C2 Human
Description
Structure and Biochemical Properties
C2 is a single-chain glycoprotein with a molecular weight of 93 kDa (native form) or ~75 kDa (recombinant form). Key structural features include:
C2 forms the proteolytic subunit of C3/C5 convertase (C4bC2 complex), enabling downstream complement activation. Deficiency disrupts this cascade, impairing microbial clearance and immune complex processing .
Biological Function
C2 is indispensable for:
-
Immune Defense: Cleavage of C3/C5 drives pathogen opsonization, phagocytosis, and membrane attack complex (MAC) formation.
-
Immune Complex Clearance: Prevents autoimmune tissue damage by solubilizing immune complexes .
-
Autoimmune Regulation: Dysfunctional C2 correlates with systemic lupus erythematosus (SLE), particularly in females .
Deficiency Pathophysiology
C2 deficiency (OMIM: 613927) arises from mutations, most commonly a 28-nucleotide deletion. This results in:
| Manifestation | Mechanism |
|---|---|
| Immunodeficiency | Impaired C3/C5 activation → Reduced bacterial clearance (e.g., Streptococcus pneumoniae) |
| Autoimmunity | Immune complex persistence → Chronic inflammation (SLE, vasculitis) |
Clinical Implications and Diagnostic Approaches
Key Findings from Research
Recombinant C2 (rhC2)
Produced via mammalian cell expression systems, rhC2 restores complement activity in C2-deficient serum. Key studies demonstrate:
| Parameter | rhC2 Efficacy | Source |
|---|---|---|
| Purity | >95% (SDS-PAGE) | |
| Activity | Restores classical pathway hemolysis, C3 fragment deposition | |
| Stability | Stable at −20°C; avoid repeated freeze-thaw cycles |
Therapeutic Applications and Future Directions
Current Therapies
-
Plasma Infusions: Provide temporary C2 replenishment but carry risks of transfusion reactions .
-
Recombinant C2: Shows promise in preclinical studies, with scalable production and reduced immunogenicity .
Emerging Strategies
-
Targeted Delivery: Nanoparticles or CRISPR/Cas9 for site-specific C2 modulation .
-
C2 Inhibitors: Experimental therapies for autoimmune diseases (e.g., SLE) .
Research Trends and Challenges
| Area | Key Advances | Gaps |
|---|---|---|
| Production | High-yield mammalian expression systems | Cost-effective scaling |
| Clinical Trials | Ex vivo validation in C2-deficient serum | In vivo efficacy data |
| Mechanistic Studies | Role in SLE pathogenesis, C2a/C2b functional dynamics | Autoimmune triggers |
Product Specs
C2, the proteolytic subunit of both the C3 and C5 convertases, plays a crucial role in the complement system's activation pathways. Upon pathway initiation, enzyme complexes form on target surfaces. C1s and MASP, components of these complexes, activate both C4 and C2. They cleave a specific peptide bond in C4, leading to the deposition of C4b on the surface. Additionally, they cleave C2 into two fragments.
Human Complement C2, derived from human plasma, has a molecular weight of 93 kDa.
This product is provided as a sterile filtered solution.
This product is formulated in a buffer containing 25mM sodium phosphate and 100mM NaCl, at a pH of 6.0.
For optimal stability, store Human C2 at 4°C. If using the entire vial, it can be stored at 4°C for 2-4 weeks. For long-term storage, freeze the product below -20°C. It is recommended to add a carrier protein (0.1% HSA or BSA) for long-term storage to enhance stability. Avoid repeated freeze-thaw cycles to preserve product integrity.
The purity of this product is greater than 95.0% as assessed by SDS-PAGE analysis.
Each plasma donor undergoes rigorous testing to ensure the absence of antibodies against HIV-1, HIV-2, HCV, and HBsAg. Only plasma that tests negative for these viruses is used for production.
Complement C2, C3/C5 convertase, C2.
Human Plasma.
Q&A
What is C2-8 and what is its mechanism of action in Huntington's disease research?
C2-8 is a small molecule inhibitor of polyglutamine aggregation that was developed based on a yeast high-throughput screen. It is a structural analog of the original C2 compound, which demonstrated ability to inhibit polyQ aggregation while maintaining relatively low toxicity in mammalian cells compared to other small molecule candidates . Mechanistically, C2-8 targets the aggregation process of mutant Huntingtin (mHTT) protein fragments containing expanded polyglutamine repeats, which are characteristic of Huntington's disease. The compound has demonstrated inhibition of mHTT polyQ fragment aggregation in multiple experimental systems including in vitro assays, transiently transfected mammalian cells, and brain slice cultures from R6/2 transgenic mice .
