Lymphotactin Human, His

What is Lymphotactin (XCL1) and what distinguishes it from other chemokines?

Lymphotactin (XCL1) belongs to the C or gamma subfamily of chemokines, with unique structural characteristics that set it apart from other chemokine families. Unlike CC and CXC chemokines that contain four conserved cysteine residues, Lymphotactin lacks two of these cysteines (specifically the 1st and 3rd), resulting in a distinctive structure with an extended carboxy terminus . Human Lymphotactin encodes a 114 amino acid residue precursor protein with a 21 amino acid residue predicted signal peptide .

The most remarkable feature of Lymphotactin is its conformational plasticity. Under physiological conditions, it exists in equilibrium between two entirely different structural states: a canonical chemokine fold consisting of a monomeric three-stranded β-sheet and carboxyl-terminal helix, and an alternative dimeric all-β-sheet arrangement with no similarity to other known proteins . This structural duality is directly linked to its dual functionality, creating a molecular switch that allows Lymphotactin to perform two distinct immunological roles with a single amino acid sequence.

How does the unique structural plasticity of Lymphotactin relate to its biological function?

Lymphotactin's structural plasticity is directly linked to its dual functionality in the immune system. The protein interconverts between two distinct structural conformations:

• The canonical chemokine fold (Ltn10): This monomeric conformation functions as an XCR1 receptor agonist, enabling T cell chemotaxis, but lacks glycosaminoglycan binding capability .

• The alternative dimeric structure (Ltn40): This conformation binds glycosaminoglycans with high affinity but fails to activate the XCR1 receptor .

These structures interconvert at a rate of approximately 1/s under physiological conditions . This conformational equilibrium is essential for Lymphotactin's complete biological activity in vivo, as each structural species displays only one of the two functional properties required for its immunomodulatory role. The chemokine-like conformation mediates cell attraction through XCR1 activation, while the alternative structure facilitates tissue localization through glycosaminoglycan binding .

What are the key immunological functions of Lymphotactin?

Lymphotactin plays several important roles in immune regulation:

• T cell and NK cell chemotaxis: It preferentially chemoattracts CD8+ T cells and NK cells, with lower efficiency for CD4+ T cells .

• Immunomodulation: It can costimulate apoptosis of CD4+ T cells but not CD8+ T cells, suggesting a role in regulating T cell population dynamics .

• Association with inflammatory diseases: Lymphotactin expression is linked to rheumatoid arthritis, acute allograft rejection, Crohn's disease, and glomerulonephritis, consistent with an immunomodulatory function .

• Cancer immunotherapy potential: Lymphotactin can recruit T cells to tumor sites, and combined expression with interleukin-2 has shown promising results in neuroblastoma treatment, including complete remission in some patients .

Lymphotactin is produced mainly through T cell receptor activation in CD4+ and CD8+ T cells, but also by NK cells and γδ T cells . Its ability to orchestrate T cell-mediated immune responses makes it a target of interest for therapeutic development in both inflammatory conditions and cancer immunotherapy.

What are the recommended methods for recombinant production of Human Lymphotactin with His-tag?

Recombinant production of His-tagged human Lymphotactin can be accomplished using several approaches, with E. coli being the most common expression system:

• Expression system selection: E. coli is typically used for structural and functional studies of Lymphotactin. The protein sequence from Val22 to Gly114 represents the mature form used for recombinant expression .

• His-tag configuration: A common approach involves N-terminal His-tagging, such as the construct "MGSSHHHHHH SSGLVPRGSH" followed by the Lymphotactin sequence .

• Expression considerations:

  • It's critical to maintain the native N-terminus of the mature protein, as modifications to this region can inactivate the chemokine .

  • Expression in E. coli generally yields protein that requires refolding to attain the native disulfide bonds.

  • The predicted molecular weight of His-tagged human Lymphotactin is approximately 12.5 kDa, though it may migrate anomalously on SDS-PAGE .

• Purification approach: Recombinant His-tagged Lymphotactin can be purified using conventional chromatography methods, including immobilized metal affinity chromatography followed by additional purification steps to achieve >95% purity .

For applications requiring post-translational modifications such as glycosylation, insect cell expression using baculovirus systems has been employed, though functional studies indicate that glycosylation of the C-terminus does not affect XCR1 activation or T cell chemotaxis .

