ASPRV1 Antibody

In which tissues is ASPRV1 predominantly expressed?

ASPRV1 exhibits a distinct expression pattern across tissues:

Recent research has expanded our understanding of ASPRV1 expression beyond epithelial tissues. It is now recognized that neutrophils in both the immune and nervous systems express ASPRV1, particularly those that have infiltrated tissues during inflammatory responses . When selecting positive control tissues for antibody validation, skin samples are most commonly used due to reliable expression, but neutrophil-rich samples may also be considered for specific research applications.

What applications are ASPRV1 antibodies validated for in research settings?

ASPRV1 antibodies have been validated for several research applications:

When conducting experiments, it's crucial to optimize antibody concentrations for your specific application and sample type. For instance, when performing Western blot on mouse skin tissue versus mouse brain tissue, different concentrations may be required for optimal results due to varying expression levels . Additionally, always include appropriate positive controls (e.g., skin tissue) and negative controls (e.g., simple epithelial tissue) to validate antibody specificity.

What is the functional role of ASPRV1 in different biological contexts?

ASPRV1 has distinct functions depending on tissue context:

• In stratified epithelia: ASPRV1 was initially characterized as processing profilaggrin, its only known substrate in the skin. This process is important for skin barrier formation, hydration, and normal skin development. ASPRV1-deficient mice show fine wrinkles and reduced skin hydration, indicating its role in maintaining skin integrity .

• In neutrophils and inflammation: Recent research has revealed a novel role for ASPRV1 in neutrophil-mediated inflammation. ASPRV1 is specifically expressed by neutrophils that infiltrate the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. These neutrophils are distinguished by their expression of ICAM1 (CD54) and acquire macrophage-like properties. ASPRV1 appears essential for the progression from acute to chronic inflammation, particularly in B cell-dependent autoimmune processes .

In experimental models, mice lacking ASPRV1 develop less severe chronic phase EAE when immunized with B cell-dependent myelin antigens, with many animals showing complete resolution of symptoms. This suggests ASPRV1 plays a critical role in perpetuating chronic inflammatory responses in certain autoimmune conditions . The substrate(s) for ASPRV1 in the inflammatory context remain to be identified and represent an important area for future investigation.

How is ASPRV1 regulated at the molecular level?

ASPRV1 is synthesized as a zymogen containing a putative transmembrane domain and a conserved catalytic domain with a key aspartic acid residue. Regulation occurs through several mechanisms:

• Activation by autocleavage: The zymogen undergoes autocleavage under slightly acidic conditions (pH ~5.5-6.5), releasing the catalytic domain that homodimerizes to form an active protease . This pH-dependent activation provides a regulatory mechanism that restricts ASPRV1 activity to specific cellular compartments or microenvironments with appropriate acidity.

• Tissue-specific expression: ASPRV1 expression is tightly regulated at the transcriptional level, with high expression in stratified epithelia but undetectable levels in simple epithelia . This suggests tissue-specific transcription factors control its expression.

• Disease-associated regulation: ASPRV1 levels are elevated in benign skin tumors but downregulated in squamous cell carcinomas , indicating complex regulatory mechanisms responding to pathological conditions.

• Inflammation-induced expression: In neutrophils, ASPRV1 expression appears to be induced during inflammatory responses, particularly in cells that have extravasated into tissue parenchyma. ICAM1+ neutrophils in the CNS during EAE show significant ASPRV1 expression compared to circulating neutrophils .

Understanding these regulatory mechanisms is crucial when designing experiments to study ASPRV1 function, as cellular conditions (particularly pH) may significantly affect enzyme activity and detection.

What is the role of ASPRV1 in experimental autoimmune encephalomyelitis (EAE) and how can ASPRV1 antibodies help study this model?

ASPRV1 plays a significant role in EAE progression, particularly in B cell-dependent forms of the disease. Research has revealed:

• Expression pattern: ASPRV1 increases in the CNS during EAE and in severe cases of multiple sclerosis. It is specifically expressed by neutrophils that have infiltrated the CNS parenchyma and express ICAM1 (CD54) .

• Functional significance: Mice lacking ASPRV1 develop a less severe chronic phase of EAE when immunized with B cell-dependent myelin antigen. Many ASPRV1-deficient animals show complete resolution of symptoms, suggesting ASPRV1 is essential for maintaining chronic inflammation .

• Cell type specificity: ASPRV1 is expressed by a specific subset of neutrophils that acquire macrophage-like properties. These ICAM1+ neutrophils can be distinguished from intravascular neutrophils that remain ICAM1-negative .

ASPRV1 antibodies can be used to:

• Identify ASPRV1-expressing cells in CNS tissues during EAE progression through immunohistochemistry

• Quantify ASPRV1 expression levels in tissue lysates via Western blot at different disease stages

• Perform co-localization studies with neutrophil markers (Ly6G) and ICAM1 to identify the specific cell population expressing ASPRV1

• Compare ASPRV1 expression in different EAE models (e.g., MOG35-55 peptide-induced versus B cell-dependent models)

When designing experiments to study ASPRV1 in EAE, consider time course analysis, as ASPRV1 expression may change throughout disease progression, with potential differences between acute and chronic phases.

How can researchers distinguish between different neutrophil populations using ASPRV1 antibodies?

