Snaclec rhodocytin subunit alpha Antibody

What is Snaclec rhodocytin and what role does the alpha subunit play in its function?

Snaclec rhodocytin is a C-type lectin-like protein found in the venom of the Malayan pit viper (Calloselasma rhodostoma). It exists as a non-disulfide-linked (αβ)₂ tetramer composed of alpha and beta subunits. The protein functions primarily as a potent inducer of platelet aggregation by binding to the C-type lectin domain family 1 member B (CLEC1B/CLEC2) on platelets.

The alpha subunit plays a critical role in the interaction with CLEC-2, with Asp4 in the α-subunit being specifically required for CLEC-2 binding . Molecular analysis indicates that during rhodocytin-CLEC-2 interaction, the alpha subunit forms five hydrogen bonds and three salt bridges with dimeric CLEC-2, demonstrating its essential role in receptor recognition and binding . The beta subunit forms fewer interactions (two hydrogen bonds and one salt bridge) .

How does the rhodocytin-CLEC-2 interaction trigger platelet activation?

The binding of rhodocytin to CLEC-2 initiates a signaling cascade that results in platelet activation and aggregation through the following mechanism:

• Rhodocytin tetramers bind to CLEC-2 dimers on platelet surfaces

• Binding leads to tyrosine phosphorylation in the cytoplasmic domain tail of CLEC-2

• This promotes the binding of spleen tyrosine kinase (Syk)

• Syk binding leads to subsequent activation of PLC-gamma-2

• This activation cascade ultimately results in platelet activation and aggregation

The interaction may promote clustering of CLEC-2 on the platelet surface, which plays a key role in triggering signaling . The binding affinity of rhodocytin with monomeric CLEC-2 has been measured at 1.01 ± 0.20 μM using surface plasmon resonance .

What are the validated applications for Snaclec rhodocytin subunit alpha Antibody?

Based on the search results, the Snaclec rhodocytin subunit alpha Antibody has been validated for the following applications:

When performing Western blotting, researchers have detected a band at approximately 31 kDa (both predicted and observed band size) . The antibody shows high specificity for the Snaclec rhodocytin subunit alpha, making it valuable for studies focusing on this specific component of the venom protein.

How can I design experiments to study the interaction between rhodocytin and CLEC-2?

To study the rhodocytin-CLEC-2 interaction, researchers can employ several methodological approaches:

• NFAT luciferase reporter assay: Transfect cells (e.g., DT40) with CLEC-2-eGFP and NFAT-luciferase reporter. Stimulate with rhodocytin (30 nM) or other compounds of interest and measure luciferase activity to assess downstream signaling .

• Surface plasmon resonance (Biacore technology): Measure binding affinity between rhodocytin and CLEC-2 (monomeric or dimeric). Previous studies have determined the affinity to be 1.01 ± 0.20 μM for rhodocytin with monomeric CLEC-2 .

• Molecular interfaces analysis: Use computational methods to analyze hydrogen bonds, salt bridges, and van der Waals forces in the rhodocytin-CLEC-2 complex .

• Platelet aggregation assays: Use light transmission aggregometry to measure the induction or inhibition of platelet aggregation by rhodocytin and its variants .

• Flow cytometry: Analyze binding of rhodocytin to CLEC-2 on cell surfaces .

What are the optimal storage and handling conditions for Snaclec rhodocytin subunit alpha Antibody?

The Snaclec rhodocytin subunit alpha Antibody is typically supplied as a liquid with specific buffer components. For optimal performance:

Storage buffer composition:

• Preservative: 0.03% Proclin 300

• Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

Storage recommendations:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles

• If working with the antibody regularly, aliquot to minimize freeze-thaw cycles

• Maintain sterile conditions when handling

The antibody is typically purified using Protein G purification methods and has a purity of >95% . Being a polyclonal antibody raised in rabbits (IgG isotype), it may show some lot-to-lot variation, so validation with positive controls is recommended for each new lot.

How can I validate the specificity of the Snaclec rhodocytin subunit alpha Antibody in my experimental system?

To validate antibody specificity:

• Western blot analysis with recombinant protein: Use recombinant Calloselasma rhodostoma Snaclec rhodocytin subunit alpha protein as a positive control. The antibody should detect a band at approximately 31 kDa .

• Peptide competition assay: Pre-incubate the antibody with excess recombinant rhodocytin subunit alpha (1-136AA) peptide before application to your experimental system. Loss of signal indicates specificity.