What are the optimal storage conditions for preserving Lymphotactin activity?

For maintaining the structural integrity and biological activity of purified recombinant Lymphotactin, the following storage guidelines are recommended:

• Short-term storage: Store at 2-8°C under sterile conditions for up to 1 month after reconstitution .

• Long-term storage:

  • Store lyophilized protein at -20°C to -70°C for up to 12 months from receipt date .

  • For extended storage after reconstitution, aliquot and store at -20°C to -70°C for up to 6 months .

  • Avoid repeated freeze-thaw cycles as these can lead to protein denaturation and activity loss .

• Buffer composition: A typical storage buffer system includes 20 mM Tris-HCl (pH 8.0) containing 30% glycerol, 2 mM DTT, and 0.2 M NaCl . The glycerol serves as a cryoprotectant, while DTT helps maintain reduced states of cysteines not involved in disulfide bonds.

• Stabilizing factors: The conformational equilibrium of Lymphotactin is influenced by salt concentration, with higher NaCl levels (≥200 mM) favoring the chemokine-like conformation that activates XCR1 .

• Quality control: Before storage, ensure proper disulfide bond formation and verify biological activity through appropriate functional assays to establish baseline activity levels for comparison after storage.

Proper storage considerations are particularly important for Lymphotactin due to its conformational plasticity, which can be affected by environmental conditions during storage.

What are the critical quality control parameters for assessing recombinant Lymphotactin preparations?

Comprehensive quality control of recombinant Lymphotactin preparations should include assessment of several critical parameters:

• Purity assessment:

  • SDS-PAGE analysis with appropriate staining (>95% purity is typically desirable)

  • High-performance liquid chromatography (HPLC)

  • Mass spectrometry to confirm molecular weight and detect potential modifications

  • Note that Lymphotactin may show anomalous migration on SDS-PAGE due to its unusual structural properties

• Structural integrity:

  • Verification of proper disulfide bond formation

  • Assessment of conformational distribution between the two structural states

  • Size exclusion chromatography to evaluate monomer-dimer distribution

• Functional activity:

  • XCR1 receptor activation using calcium flux assays

  • Chemotaxis assays with XCR1-expressing cells, such as BaF3 mouse pro-B cells transfected with human XCR1

  • Heparin binding assays to assess the glycosaminoglycan-binding conformation

  • Neutralization tests using specific antibodies (ND50 typically 3.00-30.0 μg/mL in the presence of 0.5 μg/mL recombinant human Lymphotactin)

• Endotoxin testing:

  • Particularly important for immunological studies where endotoxin contamination could confound results

A typical specification for high-quality recombinant human Lymphotactin would include: >95% purity by SDS-PAGE, confirmation of predicted molecular weight (approximately 12.5 kDa for His-tagged constructs), proper disulfide bond formation, and detectable XCR1 activation at concentrations comparable to published standards .

What techniques are most effective for investigating the conformational equilibrium of Lymphotactin?

Several complementary techniques have proven effective for investigating Lymphotactin's unique conformational equilibrium:

• NMR Spectroscopy: NMR has been instrumental in resolving the two distinct structural species and characterizing their interconversion. 2D 1H-15N HSQC spectra show characteristic patterns for each conformation. Longitudinal (T1) relaxation rates can be measured to determine the rate of structural interconversion (approximately 1/s) .

• Heparin Affinity Chromatography: Since the Ltn40 conformation binds heparin with high affinity while Ltn10 does not, heparin affinity chromatography provides a functional means to assess the conformational equilibrium .

• Time-Resolved Fluorescence: This technique can monitor conformational changes in real-time, especially when strategic fluorescent labels are incorporated at positions that experience different environments in the two conformations .

• Mutagenesis Studies: Creating amino acid substituted variants can identify residues critical for stabilizing each conformation. Mutations of residues required for glycosaminoglycan binding have been shown to shift the equilibrium toward the chemokine-like fold .

It's important to note that the interconversion occurs rapidly (on the order of seconds), making it challenging to isolate pure populations of either conformer under physiological conditions. Temperature, pH, and salt concentration can all influence the equilibrium position, with charge repulsion between arginines 23 and 43 potentially destabilizing the chemokine fold and promoting conversion to the novel Lymphotactin dimer .

How can researchers effectively assess the biological activity of recombinant Lymphotactin preparations?

