C4c Human
Description
Introduction to C4c Human
Complement C4c is a degradation product derived from complement component C4, a critical player in the human immune system that functions as a multifunctional β1-globulin present in plasma . C4c is specifically formed when C4b (another fragment of C4) is cleaved by factor I in the presence of cofactors . This proteolytic process generates the soluble C4c fragment alongside the membrane-bound C4d fragment .
As a component of the complement system, C4c plays important roles in both innate and adaptive immune responses, contributing to the body's ability to produce inflammatory and protective reactions against pathogens before adaptive responses can occur . Understanding C4c's structure, function, and clinical relevance provides valuable insights into immune system regulation and disease pathogenesis.
Molecular Structure and Properties
Human complement C4c is characterized by specific physical and biochemical properties that define its function in the immune system:
The parent molecule, complement C4, circulates in human plasma as a heterotrimer with a total molecular weight of approximately 205,000 Da . The three disulfide-linked chains have molecular weights of 93 kDa (alpha), 75 kDa (beta), and 32 kDa (gamma) . When C4 is activated, it undergoes proteolytic cleavage to generate C4a and C4b . Subsequently, C4b can be inactivated to an intermediate form called iC4b through cleavage by the serine protease factor I together with cofactors such as CD46 . Further proteolytic processing of iC4b produces the thioester-linked C4d (45 kDa) and soluble C4c (146-210 kDa, depending on the source) .
3.1. Role in Complement Pathways
C4c is intricately involved in the classical and lectin pathways of complement activation, two of the three main pathways through which the complement system operates:
The complement cascade initiated through these pathways leads to the formation of the membrane attack complex (MAC), which can lyse target cells . Throughout this process, regulators ensure that complement activation is controlled, with C4c being one of the products of this regulation.
3.2. Interactions with Other Complement Components
C4c interacts with several other components of the complement system:
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Interaction with C1 and C2: C4c interacts with C1 and C2 to form C3 convertase (C4b2b) of the classical activation pathway . This enzyme complex is crucial for the progression of the complement cascade.
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Formation of C3 Convertase: The C3 convertase (C4b2a) generated through the interaction of C4b with C2a cleaves C3 into C3a and C3b, further propagating the complement cascade .
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Structural Similarities with C3b: Comparative studies have revealed that the conformations of C4b and its paralog C3b are surprisingly conserved, which is reflected in the striking similarity between the C3 convertases (C4b2a versus C3bBb) from the classical/lectin and alternative pathways .
4.1. C4c as a Biomarker
C4c serves as a valuable biomarker for complement activation in both the classical and lectin pathways . Unlike C4d, which adheres to the plasma membrane, C4c is released into the extracellular environment after C4 fragmentation, making it potentially more accessible for detection in plasma .
The quantitative measurement of C4c in biological fluids can provide insights into the degree of complement activation in various clinical scenarios:
Modern diagnostic tools, such as ELISA kits, allow for the precise quantification of C4c in various sample types:
| ELISA Kit Parameter | Value | Reference |
|---|---|---|
| Sensitivity | 21 ng/ml | |
| Detection Range | 21-5000 ng/ml | |
| Sample Types | Serum, Plasma, Cell culture supernatant | |
| Detection Method | Colorimetric | |
| Assay Type | Sandwich (quantitative) |
4.2. Association with Diseases
The levels and activity of C4c have been linked to various pathological conditions:
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Systemic Lupus Erythematosus (SLE): SLE is often associated with congenital C4 deficiency. Reduced levels of components of the classical pathway (C1, C2, C4, C3) are common after complement activation in SLE . Monitoring C4c levels can help track disease activity in SLE patients .
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Acute Serum Sickness: This condition, characterized by immune complex formation, is associated with changes in complement levels, including C4c .
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Infections: Complement activation plays a crucial role in defense against microbial infections. Deficiencies in C4 and, consequently, alterations in C4c levels, can increase susceptibility to various infections .
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Kidney Diseases: C4c has been implicated in kidney diseases such as glomerulonephritis, where complement activation contributes to tissue damage .
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Lung Cancer: Recent studies suggest that C4c may serve as a valuable marker for the early diagnosis of lung cancer, potentially offering advantages over other complement fragments such as C4d .
Laboratory Methods for C4c Detection
Several laboratory techniques are employed for the detection and quantification of C4c in clinical and research settings:
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ELISA (Enzyme-Linked Immunosorbent Assay): ELISA kits specific for human C4c are widely used for quantitative measurement in serum, plasma, and cell culture supernatants . These assays typically employ antibodies specific for human C4c coated on a 96-well plate and offer high sensitivity and reproducibility.
