Recombinant Rat Cxcl17 protein (Cxcl17) (Active)

What is CXCL17 and what are its key molecular properties?

CXCL17, also known as VEGF co-regulated chemokine 1 (VCC-1), is a secreted protein belonging to the CXC chemokine family. The rat CXCL17 protein exhibits the following molecular characteristics:

• Amino acid sequence: SPNQEVARHH GDQHQAPRRW LWEGGQECDC KDWSLRVSKR KTTAVLEPPR KQCPCDHVKG SEKKNRRQKH HRKSQRPSRT CQQFLKRCQL ASFTLPL

• Synonyms: Protein Cxcl17, Cxcl17, RGD1304717, C-X-C motif chemokine 17, VEGF co-regulated chemokine 1, Vcc1, VCC-1

• UniProt code: D4A875

• Physical appearance: Sterile filtered white lyophilized (freeze-dried) powder when in recombinant form

CXCL17 is constitutively produced by airway and intestinal epithelium and has been implicated in various immune functions, particularly as a chemoattractant for specific immune cell types .

How is recombinant Rat CXCL17 protein typically produced?

Recombinant Rat CXCL17 is typically produced using the following methodologies:

• Expression system: Escherichia coli bacterial expression systems are commonly used for production

• Purification: Multi-step purification processes including affinity chromatography and RP-HPLC to achieve >95% purity

• Formulation: The protein is typically lyophilized from a 0.2μm filtered concentrated solution in 20mM phosphate buffer with 300mM NaCl at pH 7.4

• Quality control: Purity is determined through:

  • Analysis by RP-HPLC

  • SDS-PAGE verification

  • Biological activity testing

What is the recommended reconstitution protocol for lyophilized Rat CXCL17?

The optimal reconstitution protocol for lyophilized Rat CXCL17 includes:

• Reconstitution in sterile 18MΩ-cm H₂O at a concentration not less than 100μg/ml

• After initial reconstitution, the solution can be further diluted in other aqueous buffers as needed for specific applications

• For short-term storage (2-7 days), store at 4°C

• For long-term storage, aliquot and store below -18°C, adding a carrier protein (0.1% HSA or BSA) to prevent protein loss through adsorption to surfaces

• Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of activity

How can biological activity of Rat CXCL17 be assessed in research settings?

The biological activity of Rat CXCL17 can be assessed through several established experimental approaches:

• VEGF Expression Induction: Measured by its ability to induce VEGF expression in mouse endothelial cells. The ED50 for this effect is typically 1-5 μg/ml .

• Chemotaxis Assays:

  • Modified Boyden chamber assays can evaluate the chemotactic response of monocytes and dendritic cells to CXCL17

  • Real-time chemotaxis assays provide dynamic measurements of neutrophil migration in response to CXCL17

• Cell Binding Studies: Flow cytometry can be used to evaluate CXCL17 binding to potential target cells, particularly monocytes and dendritic cells

• GAG Binding Assays:

  • Solid-phase binding assays can quantify CXCL17's interaction with glycosaminoglycans

  • Bio-layer interferometry techniques provide high-sensitivity measurements of binding kinetics between CXCL17 and GAGs

What are the optimal storage conditions for maintaining CXCL17 activity?

To maintain optimal activity of Rat CXCL17 protein:

• Lyophilized form:

  • Store desiccated below -18°C

  • Stable at room temperature for up to 3 weeks, but refrigerated storage is preferable

• Reconstituted protein:

  • Short-term (2-7 days): Store at 4°C

  • Long-term: Store below -18°C in small aliquots

  • Add carrier protein (0.1% HSA or BSA) to minimize adsorption and denaturation

  • Strictly avoid repeated freeze-thaw cycles

• Working solutions:

  • Prepare fresh on the day of experiment when possible

  • Use sterile technique to prevent microbial contamination

  • Consider the addition of protease inhibitors when working with biological samples

How does CXCL17's GAG-binding capacity affect its function in experimental systems?

CXCL17 demonstrates robust glycosaminoglycan (GAG) binding properties that significantly impact its experimental applications:

• Superior GAG-binding capacity:

  • CXCL17 binds to multiple GAGs including heparin, heparan sulfate, and chondroitin sulfate with higher capacity than established GAG-binding chemokines like CXCL4

  • In solid-phase binding assays, immobilized CXCL17 consistently shows greater maximal recovered binding signal for GAGs compared to CXCL4

• Functional implications:

  • CXCL17's strong GAG-binding ability allows it to modulate the activity of other chemokines that depend on GAG binding for their function

  • CXCL17 has been observed to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner, likely through competitive GAG binding

• Structure-function relationship:

  • Key C-terminal motifs in CXCL17 have been implicated in GAG binding

  • The relatively high isoelectric point of CXCL17 contributes to its robust interaction with negatively charged GAGs

Table derived from experimental data in

What structural modeling approaches have been applied to understand CXCL17 conformation?

