Recombinant Mouse C-C motif chemokine 28 protein (Ccl28), partial (Active)

What is Mouse CCL28 and what are its primary biological functions?

Mouse CCL28 (also known as mucosae-associated epithelial chemokine or MEC) is a chemokine that regulates the chemotaxis of cells expressing the chemokine receptors CCR3 and CCR10. It is primarily involved in immune cell trafficking and mucosal immunity. CCL28 drives the mucosal homing of T and B lymphocytes that express CCR10 and facilitates the migration of eosinophils expressing CCR3 . It is chemotactic for resting CD4+ and CD8+ T-cells and eosinophils, and it induces calcium mobilization in target cells in a dose-dependent manner . Importantly, CCL28 is a key regulator of IgA antibody-secreting cells (ASCs) accumulation in the mammary gland, controlling the passive transfer of IgA antibodies from mother to infant .

How does mouse CCL28 compare structurally to human CCL28?

Mouse CCL28 shares 83% amino acid identity with human CCL28 in their mature regions, indicating high evolutionary conservation . This substantial homology suggests functional similarity between species, making mouse models relevant for studying human CCL28-related biology. Among CC chemokines, CCL28 shares the most sequence homology with CCL27/CTACK in both species . The mouse protein has a predicted molecular weight of approximately 12.3-12.6 kDa, slightly less than the human protein which has a reported mass of 14.3 kDa .

Where is CCL28 primarily expressed in mouse tissues?

Based on Northern blot analysis, mouse CCL28 is mainly expressed in testes, kidney, and brain . It is produced by epithelial cells, particularly columnar epithelial cells in the gut, lung, breast, and salivary glands . CCL28 is constitutively expressed in the colon, though its expression can be upregulated by pro-inflammatory cytokines and certain bacterial products, suggesting its involvement in recruiting effector cells to sites of epithelial injury . This expression pattern aligns with its role in mucosal immunity and is important to consider when designing experiments involving tissue-specific functions.

What are the key structural features of recombinant mouse CCL28?

Recombinant mouse CCL28 is typically produced as a partial protein covering amino acids 20-130 of the native sequence . The mature protein sequence begins with Ile23 and contains multiple crucial structural elements typical of CC chemokines . The amino acid sequence is:

ILPMASSCCTEVSH​HVSGRLLERVSSCS​IQRADGDCDLAAVI​LHVKRRRICISPHN​RTLKQWMRASEKVKK​NGRENVCS​GKKQPSRKDR​KGHTTRKHRT​RGTHRHEASR

The protein belongs to the intercrine beta (chemokine CC) family and has a predicted molecular weight of 12.3-12.6 kDa . Its structure contains conserved cysteine residues crucial for disulfide bond formation and tertiary structure stabilization, which are characteristic of CC chemokines.

What receptors does mouse CCL28 interact with and what cellular responses does it trigger?

Mouse CCL28 primarily interacts with two chemokine receptors:

Upon binding to CCR10, CCL28 induces calcium mobilization in a dose-dependent manner . The biological activity of mouse CCL28 is typically determined by its ability to induce chemotaxis of mouse BaF/3 cells transfected with mCCR10, with an expected ED₅₀ of 0.3-1 μg/ml . CCL28 is a key mediator in intestinal extravasation of IgA antibody-secreting cells through its interaction with CCR10 . These receptor interactions are crucial for understanding experimental design when studying CCL28-mediated cellular responses.

How does the production method affect recombinant mouse CCL28 properties?

Recombinant mouse CCL28 is typically expressed in E. coli expression systems . This prokaryotic expression system offers high protein yield but lacks the post-translational modification machinery present in mammalian cells. Consequently, E. coli-produced CCL28 may lack glycosylation that has been described for native CCL28 .

