Recombinant Human C-C motif chemokine 16 protein (CCL16) (Active)

What is the basic structure of human CCL16 protein?

Human CCL16 is a member of the CC chemokine family with unique structural features. The CCL16 cDNA encodes a 120 amino acid protein with a 23 amino acid signal peptide that is cleaved to yield a 97 amino acid mature protein. One of its distinctive characteristics is a large cleavable C-terminal extension, which is not commonly found in other chemokines. This protein is distantly related to other CC chemokines, sharing less than 30% sequence identity with them. Crystal structure analysis confirms the presence of a canonical chemokine domain, while molecular dynamics simulations suggest that the C-terminal extension may serve as an intrinsic modulator of biological activity by impairing accessibility to glycosaminoglycan binding sites .

What are the primary receptors for CCL16 and what cell types respond to it?

CCL16 elicits its biological effects by interacting with multiple cell surface chemokine receptors, primarily CCR1, CCR2, CCR5, and CCR8. In terms of cellular responses, recombinant CCL16 has been shown to attract human monocytes and THP1 cells but does not appear to attract resting lymphocytes or neutrophils. This chemokine also activates calcium influx in THP1 cells, an effect that can be desensitized by prior RANTES treatment, suggesting that CCL16 and RANTES may activate the same receptor in these cells. The receptor profile explains its selective activity on specific immune cell populations, particularly those of myeloid lineage .

Where is CCL16 primarily expressed and is its expression regulated by inflammatory stimuli?

Unlike many other chemokines that are broadly expressed and strongly upregulated during inflammation, CCL16 shows a more restricted expression pattern. Research indicates that CCL16 is chiefly synthesized by hepatocytes in the liver and circulates in the blood as a full-length protein. Interestingly, CCL16 does not show appreciable response to mediators of inflammation, distinguishing it from many other chemokines whose expression is typically induced during inflammatory conditions. This constitutive expression pattern suggests that CCL16 may have homeostatic functions under steady-state conditions rather than primarily serving as an inflammatory mediator .

What are the optimal buffer conditions for maintaining CCL16 stability in functional assays?

For optimal stability and functionality of recombinant human CCL16 in experimental settings, the protein is typically formulated in a buffer consisting of 20mM phosphate buffer (PB) with 150mM NaCl at pH 7.4. This formulation helps maintain the structural integrity and biological activity of the chemokine. For long-term storage, lyophilization is recommended, and the lyophilized product should be shipped with ice packs to prevent degradation. When reconstituting lyophilized CCL16 for experimental use, it's advisable to use the same or similar buffer conditions and avoid repeated freeze-thaw cycles that may compromise protein functionality. Stability testing should be performed before advanced functional assays to ensure the protein maintains its native conformation and activity .

How can I validate the biological activity of recombinant CCL16 in cell-based assays?

To validate the biological activity of recombinant CCL16, chemotaxis assays using human monocytes or THP1 cells represent the gold standard. These can be performed using Transwell migration systems where cells are placed in the upper chamber and CCL16 is added to the lower chamber as a chemoattractant. Cell migration is typically assessed after 2-4 hours of incubation. Additionally, calcium mobilization assays can be employed, where intracellular calcium flux is measured in real-time after CCL16 stimulation using fluorescent calcium indicators. For receptor binding validation, competition assays with known ligands of CCR1, CCR2, CCR5, or CCR8 can determine receptor specificity. In more complex models, co-culture systems can be established to assess the impact of CCL16 on cellular interactions, such as those between cancer cells and macrophages, where flow cytometry can be used to evaluate resulting phenotypic changes in target cells .

What techniques are available for detecting CCL16-receptor interactions in tissue samples?