What experimental models have been used to evaluate C2-8 efficacy?
C2-8 has been evaluated in several experimental models with increasing biological complexity:
-
Cell-free in vitro systems: Initial screening and validation of aggregation inhibition properties
-
PC12 cell models: Overexpressing mHTT-exon1 for cellular toxicity and aggregation studies (IC50 value of 50 nM)
-
R6/2 hippocampal slice models: Ex vivo tissue preparations to assess aggregate reduction
-
Drosophila models: Demonstrating dose-dependent reduction of photoreceptor neurodegeneration
-
R6/2 transgenic mouse models: In vivo assessment of pharmacokinetics, aggregate reduction, and potential therapeutic effects on motor performance and neuropathology
These diverse models provide complementary insights into both the pharmacodynamic properties and potential therapeutic applications of C2-8.
What are the key pharmacokinetic properties of C2-8 relevant to researchers?
C2-8 demonstrates favorable pharmacokinetic properties that make it suitable for in vivo research:
These properties suggest C2-8 has suitable drug-like characteristics for preclinical research, though researchers should conduct specific pharmacokinetic analyses for their particular experimental conditions and models .
How should researchers design robust preclinical studies to evaluate C2-8 efficacy?
Based on lessons from independent replication studies, researchers should consider these methodological approaches:
-
Randomization protocol: Implement stringent randomization to reduce selection bias and ensure comparable experimental groups
-
Blinding procedures: Ensure experimenters are blinded to treatment groups during data collection and analysis
-
Allocation concealment: Prevent knowledge of upcoming allocations to reduce bias
-
Sample size calculation: Perform power analysis based on expected effect sizes from previous studies to ensure adequate statistical power
-
Multiple administration routes: Consider comparing different delivery methods (oral gavage vs. intraperitoneal injection) as route may affect efficacy
-
Dosage optimization: Test multiple doses based on pharmacokinetic data (e.g., 10 mg/kg and 20 mg/kg twice daily were used in the replication study)
-
Predetermined stopping points: Establish clear experimental endpoints before beginning studies
-
Comprehensive statistics: Use appropriate statistical software and methods to analyze all outcome measures
These methodological considerations are especially important when evaluating compounds that showed promise in initial studies but require independent verification .
What behavioral and neuropathological endpoints are most appropriate for evaluating C2-8 efficacy in HD models?
Researchers should consider multiple complementary endpoints to comprehensively assess potential therapeutic effects:
Behavioral assessments:
-
Accelerating Rotarod: Measures motor coordination and learning
-
Wire-hang test: Evaluates muscle strength and coordination
-
Additional tests to consider: Open field activity, grip strength, and cognitive assessments specific to the model being used
Neuropathological assessments:
-
Brain weight (total, forebrain, and cerebellum)
-
Striatal volume measurement using unbiased stereology
-
Striatal neuronal volume assessment
-
mHTT aggregate quantification (both number and volume)
-
Neuronal cell counts in affected brain regions
The replication study demonstrated that while C2-8 significantly reduced nuclear mHTT aggregate volume, this did not translate to improvements in motor behavior or prevention of striatal atrophy in the R6/2 model, highlighting the importance of comprehensive endpoint assessment .
What explains the discrepancy between C2-8's consistent aggregate reduction and variable therapeutic efficacy?
The independent replication study revealed an important finding: despite consistently demonstrating reduction in mHTT nuclear aggregate volume, C2-8 treatment did not consistently translate to improvements in behavioral deficits or prevention of striatal neuron atrophy across studies . Several factors may explain this discrepancy:
-
Disease model characteristics: The R6/2 mouse expresses a fragment of mHTT (exon 1) and exhibits particularly aggressive phenotypes that may be less responsive to aggregate reduction alone
-
Timing of intervention: Aggregate formation may trigger cascading pathological processes that become independent of continued aggregation
-
Aggregate species toxicity: Nuclear inclusions (measured in the studies) may be less toxic than smaller, soluble oligomeric species
-
Subcellular localization: The differential impact of nuclear versus cytoplasmic aggregates may influence therapeutic outcomes
-
Background strain influences: Genetic background differences between mouse colonies may affect phenotypic expression and treatment response
Researchers should consider these factors when designing studies and interpreting results that show pharmacodynamic effects (aggregate reduction) without corresponding functional improvements .
How should researchers select appropriate HD mouse models for evaluating aggregate-targeting compounds like C2-8?