• Negative controls: Test the antibody against samples known not to express the target protein.

• Cross-reactivity testing: If working with samples from different snake species, test the antibody against venom samples from related and unrelated species to determine cross-reactivity profiles.

• SMA-PAGE analysis: For membrane protein complexes, SMA-PAGE (styrene-maleic acid copolymer-polyacrylamide gel electrophoresis) can be used to assess antibody specificity under native-like conditions .

How can rhodocytin and its antibodies be used to study CLEC-2-mediated cellular signaling beyond platelets?

While CLEC-2 is well-studied in platelets, it also plays roles in other cell types. Research approaches include:

• Leukocyte studies: Investigate CLEC-2 signaling in monocytes and granulocytes using rhodocytin as a specific ligand. The downstream signaling shows similarities between platelets and these cells .

• Endothelial cell interaction: Examine the effects of rhodocytin on endothelial cells to understand mechanisms of vascular permeability and inflammation associated with snake envenomation .

• Tumor cell research: Study the role of CLEC-2 in tumor progression and metastasis. The interaction between CLEC-2 and podoplanin (expressed on some tumor cells) is involved in tumor cell-induced platelet aggregation and metastasis .

• Comparative signaling analysis: Compare downstream signaling pathways activated by rhodocytin versus endogenous CLEC-2 ligands like podoplanin to identify shared and unique signaling components.

How can mutational studies of rhodocytin inform the development of CLEC-2 targeting therapeutics?

Mutational studies have revealed:

• Critical binding residues: Asp4 in the α-subunit of rhodocytin is required for binding to CLEC-2 . Mutations at this position can create variants that either enhance or abolish CLEC-2 binding.

• Development of inhibitory mutants: Certain rhodocytin mutants have been shown to block CLEC-2/podoplanin-dependent platelet aggregation and may inhibit tumor metastasis . These mutants could serve as templates for developing targeted therapeutics.

• Structure-function relationships: Understanding which residues are essential for CLEC-2 binding allows for rational design of small molecule inhibitors or peptide mimetics that could function as antiplatelet or antimetastatic agents.

• Multimeric structure importance: Recombinant wild-type rhodocytin forms a heterooctamer with four α- and β-subunits . This multimeric structure may be important for its function and could inform the design of multivalent inhibitors.

What are common issues when working with snake venom proteins like rhodocytin and how can they be addressed?

Common challenges and solutions include:

• Protein stability issues: Venom proteins can be unstable. Store purified rhodocytin in buffers containing glycerol (50%) and maintain at appropriate temperature (-20°C or -80°C for long-term) .

• Species variation: Different snake species produce variations of similar proteins. When using antibodies, verify cross-reactivity with the specific species being studied. For example, snaclec proteins from C. rhodostoma and C. purpureomaculatus show differences in their interaction profiles with various antivenoms .

• Functional heterogeneity: Different snaclec proteins can have opposing effects. For instance, rhodocetin (four subunits) inhibits platelet aggregation, while rhodocytin (two subunits) induces it .

• Recombinant protein expression challenges: Functional recombinant rhodocytin has been successfully produced by coexpressing both alpha and beta subunits in Chinese hamster ovary cells , addressing previous difficulties in expressing functional protein.

• Batch variation in native venom: When using native venom, variation between batches can occur. Using recombinant protein can help standardize experiments.

How does rhodocytin compare to other snake venom proteins with similar functions?

Different snaclecs have distinct effects on platelet function. Rhodocetin binds to the integrin α2A domain, preventing collagen from binding to the integrin and thus inhibiting collagen-induced platelet aggregation . In contrast, rhodocytin binds to CLEC-2, activating platelets. This functional diversity highlights the importance of precise identification and characterization of specific venom components in research and potential therapeutic applications.

What are the potential applications of rhodocytin antibodies in developing universal antivenoms?

Recent research suggests several promising directions:

• Targeting conserved epitopes: Antibodies that recognize conserved regions of venom proteins could potentially neutralize components from multiple snake species. For example, scientists at Scripps Research have developed antibodies that can block effects of lethal toxins from various snake venoms found across Africa, Asia, and Australia .

• Antibody cocktails: A combination of antibodies targeting different toxin families could provide broad coverage. Researchers suggest that "a cocktail of four antibodies could potentially work as a universal antivenom against any medically relevant snake in the world" .

• Synthetic antibody development: Modern approaches allow for developing effective antibodies without animal immunization, as demonstrated in recent research: "we could make an effective antibody entirely synthetically -- we did not immunize any animals nor did we use any snakes" .