The recombinant protein is typically purified to >97% as determined by SDS-PAGE and silver staining . Quality control includes ensuring low endotoxin levels (<1.0 EU/μg or <0.01 EU/μg depending on manufacturer) as determined by the LAL (Limulus Amebocyte Lysate) method . These production characteristics are important to consider when interpreting experimental results, particularly when comparing to in vivo-derived CCL28 which would contain post-translational modifications.

What are validated methodologies for assessing mouse CCL28 activity in vitro?

Several validated methodologies exist for assessing mouse CCL28 activity:

For optimal results when measuring chemotaxis, researchers should use mouse BaF/3 cells transfected with mCCR10 and expect an ED₅₀ in the range of 0.3-1 μg/ml . The biological activity assays should be performed with freshly reconstituted protein under appropriate buffer conditions to maintain protein integrity and ensure reproducible results.

How should recombinant mouse CCL28 be stored and handled for maximum stability and activity?

Recombinant mouse CCL28 is typically supplied as a lyophilized protein. Based on available data, the following handling recommendations apply:

• Storage: Store lyophilized protein desiccated at -20°C or lower for 6-12 months

• Formulation: Typically lyophilized from solutions containing acetonitrile (35%) and trifluoroacetic acid (0.1%)

• Reconstitution: Reconstitute in sterile water or appropriate buffer with carrier protein (e.g., 0.1% BSA) to prevent adhesion to tubes

• Working solutions: Prepare fresh dilutions for each experiment

• Avoid repeated freeze-thaw cycles: Aliquot reconstituted protein

• Quality control: Verify activity periodically, especially after prolonged storage

Proper handling is crucial as improper storage or excessive freeze-thaw cycles can lead to protein degradation and loss of biological activity, compromising experimental results.

What are appropriate experimental controls when studying CCL28 function in immunological assays?

When designing experiments to study CCL28 function, several controls should be incorporated:

Additionally, when performing in vivo experiments, vehicle-treated controls and irrelevant chemokine controls should be included. For studies involving mucosal immunity, tissue-specific controls are essential to account for local microenvironmental factors that might influence CCL28 function.

How can recombinant mouse CCL28 be utilized to study mucosal immunity mechanisms?

Recombinant mouse CCL28 serves as a valuable tool for investigating mucosal immunity through multiple experimental approaches:

• Ex vivo migration studies: Using explanted mucosal tissues to study CCL28-mediated immune cell trafficking within tissue-specific microenvironments

• CCL28-dependent IgA transport models: Investigating the role of CCL28 in regulating IgA antibody-secreting cell accumulation in the mammary gland and subsequent passive immunity transfer from mother to infant

• Intestinal extravasation studies: Examining CCL28's role in intestinal extravasation of IgA antibody-secreting cells, as demonstrated in previous research

• Hormone regulation studies: Investigating how estrogen controls CCL28 expression in uterine tissues, which has been shown to attract CCR10+ IgA plasma cells following mucosal vaccination

• Mucosal vaccination strategies: Exploiting CCL28's chemotactic properties to enhance vaccine-induced immunity at mucosal surfaces

These applications highlight CCL28's significance in understanding the regulation of mucosal immune responses, which is critical for developing interventions for infectious diseases, autoimmunity, and cancer affecting mucosal tissues.

What experimental approaches can assess the interplay between CCL28 and inflammation in mouse models?

Several sophisticated approaches can be employed to study CCL28's role in inflammation:

• Cytokine-induced expression analysis: Treating epithelial cells or tissues with pro-inflammatory cytokines (TNF-α, IL-1β) and measuring CCL28 upregulation using qPCR, ELISA, or immunohistochemistry

• Bacterial product stimulation: Exposing epithelial cells to bacterial products and assessing CCL28 production to model infection-associated inflammatory responses

• Inflammatory disease models: Using established mouse models of colitis, asthma, or other inflammatory conditions to assess CCL28 expression kinetics and function

• Conditional gene manipulation: Employing tissue-specific or inducible CCL28 knockout or overexpression systems to dissect its role in different inflammatory contexts

• Therapeutic intervention studies: Testing neutralizing antibodies against CCL28 or its receptors in inflammatory disease models

• Single-cell analysis: Applying scRNA-seq to identify cellular sources and targets of CCL28 during inflammation in relevant tissues

These approaches provide comprehensive insights into CCL28's function during inflammatory processes, potentially revealing therapeutic targets for inflammatory disorders affecting mucosal surfaces.

