Recombinant Cat Tumor necrosis factor ligand superfamily member 6 (FASLG), partial

What is the basic structure and function of feline FASLG?

Feline FASLG (Fas Ligand) is a type II transmembrane protein belonging to the tumor necrosis factor superfamily. Similar to its human and mouse homologs, feline FASLG typically consists of an extracellular domain (ECD), a transmembrane segment, and a cytoplasmic domain. The protein forms a homotrimer and binds to the Fas receptor (CD95) on target cells to trigger apoptosis.

When working with recombinant feline FASLG, researchers should be aware that:

• The protein naturally exists as a non-disulfide-linked homotrimer

• The molecular weight of the full-length protein is approximately 40 kDa

• The extracellular domain contains the receptor-binding region

• Sequence identity between feline FASLG and human FASLG is approximately 78-81%

For optimal characterization, techniques such as SDS-PAGE under non-reducing conditions, size exclusion chromatography, and analytical ultracentrifugation are recommended to confirm the trimeric state of the recombinant protein.

How does recombinant feline FASLG compare functionally to human and mouse FASLG?

Cross-species comparisons have demonstrated that both mouse and human FASLG can be active on cells from other species. Methods for functional comparison include:

• Apoptosis assays: Using cell lines sensitive to FASLG-induced apoptosis from different species

• Receptor binding studies: Comparing binding affinities to Fas receptors across species

• Intracellular signaling analysis: Examining downstream signaling pathways

Research has shown that within the extracellular domain, mouse FASLG shares 81% and 93% amino acid sequence identity with human and rat FASLG, respectively . This high level of conservation contributes to the cross-species reactivity often observed in functional assays.

For experimental validation, include both species-matched and cross-species controls when performing functional assays with recombinant feline FASLG to accurately assess comparative activity.

What are the optimal expression systems for producing recombinant feline FASLG?

The choice of expression system significantly impacts yield, functionality, and purity of recombinant feline FASLG. Research evidence suggests several viable approaches:

Mammalian Expression Systems:

• PEAK™ cells: Successfully used for expression of feline proteins with yields of at least 1 μg/ml

• HEK293T cells: Effective for producing soluble FASLG fusion proteins with proper folding

Bacterial Expression Systems:
Caution is warranted when considering bacterial expression, as evidence suggests toxicity issues. Research has shown that recombinant feline TNF (another TNF superfamily member) is toxic to E. coli, whereas equine and porcine TNF are better tolerated . The mechanism may involve:

• Alterations in protein folding

• Prevention of secretion of the feline protein

Growth curves demonstrated that E. coli cultures transformed with feline TNF reached peak densities at 3-4 hours and then decreased to near initial densities prior to recovery of growth . This pattern was observed in multiple E. coli strains (LL308 and JM101).

Recommended approach: Start with mammalian expression systems (particularly HEK293T cells) using vectors with strong promoters like CMV and include appropriate secretion signals and fusion tags to facilitate purification.

What purification strategies yield highest purity and bioactivity for recombinant feline FASLG?

Effective purification of recombinant feline FASLG typically involves multi-step processes:

• Affinity chromatography:

  • Fusion tags: Studies demonstrate one-step purification using anti-FLAG immunoaffinity column chromatography achieves high purity for feline glycoproteins

  • Receptor-based affinity: Immobilized Fas receptor can be used for functional protein selection

• Additional purification steps:

  • Size exclusion chromatography to separate trimeric from monomeric forms

  • Ion exchange chromatography for removing contaminating proteins

Critical considerations:

• Buffer composition significantly impacts stability and activity

• The presence of 10 mM reduced glutathione in elution buffer can help maintain proper folding

• Aliquoting and storing at -80°C prevents repeated freeze-thaw cycles that reduce activity

Bioactivity assessment:
For confirmation of activity, cross-link recombinant FASLG with antibodies against its tag. Published protocols show activity in the range of 0.3-1.5 ng/mL when cross-linked with 10 μg/mL of anti-polyHistidine monoclonal antibody .

How can researchers develop reliable bioassays to measure recombinant feline FASLG activity?

Development of robust bioassays for recombinant feline FASLG requires several considerations:

Cytotoxicity/apoptosis assays:

• Cell selection: Jurkat cells (particularly Rapo C2, I2.1, and I9.2 variants) have been extensively used for FASLG assays

• Cross-linking requirement: Studies show soluble FASLG requires cross-linking for optimal activity:

  • EC50 for human recombinant FASLG: 0.3-1.5 ng/mL (with 10 μg/mL cross-linking antibody)

  • EC50 for mouse recombinant FASLG: 1-8 ng/mL (with 2.5 μg/mL cross-linking antibody)

Apoptosis detection methods:

• Flow cytometry with Annexin V/PI staining

• Caspase activation assays (particularly caspase-3/8)

• Nuclear fragmentation quantification

• ROS measurement (a characteristic feature of FASLG-induced cell death)

Receptor binding assays:

• Displacement assays: Using labeled bait proteins to detect binding to cellular receptors

• Surface plasmon resonance (SPR): For determining binding kinetics and affinity constants

Assay validation guidelines:

• Include positive controls (commercial human/mouse FASLG)

• Include negative controls (inactive FASLG mutants)

• Demonstrate dose-dependency

• Confirm specificity by blocking with Fas-Fc fusion proteins

What are the key considerations for designing mutation studies in feline FASLG?