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Western Blotting: This technique can be used to detect C4c under non-reduced conditions, with an expected band size of approximately 146 kDa .
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Immunohistochemistry: This method allows for the visualization of C4c deposition in tissues, which can be particularly relevant in diseases involving complement activation and tissue damage.
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Mass Spectrometry: Advanced mass spectrometric techniques can be used for the precise identification and characterization of C4c and other complement components in complex biological samples.
Current Research and Future Perspectives
Research on human C4c continues to expand our understanding of its roles in health and disease:
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Multivariate Diagnostic Models: Recent studies have explored the development of multivariate diagnostic models incorporating C4c alongside other biomarkers, such as CYFRA 21-1 and CRP, for more accurate disease detection, particularly in lung cancer .
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Genetic Variations: Research on genetic variations affecting C4 expression and function has revealed that individuals with higher copy numbers of C4 genes (3 or 4 total copies) demonstrate superior graft survival in kidney transplantation scenarios .
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Therapeutic Targeting: Understanding the roles of C4c and related complement components in disease pathogenesis opens avenues for therapeutic intervention. Targeting specific steps in the complement cascade could potentially modulate disease progression in conditions where complement activation contributes to pathology.
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Biomarker Development: Ongoing research aims to refine the use of C4c as a biomarker for various diseases, potentially enabling earlier diagnosis, more accurate prognosis, and better monitoring of treatment responses.
Product Specs
Q&A
What is C4c and what is its role in the complement cascade?
C4c is a specific degradation product formed during activation of the classical or lectin complement pathways. The complement 1 complex first cleaves complement component 4 (C4) to form C4b and C4a. C4b is then regulated through cleavage by Factor I, which forms iC4b and blocks C3 convertase, inhibiting opsonization and activation of the classical pathway. iC4b is further degraded into the inactive fragments C4d and C4c .
Structurally, C4c consists of two a-chain fragments - alpha 4 (13 kDa) and alpha 3 (25 kDa) - linked to a beta chain through disulphide bridges and a gamma chain. It is expressed in blood plasma and serves as an important marker of complement activation .
How does C4c differ from other C4 fragments in structure and function?
The various C4 fragments have distinct structures and functions within the complement cascade:
| C4 Fragment | Molecular Structure | Functional Role | Detection Significance |
|---|---|---|---|
| C4 (intact) | Complete protein with alpha, beta, and gamma chains | Precursor molecule, inactive until cleaved | Indicates complement reservoir |
| C4b | C4 minus C4a fragment | Active in formation of C3 convertase | Marker of early complement activation |
| iC4b | Inactivated C4b (Factor I cleaved) | Reduced functional activity | Intermediate degradation marker |
| C4c | Two a-chain fragments (α3, α4), beta chain, gamma chain | Inactive end product | Stable marker of complement activation |
| C4d | Single a-chain fragment | Inactive, remains bound to cell surfaces | Tissue-bound activation marker |
Specific monoclonal antibodies have been developed that can distinguish C4c from these other fragments without cross-reactivity, enabling precise measurements of complement activation .
What are the established normal reference ranges for C4c in human plasma?
According to research conducted on Danish blood donors, the normal range of C4c in EDTA/futhan plasma has been established:
| Parameter | Value (mg/L) |
|---|---|
| Mean C4c concentration | 0.85 |
| Range | 0.19-2.21 |
| Sample size | 100 blood donors |
| Sample type | EDTA/futhan plasma |
These values provide an important baseline for comparison in both clinical and research settings when evaluating potential complement dysregulation .
What are the most specific methods for quantifying human C4c in biological samples?
The gold standard for specific C4c quantification is sandwich ELISA using monoclonal antibodies that recognize C4c without cross-reactivity with intact C4 or other C4 activation products. The methodology typically involves:
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Coating microtiter plates with a capture antibody specific for C4c
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Adding samples containing C4c, which binds to the immobilized antibody
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Adding a biotinylated anti-human C4c detection antibody
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Using HRP-conjugated streptavidin and TMB substrate for color development
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Measuring absorbance at 450 nm and calculating concentrations using a standard curve
For optimal specificity, researchers should select antibodies like clone 033D-69.4.1 (10-12) that have been validated to recognize human C4c without cross-reactivity with native C4, C4b, iC4b, or C4d .
How can researchers differentiate between in vivo activation and ex vivo artifact when measuring C4c?
