Recombinant Human C-X-C motif chemokine 17 protein (CXCL17) (Active)

What is CXCL17 and how is it categorized among chemokines?

CXCL17, also known as VEGF Coregulated Chemokine 1 (VCC1), Small Inducible Cytokine Subfamily B Member 17 (SCYB17), or Dendritic Cell and Monocyte Chemokine-like Protein (DMC), is the most recently identified human chemokine . Its classification as a CXC chemokine has been the subject of significant debate in the scientific community. Unlike most chemokines, CXCL17 contains six conserved cysteine residues, a feature shared only with CXCL16 and the 6-Cys subset of CC chemokines (CCL1, CCL15, CCL21, CCL23, and CCL28) . Several studies have questioned its structural credentials as a true chemokine based on sequence analysis and structural predictions . When subjected to secondary structure prediction tools such as DSC, CXCL17 appears to contain only α-helical regions, which contrasts with the canonical chemokine structure comprising three β-strands and one α-helix .

What are the main physiological functions of CXCL17?

CXCL17 is primarily expressed at mucosal surfaces including the proximal digestive tract, lung, stomach, urethra, and parts of the female reproductive system . While its precise function is not fully understood, research indicates it plays important roles in:

• Regulation of inflammatory responses on mucosal surfaces

• Recruitment of immune cells, particularly neutrophils to inflammatory sites

• Antimicrobial activity against bacteria and fungi

• Possible involvement in tumorigenesis through angiogenesis mechanisms

Recent studies demonstrate that CXCL17 knockout mice show significantly impaired neutrophil recruitment after lipopolysaccharide (LPS) challenge, supporting its role as a proinflammatory chemokine .

What is the molecular weight and basic structural features of recombinant human CXCL17?

Recombinant human CXCL17 typically has a molecular weight of approximately 11.5 kDa and consists of 98 amino acids in its mature form . The protein contains six cysteine residues, though there is evidence of posttranslational cleavage resulting in a mature 4-cysteine protein . There is ongoing debate about the disulfide bond connectivity of these cysteines, with different studies proposing either [C1-C3; C2-C4] or [C3-C5; C4-C6] linkage patterns . The protein tends to dimerize as a function of concentration, which is a characteristic shared with several other chemokines .

How should CXCL17 be stored and handled for optimal activity in experiments?

Based on manufacturer specifications, recombinant human CXCL17 should be stored desiccated at -20°C to maintain stability . The lyophilized powder form is generally preferred for long-term storage. When working with CXCL17 in laboratory settings, researchers should consider its tendency to dimerize at higher concentrations, which may affect experimental outcomes . For functional assays, it's important to use sterile, filtered preparations that have been properly tested for biological activity. The specific activity of high-quality recombinant CXCL17 preparations is typically >200 IU/mg, with an ED50 of less than 5 μg/ml when measuring its ability to induce VEGF expression in murine endothelial cells .

What are the recommended methods for assessing CXCL17 chemotactic activity?

Multiple experimental approaches can be used to evaluate the chemotactic activity of CXCL17:

• Modified Boyden Chamber Assays: These traditional chemotaxis assays have been used to assess CXCL17-induced migration of various cell types including monocytes, dendritic cells, and neutrophils .

• Real-time Chemotaxis Assays: More sophisticated than Boyden chambers, these assays allow continuous monitoring of cell migration in response to CXCL17 gradients, providing temporal information about chemotactic responses .

• In vivo Recruitment Models: The peritoneal LPS challenge model in mice has been used to demonstrate CXCL17's role in neutrophil recruitment. Researchers can compare neutrophil infiltration between wild-type and CXCL17 knockout mice to assess its function .

When designing chemotaxis experiments, it's important to note that CXCL17 has been observed to be a low-potency chemoattractant for human neutrophils, requiring micromolar concentrations - several orders of magnitude higher than those needed for CXCL8 (IL-8) .

How can researchers effectively measure CXCL17 binding to glycosaminoglycans (GAGs)?

CXCL17 has been shown to bind efficiently to glycosaminoglycans (GAGs), which may be crucial for its biological functions. Two primary methods have been validated for studying these interactions:

• Solid-phase Binding Assays: These assays allow quantification of CXCL17-GAG interactions using immobilized GAGs and labeled CXCL17 .

• Bio-layer Interferometry Techniques: This label-free technology can measure the kinetics of CXCL17-GAG binding in real-time, providing association and dissociation constants .

When conducting GAG binding studies with CXCL17, researchers should pay particular attention to the C-terminal motifs of the protein, as these regions have been implicated as key determinants of GAG binding efficacy .

How does CXCL17 modulate the activity of other chemokines?

An intriguing aspect of CXCL17 biology is its ability to modulate the activity of other chemokines. Research has demonstrated that CXCL17 can inhibit CXCR1-mediated chemotaxis to CXCL8 in a dose-dependent manner, despite not binding directly to CXCR1 itself . This modulatory effect appears to be a consequence of CXCL17's superior GAG-binding capability, which may disrupt chemokine gradients necessary for directed cell migration . The inhibitory effect suggests that CXCL17 or its fragments could potentially serve as prototype inhibitors of specific chemokine functions, which has significant implications for anti-inflammatory therapeutic strategies .