Multiple complementary techniques can be used to detect and characterize CCL16-receptor interactions in tissue samples. Immunofluorescence co-staining with antibodies against CCL16 and its receptors (particularly CCR1) allows visualization of potential interaction sites at the cellular level. For this purpose, tissue sections should be deparaffinized, hydrated, and subjected to antigen retrieval before blocking with serum and incubation with primary antibodies against CCL16 and its receptors. Proximity ligation assays provide higher specificity by detecting proteins that are in close proximity (<40 nm), suggesting direct interaction. For protein-level validation, co-immunoprecipitation can be performed on tissue lysates using antibodies against CCL16 or its receptors, followed by immunoblotting. At the transcriptional level, single-cell RNA sequencing can identify cell populations expressing both CCL16 and its receptors, as demonstrated in studies of hepatocellular carcinoma where CCL16 was found in cancer cells while CCR1 was predominantly expressed in macrophages .

How does the C-terminal extension of CCL16 affect its biological functions compared to other CC chemokines?

The C-terminal extension of CCL16 represents a unique structural feature that distinguishes it from other CC chemokines and significantly impacts its biological functions. Molecular dynamics simulations indicate that this extension impairs the accessibility of glycosaminoglycan (GAG) binding sites, which are crucial for chemokine localization and gradient formation in tissues. This structural characteristic may serve as an intrinsic regulatory mechanism that modulates CCL16 activity in vivo. Comparative functional assays between full-length CCL16 and truncated variants lacking the C-terminal extension have shown differences in receptor binding affinities, signaling potency, and chemotactic activity. Researchers investigating this aspect should consider expressing both full-length and truncated recombinant CCL16 variants, followed by comparative binding assays with potential receptors, chemotaxis assays with responsive cell types, and in vitro signaling studies to elucidate how this extension affects each aspect of CCL16 function .

What is the role of CCL16 in tumor microenvironment and cancer progression?

CCL16 appears to play a significant role in shaping the tumor microenvironment, particularly in hepatocellular carcinoma (HCC). Recent research has identified a CCL16-CCR1 axis in HCC, where tumor cells express and secrete CCL16 while macrophages in the tumor microenvironment express the CCR1 receptor. Functional studies have demonstrated that CCL16 secreted by tumor cells promotes the recruitment of macrophages and facilitates their polarization toward an M2 phenotype, which is generally associated with tumor-promoting functions. In experimental models, knockdown of CCL16 in HCC cells significantly reduced their ability to recruit THP1 cells (a monocytic cell line), while CCL16 overexpression enhanced this recruitment. Furthermore, co-culture experiments revealed that CCL16-expressing cancer cells increased the proportion of CD80+CD206+ cells (indicative of M2 polarization) among THP1-derived macrophages. Clinical tissue analyses have shown positive correlations between CCL16 and CCR1 expression, as well as between CD206 and CD68 expression at the protein level, further supporting the role of this chemokine in macrophage recruitment and polarization in the tumor context .

What are the methodological approaches to investigate CCL16-mediated signaling pathways?

Investigating CCL16-mediated signaling pathways requires a multi-faceted approach combining molecular, cellular, and computational techniques. For initial signaling pathway activation studies, phosphorylation assays for key signaling molecules (like MAP kinases, PI3K/Akt, JAK/STAT) can be performed using phospho-specific antibodies in Western blot or ELISA formats after treating cells with recombinant CCL16. Calcium flux assays using fluorescent calcium indicators provide real-time readouts of early receptor activation. For more comprehensive pathway analysis, phosphoproteomics can identify changes in the phosphorylation status of hundreds of proteins simultaneously. Gene expression analyses using RNA-seq or qPCR arrays can reveal downstream transcriptional effects of CCL16 stimulation. To establish causality, pharmacological inhibitors or genetic approaches (siRNA, CRISPR) targeting specific signaling components can be employed to determine their necessity in CCL16-mediated responses. In more complex biological contexts, like the tumor microenvironment, single-cell analyses combined with spatial transcriptomics can map signaling events within specific cell populations. Computational approaches, including network analysis and pathway enrichment, can then integrate these diverse datasets to construct comprehensive signaling models .

How does CCL16 differ functionally from other chemokines that signal through the same receptors?

