Recombinant Rat Lymphotactin protein (Xcl1) (Active)

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

Rat Lymphotactin (XCL1) is a C-motif chemokine that uniquely possesses only a single disulfide bond, distinguishing it from all other chemokines which typically contain multiple disulfide bonds . This structural characteristic enables XCL1 to access two completely different native state structures, making it a "metamorphic" protein. The full-length mature protein spans amino acids 22-114 with a molecular weight of approximately 10.0 kDa . As a member of the C class of chemokines, XCL1 functions primarily through binding to its receptor XCR1 and plays crucial roles in immune cell chemotaxis and signaling pathways.

How does the structural dynamics of XCL1 relate to its function?

Unlike conventional proteins with a single stable conformation, XCL1 demonstrates remarkable conformational heterogeneity, interconverting between two entirely different native states at a rate of approximately 1/s . These include:

• The chemokine-like fold: Functions as an XCR1 agonist but lacks high-affinity glycosaminoglycan (GAG) binding capacity

• The alternative dimeric structure: Binds GAGs with high affinity but cannot activate XCR1

This structural interconversion is essential for XCL1's biological activity in vivo, as each conformation performs distinct but complementary functions . Mutation studies suggest that charge repulsion between specific arginine residues (R23 and R43) may destabilize the chemokine fold and promote conversion to the novel dimeric form, while binding of anions like chloride can stabilize the chemokine conformation .

What expression systems are used for producing Recombinant Rat XCL1 and how does this affect functionality?

Recombinant Rat XCL1 is typically produced in E. coli expression systems . While mammalian expression systems might offer post-translational modifications, bacterial expression provides high yields of the core protein structure. For XCL1 specifically, E. coli-expressed protein maintains full biological activity when properly folded, as demonstrated by functional assays showing ED50 values of less than 100 ng/ml in chemotaxis bioassays using human XCR1-transfected murine BaF3 cells .

For researchers studying XCL1's structural dynamics, it's worth noting that bacterial expression can sometimes benefit structural studies by eliminating heterogeneity from variable glycosylation, allowing clearer assessment of the protein's intrinsic conformational properties .

Purity Assessment:

• SDS-PAGE analysis: The standard method showing >97% purity for high-quality preparations

• HPLC: For more sensitive purity assessment and detection of minor contaminants

Activity Assessment:

• Chemotaxis bioassay: Using human XCR1-transfected murine BaF3 cells, with active XCL1 showing ED50 < 100 ng/ml

• Calcium flux assays: Measuring intracellular calcium mobilization upon XCR1 activation

• Reporter gene assays: Similar to those used for other chemokines, measuring downstream signaling activation

Endotoxin Testing:

• LAL method: Ensuring preparations contain less than 1.0 EU/μg endotoxin

It's critical to verify both structural integrity and functional activity, especially given XCL1's conformational plasticity, which directly impacts its receptor activation properties.

Storage Recommendations:

• Store lyophilized protein at -20°C/-80°C for up to 12 months

• For reconstituted protein, store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this can compromise protein integrity and activity

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to collect all material at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Consider adding glycerol to a final concentration of 5-50% for long-term storage (50% is often recommended)

• Prepare small working aliquots to minimize freeze-thaw cycles

For experiments investigating XCL1's conformational dynamics, buffer conditions are particularly important, as anions like chloride can influence the conformational equilibrium between the two native states .

Buffer Composition:

• Consider that buffer ions (particularly chloride) may affect XCL1's conformational equilibrium

• pH can significantly impact structural stability - standard assays typically use PBS at pH 7.4

Concentration Range:

• For biological assays, start with concentrations based on the known ED50 (<100 ng/ml for chemotaxis assays)

• Use concentration gradients to establish dose-response relationships

Time Course:

• When studying cellular uptake, consider that efficient uptake by PBMCs has been demonstrated within 24-40 hours of in vitro culture

• For structural studies, remember that XCL1 conformational interconversion occurs at ~1/s

Controls:

• Include appropriate negative controls (buffer only, inactive protein variants)

• Consider using mutants that preferentially adopt one conformation to distinguish conformation-specific functions

What is known about the molecular basis of XCL1-XCR1 interaction?

The XCL1-XCR1 signaling axis involves specific molecular interactions that have been characterized through structural and functional studies. Key insights include:

• XCR1 is the only known G protein-coupled receptor (GPCR) for XCL1

• The chemokine-like conformation of XCL1 functions as an XCR1 agonist, while the alternative dimeric structure does not

• The N-terminal segment of XCL1 is critical for XCR1 activation

• High-resolution cryoelectron microscopy studies have revealed that XCL1 binding induces structural alterations in key residues at the bottom of the XCL1 binding pocket in XCR1

• The unique arrangement within the XCL1 binding site confers specificity for XCL1 in XCR1

This molecular understanding provides a foundation for designing optimized XCR1 modulators that could potentially enhance XCR1 signaling in specific contexts, such as the tumor microenvironment, to promote anti-tumor immunity .

What cell types express XCR1 and how does this influence experimental design?

XCR1 expression has been identified in various cell types, which should inform experimental design:

Immune Cells:

• Primarily expressed in conventional dendritic cells subtype 1 (cDC1s)

• Initially thought to be expressed in various immune cells, but more recent work indicates selective expression in dendritic cell subsets

Nervous System:

• Expressed in the peripheral and central nervous systems

• Present in nerve fibers, CD45-positive leucocytes, and Schwann cells in injured nerve

• In the trigeminal nucleus caudalis (Vc), XCR1 labeling is consistent with expression in terminals of Aδ- and C-fiber afferents and excitatory interneurons

Other Systems:

• Upregulated in disease conditions such as rheumatoid arthritis

• Present in oral mucosal endothelial cells in oral cancer

When designing experiments, researchers should consider:

• Cell-type specific responses to XCL1

• Potential upregulation of XCR1 in pathological conditions

• Appropriate cellular models that recapitulate the XCR1 expression patterns relevant to the research question

How can researchers investigate the dual conformational states of XCL1 in experimental settings?

