I 309 Human, His

What is human I-309 and what is its primary biological function?

Human I-309 is a small glycoprotein (15-16 kDa) secreted by activated T lymphocytes that belongs to the CC subclass of chemokines. Its primary biological function is serving as a monocyte chemoattractant, stimulating the directed migration of monocytes but not neutrophils when tested in vitro. Additionally, I-309 transiently increases cytoplasmic free calcium concentration specifically in human peripheral blood monocytes but not in lymphocytes or neutrophils .

What are the key structural characteristics of I-309?

I-309 possesses a distinctive structure characterized by:

• A disordered N-terminal region (similar to eotaxin but unlike MCP-1 and RANTES)

• A well-ordered region between residues 13 and 69 consisting of:

  • A 3₁₀-helix

  • A triple-stranded antiparallel β-sheet

  • A C-terminal α-helix

• An additional third disulfide bond (one of only three human chemokines with this feature)

• Root-mean-square deviations of 0.61 and 1.16 observed for the backbone and heavy atoms, respectively

• A significant structural deviation in the C-terminal region where the α-helix terminates early and is followed by a short extended strand

How was I-309 first isolated and characterized?

I-309 was initially characterized through the development of a stable Chinese hamster ovary cell transfectant (CDI.10) that constitutively secretes I-309 protein. The protein was purified to homogeneity using affinity chromatography on a heparin-Sepharose matrix followed by reverse-phase HPLC, yielding a glycoprotein doublet of 15-16 kDa from culture supernatant. Biochemical analysis confirmed that the purified recombinant I-309 glycoprotein was indistinguishable from the natural I-309 glycoprotein constitutively secreted by the T-cell line IDP2 .

How does the additional disulfide bond in I-309 affect its structure and function?

The additional disulfide bond in I-309 produces significant structural changes compared to standard CC chemokines. This structural feature directly causes:

• Premature termination of the C-terminal α-helix that is normally present in chemokines

• Formation of a short section of extended strand following the shortened α-helix

• Altered protein folding that may affect receptor recognition and binding kinetics

• Potentially increased stability in physiological conditions

These structural alterations influence the specificity of I-309 for its receptor, CCR8, suggesting the additional disulfide bond plays a critical role in determining receptor selectivity and downstream signaling pathways .

What experimental methods have been used to determine I-309's three-dimensional structure?

The three-dimensional solution structure of I-309 was determined using:

• ¹H nuclear magnetic resonance (NMR) spectroscopy

• Dynamic simulated annealing based on 978 experimental restraints

• Structural comparisons with other CC chemokines (eotaxin and HCC-2)

The analysis revealed that I-309 remains monomeric even at high concentrations, unlike some other chemokines that tend to dimerize. This structural determination process allowed researchers to identify the unique features of I-309 compared to other chemokines lacking the third disulfide bond .

What is the relationship between I-309 and its receptor CCR8?

I-309 specifically binds to the CCR8 receptor, and this interaction involves:

• Recognition of the distinctive structural features of I-309, particularly in the C-terminal region

• Specificity determined in part by the additional disulfide bond's effect on protein conformation

• Activation of monocyte-specific signaling pathways upon binding

• Potential for different binding kinetics compared to other chemokine-receptor pairs

Understanding the I-309/CCR8 interaction is crucial for developing targeted therapeutics that might modulate this pathway in inflammatory or immune-related conditions .

What are effective methods for producing and purifying recombinant I-309?

Research indicates that the most effective methods for producing and purifying recombinant I-309 include:

Production Systems:

• Stable Chinese hamster ovary (CHO) cell transfectants (e.g., CDI.10 line)

• Expression in bacterial systems with appropriate refolding protocols

• Mammalian expression systems for properly glycosylated variants

Purification Protocol:

• Collection of culture supernatant containing secreted I-309

• Affinity chromatography using heparin-Sepharose matrix

• Further purification via reverse-phase HPLC

• Verification of purity through SDS-PAGE, yielding a characteristic glycoprotein doublet of 15-16 kDa

• Confirmation of biological activity through monocyte chemotaxis assays

How should researchers design chemotaxis assays to measure I-309 activity?

Effective chemotaxis assay design for I-309 should include:

Essential Components:

• Boyden chamber or transwell migration system

• Freshly isolated human monocytes (not neutrophils or lymphocytes)

• Appropriate positive controls (other known monocyte chemoattractants)

• Negative controls (buffer only, inactive proteins)

• Dose-response analysis with multiple I-309 concentrations

Data Analysis Approach:

• Quantification of migrated cells (microscopy cell counting or flow cytometry)

• Statistical comparison between experimental and control conditions

• Calculation of chemotactic index (ratio of cells migrating toward I-309 versus random migration)

• Assessment of dose-dependent responses

What experimental controls are crucial in calcium mobilization studies with I-309?