Despite its classification as a chemokine, the structural characterization of CXCL17 remains challenging. Several sophisticated in silico modeling approaches have been employed:

• AlphaFold2 Modeling:

  • CXCL17 (24-119) tertiary structure prediction using ColabFold interface

  • Multiple sequence alignment performed by MMSeqs2 against Uniref and Environmental structure libraries

  • Structural modeling with AlphaFold2-ptm and AlphaFold multimer v2 predictions

  • Model confidence assessed via predicted local distance test (plDDT) scores and Predicted Aligned Error (PAE)

• De novo Folding Approaches:

  • RoseTTAFold with ColabFold for main-chain predictions

  • Scrwl4 for side-chain predictions

  • C-I-TASSER integrated modeling using I-TASSER hierarchical structure with deep learning-based contact predictions

• Homodimer Structural Analysis:

  • CXCL17 (24-119) homodimer structure was modeled using 12 iterances of model recycling

  • This approach is significant as dimerization is a characteristic feature of several chemokines

Notably, all modeling efforts have failed to conclusively support the classification of CXCL17 as a chemokine based on its predicted conformation, raising important questions about its structural classification .

How do CXCL17 knockout models inform our understanding of its physiological functions?

Studies with CXCL17 knockout (Cxcl17−/−) mice have provided valuable insights into the protein's biological functions:

• T cell population alterations:

  • Cxcl17−/− mice show significant abnormalities in T cell populations

  • Both the spleen and lymph nodes of knockout mice contain higher numbers of CD4+ and CD8+ T cells compared to wild-type controls

  • The difference is more pronounced in CD4+ T cells (~50% increase) than in CD8+ T cells (~25% increase)

• Inflammatory response modulation:

  • Knockout mice develop exacerbated disease in T cell-dependent inflammation models

  • The absence of CXCL17 creates a proinflammatory immune microenvironment

  • This enhanced inflammatory state likely contributes to the increased susceptibility to inflammatory conditions observed in these animals

• Macrophage recruitment:

  • Deletion of the cxcl17 gene in mice results in reduced numbers of alveolar macrophages within the lungs

  • This observation suggests a potential role for CXCL17 in macrophage recruitment to mucosal tissues

What are the key experimental controls when assessing CXCL17's chemotactic activity?

When designing experiments to evaluate CXCL17's chemotactic properties, researchers should implement the following controls:

• Positive Controls:

  • CXCL8/IL-8 for neutrophil chemotaxis assays (effective at nanomolar concentrations)

  • CCL2/MCP-1 for monocyte chemotaxis assays

  • Appropriate chemokines specific to the cell type being studied

• Negative Controls:

  • Heat-inactivated CXCL17 to confirm activity is protein-specific

  • Irrelevant proteins of similar molecular weight

  • Buffer-only conditions to establish baseline migration

• Concentration Series:

  • Test CXCL17 across a broad concentration range (nanomolar to micromolar)

  • CXCL17 has been shown to have neutrophil chemotactic activity at micromolar concentrations, several orders of magnitude higher than those required for CXCL8

• Time Course Analysis:

  • Monitor chemotactic responses at multiple time points

  • Implement real-time assay methods when possible to capture the full dynamics of the response

• Cell Activation Status Controls:

  • Test both quiescent and activated cells (e.g., LPS-activated vs. quiescent monocytes)

  • CXCL17 has been reported to induce chemotaxis of quiescent, but not LPS-activated peripheral blood monocytes

How can researchers address the issue of species-specific differences in CXCL17 function?

Species-specific differences can significantly impact experimental outcomes when studying CXCL17. Researchers should consider:

• Cross-species reactivity validation:

  • Confirm whether rat CXCL17 affects human or mouse cells in your system

  • Perform species-specific positive controls in parallel

  • Consider using species-matched recombinant proteins when available

• Comparative analysis approaches:

  • When studying rodent models, compare effects of both human and rodent CXCL17

  • Document species-specific differences in dose-response relationships

  • Consider sequence homology analysis between species variants

• Cell source considerations:

  • The chemotactic response to CXCL17 varies between species and cell types

  • Contrary to some reports, CXCL17 has not been consistently shown to be chemotactic for murine splenocytes

  • Human neutrophils respond to CXCL17 but at much higher concentrations than required for other chemokines

• Receptor expression analysis:

  • Confirm expression of putative CXCL17 receptors in target cells

  • Although GPR35 was initially proposed as a CXCL17 receptor, this has been challenged by multiple studies

  • Consider receptor expression differences between species when interpreting results

What are the latest findings regarding CXCL17's antimicrobial properties?