Investigating XCL1's unique conformational dynamics requires specialized approaches:

Biophysical Methods:

• NMR spectroscopy: The gold standard for resolving and characterizing the two conformational species in solution

• Heparin affinity chromatography: Can be used to separate the two conformational species based on their differential GAG-binding properties

• Time-resolved fluorescence: Useful for tracking the kinetics of conformational interconversion

Mutagenesis Approaches:

• Generate variants with mutations at residues involved in the conformational equilibrium (e.g., R23, R43)

• Create lymphotactin variants that preferentially adopt one conformation over the other

Functional Discrimination:

• Assess XCR1 activation (chemotaxis, calcium flux) to detect the chemokine-like conformation

• Measure GAG binding to detect the alternative dimeric conformation

A combined approach using these methods allows researchers to correlate structural states with specific functions and understand how environmental factors modulate the conformational equilibrium.

What is the emerging role of XCL1-XCR1 signaling in the nervous system?

Recent research has revealed previously unknown roles for XCL1-XCR1 signaling in the nervous system:

Expression Patterns:

• XCR1 is expressed in both peripheral and central nervous systems

• Expression is upregulated following nerve injury, particularly evident 3 days after chronic constriction injury (CCI)

• Co-expression with neuronal marker PGP9.5, leukocyte common antigen CD45, and Schwann cell marker S-100 in the mental nerve

• In the trigeminal root and brainstem white matter, XCR1-positive cells co-express the oligodendrocyte marker Olig2

Functional Effects:

• XCL1 increases neuronal excitability in the trigeminal nucleus caudalis (Vc), a pain-processing region of the CNS

• Activates intracellular signaling pathways in Vc

• May play a significant role in trigeminal pain pathways

This emerging evidence suggests that the XCL1-XCR1 signaling axis may be a novel target for pain management strategies, particularly in neuropathic pain conditions affecting the trigeminal system. Researchers investigating neuroimmune interactions should consider incorporating XCL1-XCR1 signaling in their experimental paradigms.

How does XCL1-XCR1 signaling contribute to cDC1 function in anti-tumor immunity?

The XCL1-XCR1 axis plays a crucial role in conventional type 1 dendritic cell (cDC1) function, particularly in the context of anti-tumor immunity:

Functional Significance:

• XCR1 serves as a marker for cDC1s

• The XCL1-XCR1 axis is crucial for cDC1 cross-presentation to activate cytotoxic T cells and natural killer (NK) cells against viral infection and tumors

• XCL1 signaling enhances the antigen presentation function of cDC1s

Therapeutic Potential:

• Modulating XCR1 signaling in cDC1s offers novel opportunities in cancer immunotherapy

• Enhanced XCR1 signaling may potentiate anti-tumor immunity in the tumor microenvironment

• XCR1-targeted therapeutics could be developed based on structural insights into XCL1-XCR1 interaction

Researchers investigating cancer immunotherapy should consider exploring the potential of targeting the XCL1-XCR1 axis to enhance cDC1-mediated anti-tumor responses. This could involve developing XCR1 agonists that mimic the active conformation of XCL1 or strategies to increase local XCL1 production in the tumor microenvironment.

Conformational Heterogeneity:

Challenge: XCL1's ability to adopt two different conformations can complicate interpretation of experimental results.
Solution: Use conformation-specific assays (XCR1 activation vs. GAG binding) to determine which conformation is responsible for observed effects. Consider buffer conditions that favor one conformation over the other.

Protein Stability:

Challenge: Maintaining XCL1 activity during storage and experimentation.
Solution: Adhere strictly to recommended storage conditions, avoid repeated freeze-thaw cycles, and use freshly reconstituted protein for critical experiments. Working aliquots should be stored at 4°C for no more than one week .

Species Specificity:

Challenge: Potential differences between rat, mouse, and human XCL1.
Solution: When designing experiments, consider species compatibility. While conserved in many aspects, there may be species-specific differences in receptor binding affinity or signaling outcomes.

Endotoxin Contamination:

Challenge: Bacterial expression systems can introduce endotoxin, affecting immune cell experiments.
Solution: Use endotoxin-tested preparations (<1.0 EU/μg) and include appropriate controls to rule out endotoxin effects .

What methodological approaches can be used to study XCL1-induced signaling pathways?

Researchers investigating XCL1-induced signaling can employ several complementary approaches:

Cellular Assays:

• Calcium flux assays: Measure intracellular calcium mobilization upon XCR1 activation

• Migration/chemotaxis assays: Quantify directional cell movement in response to XCL1 gradients

• Reporter gene assays: Monitor activation of downstream signaling pathways

Biochemical and Molecular Techniques:

• Phosphorylation studies: Assess activation of kinases downstream of XCR1

• Co-immunoprecipitation: Identify protein interaction partners

• RNAseq/transcriptomics: Characterize global changes in gene expression profiles following XCL1 stimulation

Neuronal Activity Assessment:

• Electrophysiological recordings: Measure changes in neuronal excitability in response to XCL1, as demonstrated in trigeminal nucleus caudalis neurons

• Calcium imaging: Visualize neuronal activation patterns

In Vivo Approaches:

• XCR1 knockout models: Determine the physiological relevance of XCL1-XCR1 signaling

• Pain behavior assessments: Evaluate potential analgesic effects of targeting XCL1-XCR1 signaling in neuropathic pain models