CCL16 Human

What is the basic structure of human CCL16 and how does it differ from other chemokines?

Human CCL16 is a CC-type chemokine distinguished by its unique structure comprising a canonical chemokine domain and an unusual large cleavable C-terminal extension of approximately 25 amino acids. The primary CCL16 translation product contains a 23-amino acid signal sequence followed by the chemokine domain and the C-terminal extension with weak secondary structure propensity .

Unlike most chemokines that typically contain 70-75 residues with two disulfide bonds in their mature forms, CCL16's additional C-terminal segment makes it structurally distinctive. Crystal structure analysis at 1.45 Å resolution confirms the canonical chemokine fold but reveals how the C-terminal extension influences its functional properties .

What methodological approaches are recommended for recombinant CCL16 production?

For successful recombinant production of human CCL16:

• Use a codon-optimized synthetic cDNA coding for mature human CCL16

• Express in E. coli SHuffle T7 cells at 30°C following induction with 1 mM isopropyl β-D-thiogalactopyranoside

• Purify using metal affinity chromatography with Ni-NTA agarose

• Process with PreScission protease to remove tags

• Perform final purification by size exclusion chromatography on a HiLoad 26/60 Superdex 75 pg column

• Verify protein identity and purity using capillary electrophoresis-mass spectrometry (CE-MS) analysis

This methodology yields properly folded, functional CCL16 protein suitable for structural and functional studies.

Where is CCL16 primarily expressed in human tissues and how can this expression be reliably detected?

Despite earlier conflicting reports, current evidence indicates that CCL16 is predominantly synthesized by hepatocytes in the liver, with minimal expression in other tissues. For reliable detection of CCL16 expression:

• RNA Analysis: Extract RNA using RNeasy Mini Kit and perform reverse transcription followed by real-time PCR with CCL16-specific TaqMan probes (Hs00171123_m1). Normalize expression to 18S rRNA .

• Protein Detection:

  • Immunohistochemistry: Use mouse anti-human CCL16 monoclonal IgG1 antibody with appropriate isotype controls on acetone-fixed tissue sections

  • ELISA: Quantify CCL16 in serum or culture supernatants using commercial kits

  • Immunoblotting: Detect using biotinylated polyclonal goat anti-human CCL16 antibody and streptavidin-coupled horseradish peroxidase

These complementary approaches help overcome the technical challenges that contributed to early discrepancies in CCL16 expression profiles.

How does inflammation affect CCL16 expression, and what experimental models best capture these regulatory effects?

Current research indicates that CCL16 shows minimal response to typical inflammatory mediators in hepatocytes, contrary to the regulation patterns of many other chemokines. For investigating potential regulatory effects:

• In vitro models: Culture primary human hepatocytes and stimulate with inflammatory cytokines (IL-1β, IL-6, TNF-α, oncostatin M) at physiologically relevant concentrations

• Patient samples: Compare CCL16 levels in normal liver versus diseased states (cirrhosis, viral hepatitis, hepatocellular carcinoma)

• Dynamic regulation: Measure CCL16 in sera from patients before and after partial liver resection to assess temporal changes

Statistical analysis of results should employ unpaired Mann-Whitney U tests, with p values <0.05 considered statistically significant, to account for non-normal distributions typically seen in cytokine expression data .

What is the functional significance of CCL16's C-terminal extension, and how can researchers investigate its role?

The C-terminal extension of CCL16 appears to modulate biological activity by impairing the accessibility of glycosaminoglycan (GAG) binding sites. To investigate this unique feature:

• Limited proteolysis studies: Compare full-length and truncated CCL16 (with the C-terminal extension removed) for:

  • GAG binding affinity

  • Receptor activation potential

  • Chemotactic activity in vitro

• Molecular dynamics (MD) simulations: Analyze how the C-terminal extension affects:

  • Protein flexibility

  • Surface accessibility

  • Binding site exposure

  • Potential conformational changes

• Structure-function analysis: Create targeted mutations within the C-terminal region to identify critical residues regulating GAG interactions

These approaches can reveal how enzymatic processing of CCL16 in tissue microenvironments might represent an activation mechanism, similar to proteolytic regulation observed in other chemokines.

How does CCL16 interact with glycosaminoglycans and what methodologies can assess these interactions?

To characterize CCL16-GAG interactions:

• Solid-phase binding assays: Measure binding of recombinant CCL16 to immobilized heparin and other GAGs

• Surface plasmon resonance (SPR): Determine binding kinetics and affinity constants for different GAG species

• Isothermal titration calorimetry (ITC): Analyze thermodynamic parameters of CCL16-GAG interactions

• Functional assays: Compare chemotactic activity of CCL16 in the presence/absence of soluble GAGs or cells with altered GAG expression

• Structural analysis: Use crystallography or NMR to identify specific CCL16 residues involved in GAG binding

Research indicates that matrix metalloproteinase processing enhances GAG binding of truncated CCL16, suggesting that controlled proteolysis may regulate CCL16 activity in vivo .

What crystallization conditions are optimal for structural studies of CCL16?