Recent research has uncovered potential antimicrobial functions of CXCL17:

• Antimicrobial spectrum:

  • In vitro data have demonstrated a broad spectrum of microbicidal activity against both bacteria and fungi

  • This suggests CXCL17 may play a role in mucosal immunity beyond its chemotactic functions

• Mechanism considerations:

  • The antimicrobial activity may be related to CXCL17's highly basic nature

  • Further research is needed to determine if this activity is direct (through interaction with microbial membranes) or indirect (through modulation of immune responses)

• Experimental approaches:

  • Researchers investigating antimicrobial properties should consider:

    • Minimum inhibitory concentration (MIC) assays against various microorganisms

    • Time-kill kinetics studies

    • Structural analysis to identify antimicrobial domains within the protein

How might CXCL17's GAG-binding properties be exploited for therapeutic applications?

The robust GAG-binding capacity of CXCL17 presents intriguing opportunities for therapeutic development:

• Chemokine inhibition strategies:

  • CXCL17 has been shown to inhibit CXCR1-mediated chemotaxis to CXCL8 in a dose-dependent manner

  • This suggests potential for CXCL17 or its derivatives as inhibitors of inflammatory chemokine activity

• C-terminal fragments as prototypes:

  • C-terminal motifs of CXCL17 have been implicated in GAG binding

  • These fragments may serve as prototypic inhibitors of chemokine function

  • Researchers could develop peptide derivatives based on these motifs for targeted therapeutic applications

• GAG-binding competitive inhibition:

  • CXCL17's superior GAG-binding compared to other chemokines (like CXCL4) suggests potential utility in competitive inhibition strategies

  • Such approaches could be explored for conditions where dysregulated chemokine activity contributes to pathology

• Research directions:

  • Structure-activity relationship studies to identify minimal GAG-binding domains

  • Development of stable peptide derivatives with enhanced GAG-binding properties

  • In vivo testing in inflammatory disease models to assess therapeutic potential

What strategies can address inconsistent results in CXCL17 chemotaxis assays?

Researchers frequently encounter variability in chemotaxis assays with CXCL17. The following strategies can improve reproducibility:

• Concentration considerations:

  • CXCL17 exhibits chemotactic activity for human neutrophils at micromolar concentrations, orders of magnitude higher than CXCL8

  • Test a broad concentration range (nanomolar to micromolar) to identify optimal conditions

• Cell preparation factors:

  • Ensure consistent cell isolation protocols

  • Account for donor variability when using primary human cells

  • Control for cell activation status (CXCL17 induces chemotaxis in quiescent but not LPS-activated monocytes)

• Assay methodology:

  • Consider real-time chemotaxis assays for more detailed kinetic information

  • Traditional Boyden chamber assays may miss subtle or transient responses

  • Multiple time points should be evaluated to capture the complete response profile

• Protein quality:

  • Verify protein activity before experiments

  • Use freshly reconstituted protein when possible

  • Consider the source of recombinant protein (mammalian vs. prokaryotic expression)

• Technical considerations:

  • Ensure uniform temperature and CO₂ conditions throughout the experiment

  • Minimize vibration and other physical disturbances during the assay

  • Implement blinded analysis of results to prevent bias

How can researchers distinguish between direct and indirect effects of CXCL17 on immune cell function?

Differentiating direct cellular effects from indirect modulatory actions of CXCL17 requires careful experimental design:

• Receptor blocking experiments:

  • Although a definitive receptor for CXCL17 remains controversial, conduct experiments with receptor antagonists (if suspected receptors are known)

  • GPR35 was previously proposed as a CXCL17 receptor, though this has been challenged

  • Consider using GPR35 inhibitors (like ML-339) as controls in experiments

• Signal transduction analysis:

  • Monitor early signaling events (calcium flux, MAPK activation)

  • Direct receptor engagement typically produces rapid signaling responses

  • Compare timing and magnitude of responses to known direct agonists

• Conditioned media experiments:

  • Compare effects of direct CXCL17 application to conditioned media from CXCL17-treated cells

  • This approach can help identify secondary mediators released in response to CXCL17

• Inhibitor studies:

  • Use specific inhibitors of candidate signaling pathways

  • Include controls for potential off-target effects of inhibitors

  • Implement concentration-response studies with inhibitors

• Genetic approaches:

  • Consider using cells from knockout models (e.g., Cxcl17−/− mice)

  • Implement gene silencing approaches (siRNA, shRNA) for suspected mediators

  • CRISPR-Cas9 gene editing of potential signaling components or receptors