Cleaved-ADAM17 (R215) Antibody

What is ADAM17 and what cellular functions does it perform?

ADAM17 (A Disintegrin And Metalloprotease 17), also known as TACE (TNF-α Converting Enzyme), is a transmembrane metalloprotease that mediates the ectodomain shedding of numerous transmembrane proteins, including adhesion proteins, growth factor precursors, and cytokines involved in inflammation and immunity . It plays a critical role in cleaving the membrane-bound precursor of TNF-alpha to its mature soluble form and is responsible for the proteolytical release of soluble JAM3 from endothelial cell surfaces . ADAM17 is initially synthesized as an inactive zymogen, with its latency maintained by an autoinhibitory pro-domain. Its activation requires proteolytic processing by proprotein convertases such as furin and PC7, which remove the inhibitory pro-domain and generate the catalytically active form of the enzyme .

What is the significance of the R215 cleavage site in ADAM17 processing?

The R215 site represents a critical boundary between the pro-domain and catalytic domain of ADAM17 . Current understanding of ADAM17 activation involves a two-step cleavage process: first at an upstream proprotein convertase (PC) site (RKR58/D59) within the pro-domain, followed by cleavage at the boundary site (RVKR214/R215) between the pro-domain and the catalytic domain . Research has demonstrated that while processing at the boundary R215 site is important, it is actually secondary to the prerequisite cleavage at the upstream PC site embedded within the pro-domain sequence . This two-step processing mechanism is crucial for proper functional maturation of ADAM17, as mutations in either site can significantly affect the enzyme's catalytic activity even if some processing still occurs .

What does the Cleaved-ADAM17 (R215) Antibody specifically detect?

The Cleaved-ADAM17 (R215) Antibody specifically detects endogenous levels of the activated form of ADAM17 resulting from cleavage adjacent to arginine 215 (R215) . This polyclonal antibody targets the amino acid region 196-245 of human ADAM17, which encompasses the cleavage site . The antibody recognizes the processed catalytic domain that begins with the N-terminal sequence 215RADPD, representing the mature, catalytically active form of the enzyme . This specificity makes the antibody valuable for distinguishing between the inactive proenzyme form and the mature, proteolytically active ADAM17 in experimental systems .

What are the standard research applications for Cleaved-ADAM17 (R215) Antibody?

The Cleaved-ADAM17 (R215) Antibody is primarily designed for Western Blot and ELISA research applications . In Western blot applications, the recommended dilution range is 1:500-1:2000, while for ELISA the suggested dilution is 1:20000 . The antibody exhibits reactivity against human, rat, and mouse samples, making it versatile for comparative studies across species . It is important to note that this antibody is strictly for research use only (RUO) and must not be used in diagnostic or therapeutic applications . Researchers commonly employ this antibody to investigate ADAM17 activation status, processing events, and to correlate ADAM17 activity with various physiological and pathological processes, including studies on Alzheimer's disease where ADAM17 variants like the R215I mutation have been implicated .

How does the maturation process of ADAM17 differ from other ADAM family members?

This discovery suggests a common activation mechanism across these family members. Mutations in the upstream PC site of ADAM17, ADAM10, and ADAM9 all resulted in reduced catalytic activity despite some processing still occurring at the canonical boundary site between the pro-domain and catalytic domain . This indicates that proper processing at both sites is crucial for optimal enzyme activity across these ADAM family members. The existence of this regulatory mechanism in multiple ADAMs points to an evolutionarily conserved activation process that may be targeted for therapeutic interventions in conditions where dysregulated ADAM activity contributes to pathology .

What is the relationship between glycosylation of ADAM17 and its processing at the R215 site?

ADAM17 undergoes N-glycosylation, which is important for its proper folding, transport, and activity . While the search results don't provide exhaustive details about the direct relationship between glycosylation and R215 processing, they do indicate that ADAM17 contains predicted N-glycosylation sites . Deglycosylation experiments with PNGase F and endoglycosidase H (Endo H) have been used to study the glycosylation status of ADAM17 .

The maturation process of ADAM17 involves both glycosylation and proteolytic processing. After initial synthesis, ADAM17 is glycosylated in the endoplasmic reticulum and Golgi apparatus, then cleaved by proprotein convertases like furin and PC7 . This processing includes cleavage at both the upstream PC site and the boundary site (R215), which is necessary for the removal of the pro-domain and generation of the catalytically active form .