For successful crystallization of CCL16:

• Protein preparation: Concentrate purified CCL16 to approximately 2.2 mg/mL

• Screening approach: Use robotic sitting-drop vapor diffusion setups at 293 K to identify initial crystallization conditions

• Optimized conditions: Use 1.6 M sodium citrate (pH 6.5) as a precipitating agent, which has yielded high-quality single crystals

• Data collection parameters:

  • Cryo-cool crystals directly in mother liquor

  • Collect diffraction data at 100 K using synchrotron radiation (wavelength ~0.979 Å)

  • Process data using XDS and XSCALE

• Structure determination workflow:

  • Use molecular replacement with related chemokine structures as search models

  • Build missing parts interactively with Coot

  • Refine structure using phenix.refine

  • Validate final model with MolProbity and Coot

This approach has yielded structures with resolution up to 1.45 Å, providing detailed insights into CCL16 conformation.

How can researchers effectively analyze the oligomerization state of CCL16?

To characterize CCL16 oligomerization:

• Size exclusion chromatography: Analyze elution profiles at different protein concentrations to detect concentration-dependent oligomerization

• Analytical ultracentrifugation: Determine sedimentation coefficients and molecular weights in solution

• Dynamic light scattering: Measure hydrodynamic radius under various conditions

• Chemical crosslinking: Use bifunctional reagents to capture transient oligomeric species

• Native mass spectrometry: Directly observe oligomeric species and determine their stoichiometry

Since chemokine oligomerization is often critical for in vivo function, comparing CCL16's oligomerization properties with other CC chemokines can provide insights into its biological activities and regulation mechanisms.

How should researchers interpret conflicting data regarding CCL16's role in inflammatory diseases?

To resolve conflicting reports about CCL16's involvement in inflammatory conditions:

• Standardize detection methods: Use validated, consistent techniques for CCL16 measurement across studies

• Distinguish forms: Develop assays that can differentiate between full-length CCL16 and proteolytically processed forms

• Contextual analysis: Consider CCL16 within broader cytokine/chemokine networks rather than in isolation

• Disease phenotyping: Stratify patients based on detailed disease characteristics and progression stages

• Multi-omics approach: Integrate CCL16 data with genomics, transcriptomics, and proteomics datasets

CCL16 has been associated with ulcerative colitis, irritable bowel syndrome, eosinophilic pneumonia, preeclampsia, cardiovascular disease, and chronic kidney disorders, suggesting complex and context-dependent roles .

What is the relationship between CCL16 blood levels and liver function, and how can this be studied longitudinally?

To investigate CCL16 as a potential liver-derived biomarker:

• Patient cohorts: Establish well-characterized cohorts with:

  • Healthy controls

  • Various stages of liver disease (cirrhosis, hepatitis, etc.)

  • Post-liver transplantation subjects

  • Post-partial hepatectomy patients

• Longitudinal sampling: Collect serial samples to track CCL16 levels over time, particularly following interventions

• Correlative analysis: Compare CCL16 levels with:

  • Standard liver function tests (ALT, AST, bilirubin)

  • Liver imaging data

  • Histological assessments

  • Clinical outcomes

• Statistical approach: Use repeated measures ANOVA with appropriate post-hoc tests and mixed-effects models to account for individual variations

The high constitutive expression of CCL16 by hepatocytes suggests potential value as a marker of functional liver mass or hepatocyte integrity.

What molecular dynamics approaches best capture the flexibility and conformational changes of CCL16's C-terminal extension?

For comprehensive molecular dynamics analysis of CCL16:

• Simulation setup:

  • Prepare both full-length and truncated CCL16 structures

  • Use explicit solvent models with physiological ion concentrations

  • Employ modern force fields optimized for proteins (AMBER ff14SB, CHARMM36m)

• Simulation strategies:

  • Conduct long-timescale (>500 ns) conventional MD simulations

  • Apply enhanced sampling methods (accelerated MD, replica exchange)

  • Perform targeted simulations of protein-GAG complexes

• Analysis methods:

  • Calculate root-mean-square fluctuations (RMSF) to identify flexible regions

  • Use principal component analysis to identify dominant motion modes

  • Analyze solvent-accessible surface area changes

  • Track distances between key residues over simulation time

  • Calculate free energy landscapes

These approaches can reveal how the C-terminal extension dynamically modulates access to functional sites on the CCL16 chemokine domain.

How do post-translational modifications affect CCL16 structure and function?

To investigate potential post-translational modifications (PTMs) of CCL16:

• PTM identification:

  • Analyze endogenous CCL16 from human plasma using high-resolution mass spectrometry

  • Focus on glycosylation, phosphorylation, and proteolytic processing

  • Compare modifications between healthy subjects and disease states

• Functional impact assessment:

  • Generate recombinant CCL16 variants with and without specific modifications

  • Compare receptor binding, signaling, and chemotactic activity

  • Assess GAG binding properties of modified proteins

• Structural consequences:

  • Determine how PTMs affect CCL16 oligomerization

  • Use NMR to detect conformational changes induced by modifications

  • Apply hydrogen-deuterium exchange mass spectrometry to map structural impacts

Understanding the PTM landscape of CCL16 may help explain its unique biological properties and resolve contradictions in previously reported functional studies.