When evaluating aggregate-targeting compounds, researchers should consider the significant differences between available HD mouse models:
| Model Type | Characteristics | Aggregate Profile | Disease Progression | Recommendation for C2-8 Testing |
|---|---|---|---|---|
| Fragment models (R6/2) | Express mHTT-exon1 | High nuclear and cytoplasmic aggregate load | Aggressive, often lethal | Good for proof-of-concept aggregate reduction studies |
| Full-length models (BACHD, YAC128) | Express full mHTT protein | Lower aggregate load | Slowly progressive | May better reveal therapeutic benefits of modest aggregate reduction |
| Knock-in models (zQ175) | Endogenous Htt with expanded CAG | Progressive aggregate formation | Slower onset, progressive | Potentially more translatable to human disease progression |
The replication study authors suggest that "a compound that could not ameliorate the more aggressive disease phenotype in a fragment model may still have efficacy in full-length mHTT mouse models with milder disease phenotypes" . Therefore, researchers should consider testing C2-8 in both fragment and full-length models, ideally on well-defined genetic backgrounds (either inbred or F1), to comprehensively evaluate its therapeutic potential .
What methodological approaches can address the challenges of independent replication in preclinical HD research?
The independent replication study of C2-8 highlights several methodological approaches to enhance reproducibility:
-
Standardized reporting: Follow rigorous standards for study design and reporting (e.g., those suggested by NINDS)
-
Shared protocols: Establish clear communication between original and replication teams to ensure methodological consistency where appropriate
-
Detailed methods publication: Include comprehensive methodological details in publications, including genetic background, CAG repeat length, housing conditions, behavioral protocols, and drug formulation/administration
-
Compound sourcing documentation: Clearly report the source, purity, and characterization of compounds being tested
-
Data sharing: Make raw data available for secondary analyses
-
Consensus endpoints: Develop field-wide consensus on the most relevant and reproducible endpoints for specific types of interventions
-
Multi-center testing: Consider collaborative, multi-center testing of promising compounds before clinical translation
These approaches can help address the "reproducibility crisis" in preclinical research and ensure that only the most robust findings advance to clinical testing .
How should researchers analyze and interpret aggregate reduction data in preclinical studies?
The analysis of aggregate reduction requires careful methodological considerations:
-
Quantification methods: Use unbiased stereology to quantify aggregate number, size, and distribution
-
Whole-brain analysis: Examine multiple brain regions, not just the striatum, to understand regional variability
-
Correlation with phenotypes: Analyze correlations between aggregate measures and behavioral/neuropathological outcomes within individual animals
-
Dimensional analysis: Consider both aggregate number and volume/size, as these may have different biological significance
-
Threshold considerations: Determine what degree of aggregate reduction is likely to be biologically meaningful
-
Temporal dynamics: Assess aggregate formation and reduction over time rather than at a single endpoint
-
Statistical approach: Use appropriate statistical tests that account for the non-normal distribution often seen with aggregate counts
In the C2-8 replication study, researchers found significant reduction in nuclear mHTT aggregate volume but not corresponding improvement in functional outcomes, highlighting the complex relationship between aggregation and disease manifestation .
What approaches can help reconcile contradictory findings between original and replication studies?
When faced with contradictory findings between studies, researchers should:
-
Identify key methodological differences: Systematically compare experimental protocols, including:
-
Animal source and genetic background
-
CAG repeat length and stability
-
Housing and environmental conditions
-
Compound formulation and administration route
-
Behavioral testing protocols and timing
-
Analysis methods and statistical approaches
-
-
Conduct meta-analysis: Pool data across studies where methodology is sufficiently similar
-
Perform sub-group analyses: Examine whether specific subsets of animals respond differently to treatment
-
Design bridging studies: Target specific methodological differences to determine their impact on outcomes
-
Consider biomarker validation: Develop and validate biomarkers that can serve as reliable indicators of target engagement and disease modification
The C2-8 replication study noted several potential factors that might explain differences from the original findings, including mouse source, CAG repeat length, housing conditions, compound source, behavioral protocols, and drug administration route .
What are the key considerations for translating aggregate-targeting compounds from preclinical to clinical studies?