Although the search results don't explicitly detail how glycosylation affects R215 site accessibility, it's established in the broader literature that proper glycosylation can influence protein conformation and thereby affect the accessibility of proteolytic cleavage sites. Researchers investigating the interplay between these post-translational modifications should consider both glycosylation status and proteolytic processing when analyzing ADAM17 activation in their experimental systems .

How does the R215I mutation affect ADAM17 function and what are its implications for neurological disorders?

The R215I mutation in ADAM17 (rs142946965) has been identified as a rare nonsynonymous variant (SNV) that co-segregates with an autosomal-dominant pattern of late-onset Alzheimer's disease (AD) . This mutation inhibits pro-protein cleavage and the formation of the active enzyme, leading to loss-of-function of ADAM17 α-secretase activity .

Mechanistically, the R215I mutation affects the boundary cleavage site between the pro-domain and catalytic domain of ADAM17, preventing proper processing and activation of the enzyme . This impaired activation results in reduced ADAM17 activity, which has downstream effects on substrate processing and signaling pathways regulated by ADAM17.

Research has identified a strong negative correlation between ADAM17 and APP gene expression in human brain tissues, and in vitro evidence indicates that ADAM17 negatively controls the expression of APP . Consequently, the p.R215I mutation leads to elevated Aβ formation in vitro, potentially explaining its association with Alzheimer's disease pathology . This connects ADAM17 dysfunction directly to the amyloid cascade that is central to AD pathogenesis.

These findings suggest that ADAM17 may be a potential therapeutic target for AD and that screening for ADAM17 variants might be valuable in identifying individuals at risk for late-onset AD. Furthermore, these insights provide a molecular mechanism linking ADAM17 processing, specifically at the R215 site, to neurodegenerative processes .

What experimental approaches can be used to distinguish between upstream PC site processing and R215 boundary site processing of ADAM17?

Distinguishing between processing at the upstream PC site (RKR58/D59) and the boundary site (RVKR214/R215) requires a combination of molecular and biochemical techniques :

• Site-directed mutagenesis: Creating mutations at either the upstream site (US mutant), the boundary site (BS mutant), or both sites (2M mutant) can help assess the contribution of each site to ADAM17 processing and activity .

• Western blotting with N-terminal sequencing: After processing by proprotein convertases like furin, wild-type ADAM17 yields two protein fragments: a 35-kDa catalytic domain fragment with N-terminal sequence 215RADPD, and a 15-kDa pro-domain fragment with N-terminal sequence 59DLQTS . By analyzing the size and N-terminal sequences of the resulting fragments, researchers can determine which cleavage sites were utilized.

• Enzymatic activity assays: Fluorogenic peptide substrates can be used to measure the catalytic activity of processed ADAM17. This approach revealed that even when the upstream site mutant (US) was cleaved at the boundary site, the resulting product could not process the fluorogenic peptide, suggesting that the pro-domain remained bound to the catalytic domain despite cleavage .

• Native gel electrophoresis: This technique can assess whether the pro-domain and catalytic domain remain associated after processing. Results showed that in the US mutant, the pro-domain remained in complex with the catalytic domain even after cleavage at the boundary site, indicating that processing at the upstream PC site is necessary for complete dissociation of the pro-domain .

• Immunodetection with specific antibodies: Antibodies that specifically recognize the cleaved form resulting from processing at either site can help track the processing status in various experimental conditions .

These approaches collectively demonstrated that full dissociation of the pro-domain, which is necessary for complete activation of ADAM17, is achieved only after cleavage at the non-canonical upstream PC site RKR58/D59 .

How do phosphorylation events interact with proteolytic processing to regulate ADAM17 activity?

ADAM17 activity is regulated by a complex interplay between proteolytic processing and phosphorylation events . Several key phosphorylation sites have been identified that significantly influence ADAM17-mediated ectodomain shedding:

• Threonine-735 (Thr-735): Phosphorylation at this residue by p38 MAP kinase (MAPK14) is required for ADAM17-mediated ectodomain shedding activity . This phosphorylation event appears to be critical for the proper function of the mature enzyme.

• Serine-819 (Ser-819) and Serine-791 (Ser-791): Stimulation by growth factors or phorbol 12-myristate 13-acetate induces phosphorylation of Ser-819 while simultaneously decreasing phosphorylation of Ser-791 . This differential phosphorylation pattern suggests a coordinated regulatory mechanism that fine-tunes ADAM17 activity in response to cellular signaling.

