Recombinant Pan troglodytes Lymphotoxin-beta (LTB), partial

What is the molecular structure of Pan troglodytes LTB and how does it compare to human LTB?

Pan troglodytes LTB is a member of the tumor necrosis factor (TNF) superfamily with high homology to human LTB. The protein's expression region typically spans amino acids 49-244, with an accession number of Q862Z7 . When comparing chimpanzee LTB to human LTB, researchers should note that the amino acid sequence shows extremely high conservation, reflecting the close evolutionary relationship between these species. The protein functions as a type II membrane protein that forms heterotrimers with Lymphotoxin-alpha, with the predominant form being the lymphotoxin-alpha 1/beta 2 complex .

For structural studies, the protein is frequently expressed with an N-terminal 6xHis-SUMO tag with a theoretical molecular weight of approximately 36.8 kDa in its tagged form . Researchers should consider this tag when designing experiments that may be affected by terminal modifications.

What are the functional similarities and differences between LTB from Pan troglodytes and other species?

Functionally, Pan troglodytes LTB shares significant similarity with human LTB, serving as an inducer of inflammatory response systems and playing roles in the development of lymphoid tissue . The functional conservation extends to the formation of heterotrimers with Lymphotoxin-alpha, where the predominant surface form is the lymphotoxin-alpha 1/beta 2 complex that acts as the primary ligand for the lymphotoxin-beta receptor .

When conducting comparative studies, researchers should be aware that while mouse and rat LTB share approximately 73% amino acid sequence identity with human LTB within common regions of their extracellular domains, these rodent orthologs contain significant insertions (66 aa in mouse, 65 aa in rat) within the extracellular domain that are not present in primate LTBs . This structural difference may impact cross-species experimental design and interpretation.

What expression systems are optimal for producing recombinant Pan troglodytes LTB, and how do they affect protein quality?

When selecting an expression system, researchers should consider the downstream applications and whether post-translational modifications are critical for their experimental questions .

What are the optimal storage and handling conditions for maintaining recombinant Pan troglodytes LTB activity?

Based on manufacturer recommendations, recombinant Pan troglodytes LTB should be stored using the following guidelines:

• The lyophilized protein should be reconstituted at approximately 500 μg/mL in sterile PBS .

• For long-term storage, aliquot the reconstituted protein and store at -20°C to -80°C in a manual defrost freezer .

• Avoid repeated freeze-thaw cycles as they can significantly reduce protein activity .

• Some preparations may include carrier proteins like BSA to enhance stability, but carrier-free versions are available when the presence of BSA would interfere with experiments .

• For formulations containing glycerol (e.g., 50% glycerol in Tris-based buffer), storage conditions may vary but generally allow greater stability at -20°C .

Researchers should empirically validate activity retention in their specific experimental systems, particularly for functional assays requiring specific protein-protein interactions.

How can recombinant Pan troglodytes LTB be effectively used in comparative immunology studies?

Recombinant Pan troglodytes LTB serves as a valuable tool for comparative immunology between humans and non-human primates. When designing such studies:

• Consider using both human and chimpanzee LTB in parallel experiments to directly compare functional properties, receptor binding, and downstream signaling pathways.

• For receptor binding studies, note that LTB forms heterotrimers with LT-alpha, and these complexes bind to specific receptors. The LT-alpha 1/beta 2 heterotrimer specifically binds and activates the Lymphotoxin beta R/TNFRSF3 (LT beta R) .

• In inflammation models, Pan troglodytes LTB can be used to investigate evolutionary conservation of inflammatory pathways between species. The protein functions as an inducer of inflammatory response systems .

• When studying lymphoid tissue development, comparative analysis can reveal subtle species-specific differences in signaling efficiency or downstream pathway activation.

• For interaction studies with other immune components, researchers should account for the possibility of species-specific interaction partners that may have diverged between humans and chimpanzees.

Researchers should verify cross-reactivity with antibodies and other detection reagents when using both human and chimpanzee proteins in the same experimental system.

What experimental controls are critical when using recombinant Pan troglodytes LTB in functional assays?

When designing functional assays using recombinant Pan troglodytes LTB, researchers should implement the following controls:

• Negative Controls:

  • Tagged protein control: Use an irrelevant protein with the same tag (e.g., 6xHis-SUMO) to control for tag-specific effects .

  • Heat-inactivated LTB: To distinguish between specific functional effects and non-specific protein effects.

  • Buffer-only control: To account for buffer components' effects.

• Positive Controls:

  • Human recombinant LTB: When available, to benchmark expected activity levels and compare functional conservation .

  • Known LTB-responsive cell lines or systems with validated readouts.

• Specificity Controls:

  • Blocking antibodies against LTB or its receptor to confirm the specificity of observed effects.

  • Competitive binding assays with unlabeled protein to verify specific interactions.

• Validation Approaches:

  • Dose-response experiments to establish appropriate working concentrations.

  • Time-course studies to determine optimal incubation periods for detecting biological responses.

Each experimental system may require additional specific controls depending on the assay format and research question.