CCL16 shares receptors (CCR1, CCR2, CCR5, and CCR8) with several other chemokines, yet exhibits distinct functional properties. Unlike many inflammation-induced chemokines that signal through these same receptors, CCL16 is constitutively expressed by hepatocytes and circulates in blood without requiring inflammatory stimuli. This suggests a potential homeostatic role rather than a primarily inflammatory function. CCL16 demonstrates potent myelosuppressive activity and inhibits the proliferation of myeloid progenitor cells, a property not shared by all chemokines binding to the same receptors. When designing comparative studies to investigate these functional differences, researchers should include parallel assays with other chemokines that bind the same receptors (such as CCL3, CCL4, and CCL5 for CCR1 and CCR5) using identical experimental conditions. These comparisons should measure receptor binding affinities, signaling pathway activation patterns, calcium mobilization kinetics, and downstream functional outcomes like chemotaxis, cell adhesion, and gene expression changes. Such comprehensive comparisons would highlight the unique aspects of CCL16 signaling and function despite shared receptor usage .

What experimental controls should be included when studying CCL16-specific effects in complex biological systems?

When investigating CCL16-specific effects in complex biological systems such as tumor microenvironments or inflammatory conditions, several critical controls must be included to ensure reliable and interpretable results. First, receptor blocking experiments using specific antibodies or antagonists against CCR1, CCR2, CCR5, and CCR8 can determine which receptors mediate the observed effects. Second, gene knockdown or knockout approaches targeting CCL16 in relevant cell types (e.g., hepatocytes or tumor cells) provide loss-of-function controls. Conversely, CCL16 overexpression systems offer gain-of-function controls. Third, structurally related chemokines that bind the same receptors serve as important comparative controls to distinguish CCL16-specific versus receptor-specific effects. Fourth, heat-inactivated or enzymatically digested CCL16 preparations provide negative controls for protein-specific effects. For in vivo studies, careful selection of animal models is crucial, noting that certain chemokines show species-specific differences in receptor binding and function. Finally, comprehensive analyses should include multiple readouts (e.g., signaling pathway activation, transcriptional responses, and functional outcomes) to build a complete picture of CCL16-specific biology in the system under investigation .

What are the common challenges in producing high-quality recombinant CCL16 and how can they be addressed?

Production of high-quality recombinant CCL16 presents several challenges that researchers should be aware of. First, ensuring proper folding and disulfide bond formation is critical for maintaining the canonical chemokine domain structure, which is essential for receptor binding and function. This can be addressed by optimizing expression systems, with E. coli being commonly used as evidenced by the recombinant protein described in the search results. For E. coli expression, inclusion of redox buffers during protein refolding and purification under denaturing conditions followed by carefully controlled refolding protocols can improve the yield of correctly folded protein. Second, the presence of the unique C-terminal extension may affect solubility and stability of the full-length protein. This can be mitigated by testing different solubilization buffers and stabilizing additives. Third, endotoxin contamination is a significant concern, especially for immunological studies. Implementing endotoxin removal steps during purification and validating levels using the LAL method (ensuring <1.0 EU per μg as mentioned in the specifications) is essential. Finally, batch-to-batch variation in activity should be addressed through rigorous quality control testing, including SDS-PAGE for purity assessment (aiming for >95% purity), mass spectrometry for identity confirmation, and functional assays to verify biological activity .

How can researchers accurately quantify CCL16-induced changes in macrophage polarization?

Accurately quantifying CCL16-induced changes in macrophage polarization requires a multi-parameter approach combining flow cytometry, transcriptional analysis, and functional assays. Flow cytometry represents a primary method, using markers such as CD80 (present on both M1 and M2 macrophages) and CD206 (specifically elevated in M2 macrophages) as demonstrated in the CCL16 studies with THP1 cells. To implement this approach, macrophages should be isolated after CCL16 exposure, stained with fluorochrome-conjugated antibodies against polarization markers, and analyzed using appropriate gating strategies to distinguish different macrophage populations. Transcriptional profiling via qPCR or RNA-seq should measure expression of M1-associated genes (IL-1β, TNF-α, iNOS) and M2-associated genes (IL-10, TGF-β, Arginase-1) to provide molecular confirmation of polarization states. Secretome analysis using multiplex cytokine assays can characterize the functional output of polarized macrophages. Functional assays, such as phagocytosis efficiency, migration capacity, and T-cell activation/suppression assays, provide additional evidence of polarization effects. Finally, imaging-based approaches, including immunofluorescence with appropriate markers, allow visualization of polarized macrophages in tissue contexts, particularly valuable for in vivo or ex vivo studies .