• Cross-reactivity profiling: Antibodies against rhodocytin should be evaluated for cross-reactivity with similar proteins from other snake species. In studies of C. purpureomaculatus venom, both snaclec rhodocetin subunit alpha and beta were identified as antigenic proteins recognized by certain antivenoms but not others .

How can understanding the structure-function relationship of rhodocytin inform therapeutic approaches to thrombotic disorders?

Insights from rhodocytin research suggest several therapeutic approaches:

• CLEC-2 targeting drugs: Understanding how rhodocytin binds CLEC-2 could lead to development of small molecules or biologics that modulate CLEC-2 activity. This could potentially serve as a novel class of antiplatelet agents for treating thrombotic disorders .

• Inhibitors based on structure: There are currently no small molecules or drugs specifically targeting snaclec rhodocetin or rhodocytin. Theoretical approaches could include developing inhibitors that target either the protein itself or its binding site (CLEC-2 for rhodocytin) .

• Anti-metastatic applications: Modified rhodocytin or rhodocytin-derived compounds could potentially serve as anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy .

• Bidirectional modulation: Understanding the molecular interfaces in rhodocytin-CLEC-2 interaction could allow development of both activators and inhibitors of this pathway, providing flexible therapeutic approaches for different medical conditions .

• Crosstalk targeting: Rhodocytin-induced reactions exemplify toxin-induced crosstalk between coagulation (platelets), endothelium, and inflammation (immunocompetent cells). Detailed understanding of this crosstalk could provide novel therapeutic targets beyond direct CLEC-2 modulation .

What molecular and structural biology techniques are most effective for studying rhodocytin-receptor interactions?

Several complementary techniques have proven valuable:

• Crystallography: Has been used to solve the structures of both CLEC-2 and rhodocytin, enabling the development of interaction models .

• Mutagenesis: Site-directed mutagenesis has helped characterize the interaction surfaces and identify critical residues like Asp4 in the α-subunit .

• Blue native PAGE: Effective for analyzing the formation of multimers, confirming that recombinant wild-type rhodocytin forms a heterooctamer similar to native rhodocytin .

• Surface plasmon resonance: Provides quantitative measurement of binding affinities between rhodocytin and CLEC-2 variants .

• Molecular dynamics simulations: Can analyze the molecular interfaces and dynamics involved in rhodocytin-CLEC-2 interactions, providing insights into conformational changes during binding .

• Recombinant protein engineering: Expression of functional rhodocytin has been achieved by coexpression of both subunits in Chinese hamster ovary cells, enabling detailed structure-function studies .

• LC-MS/MS analysis: Useful for protein identification and characterization, as demonstrated in studies identifying snaclec components in snake venoms .

By combining these approaches, researchers can gain comprehensive insights into the structural basis and functional consequences of rhodocytin-receptor interactions.

How do findings from rhodocytin studies translate to understanding snake envenomation pathology in humans?

The clinical picture of Calloselasma rhodostoma envenomation includes:

• Local reactions: Swelling, bleeding, and eventually necrosis

• Systemic effects: Coagulation disturbances and distant bleeding in various organs

These symptoms suggest toxins in the venom affect:

• Endothelial cells and vessel permeability

• Extravasation and activation of immunocompetent cells

• Platelets and the coagulation cascade

The in vivo reactions to rhodocytin represent examples of toxin-induced crosstalk between coagulation (platelets), endothelium, and inflammation (immunocompetent cells). Understanding this complex interplay has implications not only for snake envenomation treatment but also for understanding similar processes in other pathological conditions .

What approaches can bridge basic rhodocytin research with potential therapeutic applications?

To translate basic research findings to clinical applications:

• Animal models: Development of animal models that mimic human pathology, such as cutaneous injections of rhodocytin to model snakebites, could supplement human studies .

• Translational research collaboration: Bringing together basic scientists studying rhodocytin mechanisms with clinical researchers studying snake envenomation could accelerate translation of findings.

• Structure-based drug design: Using the crystal structures of rhodocytin and CLEC-2 to design small molecule inhibitors or biologics that could modulate this interaction in therapeutically useful ways.

• Biomarker identification: Identifying biomarkers of CLEC-2 activation that could be used in clinical settings to monitor treatment efficacy or disease progression.

• Differential targeting: Developing approaches that can selectively target pathological CLEC-2 activation while preserving physiological functions, potentially by exploiting differences in signal intensity or cellular context.