How can heterotrimeric complexes of Pan troglodytes LTB with LT-alpha be effectively reconstituted for functional studies?

Reconstituting functional LT-alpha/LTB heterotrimers represents a significant technical challenge. Based on available research methodologies:

• Co-expression Strategy:

  • The most effective approach involves co-expressing LT-alpha and LTB in a suitable expression system.

  • For the LT-alpha 1/beta 2 heterotrimer, researchers can use a construct design similar to that described for human proteins: "rhLT alpha (Leu35-Leu205) Accession # P01374|GGGGS|rhLT beta (Gln49-Gly244) Accession # Q06643|GGGGS|rhLT beta (Gln49-Gly244) Accession # Q06643" .

  • This linked construct ensures the correct 1:2 stoichiometry of alpha to beta subunits.

• In vitro Assembly Protocol:

  • If separately expressed proteins must be used, researchers should:
    a. Combine purified LT-alpha and LTB at a 1:2 molar ratio
    b. Incubate under mild refolding conditions (typically involving gradual dialysis)
    c. Confirm complex formation via size exclusion chromatography and/or native PAGE

• Activity Verification:

  • Test binding to recombinant LT beta R using surface plasmon resonance (SPR) or ELISA-based methods

  • Verify biological activity in cell-based assays showing activation of NF-κB signaling pathways

  • Dose-dependent induction of inflammatory responses in appropriate cell types, with EC50 values typically in the range of 3-15 ng/mL for properly formed heterotrimers

Researchers should note that the biological potency of reconstituted heterotrimers can vary significantly based on the reconstitution method and should be empirically validated for each preparation.

What are the current methodologies for investigating the role of Pan troglodytes LTB in lymphoid tissue development and homeostasis?

Advanced research into the role of LTB in lymphoid tissue development employs several sophisticated approaches:

• Ex vivo Organ Culture Systems:

  • Lymph node anlagen or splenic tissue sections can be cultured with recombinant LTB to assess its direct effects on stromal cell networks and lymphoid organization.

  • These systems allow for the assessment of species-specific differences in developmental signaling.

• 3D Organoid Models:

  • Researchers are increasingly developing organoid systems incorporating lymphoid stromal cells and hematopoietic components.

  • These models can be used to test the specific contribution of LTB to lymphoid tissue architecture when supplemented with recombinant LTB or when LTB signaling is blocked.

• Pathway Analysis Techniques:

  • Phospho-flow cytometry to quantify activation of downstream signaling components (particularly NF-κB pathway members).

  • Single-cell RNA sequencing to identify LTB-responsive cell populations and characterize transcriptional programs initiated by LTB signaling.

  • ChIP-seq approaches to map LTB-dependent changes in chromatin accessibility and transcription factor binding.

• Comparative Cross-Species Approaches:

  • Using both human and Pan troglodytes LTB on cells from both species to identify potential species-specific signaling differences.

  • Combining with pathway inhibitors to dissect conservation of downstream signaling networks.

Researchers investigating these questions should consider the important cross-talk between LTB signaling and other developmental pathways, particularly in the context of emerging research showing connections to KDM6B-driven epigenetic reprogramming associated with lymphoid stromal cell commitment .

What are common issues encountered when using recombinant Pan troglodytes LTB in cellular assays, and how can they be addressed?

Researchers commonly encounter several technical challenges when working with recombinant LTB in cellular systems:

• Low Biological Activity:

  • Potential causes: Protein misfolding, aggregation, or degradation during storage/handling.

  • Solutions:
    a. Verify protein integrity via SDS-PAGE before use
    b. Optimize reconstitution protocols (buffer composition, protein concentration)
    c. Include protein stabilizers (e.g., BSA) for dilute working solutions
    d. Consider alternative expression systems if E. coli-expressed protein shows limited activity

• High Background in Control Samples:

  • Potential causes: Endotoxin contamination, non-specific effects of tags or buffer components.

  • Solutions:
    a. Use endotoxin-tested preparations or perform additional purification
    b. Include appropriate tag-only control proteins
    c. Test multiple buffer formulations for reconstitution
    d. Implement more stringent washing protocols in binding assays

• Inconsistent Results Between Experiments:

  • Potential causes: Protein instability, variation in cell responsiveness.

  • Solutions:
    a. Prepare single-use aliquots to avoid freeze-thaw cycles
    b. Standardize cell culture conditions, including passage number and seeding density
    c. Develop internal standard curves with each experiment
    d. Consider using stable reporter cell lines for functional readouts

• Difficulties in Detecting Protein-Protein Interactions:

  • Potential causes: Insufficient protein concentration, improper buffer conditions, interfering tags.

  • Solutions:
    a. Titrate protein concentrations over a wide range
    b. Optimize buffer conditions (pH, salt concentration, detergents)
    c. Consider tag removal if steric hindrance is suspected
    d. Try alternative detection methods (e.g., SPR vs. co-IP)

Each of these challenges requires empirical optimization within the specific experimental context.