Comprehensive assessment of Lymphotactin's biological activity requires evaluation of both its XCR1 activation capability and glycosaminoglycan binding properties:

• XCR1 Activation Assays:

  • Calcium flux assays using XCR1-transfected cell lines (such as BaF3 mouse pro-B cells expressing human XCR1)

  • Chemotaxis assays measuring the migration of XCR1-expressing cells through a membrane in response to a Lymphotactin gradient

  • The chemotactic response can be quantified using methods such as Resazurin-based cell counting

• Glycosaminoglycan Binding Assays:

  • Heparin affinity chromatography to measure binding affinity

  • Surface plasmon resonance to determine binding kinetics to immobilized glycosaminoglycans

• Neutralization Studies:

  • Specific antibodies like the Goat Anti-Human XCL1/Lymphotactin Antibody can be used to block Lymphotactin activity in a dose-dependent manner

  • The neutralization dose (ND50) typically ranges from 3.00-30.0 μg/mL in the presence of 0.5 μg/mL recombinant human Lymphotactin

• Data Analysis:

  • For chemotaxis data, plot the number of migrated cells versus Lymphotactin concentration to generate dose-response curves

  • For neutralization studies, plot the percent inhibition of migration versus antibody concentration

These functional assays are essential to confirm that recombinant Lymphotactin preparations maintain both conformational states and their respective activities, particularly after storage or manipulation.

How can researchers optimize experimental conditions to study one specific conformation of Lymphotactin?

Optimizing conditions to favor one specific conformation of Lymphotactin requires strategic manipulation of factors that influence the conformational equilibrium:

• To favor the chemokine-like Ltn10 conformation:

  • Increase sodium chloride concentration (≥200 mM NaCl)

  • Lower temperatures (10-15°C)

  • Neutral to slightly alkaline pH (7.0-8.0)

  • Use specific mutations that destabilize the Ltn40 conformation

  • Addition of XCR1 receptor fragments that selectively bind Ltn10

• To favor the dimeric Ltn40 conformation:

  • Reduce sodium chloride concentration (≤50 mM)

  • Higher temperatures (30-37°C)

  • Slightly acidic pH (6.0-6.5)

  • Use mutations that disrupt the chemokine fold

  • Addition of specific glycosaminoglycans that selectively bind Ltn40

  • Higher protein concentrations to favor dimerization

• Mutagenesis approaches:

  • CC3 mutant (with two additional cysteines forming a third disulfide bond) stabilizes the Ltn10 conformation

  • Mutation of key arginine residues involved in GAG binding can shift the equilibrium toward Ltn10

• Experimental design considerations:

  • Include conformational state controls in experiments

  • Monitor the conformational distribution under experimental conditions

  • Account for the dynamic nature of the equilibrium when interpreting results

  • Consider temperature control during experiments, as temperature shifts can alter the conformational distribution

These approaches can help researchers enrich for a specific conformational state, though achieving complete separation under physiological conditions remains challenging due to the dynamic equilibrium between the two forms.

What are the critical considerations for designing experiments to study Lymphotactin's role in T cell-mediated immune responses?

When designing experiments to investigate Lymphotactin's immunomodulatory functions, researchers should consider several key factors:

• Choice of experimental system:

  • Primary human T cells offer physiological relevance but exhibit donor variability

  • Cell lines provide consistency but may lack complete signaling pathways

  • Animal models should be approached with caution due to the approximately 60% sequence homology between human and mouse Lymphotactin

• Conformational state considerations:

  • Buffer conditions can influence the conformational equilibrium (especially salt concentration)

  • Experimental temperatures should be physiologically relevant

  • Consider using mutants that preferentially stabilize one conformation for dissecting specific functions

• Functional readouts:

  • For chemotactic responses, both transwell migration assays and real-time visualization of cell migration should be employed

  • When studying CD8+ vs CD4+ T cell responses, note that Lymphotactin preferentially chemoattracts CD8+ T cells and may costimulate apoptosis in CD4+ T cells

  • For NK cell studies, consider both chemotactic responses and potential effects on cytolytic activity

• Relevant disease models:

  • For autoimmune conditions (rheumatoid arthritis, Crohn's disease), evaluate both pro-inflammatory and immunomodulatory effects

  • In cancer immunotherapy models, assess T cell recruitment to tumor sites and potential synergy with cytokines like IL-2

• Controls and validation:

  • Include neutralizing antibodies to confirm specificity

  • Use XCR1 antagonists or XCR1-deficient cells as controls

  • Compare results with other chemokines to establish Lymphotactin-specific effects

These considerations help ensure that experiments accurately capture Lymphotactin's complex immunomodulatory functions while accounting for its unique structural plasticity.