Differentiating between genuine in vivo activation and ex vivo artifacts is critical for accurate C4c measurement. Recommended approaches include:
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Using appropriate anticoagulants and protease inhibitors (e.g., EDTA/futhan combination) during sample collection
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Processing samples immediately or preserving at 4°C if immediate processing is not possible
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Including specialized controls:
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Samples from factor I deficient patients (which should not contain C4c)
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Deliberately activated samples as positive controls
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Samples with minimal handling to establish baseline
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Comparing patterns across multiple timepoints or conditions
Sample handling procedures must be standardized and documented to ensure that measured C4c levels accurately reflect physiological complement activation rather than handling artifacts .
What validation steps are essential when developing new C4c-specific antibodies?
Developing highly specific antibodies for C4c requires rigorous validation:
| Validation Step | Methodology | Expected Outcome |
|---|---|---|
| Epitope specificity | Testing against purified C4c, C4, C4b, iC4b, C4d | Reaction with C4c only |
| Cross-reactivity assessment | Western blotting with different C4 fragments | Single band at expected C4c MW |
| Biological validation | Testing with factor I deficient samples | No reactivity (these samples lack C4c) |
| Immunoprecipitation | Pull-down assays followed by fragment analysis | Specific isolation of C4c |
| Immunoaffinity chromatography | Analysis of eluted fragments | Confirmation of C4c specificity |
These validation steps are critical to ensure that the antibody specifically recognizes C4c without interference from other complement components or C4 fragments .
How should experiments be designed to investigate C4c formation kinetics in different activation pathways?
To effectively study C4c formation kinetics:
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Design time-course experiments with sampling at multiple timepoints (0, 15, 30, 60, 120 minutes, etc.)
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Implement pathway-specific activators:
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Classical pathway: Immune complexes or heat-aggregated immunoglobulins
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Lectin pathway: Mannan or specific carbohydrate ligands
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Alternative pathway: Zymosan or bacterial lipopolysaccharides
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Include pathway-specific inhibitors as controls
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Employ C4c-specific assays alongside measurements of other complement components
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Consider both fluid-phase and surface-bound C4c detection methods
This comprehensive approach allows for detailed characterization of C4c formation dynamics under different activation conditions, providing insights into pathway-specific complement activation profiles .
What strategies can researchers use to study C4c in tissues versus circulation?
Studying C4c in tissues requires different approaches than serum or plasma analysis:
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Tissue extraction protocols must be optimized to preserve C4c epitopes while efficiently releasing the fragment from tissue
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Immunohistochemistry using C4c-specific antibodies can localize sites of complement activation
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Laser capture microdissection combined with mass spectrometry can identify C4c in specific tissue regions
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Perfusion experiments can distinguish between tissue-deposited and circulating C4c
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Paired analysis of circulation and tissue compartments provides comprehensive activation profiles
Researchers should be aware that C4c dynamics may differ significantly between circulation and tissues, requiring specialized methodological approaches for each compartment .
How can researchers integrate C4c measurements with other complement markers for comprehensive assessment?
A multi-marker approach provides the most complete assessment of complement activation:
| Pathway | Recommended Markers | Rationale for Combination |
|---|---|---|
| Classical/Lectin | C4c, C4d, C1q, MBL | Distinguishes between activation routes and progression |
| Alternative | Factor B, properdin, Ba | Identifies alternative pathway involvement |
| Terminal | C5a, SC5b-9 (TCC) | Determines extent of terminal pathway activation |
| Regulatory | Factor H, Factor I, C4BP | Assesses regulatory capacity |
This integrated approach provides a nuanced understanding of the complement activation status that single-marker assessments cannot achieve. Data integration should employ multivariate statistical approaches to identify activation patterns characteristic of specific conditions .
How do genetic variants of complement components affect C4c measurements?
Genetic variations in complement components can significantly impact C4c measurements and interpretation:
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C4A and C4B gene copy number variations affect baseline C4 levels and subsequently C4c generation
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Polymorphisms in factor I can alter the rate of C4b cleavage and C4c formation
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Genetic variations in regulatory proteins may influence C4c clearance rates
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Ethnic differences in complement genetics necessitate population-specific reference ranges
Researchers should consider genotyping key complement components when conducting C4c studies in heterogeneous populations to account for these genetic influences on measurements .
What approaches can resolve discrepancies between C4c levels and clinical manifestations?
