Recombinant Schistosoma haematobium Tetraspanning orphan receptor (TOR)

What is the structural composition of Schistosoma haematobium TOR protein?

Schistosoma haematobium TOR is a tetraspanning protein characterized by four transmembrane domains. The complete amino acid sequence consists of 313 amino acids with the expression region at 1-313, encompassing the following sequence:

PQCESETNFHYDIPPGYKDDVLVDVNNMSPSLVSDTQKHERGSHEVKIKHFSPYIAVCVTTFSLAFCCFMVHAAITRQPTHLLPFFFIQVFDLIICLIHILGFMSSTSDIRLVIHTKTGPIYIKSTGLTFIILSISCMMLAFKAYCLGMVWDCYKYLMLNRRGNLDDWYSDQWGHLSTFWSLLRTGRNRGNNSIGNSGSPNEPNTRPRPDTITYDPANDLPKYEDILKIRNAYAPPPYYCSNTNGNVNTTTTDAVTTNTTITSATTANATTTITTNANTNTSTTTSVISPLTTTNKDDTQINNASSNAHSSC

The protein contains conserved regions typical of tetraspanin proteins, including hydrophobic transmembrane domains and extracellular loops that are critical for its function in host-parasite interactions. Based on structural analysis, researchers have identified specific regions that may be involved in immunomodulation and host receptor binding.

How does S. haematobium TOR differ from similar proteins in other Schistosoma species?

While S. haematobium TOR shares significant homology with tetraspanin orphan receptors from other Schistosoma species, such as S. japonicum (SjTOR), there are notable structural and functional differences. S. haematobium TOR (Sh-TOR) is also known by alternative names including Complement C2 receptor inhibitor tetraspanning and trispanning orphan receptor .

Comparative sequence analysis reveals species-specific variations in the extracellular domains, which likely contribute to different immunomodulatory capabilities. These variations may explain the distinct pathological manifestations associated with different Schistosoma infections. S. haematobium is specifically associated with urogenital schistosomiasis and has been linked to bladder cancer promotion and angiogenesis, whereas other species primarily affect intestinal systems .

What are the recommended protocols for expressing recombinant S. haematobium TOR?

The expression of functional recombinant S. haematobium TOR requires careful consideration of expression systems that maintain proper protein folding and post-translational modifications. Based on current methodologies, the following protocol is recommended:

• Gene synthesis or amplification: Optimize the codon usage for the selected expression system while maintaining the complete coding sequence (amino acids 1-313).

• Vector selection: Choose a vector that includes appropriate purification tags (His-tag or GST-tag) that can be later removed if necessary for functional studies.

• Expression system: Eukaryotic expression systems (particularly insect cells or mammalian cells) are preferable to prokaryotic systems to ensure proper folding of the transmembrane domains.

• Purification approach: Use a two-step purification process involving affinity chromatography followed by size exclusion chromatography to isolate properly folded protein.

• Storage: Store the purified protein in Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for long-term storage to maintain stability .

The expression and purification process must be carefully monitored to ensure that the recombinant protein maintains its native conformation, particularly in the transmembrane regions crucial for function.

What detection methods are most effective for S. haematobium TOR in experimental settings?

For reliable detection of S. haematobium TOR in experimental settings, several complementary approaches are recommended:

• Immunological detection:

  • Western blotting using polyclonal antibodies raised against specific epitopes of S. haematobium TOR

  • ELISA assays utilizing recombinant TOR as a standard for quantification

  • Immunofluorescence for localization studies in tissue samples

• Molecular detection:

  • RT-PCR for mRNA expression analysis

  • qPCR for quantitative assessment of gene expression levels

• Proteomic approaches:

  • Mass spectrometry for identification and characterization of TOR in complex protein mixtures

  • Protein arrays for high-throughput detection in multiple samples

The choice of detection method should be tailored to the specific research question, with consideration for sensitivity requirements and the complexity of the biological sample being analyzed.

How does S. haematobium TOR contribute to immune modulation during parasitic infection?