What are the current controversies regarding CXCL17's receptor interactions?

The identification of CXCL17's receptor(s) has been a subject of ongoing debate:

• GPR35 Controversy: GPR35 was initially proposed as a receptor for CXCL17 , but this has been challenged by multiple independent studies . Recent research has identified 5-hydroxyindoleacetic acid, a serotonin metabolite, as the ligand for GPR35, which recruits GPR35+ neutrophils at nanomolar concentrations .

• Receptor-Independent Functions: Some studies suggest that CXCL17 may exert certain functions through receptor-independent mechanisms, particularly via its interactions with GAGs in the extracellular matrix .

• Receptor Nomenclature Confusion: The controversy has led to confusion in the literature, with some studies prematurely designating GPR35 as "CXCR8" despite insufficient evidence for it being the definitive CXCL17 receptor .

Researchers investigating CXCL17-receptor interactions should design experiments that can distinguish between direct receptor activation and indirect effects mediated through GAG binding or other mechanisms.

How do CXCL17 knockout models help elucidate its function in vivo?

Studies using CXCL17 knockout (KO) mice have provided valuable insights into the protein's physiological functions. In comparison with wild-type littermates, CXCL17 KO mice exhibit:

• Impaired Neutrophil Recruitment: Significantly reduced peritoneal neutrophil recruitment following lipopolysaccharide (LPS) challenge .

• Dysregulated Inflammatory Mediators: Altered expression levels of Cxcl1, Cxcr2, and interleukin-6, all of which directly impact neutrophil recruitment .

• Selective Impact on Immune Cell Types: Interestingly, CXCL17 KO mice show no difference in monocyte recruitment post-LPS challenge or in peritoneal macrophage levels in both unchallenged and LPS-challenged conditions .

These findings support the classification of CXCL17 as a proinflammatory chemokine with a specific role in promoting neutrophil trafficking during the early stages of inflammatory response .

What evidence challenges the classification of CXCL17 as a true chemokine?

Several lines of evidence have raised questions about CXCL17's classification as a true chemokine:

• Secondary Structure Predictions: When analyzed using tools like the DSC server, CXCL17 is predicted to contain only α-helical regions, contrasting with the canonical chemokine fold (three β-strands and one α-helix) .

• Sequence Alignment Issues: Sequence alignment demonstrates that 4-Cys CXCL17 does not follow the typical CXC-type cysteine pattern and appears too short to adopt a conventional chemokine fold .

• 3D Modeling Results: Recent modeling efforts yielded a structure with four α-helices for CXCL17, which differs substantially from the characteristic chemokine structure .

• Homology Search Results: When analyzed using linear hidden Markov models, only two CC-type chemokines were identified as homologs of CXCL17, rather than the expected CXC-type chemokines .

This controversy has significant implications for research directions, as classification impacts expectations about receptor interactions, signaling mechanisms, and potential therapeutic applications.

How do circular dichroism (CD) spectra inform our understanding of CXCL17 structure?

Circular dichroism (CD) spectroscopy has been used to analyze CXCL17's secondary structure, with results adding to the classification debate:

These spectroscopic differences contribute to the ongoing debate about whether CXCL17 truly belongs to the chemokine family from a structural perspective.

What is the significance of CXCL17's microbicidal activity?

Beyond its chemotactic functions, CXCL17 has demonstrated broad-spectrum antimicrobial activity against bacteria and fungi in vitro . This activity positions CXCL17 within the growing class of chemokines with dual immunoregulatory and antimicrobial properties. For researchers interested in this aspect of CXCL17 biology, important considerations include:

• Effective Concentrations: Determining the physiologically relevant concentrations at which CXCL17 exerts antimicrobial effects in mucosal tissues.

• Structural Determinants: Identifying which domains or peptide fragments of CXCL17 are responsible for its microbicidal properties.

• Mechanism of Action: Investigating whether CXCL17 acts through membrane disruption, similar to other antimicrobial peptides, or through alternative mechanisms.

• Potential Therapeutic Applications: Exploring CXCL17-derived peptides as templates for novel antimicrobial agents, particularly for mucosal infections.

How does CXCL17 contribute to mucosal immunity?

CXCL17 is constitutively expressed at mucosal surfaces, suggesting a specialized role in mucosal immunity . Current research indicates several potential functions:

• Homeostatic Immune Cell Recruitment: CXCL17 may help maintain appropriate populations of immune cells at mucosal sites under steady-state conditions.

• First-line Pathogen Defense: Through its dual chemotactic and antimicrobial properties, CXCL17 could provide immediate protection at vulnerable mucosal interfaces.

• Inflammatory Response Regulation: The ability of CXCL17 to modulate neutrophil recruitment and potentially interfere with other chemokine functions suggests a role in fine-tuning inflammatory responses at mucosal sites .

• Tissue-specific Immune Programming: Expression of CXCL17 may contribute to the unique immunological environments of different mucosal tissues.

Future research should investigate tissue-specific knockout models and mucosal infection challenges to better define CXCL17's role in maintaining mucosal barrier integrity and coordinating immune responses.