What are the potential applications of Lymphotactin in cancer immunotherapy research?

Lymphotactin has shown promising potential in cancer immunotherapy research through several mechanisms:

• T Cell Recruitment to Tumors:

  • Lymphotactin can be expressed at tumor sites to recruit CD8+ cytotoxic T lymphocytes

  • Animal studies have demonstrated that Lymphotactin expression can attract T cells to tumors, enhancing anti-tumor immune responses

• Combination Therapies:

  • Synergistic effects have been observed when Lymphotactin is combined with cytokines like IL-2

  • A neuroblastoma tumor vaccine combining Lymphotactin and IL-2 expression induced measurable antitumor immune responses, including complete remission in some patients

  • These combination approaches potentially enhance both the recruitment and activation of tumor-specific T cells

• Gene Therapy Approaches:

  • Tumor cells can be engineered to express Lymphotactin to create an in situ vaccine effect

  • Dendritic cells expressing Lymphotactin have been explored as cellular vaccines

• Research Considerations:

  • The dual conformational states of Lymphotactin may contribute to its efficacy by promoting both T cell recruitment (via XCR1 activation) and retention at tumor sites (via glycosaminoglycan binding)

  • Strategic modulation of the conformational equilibrium might optimize anti-tumor responses

A number of animal studies have shown that Lymphotactin can effectively recruit T cells to the site of a tumor, and combined expression of Lymphotactin and interleukin-2 has demonstrated clinical promise . These findings suggest that Lymphotactin-based approaches could complement existing immunotherapy strategies by enhancing T cell trafficking to tumors.

How does the interaction between Lymphotactin and glycosaminoglycans influence its biological functions?

The interaction between Lymphotactin and glycosaminoglycans (GAGs) represents a critical aspect of its biology and has profound implications for its function:

• Structural Basis of Interaction:

  • The alternative dimeric Ltn40 conformation exhibits high-affinity GAG binding, while the canonical Ltn10 chemokine fold does not

  • Key basic residues, particularly arginines, form the GAG binding site in the Ltn40 structure

  • Mutation of these residues shifts the conformational equilibrium toward the chemokine-like fold

• Functional Consequences:

  • GAG binding facilitates the localization and concentration of Lymphotactin on cell surfaces and extracellular matrix

  • This localization may create chemotactic gradients necessary for directed cell migration

  • The binding may also protect Lymphotactin from proteolytic degradation, extending its half-life in tissues

• Regulatory Mechanisms:

  • The conformational equilibrium between Ltn10 and Ltn40 effectively regulates GAG binding

  • Environmental factors that influence this equilibrium (ionic strength, pH) can therefore modulate Lymphotactin's tissue distribution

  • Charge repulsion between arginines 23 and 43 has been proposed to destabilize the chemokine fold and promote conversion to the GAG-binding dimeric form

Understanding this interaction provides insights into how Lymphotactin functions in tissues and suggests strategies for therapeutic modulation of its activity in various disease contexts.

What is the comparative analysis of the two structural conformations of Lymphotactin?

The following table presents a comprehensive comparison between the two structural conformations of human Lymphotactin:

This structural interconversion represents a complete rearrangement of tertiary contacts, resulting in two entirely different folds from the same amino acid sequence . The equilibrium between these states is influenced by solution conditions and can be shifted by specific mutations, particularly those affecting charged residues involved in glycosaminoglycan binding .

What are the key resources and reagents available for Lymphotactin research?

The following table summarizes key resources and reagents available for Lymphotactin research:

These resources provide researchers with the tools necessary to investigate Lymphotactin's unique structural properties and immunological functions across structural biology, protein biochemistry, and immunology fields.

What are the specifications for commercially available recombinant human Lymphotactin with His-tag?

The following table details the specifications for commercially available recombinant human Lymphotactin with His-tag:

This recombinant protein is suitable for a wide range of research applications, including structural studies, receptor binding assays, chemotaxis studies, and investigation of Lymphotactin's conformational dynamics.