S. haematobium TOR plays a complex role in modulating host immune responses during infection. As a tetraspanning membrane protein, it is likely exposed on the parasite surface and interacts directly with host immune components. Current research suggests several mechanisms of action:

• Complement regulation: S. haematobium TOR may function as a complement C2 receptor inhibitor, as indicated by one of its alternative names . This function would protect the parasite from complement-mediated attack by inhibiting a key step in the complement cascade.

• T-cell response modulation: Similar to other Schistosoma antigens, TOR likely contributes to shifting the immune response toward a Th2-dominated profile. This immune deviation helps create an environment favorable for parasite survival by downregulating potentially harmful Th1 and Th17 responses .

• Regulatory T-cell induction: TOR may induce regulatory T cells that actively suppress immune responses against the parasite, contributing to chronic infection.

These immunomodulatory properties make S. haematobium TOR a promising candidate for therapeutic applications in autoimmune disorders, where downregulation of inflammatory responses would be beneficial.

What are the methodological challenges in studying TOR's role in S. haematobium-induced bladder cancer?

Investigating the relationship between S. haematobium TOR and bladder cancer development presents several methodological challenges:

• Appropriate model systems: Traditional rodent models may not fully recapitulate human bladder cancer progression following S. haematobium infection. Researchers should consider:

  • Humanized mouse models

  • 3D bladder organoid cultures

  • Patient-derived xenografts

• Temporal considerations: The long latency period between infection and cancer development necessitates extended study timelines or the identification of reliable early biomarkers.

• Molecular mechanisms: Determining whether TOR directly promotes carcinogenesis requires sophisticated approaches:

  • Conditional expression systems in bladder epithelial cells

  • CRISPR/Cas9-mediated manipulation of TOR expression

  • Proteomic analysis of TOR-interacting partners in bladder tissue

• Separating TOR effects from other S. haematobium factors: Since S. haematobium has been shown to promote bladder cancer cell proliferation and angiogenesis , isolating the specific contribution of TOR requires careful experimental design with appropriate controls.

Researchers must integrate multiple experimental approaches to establish causality between TOR expression and oncogenic transformation in bladder epithelium.

How can structure-function relationships in S. haematobium TOR be leveraged for therapeutic development?

Understanding the structure-function relationships of S. haematobium TOR provides opportunities for therapeutic development through the following methodological approaches:

• Epitope mapping: Identifying immunodominant epitopes within TOR that elicit protective immune responses versus those that induce immunomodulatory effects.

• Functional domain analysis: Determining which domains are responsible for specific activities:

  • Transmembrane domains for membrane insertion and stability

  • Extracellular loops for interaction with host receptors

  • Cytoplasmic domains for signaling functions

• Rational drug design: Using the amino acid sequence information to predict three-dimensional structures and identify potential binding pockets for small molecule inhibitors.

• Peptide-based therapeutics: Developing peptide mimetics based on functional regions of TOR that could either:

  • Block parasite-host interactions in schistosomiasis treatment

  • Harness immunomodulatory properties for autoimmune disease therapy

The complete amino acid sequence provided in the product information serves as the foundation for these structure-function analyses, enabling the design of targeted experimental approaches.

What is the current evidence for using S. haematobium TOR in autoimmune disease therapy?

The potential application of S. haematobium TOR in autoimmune disease therapy is supported by emerging evidence on the immunomodulatory properties of Schistosoma antigens:

• Mechanism of action: Schistosoma soluble egg antigens (SEAs) enhance Th2 immunity while alleviating outcomes of Th1 and Th17 responses , which are often pathogenic in autoimmune conditions. TOR likely contributes to this immunomodulation.

• Experimental evidence: Studies with related Schistosoma antigens have demonstrated efficacy in animal models of:

  • Multiple sclerosis

  • Inflammatory bowel disease

  • Rheumatoid arthritis

  • Type 1 diabetes

• Translational challenges:

  • Isolating the specific contributions of TOR versus other Schistosoma antigens

  • Ensuring consistent immunomodulatory effects across patient populations

  • Addressing safety concerns regarding potential oncogenic effects, given the association between S. haematobium and bladder cancer

• Delivery approaches:

  • Recombinant protein administration

  • Peptide-based formulations

  • Gene therapy approaches for localized expression

While promising, significant research is still needed to establish S. haematobium TOR as a viable therapeutic for autoimmune diseases, with careful consideration of both efficacy and safety profiles.

