IL 18 Human, His

What is the structural composition of IL-18 Human, His?

IL-18 Human, His refers to recombinant human Interleukin-18 with a histidine tag. It is a single, non-glycosylated polypeptide chain containing 157 amino acids fragment (positions 37-193) with a molecular weight of 20kDa, fused with a 4.5kDa amino-terminal hexahistidine tag . Structurally, IL-18 adopts a β-trefoil fold comprising 12 β-strands (β1-β12) and 2 α-helices (α1-α2) . This specific three-dimensional arrangement is critical for receptor recognition and biological function. The protein maintains its structure during complex formation, with minimal conformational changes observed upon binding to receptors .

How should IL-18 Human, His be stored to maintain stability?

For short-term use (2-4 weeks), IL-18 Human, His can be stored at 4°C. For longer periods, it should be stored frozen at -20°C . It's crucial to avoid repeated freeze-thaw cycles as they can compromise protein integrity and biological activity . The protein is typically supplied in a buffer containing 20mM Tris-HCl pH-8 and 50% glycerol, which helps maintain stability . Some formulations are lyophilized from filtered solutions with trehalose as a protectant, requiring proper reconstitution according to the Certificate of Analysis for optimal performance . This careful storage approach ensures that the protein retains its structural integrity and functional properties for experimental use.

What are the key biological activities of IL-18 Human, His?

IL-18 Human, His functions as a potent proinflammatory cytokine that induces interferon-gamma (IFN-γ) production from T helper 1 (Th1) cells, natural killer (NK) cells, and activated macrophages . When combined with IL-12, it inhibits IL-4 dependent IgE and IgG1 production while enhancing IgG2a production by B cells . IL-18 activates the NF-κB and mitogen-activated protein kinase pathways, which upregulate the expression of various inflammatory cytokines . This activation occurs through a signaling cascade involving the adaptor molecule MyD88, IRAK4, IRAK1/2, and TRAF6, ultimately forming a complex called the Myddosome . The recombinant His-tagged protein retains these biological activities, making it suitable for studying IL-18-mediated immune responses in experimental systems.

What is the mechanism of IL-18 receptor complex formation?

The IL-18 receptor complex formation involves a sequential binding mechanism:

• IL-18 first binds to IL-18Rα with moderate affinity (KD ≈ 4.7 nM) .

• The IL-18/IL-18Rα binary complex then recruits the co-receptor IL-18Rβ .

• This ternary complex formation juxtaposes the intracellular Toll-Interleukin-1 receptor domains of both receptors .

The crystallographic data reveals that IL-18Rα curls around IL-18, while IL-18Rβ contacts the lateral portion of the IL-18/IL-18Rα binary complex . Interestingly, the second domain (D2) of both IL-18 receptors lacks one β-strand (d2) that is conserved among other IL-1-related receptors . Additionally, N-linked glycans play a role in bridging the two receptors, contributing to the binding affinity and stability of the complex . This structural arrangement is critical for proper signal transduction and subsequent activation of downstream inflammatory pathways.

How does IL-18BP sequester IL-18 and prevent its activity?

IL-18BP (IL-18 Binding Protein) is a natural inhibitor that sequesters IL-18 through a high-affinity interaction:

• IL-18BP binds to IL-18 at the same epitope as IL-18Rα, acting as a direct competitive inhibitor .

• The binding interface involves a large hydrophobic patch flanked by two tightly fitting hydrophobic pockets complemented by salt bridges .

• IL-18BP binds IL-18 with significantly higher affinity (sub-nanomolar KD) than IL-18Rα (KD ≈ 4.7 nM), effectively preventing receptor binding .

The crystal structure of the IL-18:IL-18BP complex reveals that structural mimicry and direct steric competition underlie the sequestration mechanism . An interesting discovery is the formation of a novel disulfide-linked interface resulting in a tetrameric assembly of human IL-18 and IL-18BP . This high-affinity interaction makes IL-18BP an effective negative regulator of IL-18 activity in vivo and a potentially useful tool in experimental settings to neutralize IL-18 function.

How does the His-tag on IL-18 affect its receptor binding and signaling capabilities?