How can researchers integrate CCL28 studies with broader chemokine network analysis?

To understand CCL28 within the context of the broader chemokine network, researchers can employ these integrative approaches:

• Chemokine/receptor expression profiling: Comprehensive analysis of chemokine and receptor expression patterns in specific tissues under various conditions using multiplex qPCR arrays or proteomics

• Competitive binding studies: Investigating how CCL28 competes with other chemokines (particularly CCL27) for binding to shared receptors (CCR10)

• Chemokine gradient modeling: Creating in vitro systems that model the complexity of multiple overlapping chemokine gradients to study cell migration decisions

• Receptor internalization and desensitization: Examining how CCL28-induced receptor internalization affects cellular responsiveness to other chemokines

• Systems biology approaches: Computational modeling of chemokine network dynamics including CCL28 signaling pathways and their integration with other immune signaling networks

• Combinatorial chemokine manipulations: Simultaneous modulation of multiple chemokines including CCL28 to observe synergistic or antagonistic effects

This integrative perspective is essential for understanding how CCL28 functions within the complex immune microenvironment and how its manipulation might affect broader immune responses.

What are common challenges when working with recombinant mouse CCL28 and their solutions?

These troubleshooting strategies can help researchers overcome common technical challenges and ensure reproducible results when working with recombinant mouse CCL28.

What methods are recommended for validating recombinant mouse CCL28 quality and activity?

A comprehensive validation approach for recombinant mouse CCL28 should include:

• Purity assessment: SDS-PAGE with silver staining to confirm >97% purity as specified by manufacturers

• Endotoxin testing: LAL assay to verify endotoxin levels below 1.0 EU/μg or 0.01 EU/μg (depending on manufacturer specifications)

• Mass spectrometry: To confirm protein identity and detect potential contaminants or degradation products

• N-terminal sequencing: Verification that the protein begins with the expected Ile23 residue

• Functional validation: Chemotaxis assay using CCR10-transfected BaF/3 cells with expected ED₅₀ of 0.3-1 μg/ml

• Calcium mobilization assay: Complementary functional test measuring intracellular calcium flux in receptor-expressing cells

• Binding assays: Surface plasmon resonance or similar techniques to determine binding kinetics to purified CCR10 or CCR3

This multi-faceted approach ensures that only high-quality, biologically active CCL28 preparations are used in experiments, supporting reproducible and reliable research outcomes.

How can researchers standardize CCL28-based experiments across different laboratory settings?

To enhance reproducibility of CCL28-related research across different laboratories, consider these standardization approaches:

• Detailed methodology reporting: Include comprehensive information on CCL28 source, lot number, reconstitution method, and storage conditions in publications

• Standard activity units: Express CCL28 concentrations in both mass units (μg/ml) and biological activity units based on standardized chemotaxis assays

• Reference standards: Establish and share well-characterized CCL28 reference preparations between laboratories

• Validated cell lines: Use authenticated CCR10/CCR3-expressing cell lines with verified receptor expression levels

• Standardized protocols: Develop and share detailed protocols for common CCL28-related assays, including all critical parameters:

  • Buffer compositions

  • Incubation times and temperatures

  • Cell densities and passage numbers

  • Analytical methods and quantification approaches

• Positive controls: Include established positive controls (e.g., other well-characterized chemokines) in experimental designs

• Interlaboratory validation: Periodically perform cross-laboratory testing of the same CCL28 preparations to identify and address sources of variability

Implementing these standardization practices will enhance data comparability across different research groups and accelerate collective understanding of CCL28 biology and its therapeutic potential.