What are the optimal conditions for maintaining stability of recombinant S. haematobium TOR?

Maintaining stability of recombinant S. haematobium TOR requires specific storage and handling conditions:

Researchers should monitor protein stability through periodic quality control testing, such as SDS-PAGE and functional assays, especially when using stored protein for extended experimental series.

How can researchers accurately quantify the immunomodulatory effects of S. haematobium TOR?

Accurate quantification of TOR's immunomodulatory effects requires multifaceted experimental approaches:

• In vitro assays:

  • Cytokine profiling: Measure shifts in Th1/Th2/Th17 cytokine production using multiplex assays

  • Flow cytometry: Quantify changes in immune cell populations (Tregs, Th2, etc.)

  • Reporter cell lines: Develop systems that indicate activation of specific immune pathways

• Ex vivo approaches:

  • Peripheral blood mononuclear cell (PBMC) stimulation assays

  • Organ culture systems for tissue-specific responses

• In vivo models:

  • Humanized mouse models for human-relevant immune responses

  • Established autoimmune disease models to assess therapeutic potential

• Data analysis methods:

  • Multiparameter analysis to identify patterns of immune modulation

  • Systems biology approaches to understand network effects

  • Longitudinal studies to capture temporal dynamics of immune responses

These methodological approaches should be complemented by appropriate statistical analyses to ensure robust and reproducible quantification of immunomodulatory effects.

What are the most promising approaches for targeting S. haematobium TOR in vaccine development?

Vaccine development targeting S. haematobium TOR should consider several strategic approaches:

• Epitope selection strategies:

  • Identify conserved epitopes across Schistosoma species to develop broadly protective vaccines

  • Focus on epitopes that induce neutralizing antibodies rather than those that trigger immunomodulation

  • Use computational approaches to predict immunodominant epitopes based on the complete amino acid sequence

• Adjuvant selection:

  • Choose adjuvants that counteract TOR's natural immunomodulatory properties

  • Consider combination adjuvants that activate multiple pathways

• Delivery platforms:

  • DNA vaccines expressing optimized TOR sequences

  • mRNA-based approaches for enhanced protein expression

  • Viral vector systems for improved immunogenicity

• Combination approaches:

  • Include TOR with other Schistosoma antigens for synergistic protection

  • Target multiple life-cycle stages simultaneously

• Evaluation metrics:

  • Measure both antibody and cell-mediated responses

  • Assess reduction in worm burden and egg production

  • Evaluate prevention of pathology in challenge studies

The full amino acid sequence of S. haematobium TOR provides the foundation for these vaccine development efforts, allowing for rational design approaches based on structural predictions and epitope analyses.

How might genetic variation in S. haematobium TOR impact diagnostic and therapeutic applications?

Genetic variation in S. haematobium TOR across geographical regions and isolates presents both challenges and opportunities for research applications:

• Diagnostic implications:

  • Conserved regions should be targeted for broad-spectrum diagnostic assays

  • Variable regions may enable strain-specific identification

  • Multiplex assays targeting multiple epitopes can improve diagnostic reliability

• Therapeutic considerations:

  • Drug development should focus on conserved functional domains

  • Immunotherapies may need to address strain-specific variations

  • Population genetics studies should inform clinical trial design and implementation

• Methodological approaches to assess variation:

  • Next-generation sequencing of field isolates

  • Comparative genomics across geographical regions

  • Functional characterization of variant proteins

• Impact on experimental design:

  • Include diverse TOR variants in preclinical studies

  • Develop validation panels representing global diversity

  • Consider adaptive clinical trial designs that can accommodate genetic heterogeneity

Researchers should systematically catalog and characterize TOR genetic diversity to ensure robust diagnostic and therapeutic development that remains effective across diverse parasite populations.