When designing mutation studies for feline FASLG, researchers should focus on functional domains and critical residues:

Critical domains for mutation analysis:

• Receptor binding region: Mutations in the binding interface between FASLG and Fas receptor

  • Key loops: AA' (residues 163-170), DE (215-221), and GH (269-274)

  • Specific residues: E163, D164, E270, E271 (equivalent human positions)

• Trimerization domain: Mutations affecting the stability of the trimer structure

• Cleavage sites: Mutations at metalloproteinase cleavage sites that generate soluble FASLG

Mutation types to consider:

• Single amino acid substitutions (e.g., E163A, D164A)

• Double mutations (e.g., ED163-164AA, EE270-271AA)

• Domain swapping (e.g., replacing segments with corresponding regions from other TNF family members)

Functional impact assessment:
Researchers have demonstrated that single-point mutations in FasL that interfere with PPCR (patch of positively charged residue epitope) engagement inhibited apoptotic signaling in tumor cells and T cells . Consider assessing:

• Receptor binding affinity

• Signaling capacity

• Apoptosis induction

• Trimer formation

Important model systems:
The ALPS (autoimmune lymphoproliferative syndrome) mutations provide natural models for FasL dysfunction. Research has revealed differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations .

How is recombinant feline FASLG being utilized in cancer immunotherapy research?

Recent research has highlighted the potential of FASLG in cancer immunotherapy, particularly in understanding tumor immune evasion mechanisms:

Key research applications:

• CAR-T cell bystander killing function:

  • Studies show Fas-mediated bystander killing is vital to the success of CAR-T therapies in tumors

  • The PPCR epitope in the cysteine-rich domain 2 of Fas is critical for agonist antibody targeting and CAR-T bystander function in cancer models

• Tumor immune escape mechanisms:

  • Tumor cells can exploit FASLG expression to kill tumor-infiltrating lymphocytes

  • Research has shown that blockade of Fas signaling in breast cancer cells suppresses tumor growth and metastasis by disrupting Fas signaling-initiated cancer-related inflammation

• Development of agonistic antibodies:

  • Targeting specific epitopes of Fas for cancer therapy

  • Structure-based design of next-generation immunotherapeutics capable of selectively targeting tumors

Methodological approaches:

• Use of recombinant FASLG to study receptor clustering and signaling in tumor cells

• Development of Fas receptor activation assays to screen potential therapeutic candidates

• Application of super-resolution microscopy to study Fas/CD95 reorganization induced by ligand binding

What role does recombinant feline FASLG play in studying inflammatory and autoimmune conditions?

Recombinant feline FASLG serves as a valuable tool in studying inflammatory and autoimmune conditions:

Neurological disease models:
Studies have examined whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and TRAIL. Research has shown that:

• After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain

• Recombinant CD95 ligand induced apoptosis in primary neurons and neuron-like cells in vitro

• In lpr mice (expressing dysfunctional CD95), reversible middle cerebral artery occlusion resulted in significantly smaller infarct volumes than in wild-type animals

Autoimmune models:

• The gld mice model, which has a FasL point mutation, develops severe lymphoproliferation and systemic autoimmunity

• ALPS (autoimmune lymphoproliferative syndrome) is directly attributed to homozygous mutations in FasL

Inflammatory response modulation:

• In the absence of TGF-beta, FasL/Fas interactions promote neutrophil-mediated inflammatory responses rather than apoptosis

• Fas Ligand-induced apoptosis plays a central role in the development of immune tolerance and maintenance of immune privileged sites

Experimental approaches:

• Using recombinant FASLG to induce controlled inflammation in cellular and animal models

• Studying the intersection of apoptotic and inflammatory pathways

• Developing targeted inhibitors of FASLG/Fas interactions for autoimmune therapy

What are the considerations for designing optimized recombinant feline FASLG constructs?

Designing optimized recombinant feline FASLG constructs requires careful consideration of several factors:

Domain selection and boundaries:

• Extracellular domain (ECD) selection: For mouse FASLG, the ECD corresponds to Gln101-Leu279

• Human FASLG: The functional domain corresponds to Pro134-Leu281

• Feline constructs should be designed with comparable boundaries based on sequence alignment

Fusion partners and tags:
Several tag options have been successfully utilized:

• Hemagglutinin tag (YPYDVPDYA): Used in mouse recombinant FASLG constructs

• Polyhistidine tag: Utilized for human FASLG with successful results

• Fc fusion: Effective for improved stability and half-life

• Trimerization domains: Addition of GCN4-IZ and (GGGS)3 linkers has proven effective

Expression optimization:

• Signal peptide selection: Optimized secretion signals for the expression system

• Codon optimization: Adjust codon usage for the intended expression host

Advanced design strategies:

• Yoked constructs: Single-chain designs linking multiple domains

  • Research demonstrates that yoked proteins can retain significant bioactivity while showing different receptor binding characteristics

• Cross-species chimeras: Replacing domains with human or mouse equivalents to study specific functions

• Stabilized variants: Engineering disulfide bonds or other stabilizing modifications to enhance shelf-life

How can advanced microscopy and imaging techniques be applied to study FASLG-mediated receptor clustering?