How can researchers distinguish between effects mediated by Pan troglodytes LTB alone versus those requiring heterotrimer formation with LT-alpha?

Distinguishing between the biological effects of LTB alone versus LT-alpha/LTB heterotrimers represents a significant challenge in experimental design. Researchers can employ the following strategies:

• Receptor-Specific Approaches:

  • LT-alpha alone (as a homotrimer) binds to TNFR1, TNFR2, HVEM, and Troy, while LT-alpha/LTB heterotrimers specifically bind to LTβR .

  • Use receptor-specific blocking antibodies to differentiate which receptor pathway mediates observed effects.

  • Employ cells with genetic knockout or knockdown of specific receptors.

• Structural Variants:

  • Utilize LTB isoform b, which is unable to complex with LT-alpha, as a control to identify LTB-specific functions independent of LT-alpha .

  • Compare effects of purified LT-alpha homotrimers, engineered LT-alpha/LTB heterotrimers, and LTB alone.

• Biochemical Verification:

  • Always verify the oligomeric state of the protein preparation using size exclusion chromatography or native PAGE before conducting functional assays.

  • For LT-alpha/LTB heterotrimers, confirm the expected 1:2 stoichiometry.

• Signaling Pathway Analysis:

  • Different downstream signaling cascades are activated by different receptor engagements.

  • Analyze pathway-specific markers (e.g., differential NF-κB subunit activation or MAPK pathway components) to infer which receptor-ligand interaction is responsible for observed effects.

• Comparative Analysis with Human System:

  • Use established human LT system components as references, where the signaling pathways are better characterized .

  • This comparison can help identify conserved versus divergent signaling mechanisms.

These approaches, often used in combination, can provide compelling evidence for distinguishing the specific contributions of different LTB-containing complexes.

What emerging applications of recombinant Pan troglodytes LTB show promise for advancing comparative immunology?

Several cutting-edge research directions utilizing recombinant Pan troglodytes LTB are currently expanding our understanding of primate immunology:

• Evolutionary Immunology:

  • Comparing binding kinetics and signaling potency of LTB between humans and non-human primates to identify subtle evolutionary adaptations in immune system regulation.

  • Investigating species-specific differences in receptor-ligand interfaces that might reflect pathogen-driven selection pressures.

• Comparative Immune System Development:

  • Using both human and chimpanzee LTB in developmental models to elucidate conserved versus species-specific aspects of lymphoid tissue organization.

  • Exploring the role of LTB in tissue-specific immune cell homing mechanisms across primate species.

• Disease Modeling:

  • Using comparative LTB signaling to understand differential susceptibility to inflammatory conditions between humans and chimpanzees.

  • Investigating species-specific responses to pathogens that interact with the LTB signaling axis.

• Single-Cell Omics Integration:

  • Applying single-cell transcriptomics and proteomics to map species-specific cellular responses to LTB stimulation.

  • Identifying cell populations with divergent responsiveness to LTB signaling between humans and chimpanzees.

• Epigenetic Regulation:

  • Recent research suggests connections between LTB signaling and KDM6B-driven epigenetic reprogramming in lymphoid stromal cells .

  • Comparative studies may reveal species-specific epigenetic responses to LTB stimulation.

These emerging research directions highlight the continuing importance of recombinant Pan troglodytes LTB as a tool for understanding both conserved immune mechanisms and species-specific adaptations.

How might recombinant Pan troglodytes LTB contribute to understanding the evolution of immune system regulation in primates?

Recombinant Pan troglodytes LTB represents a valuable tool for investigating evolutionary aspects of immune regulation in primates:

• Molecular Evolution Analysis:

  • Comparing the fine molecular interactions of LTB with its receptors across primate species can reveal selective pressures that have shaped immune signaling.

  • Researchers can use recombinant proteins from multiple primate species in cross-binding experiments to create interaction matrices identifying species-specific binding preferences.

• Signaling Network Conservation:

  • Mapping downstream signaling cascades activated by LTB across primates can identify both conserved "core" pathways and species-specific regulatory adaptations.

  • This approach may reveal how different primates have evolved to fine-tune inflammatory responses through modifications to the LTB signaling axis.

• Developmental Immunology:

  • LTB plays crucial roles in lymphoid tissue development and organization .

  • Comparative studies using recombinant LTB from different primates can illuminate how evolutionary changes in this signaling pathway might contribute to species-specific immune architecture.

• Pathogen Response Specialization:

  • Different primate species have co-evolved with distinct pathogen repertoires.

  • Studying how LTB-mediated responses differ between species may provide insights into host-pathogen co-evolution.

• Methodological Framework:

  • For rigorous evolutionary studies, researchers should:
    a. Use recombinant proteins from multiple primate species (not just human and chimpanzee)
    b. Include appropriate phylogenetic controls in experimental design
    c. Employ both biochemical and cellular readouts to capture different aspects of functional conservation
    d. Consider the influence of other interacting components that may have co-evolved

By systematically applying these approaches, researchers can contribute to our understanding of how natural selection has shaped primate immune systems through modifications to conserved signaling pathways like the LTB axis.