The relationship between phosphorylation and proteolytic processing appears to be sequential and interdependent. While proteolytic processing at both the upstream PC site and the R215 boundary site is necessary for removing the inhibitory pro-domain and generating the potentially active enzyme , subsequent phosphorylation events, particularly at Thr-735, are required for the functionally mature enzyme to efficiently perform its shedding activities .

This dual regulation through proteolytic processing and phosphorylation provides cells with multiple checkpoints to control ADAM17 activity, allowing for precise modulation in different physiological and pathological contexts. Researchers investigating ADAM17 regulation should consider monitoring both processing status at R215 and phosphorylation status at key regulatory sites to fully understand the activation state of the enzyme in their experimental systems .

What are the optimal conditions and protocols for using Cleaved-ADAM17 (R215) Antibody in Western blot experiments?

When conducting Western blot experiments with the Cleaved-ADAM17 (R215) Antibody, researchers should follow these optimized protocols for best results:

Sample Preparation:

• Wash cells with ice-cold PBS before lysis to remove media components that might interfere with detection .

• Use ice-cold standard RIPA buffer containing 10 mM 1,10-phenanthroline (to inhibit metalloproteases) and protease inhibitor cocktail (e.g., Complete, Roche) .

• Include 10 mM 1,10-phenanthroline in lysis buffers to prevent auto-catalytic degradation of ADAM17 .

Western Blot Protocol:

• Dilution Range: Use the antibody at a dilution of 1:500-1:2000 in appropriate blocking buffer .

• Detection System: SuperSignal West Femto Chemiluminescent Substrate (Pierce) has been successfully used for visualization .

• Controls: Include beta-Actin or alpha-Tubulin as loading controls for normalization .

• Expected Bands:

  • Mature/cleaved ADAM17: ~85-90 kDa

  • Processed catalytic domain after furin cleavage: ~35 kDa (N-terminal sequence 215RADPD)

  • Pro-domain fragment: ~15 kDa (N-terminal sequence 59DLQTS)

Data Analysis:

• Normalize signals to loading controls for quantitative analysis .

• For statistical comparisons, non-parametric tests like Kruskal-Wallis with Dunn's multiple comparison test are appropriate for Western blot data .

Troubleshooting Tips:

• If detecting multiple bands, consider using deglycosylation with PNGase F to remove N-glycans that might cause heterogeneity in band patterns .

• Store the antibody at -20°C for up to 1 year from the date of receipt, and avoid repeat freeze-thaw cycles to maintain activity .

• The antibody formulation (liquid in PBS containing 50% Glycerol, 0.5% BSA, and 0.02% Sodium Azide) helps maintain stability during storage .

What controls and validation steps should be implemented when using Cleaved-ADAM17 (R215) Antibody in experimental settings?

Implementing appropriate controls and validation steps is crucial for ensuring the reliability and specificity of results when using the Cleaved-ADAM17 (R215) Antibody:

Positive Controls:

• Cell lines or tissues known to express high levels of activated ADAM17.

• Recombinant ADAM17 catalytic domain with the correct N-terminal sequence (215RADPD) .

• Samples treated with phorbol 12-myristate 13-acetate (PMA), which stimulates ADAM17 activation through phosphorylation .

Negative Controls:

• ADAM17-knockout cell lines or tissues.

• Samples treated with broad-spectrum metalloprotease inhibitors like 1,10-phenanthroline .

• Cell lines expressing the 2M mutant (mutations in both upstream and boundary PC sites), which remains as an uncleaved inactive zymogen .

Specificity Validation:

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (amino acids 196-245 of human ADAM17) to confirm binding specificity .

• Cross-reactivity Assessment: Test the antibody against related ADAM family members (particularly ADAM10) to ensure specificity for ADAM17.

• siRNA Knockdown: Reduce ADAM17 expression using siRNA and confirm corresponding reduction in antibody signal.

Functional Validation:

• Correlate with Activity Assays: Use fluorogenic peptide substrates to measure ADAM17 activity and correlate with antibody detection of cleaved ADAM17 .

• Substrate Shedding Assays: Monitor shedding of known ADAM17 substrates (e.g., TNF-α) and correlate with detection of cleaved ADAM17.

Processing State Validation:

• Native Gel Electrophoresis: Complement denaturing SDS-PAGE with native gel electrophoresis to assess whether the pro-domain has fully dissociated from the catalytic domain .

• N-terminal Sequencing: When feasible, confirm the identity of detected bands by N-terminal sequencing to verify cleavage at the R215 site .