When C4c levels do not correlate with clinical manifestations, consider:
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Compartmentalization of complement activation (systemic vs. local)
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Temporal relationship between complement activation and sampling
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Presence of autoantibodies against complement components
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Compensatory mechanisms in chronic complement activation
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Consumption of complement components in severe activation
These factors may explain why C4c measurements sometimes diverge from clinical expectations. Multi-compartment sampling and longitudinal measurements can help resolve such discrepancies .
How can researchers address sample stability issues when measuring C4c?
C4c measurements can be affected by sample handling and storage. Recommended approaches include:
| Challenge | Solution | Rationale |
|---|---|---|
| Ex vivo activation | EDTA/futhan anticoagulants | Inhibits complement activation during processing |
| Freeze-thaw degradation | Single-use aliquots | Prevents multiple freeze-thaw cycles |
| Long-term storage | -80°C storage | Minimizes degradation of complement components |
| Shipping conditions | Temperature-controlled shipping | Maintains sample integrity during transport |
| Batch effects | Include inter-assay controls | Ensures comparability across measurement batches |
These precautions help ensure that measured C4c levels accurately reflect in vivo activation states rather than artifacts introduced during sample handling .
What statistical approaches are most appropriate for analyzing C4c data in clinical research?
Statistical analysis of C4c data should be tailored to the research question:
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For comparing groups: Non-parametric tests (Mann-Whitney U or Kruskal-Wallis) are often appropriate as C4c values typically do not follow normal distribution
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For correlation with clinical parameters: Spearman's rank correlation is recommended for similar reasons
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For predictive modeling: Logistic regression or machine learning approaches can identify C4c cut-offs with clinical relevance
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For longitudinal studies: Mixed-effects models account for repeated measures and individual variation
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For multivariate analysis: Principal component analysis can identify patterns across multiple complement markers including C4c
How should researchers interpret changes in C4c levels during therapeutic complement inhibition?
Monitoring C4c during complement-targeted therapies requires specific interpretative frameworks:
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Early decrease in C4c may indicate successful inhibition of classical/lectin pathway activation
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Persistent elevation despite therapy may suggest:
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Insufficient dosing
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Alternative pathway bypass
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Ongoing strong activating stimulus
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Drug neutralizing antibodies
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Rebound increases after therapy cessation provide insights into underlying disease activity
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Differential effects on C4c versus other markers may reveal pathway-specific effects of interventions
These interpretations help assess therapeutic efficacy and understand the mechanisms of complement-targeting treatments .
What are the emerging applications of C4c as a biomarker in precision medicine?
C4c is increasingly being investigated as a precision medicine biomarker:
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As a companion diagnostic for complement-targeting therapies
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For stratifying patients in clinical trials based on complement activation patterns
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In monitoring transplant rejection where complement activation plays a role
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For assessing autoimmune disease activity and predicting flares
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In combination with genetic information to create personalized complement profiles
The specificity of C4c for classical/lectin pathway activation makes it valuable for precision approaches to conditions where these pathways play pathogenic roles. Integration with genetic and other biomarker data enhances its utility in personalized medicine approaches .
Product Science Overview
Complement Component C4c is a fragment of the complement system, which is a crucial part of the immune system. The complement system consists of a series of small proteins that enhance (or “complement”) the ability of antibodies and phagocytic cells to clear pathogens from an organism. Complement Component C4c is derived from Complement Component 4 (C4), which plays a significant role in the classical and lectin pathways of the complement system .
Complement Component 4 (C4) is a glycoprotein composed of three polypeptide chains: alpha (α), beta (β), and gamma (γ). The C4 protein is cleaved into C4a and C4b by the enzyme C1s during the activation of the complement system. C4b then binds to the pathogen surface and forms a complex with C2a, creating the C3 convertase (C4b2a), which is essential for the activation of the complement cascade .
C4c is a degradation product of C4b. It is generated when C4b is cleaved by factor I in the presence of cofactors such as complement receptor 1 (CR1) or membrane cofactor protein (MCP). The cleavage of C4b results in the formation of C4c and C4d. C4c is released into the plasma, while C4d remains bound to the cell surface .
Complement Component C4c, as part of the complement system, plays a vital role in the immune response. The complement system helps to clear pathogens, promote inflammation, and enhance the ability of antibodies to neutralize pathogens. C4c, being a fragment of C4b, indirectly contributes to these processes by being a marker of complement activation .
The levels of Complement Component C4c in the blood can be used as a biomarker for various diseases. Elevated levels of C4c can indicate increased complement activation, which is often seen in autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. Conversely, low levels of C4c may suggest a deficiency in the complement system, which can lead to increased susceptibility to infections .