The His-tag on IL-18 is typically positioned at the N-terminus, which is distinct from the primary receptor binding sites. While the tag generally doesn't significantly interfere with receptor binding interfaces, researchers should consider several factors:

• The His-tag may introduce minor steric effects that slightly alter binding kinetics, though these effects are usually minimal .

• In structural studies such as crystallography, the tag provides a useful tool for protein purification but may need to be removed to eliminate potential artifacts .

• When conducting binding affinity studies, comparing tagged and untagged versions can help determine if the tag influences receptor interactions.

Binding studies using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) suggest that His-tagged IL-18 maintains high-affinity binding to its receptors, but validation in specific experimental systems is recommended . The retention of biological activity in cell-based assays further indicates that the tag doesn't significantly impair functional interactions .

How can the biological activity of IL-18 Human, His be quantitatively measured?

The biological activity of IL-18 Human, His can be quantified through several established approaches:

The specific activity of premium grade Human IL-18 can be >3.00×10^6 IU/mg, calibrated against WHO Reference Reagent Interleukin-18 . Functional assays typically involve dose-response experiments where cells are treated with varying concentrations of IL-18, followed by measurement of the appropriate readout. The binding of Human IL-18 to IL-18BP can also be measured, with linear binding observed in the range of 0.6-20 ng/mL .

What are recommended methods for validating IL-18 Human, His preparations?

To validate IL-18 Human, His preparations, researchers should employ multiple analytical methods:

• SDS-PAGE: To assess purity (should be >95%) and molecular weight (approximately 17-20 kDa under reducing conditions) .

• Western blotting: Using specific anti-IL-18 or anti-His antibodies to confirm identity.

• Binding assays: Testing interaction with known binding partners such as IL-18BP or IL-18Rα using ELISA or surface plasmon resonance .

• Bioactivity assays: Confirming functional activity through cell-based assays as described above.

• Mass spectrometry: For precise molecular weight determination and sequence verification.

The protein typically migrates as 17 kDa±3 kDa when calibrated against protein markers under reducing conditions in SDS-PAGE . Comparing your results with reference standards and product specifications ensures quality and consistency across experiments. Biophysical methods like multi-angle light scattering (MALLS) can also be used to determine the molecular weight, with values of approximately 18.2 ± 0.5 kDa for unglycosylated IL-18 .

What controls should be included when using IL-18 Human, His in functional assays?

When designing functional assays with IL-18 Human, His, include the following controls:

• Negative controls:

  • Untreated cells (baseline response)

  • Heat-inactivated IL-18 (to confirm specificity of biological effects)

  • Irrelevant His-tagged protein (to rule out His-tag effects)

• Positive controls:

  • Known IL-18 responsive cell line (e.g., KG-1)

  • Alternative cytokine that induces similar responses

• Specificity controls:

  • IL-18BP to neutralize IL-18 activity

  • Anti-IL-18 or anti-IL-18R antibodies

  • IL-18 receptor knockout or knockdown cells

• Dose-response controls:

  • Serial dilutions of IL-18 to establish dose-dependence

  • Time-course experiments to determine optimal stimulation time

These controls help validate experimental outcomes and provide context for interpreting results. When using the KG-1 cell line, measuring IL-8 secretion provides a reliable readout for IL-18 activity that can be inhibited by IL-18BP, confirming specificity .

How can IL-18 Human, His be used to study the structural basis of receptor recognition?

IL-18 Human, His provides a valuable tool for structural studies of receptor interactions through several approaches:

• Crystallography methods:

  • Co-crystallization with receptor extracellular domains

  • Phase determination using the His-tag for metal ion binding

  • Structure determination of ternary complexes with both IL-18Rα and IL-18Rβ

• Binding interface analysis:

  • Mapping binding epitopes through mutagenesis studies

  • Identifying critical residues for receptor recognition

  • Comparing with related cytokine-receptor complexes

The crystal structure of human IL-18 bound to its receptor domains reveals important details about the binding interface. For example, the IL-18 Site III comprises the prominent β8-β9 hairpin and β11-α2 loop, where the aromatic ring of His109 forms π-π stacking with Tyr212 of IL-18Rβ at a 3.4 Å distance, surrounded and stabilized by multiple hydrogen bonds . These structural insights can guide the development of antagonists or agonists with therapeutic potential.