Implementing these control and validation steps will enhance confidence in experimental results and facilitate accurate interpretation of data generated using the Cleaved-ADAM17 (R215) Antibody.

How can researchers effectively use Cleaved-ADAM17 (R215) Antibody in ELISA applications?

For effective use of Cleaved-ADAM17 (R215) Antibody in ELISA applications, researchers should follow these recommendations:

ELISA Protocol Optimization:

• Antibody Dilution: Use the recommended dilution of 1:20000 for ELISA applications . This high dilution reflects the sensitivity of the antibody in this format and helps minimize background.

• Coating Buffer: For direct ELISA, use standard carbonate/bicarbonate buffer (pH 9.6) or phosphate buffer (pH 7.4) for coating the plate with target protein.

• Blocking Buffer: Use PBS containing 1-5% BSA and 0.05% Tween-20 to minimize non-specific binding. The formulation already contains 0.5% BSA, which helps reduce background .

• Sample Preparation: Prepare cell or tissue lysates using RIPA buffer containing protease inhibitors as described for Western blot applications .

• Detection System: Use an appropriate secondary antibody (anti-rabbit IgG) conjugated to HRP or other detection enzymes, as this is an unconjugated rabbit polyclonal antibody .

Assay Designs:

• Sandwich ELISA: This format can be used when pairing with a capture antibody that recognizes a different epitope of ADAM17.

  • Capture antibody: Use antibodies targeting conserved regions outside the 196-245 aa range.

  • Detection: Use the Cleaved-ADAM17 (R215) Antibody followed by HRP-conjugated anti-rabbit IgG.

• Direct ELISA: Useful for measuring levels of cleaved ADAM17 in purified preparations or simple sample matrices.

  • Coat wells directly with sample containing ADAM17.

  • Detect with Cleaved-ADAM17 (R215) Antibody followed by appropriate secondary antibody.

• Competitive ELISA: Beneficial for complex samples where interference might be an issue.

  • Pre-incubate samples with Cleaved-ADAM17 (R215) Antibody.

  • Add to wells coated with purified cleaved ADAM17 or immunizing peptide.

  • Detect bound antibody using appropriate secondary antibody.

Quantification and Standards:

• Generate a standard curve using recombinant ADAM17 catalytic domain with the correct N-terminal sequence (215RADPD) .

• Include a range of standards to ensure sample measurements fall within the linear range of the assay.

• Normalize data to total protein concentration in samples to account for sample-to-sample variation.

Controls and Validation:

• Include positive and negative controls as described in the previous section.

• Perform spike-and-recovery experiments to assess matrix effects in complex samples.

• Validate ELISA results against Western blot data when possible to confirm detection of the correctly processed form of ADAM17.

Following these guidelines will help researchers develop sensitive and specific ELISA-based assays for detecting cleaved ADAM17 in various research contexts.

What are the key considerations when investigating the relationship between ADAM17 processing at R215 and its role in disease models?

When investigating the relationship between ADAM17 processing at R215 and its role in disease models, researchers should consider several key aspects:

Experimental Design Considerations:

• Disease-Relevant Cell and Tissue Models:

  • Select cell types that express ADAM17 and are relevant to the disease being studied.

  • For Alzheimer's disease studies, neuronal and glial cell models are appropriate .

  • Consider using patient-derived cells or tissues when available, particularly for genetic variants like R215I .

• Genetic Manipulation Approaches:

  • Site-directed mutagenesis to create disease-associated mutations (e.g., R215I) in ADAM17 .

  • CRISPR/Cas9 genome editing to introduce or correct mutations at endogenous loci.

  • siRNA or shRNA knockdown of ADAM17 to assess loss-of-function effects.

• Functional Readouts:

  • Monitor shedding of disease-relevant ADAM17 substrates.

  • For Alzheimer's disease, measure APP processing and Aβ formation .

  • Assess downstream signaling pathways affected by ADAM17-mediated shedding.

• Correlative Analyses:

  • Examine correlations between ADAM17 expression/activity and disease biomarkers.

  • The negative correlation between ADAM17 and APP gene expression in human brain provides a model for such analyses .

Analytical Approaches:

• Processing State Assessment:

  • Use the Cleaved-ADAM17 (R215) Antibody to monitor processing status in disease models .

  • Complement with measurements of enzymatic activity using fluorogenic substrates .

  • Assess pro-domain dissociation using native gel electrophoresis .