When conducting calcium mobilization studies to assess I-309 activity, researchers should implement these critical controls:

Cell Population Controls:

• Test purified monocytes, lymphocytes, and neutrophils separately

• Include positive controls for each cell type (known calcium mobilizers)

• Verify cell viability and loading of calcium indicators

Technical Controls:

• Baseline measurements before stimulation

• Vehicle-only controls

• Concentration gradient responses

• Receptor blocking experiments (anti-CCR8 antibodies)

• Cross-desensitization studies with other chemokines

These controls help establish the specificity of I-309's effects on monocytes versus other leukocyte populations .

What experimental designs are most appropriate for studying I-309's effects in vitro?

When investigating I-309's effects in vitro, researchers should consider:

Recommended Designs:

• Pretest-posttest control group design for measuring cellular responses

• Time-series experiments for evaluating duration of calcium flux or signaling

• Factorial designs for examining interactions between I-309 and other cytokines

• Dose-response studies with multiple concentrations

Design Considerations:

• Randomization of treatment conditions

• Blinding of outcome assessments where possible

• Appropriate sample sizes based on power analysis

• Inclusion of positive and negative controls

• Technical replicates to account for measurement variability

• Biological replicates to account for donor-to-donor variation

How can quasi-experimental designs be applied to human studies involving I-309?

Quasi-experimental designs may be necessary when studying I-309 in human populations where full experimental control is not possible:

Applicable Quasi-Experimental Approaches:

• Time-series designs: Measuring I-309 levels before and after an intervention or event

• Nonequivalent control group designs: Comparing I-309 levels or responses between similar but not randomly assigned groups

• Multiple time-series designs: Comparing I-309 patterns over time in different populations

• Regression-discontinuity analysis: Especially useful when studying I-309 in relation to threshold-based clinical decisions

Implementation Considerations:

• Careful matching of comparison groups on relevant variables

• Statistical adjustment for confounding factors

• Transparent reporting of design limitations

• Combining multiple quasi-experimental approaches to strengthen inference

What statistical approaches are most appropriate for analyzing I-309 functional data?

When analyzing complex datasets involving I-309:

• Consider both parametric and non-parametric approaches based on data distribution

• Implement appropriate post-hoc tests when using ANOVA

• Use statistical software capable of handling repeated measures

• Report effect sizes alongside p-values

• Consider developing predictive models for I-309 responses when multiple variables are involved

How should researchers design experiments to investigate I-309's role in specific diseases?

When investigating I-309's role in disease contexts, researchers should:

Study Design Elements:

• Begin with observational studies comparing I-309 levels in patient cohorts versus controls

• Implement case-control designs with careful matching of demographic and clinical variables

• Progress to longitudinal studies tracking I-309 levels during disease progression

• Consider intervention studies targeting the I-309/CCR8 axis when appropriate

Methodological Considerations:

• Standardize sample collection protocols (timing, processing, storage)

• Account for confounding factors that may influence I-309 expression

• Incorporate multiple biomarkers to establish specificity of I-309's role

• Combine clinical measurements with functional in vitro assays

• Consider genetic analyses of I-309 and CCR8 polymorphisms

What are the challenges in developing antagonists or inhibitors of I-309?

Developing effective I-309 antagonists presents several challenges:

Technical Challenges:

• The unique structural features of I-309, particularly the additional disulfide bond, may require specialized approaches to antagonist design

• Achieving specificity for I-309 without affecting other chemokines

• Determining the precise binding interface between I-309 and CCR8

• Developing assays that accurately measure antagonist efficacy

Strategic Approaches:

• Structure-based drug design utilizing the NMR structure of I-309

• Peptide-based antagonists derived from CCR8 binding regions

• Antibody or aptamer development targeting I-309-specific epitopes

• Small molecule screening focused on disrupting the I-309/CCR8 interaction

What are emerging research questions about I-309 that remain to be addressed?

Despite significant progress in understanding I-309, several critical questions remain:

• How does the expression of I-309 change during different immune responses and inflammatory conditions?

• What is the complete signaling network activated by I-309/CCR8 interaction in different cell types?

• How does post-translational modification of I-309 affect its bioactivity?

• What is the evolutionary significance of the additional disulfide bond in I-309?

• How might I-309 function be targeted therapeutically in specific disease contexts?

Addressing these questions will require innovative experimental approaches combining structural biology, cell signaling analysis, and translational research in relevant disease models .

How can researchers integrate new technologies into I-309 studies?

Advancing I-309 research will benefit from integrating cutting-edge technologies:

• Single-cell analysis to determine cellular sources and responses to I-309 with unprecedented resolution

• CRISPR-Cas9 genome editing to study the effects of I-309 or CCR8 modification in relevant cell types

• Advanced imaging techniques to visualize I-309/CCR8 interactions in real-time

• Systems biology approaches to position I-309 within broader cytokine networks

• Computational modeling to predict I-309 interactions and function based on its unique structural features