What structural features distinguish IL-18 from other IL-1 family members?

Structural comparison between IL-18 and other IL-1 family members reveals both conserved elements and unique features:

The second domain (D2) of the two IL-18 receptors lacks one β-strand (d2) that is conserved among other IL-1-related receptors, which was previously shown to contribute to inter-receptor interaction . Additionally, N-linked glycans play a role in bridging the two IL-18 receptors, which was observed in the signaling IL-1β receptor complex but was absent in its decoy complex . These subtle structural differences underlie the specificity of different cytokine-receptor systems within the IL-1 family.

What crystallographic conditions have successfully yielded IL-18 complex structures?

The successful crystallization of IL-18 complexes has been achieved under specific conditions, providing valuable information for researchers attempting similar structural studies:

For the IL-18:IL-18BP complex, important advances in crystallization efforts included:

• Elimination of the flexible N-terminus of IL-18BP

• Minimization of heterogeneous glycosylation patterns while retaining functional activity

• Development of a construct (IL-18BP ΔN-EH) that maintained sub-nanomolar affinity toward IL-18

The resulting crystal structure had unit cell dimensions of a=109.82 Å, b=44.52 Å, c=60.28 Å, with angles α=90°, β=99.86°, γ=90° . These details provide a starting point for researchers attempting to crystallize IL-18 in complex with its binding partners or receptors.

How do viral IL-18 binding proteins differ from human IL-18BP?

Viral IL-18 binding proteins (vIL-18BPs) offer unique insights into IL-18 biology through comparative analysis with human IL-18BP (hIL-18BP):

• Structural comparison:

  • vIL-18BPs from poxviruses share the core immunoglobulin-like fold with hIL-18BP

  • Despite low sequence identity, key binding interface elements are functionally conserved

  • Major variations exist in the CD loop mediating IL-18 binding and in the AB loop

• Binding mechanism:

  • Both vIL-18BPs and hIL-18BP bind to the same epitope on IL-18

  • Both use structural mimicry and direct steric competition to prevent receptor binding

  • Subtle differences in binding interfaces may confer different affinities or specificities

Structure-based sequence alignments and structural superpositions of human IL-18BP against viral IL-18BPs (ectvIL-18BP and yldvIL-18BP) establish the strong conservation of the adopted fold, despite significant sequence divergence . This evolutionary convergence highlights the critical importance of IL-18 regulation in both host immune defense and viral immune evasion strategies, and provides insights for designing therapeutic IL-18 antagonists.

How does the binding affinity of IL-18 to its receptors compare with its binding to IL-18BP?

The binding affinities of IL-18 to its various binding partners show significant differences that have important functional implications:

Human IL-18BP binds to IL-18 with significantly higher affinity than IL-18Rα, which explains its effectiveness as a natural inhibitor . The binary complex affinity measured between IL-18 and IL-18Rα (KD = 4.7 nM) is markedly higher than previously reported affinities (KD∼60 nM) measured by surface plasmon resonance .

The thermodynamic binding profiles obtained through isothermal titration calorimetry (ITC) show that different versions of IL-18BP (full-length and truncated) maintain similarly high affinities toward IL-18 . This high-affinity interaction makes IL-18BP an effective negative regulator of IL-18 activity in vivo and a potentially useful tool in experimental settings to neutralize IL-18 function.

What role do glycosylation patterns play in IL-18 receptor interactions?