Advanced microscopy and imaging techniques have revolutionized our understanding of FASLG-mediated receptor clustering:

Super-resolution microscopy approaches:
Research has utilized super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction with FasL on planar lipid bilayers, revealing:

• Rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL

• FADD recruitment dependent on an intact Fas death domain

• Lipid raft association playing a secondary role in receptor clustering

Methodological considerations:

• Sample preparation:

  • Suitable cell lines: Rapo C2 (Fas-deficient), I2.1 (FADD-deficient), and I9.2 (caspase-8-deficient) Jurkat T cells

  • Transfection protocols optimized for minimal cellular stress

  • Appropriate fluorescent protein tagging (mCerulean3, mVenus)

• Imaging platforms:

  • PALM (Photoactivated Localization Microscopy)

  • Fluorescence correlation imaging

  • Flow-cytometric FRET analysis

• Data analysis:

  • Cluster size quantification

  • Colocalization analysis

  • Temporal dynamics assessment

Critical controls:

• ALPS mutants (A257D and E261K) as negative controls for clustering

• Point mutations in the death domain to assess recruitment specificity

• Constructs lacking preligand assembly domain (ΔPLAD) or death domain (ΔDD)

Future applications:
This methodology can be applied to feline FASLG to determine species-specific differences in:

• Clustering kinetics

• Threshold requirements for signal initiation

• Differential recruitment of signaling molecules

How does mRNA toxicity relate to FASLG function, and what are the implications for recombinant protein design?

Recent groundbreaking research has revealed unexpected aspects of FASLG mRNA toxicity:

Key findings:

• CD95L mRNA itself is toxic to cells even without prior conversion to small (s)RNAs

• When expressed, full-length CD95L mRNA is highly toxic to cells and induces a form of cell death similar to apoptosis

• Small RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC), which is required for the toxicity

• Processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha

Mechanistic insights:
The CD95L mRNA harbors sequences that when converted into small interfering (si) or short hairpin (sh)RNAs, cause toxicity in cancer cells by targeting a network of survival genes through RNA interference (RNAi), a process termed DISE (Death Induced by Survival gene Elimination) .

Experimental evidence:

• HeyA8 cells expressing CD95L constructs with mutations preventing protein translation (CD95L MUTNP) showed toxicity comparable to wild-type CD95L

• This effect persisted in CD95 knockout cells, confirming it was independent of CD95L-CD95 protein interaction

• Nuclear fragmentation and ROS production indicated cell death mechanisms similar to DISE

Implications for recombinant protein design:

• Consider codon optimization not only for expression efficiency but also to minimize potential toxic RNA sequences

• Design expression constructs with appropriate 5' and 3' UTRs to enhance stability and translation

• Include RNA stabilizing elements to prevent processing into toxic sRNAs

• Consider inducible expression systems for toxic constructs

What are the emerging interactions between coronaviruses and TNFSF pathways that might influence feline FASLG research?

Recent research has uncovered interesting connections between coronaviruses and TNF superfamily pathways:

Feline infectious peritonitis (FIP) and coronavirus recombination:
A novel, highly pathogenic FCoV-CCoV recombinant has emerged, responsible for a rapidly spreading outbreak of feline infectious peritonitis:

• The recombination spans the spike protein region, showing 97% sequence identity to pantropic canine coronavirus CB/05

• This recombination has resulted in altered cell tropism and increased pathogenicity

• The outbreak shows evidence of direct cat-to-cat transmission

Potential interactions with TNF superfamily pathways:
While direct interactions between FCoV and FASLG have not been extensively studied, several research directions emerge:

• How coronavirus infection modulates apoptotic pathways involving FASLG/Fas

• Whether inflammatory responses in FIP involve dysregulation of death ligand expression

• Potential therapeutic targeting of TNFSF pathways in coronavirus infections

Future research directions:

• Investigation of FASLG/Fas expression patterns in tissues affected by FCoV

• Examination of how coronavirus infection alters susceptibility to FASLG-induced apoptosis

• Development of therapeutic strategies targeting TNFSF pathways to modulate inflammatory responses in coronavirus infections

Methodological approaches:

• Single-cell RNA sequencing to map expression changes during infection

• Proteomic analysis of receptor-ligand interactions

• Functional assays to assess apoptotic pathway sensitivity during infection