• Phosphorylation Status:

  • Monitor disease-relevant phosphorylation events that regulate ADAM17 activity.

  • Assess phosphorylation at Thr-735, Ser-819, and Ser-791 in disease contexts .

• Gene Expression Analysis:

  • Examine correlations between ADAM17 and target gene expression.

  • Use tools like the MERAV database for gene expression correlation analyses .

  • Apply appropriate statistical methods (e.g., linear model fit, correlation tests in R) .

Translational Considerations:

• Genetic Screening:

  • Screen for ADAM17 variants in patient populations.

  • The identification of R215I mutation in late-onset AD patients provides a precedent .

• Biomarker Development:

  • Assess whether cleaved ADAM17 levels correlate with disease progression.

  • Determine if the ratio of processed to unprocessed ADAM17 has diagnostic value.

• Therapeutic Targeting:

  • Explore approaches to modulate ADAM17 processing at R215 as potential interventions.

  • Consider the dual regulation through both proteolytic processing and phosphorylation when designing therapeutic strategies .

By addressing these considerations, researchers can develop comprehensive approaches to understanding how ADAM17 processing at R215 contributes to disease pathogenesis and identify potential therapeutic avenues based on this mechanism.

How can researchers differentiate between the effects of mutations at the upstream PC site versus the R215 boundary site in experimental systems?

To differentiate between the effects of mutations at the upstream PC site (RKR58/D59) versus the R215 boundary site (RVKR214/R215) in experimental systems, researchers can implement the following comprehensive approach:

Mutant Construction and Expression:

• Generate a panel of ADAM17 mutants:

  • Wild-type (WT) ADAM17

  • Upstream site mutant (US): mutation at RKR58/D59

  • Boundary site mutant (BS): mutation at RVKR214/R215

  • Double mutant (2M): mutations at both sites

• Expression systems:

  • Transiently transfect constructs into appropriate cell lines

  • Consider stable cell lines for long-term studies

  • Use inducible expression systems to control timing and level of expression

Biochemical Characterization:

• Processing Pattern Analysis:

  Mutant	Upstream Processing	Boundary Processing	Full-Length Protein	Catalytic Domain	Pro-Domain	Activity
  WT	Yes	Yes	Minimal	35 kDa (215RADPD)	15 kDa (59DLQTS)	High
  US	No	Yes	Partial	35 kDa (215RADPD)	20 kDa (intact)	Low
  BS	Yes	No	Partial	Modified size	15 kDa (59DLQTS)	Moderate
  2M	No	No	Predominant	Not detected	Not cleaved	None

• Native Gel Electrophoresis:

  • Assess pro-domain dissociation patterns in different mutants

  • WT and BS mutants show similar migration patterns after furin treatment

  • US and 2M mutants show distinct patterns indicating pro-domain remains associated with catalytic domain

Functional Assays:

• Enzymatic Activity Measurements:

  • Use fluorogenic peptide substrates to quantify catalytic activity

  • WT and BS mutants show activity after furin treatment

  • US and 2M mutants show minimal to no activity despite partial processing of US

• Substrate Shedding Assays:

  • Monitor shedding of known ADAM17 substrates (e.g., TNF-α)

  • Compare shedding efficiency between wild-type and mutant constructs

  • Correlate with processing status at each site

Structural and Interaction Studies:

• Co-immunoprecipitation:

  • Assess interaction between pro-domain and catalytic domain in different mutants

  • US mutant should show persistent interaction despite boundary site cleavage

• Protein Conformation Analysis:

  • Limited proteolysis to assess conformational differences

  • Circular dichroism spectroscopy to detect structural changes

  • Thermal shift assays to measure protein stability differences

Cellular Localization and Trafficking:

• Immunofluorescence microscopy:

  • Track cellular localization of different mutants

  • Assess co-localization with compartment markers

• Cell surface biotinylation:

  • Quantify surface expression of different mutants

  • Compare surface/total ratios to assess trafficking efficiency

Comparative Data Analysis:

The following comparative approach will clearly distinguish the effects of mutations at different sites:

• Processing efficiency: Compare the ratio of processed to unprocessed ADAM17 for each mutant using Western blot with densitometry.

• Structure-function correlation: Plot enzymatic activity against processing status for each mutant to visualize the relationship between processing at each site and functional outcomes.

• Statistical analysis: Apply appropriate statistical tests (e.g., non-parametric Kruskal-Wallis test and Dunn's multiple comparison test) to determine significant differences between mutants .