Glycosylation patterns significantly influence IL-18 receptor interactions through several mechanisms:

• Inter-receptor bridging:

  • N-linked glycans play a role in bridging the two IL-18 receptors

  • This glycan-mediated bridging was observed in the signaling IL-18 receptor complex

  • Similar bridging occurs in the IL-1β receptor complex but is absent in its decoy complex

• Binding affinity contribution:

  • IL-18Rα N-linked glycans proximal to IL-18 in the complexes contribute to binding affinity

  • Manipulation of glycosylation patterns can alter receptor binding kinetics

• Experimental considerations:

  • Heterogeneous glycosylation can complicate structural studies

  • For crystal structure determination of IL-18:IL-18BP, minimization of heterogeneous glycosylation patterns while retaining functional activity was crucial

  • The human IL-18BP full-length protein contains approximately 14.8 ± 0.6 kDa of glycans on a 17.6 ± 0.1 kDa protein backbone

These findings emphasize the importance of glycosylation in IL-18 biology and should be considered when designing experiments or interpreting results from studies using differently glycosylated forms of IL-18 or its receptors.

What factors can affect the reproducibility of IL-18 Human, His experiments?

Several factors can influence the reproducibility of experiments using IL-18 Human, His:

• Protein stability and storage:

  • Repeated freeze-thaw cycles can degrade protein quality

  • Improper storage temperatures affect long-term stability

  • Buffer composition impacts protein folding and activity

• Cellular factors:

  • Receptor expression levels on target cells

  • Presence of endogenous IL-18BP in culture systems

  • Cell passage number and culture conditions

  • Co-stimulatory cytokine requirements (e.g., IL-12)

• Technical considerations:

  • Batch-to-batch variation in recombinant protein preparation

  • Differences in protein purity (should be >95% as determined by SDS-PAGE)

  • Presence of contaminants like endotoxin that can activate cells independently

• Assay-specific factors:

  • Sensitivity and dynamic range of detection methods

  • Timing of measurements relative to stimulation

  • Dose-response relationships that may not be linear

Careful experimental design with appropriate controls, consistent protocols, and thorough documentation of protein characteristics can help address these factors and improve reproducibility.

How can researchers optimize cell-based assays using IL-18 Human, His?

Optimizing cell-based assays with IL-18 Human, His requires attention to several key parameters:

• Cell selection and preparation:

  • Use well-characterized IL-18-responsive cells (e.g., KG-1)

  • Maintain consistent cell density and passage number

  • Verify receptor expression levels before experiments

  • Consider co-stimulation with IL-12 for optimal responses

• Assay conditions:

  • Determine optimal concentration range through dose-response studies

  • Establish appropriate time-course for measuring responses

  • Select appropriate readouts (e.g., IFN-γ, IL-8 secretion, NF-κB activation)

  • Include positive controls (known IL-18 responsive systems)

• Quality control:

  • Use freshly prepared or properly stored IL-18 preparations

  • Include activity standards in each experiment

  • Verify protein concentration using validated methods

  • Test each new lot of IL-18 against reference standards

• Data analysis:

  • Use appropriate statistical methods for dose-response data

  • Compare EC50 values rather than single-point measurements

  • Consider normalizing data to maximum response

  • Account for background activity in control samples

By systematically optimizing these parameters, researchers can develop robust and reproducible cell-based assays for studying IL-18 biology or screening for modulators of IL-18 activity.

What are the challenges in studying IL-18 in complex biological systems?

Studying IL-18 in complex biological systems presents several methodological challenges:

• Receptor expression heterogeneity:

  • Variable expression levels of IL-18Rα and IL-18Rβ across cell types

  • Differential expression of inhibitory IL-18BP

  • Presence of soluble receptor forms that modulate responses

• Signaling pathway complexity:

  • Cell-specific adaptor protein availability

  • Cross-talk with other cytokine signaling pathways (especially IL-12)

  • Variations in downstream transcriptional regulation

• Experimental design considerations:

  • Need for appropriate primary cell models versus cell lines

  • Controlling for endogenous IL-18 and IL-18BP production

  • Accounting for differential glycosylation patterns that affect binding

• Analytical challenges:

  • Requiring multi-parametric readouts beyond single cytokine production

  • Temporal dynamics of response that vary between cell types

  • Dose-response relationships that differ by cell lineage

To address these challenges, researchers should consider using multi-omics approaches, single-cell analysis techniques to capture heterogeneity, and systems biology models to integrate complex datasets. Genetic manipulation to standardize receptor expression levels can also provide more controlled experimental systems.