How might CCL16 function in the context of liver diseases beyond hepatocellular carcinoma?

Given CCL16's predominant expression in hepatocytes and its emerging role in hepatocellular carcinoma (HCC), there is significant potential for its involvement in other liver pathologies. In non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), CCL16 may influence the recruitment and polarization of macrophages, potentially contributing to disease progression through modulation of inflammation and fibrosis. Research approaches should include quantifying CCL16 expression in patient liver biopsies across different stages of these diseases, correlating levels with disease severity and macrophage infiltration patterns. In viral hepatitis, CCL16 might play a role in shaping immune responses to infection, particularly given its interaction with CCR1 and CCR5, which are also relevant in viral immunity. For autoimmune liver diseases like primary biliary cholangitis or autoimmune hepatitis, investigations should focus on whether CCL16 contributes to breaking immune tolerance or modulates autoimmune responses. Methodologically, single-cell RNA sequencing of liver samples from diverse liver pathologies could identify cell-specific expression patterns of CCL16 and its receptors, while functional studies in relevant animal models or ex vivo human liver slices could elucidate its causal roles .

What techniques can be used to investigate the potential therapeutic applications of modulating the CCL16-CCR1 axis?

Investigating the therapeutic potential of modulating the CCL16-CCR1 axis requires a comprehensive set of techniques spanning from molecular to clinical approaches. In preclinical development, high-throughput screening of small molecule libraries can identify CCL16-CCR1 interaction inhibitors, while structure-based drug design utilizing the CCL16 crystal structure data can guide rational design of antagonists or agonists. Neutralizing antibodies against CCL16 or CCR1 represent alternative biological approaches for axis inhibition. For validation, binding assays using surface plasmon resonance or bioluminescence resonance energy transfer can confirm target engagement, while cell-based reporter systems can assess functional effects on signaling. In disease models, genetic approaches using CRISPR/Cas9 to modulate CCL16 or CCR1 expression in specific cell types can evaluate the axis contribution to pathology. Translational studies should include ex vivo testing using patient-derived samples (such as tumor organoids or immune cells) treated with CCL16-CCR1 modulators to predict clinical responses. Finally, biomarker development identifying patients most likely to benefit from CCL16-CCR1 targeting therapies could include tissue microarrays to correlate expression patterns with clinical outcomes, or liquid biopsies to monitor circulating CCL16 levels .

How can computational approaches enhance our understanding of CCL16 structure-function relationships?

Computational approaches offer powerful tools for exploring CCL16 structure-function relationships, particularly given the availability of its crystal structure. Molecular dynamics simulations can reveal how the unique C-terminal extension of CCL16 influences the protein's conformational dynamics and affects accessibility of the glycosaminoglycan binding sites, as mentioned in the search results. These simulations should explore the protein under various conditions, including different pH, salt concentrations, and in the presence of potential binding partners. Protein-protein docking algorithms can model interactions between CCL16 and its receptors (CCR1, CCR2, CCR5, and CCR8), identifying key residues involved in binding specificity and affinity. Machine learning approaches applied to chemokine sequence-structure-function datasets can predict functional consequences of specific CCL16 mutations or identify unique structural features that distinguish it from other chemokines. Systems biology modeling can integrate CCL16 signaling into broader pathway networks, potentially revealing emergent properties in complex systems like the tumor microenvironment. Finally, virtual screening of compound libraries against CCL16 or its receptors can accelerate the discovery of modulators with therapeutic potential. The integration of these computational approaches with experimental validation creates a powerful framework for understanding this unique chemokine .