CCR10-positive T cells . This process is time-dependent, with CCR10 expression returning to baseline levels by 24 hours post-stimulation, coinciding with Ccl27 protein level normalization .

What methodological approaches best characterize Ccl27's role in tumor immune escape mechanisms?

To characterize Ccl27's role in tumor immune escape, researchers should implement a comprehensive approach combining molecular, cellular, and in vivo methods. The experimental workflow should include:

• Quantitative PCR analysis comparing Ccl27 expression across tissue samples representing different stages of cutaneous carcinogenesis

• Immunohistochemical assessment of phosphorylated ERK levels as an indicator of EGFR-Ras pathway activation

• In vitro modulation of EGFR-Ras signaling using EGF stimulation, H-RasV12 transfection, and EGFR tyrosine kinase inhibitors

• In vivo neutralization of Ccl27 with antibodies to assess effects on leukocyte recruitment and tumor growth

These methodological approaches reveal that keratinocyte-derived skin tumors may evade T cell-mediated antitumor immune responses by down-regulating Ccl27 expression through activation of the EGFR-Ras-MAPK signaling pathway . In vivo neutralization experiments demonstrate that blocking Ccl27 leads to decreased leukocyte recruitment to cutaneous tumor sites and significantly enhances primary tumor growth .

How can researchers effectively study the dynamics of Ccl27 transport in the lymphatic system?

Studying Ccl27 lymphatic transport dynamics requires temporal and spatial tracking methodologies. Researchers should implement:

• Time-course experiments following inflammatory stimulus (e.g., DNFB application) to track Ccl27 accumulation in skin-draining lymph nodes

• Fluorescently labeled Ccl27 for direct visualization of protein trafficking

• Quantitative RT-PCR to measure changes in CCR10 mRNA expression in lymph nodes, normalized to CD2 (T cell marker)

• Correlative analysis between Ccl27 protein levels and CCR10+ T cell influx

This methodological approach reveals that Ccl27 rapidly accumulates in skin-draining lymph nodes following inflammatory stimuli, with a corresponding transient increase in CCR10+ T cells . The temporal relationship shows that CCR10 mRNA expression increases approximately 5-fold following inflammation but returns to baseline levels by 24 hours post-stimulation, coinciding with normalization of Ccl27 protein levels . This methodology provides crucial insights into the kinetics of chemokine-directed lymphocyte trafficking in cutaneous immune responses.

What are the most common challenges in maintaining Ccl27 biological activity during purification and storage?

Maintaining Ccl27 biological activity presents several challenges that researchers must address through careful methodology. The protein's tendency to form different oligomeric states (monomer, dimer, and tetramer) across concentration gradients complicates purification and stability . Key methodological considerations include:

• Concentration management: Maintain protein concentration within the monomeric-favoring range (typically <100 μM) when monomer is the desired form

• Buffer optimization: Use buffers containing low concentrations of reducing agents to prevent disulfide-mediated aggregation

• Glycosaminoglycan contamination control: Remove GAG contaminants as they induce oligomerization

• Storage temperature regulation: Store at -80°C in single-use aliquots to prevent freeze-thaw cycles

When performing solubility assays, researchers should carefully monitor precipitation thresholds . For activity measurements, use the beta-arrestin recruitment assay on CCR10 as the gold standard for confirming biological function . Since Ccl27 is "fully biologically active," researchers should verify activity batch-to-batch using functional assays rather than relying solely on structural integrity tests .

How should researchers address experimental discrepancies when comparing in vitro and in vivo Ccl27 functionality?

When confronting discrepancies between in vitro and in vivo Ccl27 functionality, researchers should implement a systematic troubleshooting approach addressing multiple variables. Key methodological considerations include:

• Oligomerization state differences: In vitro studies typically use defined concentrations favoring particular oligomeric states, while in vivo environments may contain variable concentrations affecting oligomerization dynamics

• Glycosaminoglycan interactions: In vivo GAG binding induces oligomerization, potentially altering function compared to in vitro systems lacking GAGs

• Receptor expression density variations: In vitro systems often use cells overexpressing CCR10, whereas in vivo models have physiological receptor levels

• Proteolytic processing differences: In vivo environments contain proteases that may process Ccl27 differently than controlled in vitro conditions

For experimental reconciliation, researchers should:

• Perform concentration-dependent studies spanning physiologically relevant ranges

• Include GAGs in in vitro systems to mimic in vivo conditions

• Use cell lines with varying receptor densities

• Analyze Ccl27 integrity in biological samples to detect potential processing events

The apparent contradictions often stem from Ccl27's complex biology, where it exists in multiple oligomeric states and interacts with different binding partners under varying conditions .

What are promising approaches for studying the differential effects of Ccl27 oligomeric states on biological function?

Studying the differential effects of Ccl27 oligomeric states requires innovative methodological approaches targeting specific oligomeric forms. Promising research strategies include:

• Engineering Ccl27 variants with stabilized oligomeric states through strategic disulfide bonds or interface mutations based on NMR structural data

• Developing oligomerization-specific antibodies that selectively recognize different quaternary structures

• Implementing advanced biophysical techniques including:

  • Single-molecule FRET to detect transitions between oligomeric states

  • Analytical ultracentrifugation with fluorescence detection for real-time monitoring

  • Advanced NMR methods such as paramagnetic relaxation enhancement (PRE) to map oligomeric interfaces

Researchers should correlate oligomeric state with specific biological functions through parallel assays of receptor activation, cell migration, and glycosaminoglycan binding . The relationship between oligomerization and function could reveal novel therapeutic strategies targeting specific Ccl27 quaternary structures to modulate immune responses in skin conditions and cancer.

How might computational modeling enhance our understanding of Ccl27-CCR10 interactions for targeted drug development?

Computational modeling offers powerful approaches for understanding Ccl27-CCR10 interactions at the molecular level, with significant implications for drug development. Advanced methodological strategies include:

• Molecular dynamics simulations of Ccl27 in different oligomeric states to identify dynamic conformational changes affecting receptor binding

• Homology modeling of CCR10 based on other chemokine receptor structures, refined through experimental constraints

• Protein-protein docking algorithms to predict Ccl27-CCR10 binding interfaces, with validation through mutagenesis data

• Virtual screening of compound libraries against the Ccl27-CCR10 interface to identify potential modulators

These computational approaches should incorporate the unique features of Ccl27, particularly the significant role of both N-terminal and C-terminal regions in receptor activation . The finding that adding a second phenylalanine at the N-terminus creates a "super-agonist" with 10-fold increased activity provides a valuable pharmacophore model for agonist design . Conversely, partial antagonist properties observed in certain mutants offer templates for developing receptor blockers. Integration of computational results with experimental validation through migration assays, calcium flux measurements, and receptor desensitization studies will accelerate the development of targeted therapeutics for inflammatory skin diseases and potentially cancer immunotherapy.