Researchers considering translation of aggregate-targeting compounds should address:
-
Target validation: Establish whether aggregate reduction is mechanistically linked to disease modification in humans
-
Biomarker development: Identify measurable indicators of aggregate burden that can be assessed in living patients
-
Therapeutic window: Determine the optimal timing for intervention in relation to disease onset
-
Dosage and exposure: Establish dose-response relationships and ensure adequate CNS exposure in humans
-
Safety profile: Thoroughly assess potential off-target effects, particularly in long-term administration
-
Patient stratification: Consider whether specific genetic or clinical subgroups might respond differently
-
Combination approaches: Evaluate whether combining aggregate-targeting with other therapeutic modalities enhances efficacy
The C2-8 studies suggest that while aggregate reduction is achievable in preclinical models, the relationship between this pharmacodynamic effect and clinical benefit requires further investigation .
How can researchers better predict which preclinical findings will translate to clinical settings?
To enhance clinical translation prediction, researchers should:
-
Use multiple complementary models: Test compounds in both fragment and full-length HD models with different aggregate profiles
-
Employ humanized models: Consider testing in humanized models expressing full-length human mHTT
-
Apply translational biomarkers: Develop biomarkers that can be measured similarly in both preclinical models and patients
-
Assess age-dependent effects: Evaluate efficacy across different disease stages
-
Consider species differences: Account for differences in brain size, metabolism, and age-scaling between models and humans
-
Establish pharmacodynamic thresholds: Determine what degree of target engagement (aggregate reduction) is needed for functional improvement
-
Validate molecular targets: Confirm that molecular targets of aggregate-targeting compounds function similarly in animal models and humans
The C2-8 studies demonstrate that target engagement (aggregate reduction) alone may be insufficient to predict therapeutic benefit, highlighting the need for more sophisticated predictive approaches .
What novel experimental approaches could enhance evaluation of C2-8 and similar compounds?
Researchers should consider these innovative approaches:
-
Patient-derived models: Test C2-8 in induced pluripotent stem cell (iPSC)-derived neurons from HD patients
-
3D organoid models: Evaluate effects in brain organoids that better recapitulate human neural architecture
-
Multi-modal imaging: Combine MRI, PET, and other imaging modalities to track aggregates and neurodegeneration in vivo
-
Single-cell analyses: Examine cell-type specific responses to aggregate reduction
-
System biology approaches: Integrate transcriptomic, proteomic, and metabolomic data to understand mechanism comprehensively
-
Computational modeling: Develop predictive models of aggregate formation and clearance to optimize dosing regimens
-
Long-term low-dose studies: Evaluate whether chronic low-dose administration provides benefits not seen in shorter studies
These approaches could provide deeper insights into both the mechanism of action and potential therapeutic applications of C2-8 beyond what conventional models have revealed .
What are the most promising research directions for advancing aggregate-targeting therapeutics for HD?
Based on current evidence, these research directions appear most promising:
-
Combination therapies: Investigate whether combining C2-8 with compounds targeting different disease mechanisms enhances efficacy
-
Prevention vs. treatment: Determine whether earlier intervention (before substantial aggregate formation) improves outcomes
-
Structural modification: Develop C2-8 analogs with enhanced brain penetration or aggregation-inhibiting properties
-
Target refinement: Identify specific aggregate species (oligomers vs. inclusions) most responsive to C2-8 treatment
-
Delivery optimization: Explore alternative delivery methods including sustained-release formulations
-
Genetic background effects: Investigate how genetic modifiers of HD influence response to aggregate-targeting compounds
-
Mechanistic studies: Elucidate the precise molecular interactions between C2-8 and mHTT protein
The findings from both original and replication studies of C2-8 provide valuable insights that can guide these future research directions, potentially leading to more effective aggregate-targeting therapeutic approaches for HD .
Product Science Overview
Complement C2 is essential for the activation of the classical and lectin pathways of the complement system. When the body detects a foreign invader, the complement system is activated. Complement C2 binds to an activated surface-bound C4b in the presence of magnesium ions (Mg²⁺), forming the C4bC2 complex. This complex is then cleaved by C1s or MASP2 into C2a and C2b . The C2a fragment remains bound to C4b, forming the C3-convertase (C4b2a), which is crucial for the subsequent steps in the complement activation cascade .
Deficiency in Complement C2 is the most common genetically determined complete complement deficiency. It is associated with a number of diseases, most notably recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults . The deficiency of C2 can lead to increased susceptibility to bacterial infections and a higher risk of developing autoimmune diseases .
Research has shown that recombinant human complement component C2 can restore classical complement pathway activity in vitro. This has potential therapeutic implications for treating C2 deficiency diseases . For instance, infusions with normal fresh frozen plasma have demonstrated therapeutic effects, but protein replacement therapy has not yet been fully evaluated .