Recent research has identified distinct neutrophil populations in inflammatory conditions, with ASPRV1 serving as a potential marker for specific subsets. To distinguish these populations:

• Combined marker approach: Use ASPRV1 antibodies in conjunction with other markers to identify specific neutrophil subsets. For example:

• Flow cytometry protocol: To distinguish these populations:

  • Isolate cells from tissue of interest (e.g., spinal cord in EAE)

  • Stain for surface markers (CD45, CD11b, Ly6G, ICAM1)

  • Fix and permeabilize cells for intracellular ASPRV1 staining

  • Analyze by flow cytometry, gating first on CD45hiCD11b+Ly6G+ cells, then separating ICAM1+ from ICAM1- populations, and finally assessing ASPRV1 expression

• Immunofluorescence approach: For tissue localization, perform multiplex immunofluorescence with antibodies against:

  • Ly6G (neutrophil marker)

  • ICAM1 (to distinguish subpopulations)

  • ASPRV1 (to confirm expression)

  • Vascular markers (e.g., CD31) to determine intravascular versus parenchymal location

This approach allows visualization of ASPRV1-expressing neutrophils in situ and confirmation of their parenchymal localization .

How can ASPRV1 be targeted to modulate chronic inflammation in experimental models?

Based on research showing ASPRV1's role in maintaining chronic inflammation, several approaches can be used to target this protease:

• Genetic approaches:

  • Generate conditional knockout models using Cre-lox systems with neutrophil-specific promoters

  • Use CRISPR/Cas9 to create point mutations in the catalytic domain to study specific activity-dependent functions

  • Implement inducible knockdown systems to modulate ASPRV1 expression at different disease stages

• Pharmacological approaches:

  • Develop specific aspartic protease inhibitors targeting ASPRV1's catalytic site

  • Screen existing aspartic protease inhibitors for activity against ASPRV1

  • Design peptide-based inhibitors mimicking known substrates

• Antibody-based approaches:

  • Develop function-blocking antibodies against ASPRV1's catalytic domain

  • Use intracellular antibody delivery methods to target ASPRV1 within neutrophils

  • Create bispecific antibodies targeting both ASPRV1 and neutrophil-specific surface markers

When evaluating ASPRV1 targeting strategies, researchers should assess:

• Effects on both acute and chronic phases of inflammation

• Cell-type specificity of intervention

• Potential off-target effects on other aspartic proteases

• Impact on normal neutrophil functions versus pathological activities

Since ASPRV1 deficiency shows minimal physiological impact under normal conditions (with only fine wrinkles and reduced skin hydration in knockout mice) , it may represent a viable therapeutic target with limited side effects.

What are the technical considerations for detecting ASPRV1 in neutrophils versus epithelial tissues?

Detection of ASPRV1 in different tissue contexts requires specific technical considerations:

When designing experiments to detect ASPRV1 in neutrophils:

• Sample preparation: Process tissues quickly to preserve neutrophil integrity. For flow cytometry, use gentle dissociation methods and keep cells cold to minimize activation.

• Fixation protocols: For immunohistochemistry, optimize fixation to preserve neutrophil morphology while maintaining ASPRV1 antigenicity. Paraformaldehyde (4%) is generally suitable, but shorter fixation times (2-4 hours) may be preferable for neutrophil studies.

• Antibody validation: Confirm specificity using both positive controls (skin tissue) and negative controls (simple epithelia). For neutrophil studies, compare ICAM1+ and ICAM1- populations to verify differential expression .

• Counter-staining: When analyzing tissue sections, co-stain with neutrophil markers (Ly6G in mice, CD66b in humans) and ICAM1 to properly identify ASPRV1-expressing neutrophil subsets.

• Western blot analysis: When analyzing inflammatory tissues, be aware that multiple cell types may be present. Consider cell sorting prior to protein extraction to specifically analyze neutrophil populations.

How can researchers address the issue of multiple band detection when using ASPRV1 antibodies?

When performing Western blot analysis with ASPRV1 antibodies, researchers often encounter multiple bands. Here's how to interpret and troubleshoot this issue:

• Expected bands and their interpretation:

  • 37 kDa: Full-length ASPRV1 zymogen with transmembrane domain

  • 28 kDa: Processed active form following autocleavage

• Common issues and solutions:

• Validation approaches:

  • Use ASPRV1 knockout tissue as a negative control

  • Compare with recombinant ASPRV1 protein (full-length and processed forms)

  • Perform peptide competition assays to confirm specificity

  • Test antibodies targeting different epitopes of ASPRV1

• Technical optimization:

  • For improved band separation, use 10-12% acrylamide gels

  • Consider gradient gels for better resolution of both forms

  • Optimize transfer conditions for proteins in this molecular weight range

  • If studying processing dynamics, consider non-reducing conditions to preserve potential dimers

Understanding the biological significance of the different forms is crucial for proper data interpretation, as the ratio between 37 kDa and 28 kDa bands may provide information about ASPRV1 activation in your experimental system .

What approaches can researchers use to study ASPRV1 enzymatic activity beyond antibody-based detection?

While antibodies are valuable for detecting ASPRV1 protein, studying its enzymatic activity requires different approaches:

• Fluorogenic substrate assays:

  • Design peptide substrates containing a quenched fluorophore pair that fluoresces upon cleavage

  • Based on known ASPRV1 cleavage sites (e.g., from profilaggrin processing)

  • Perform assays at slightly acidic pH (5.5-6.5) to mimic ASPRV1 activation conditions

  • Include controls with specific aspartic protease inhibitors to confirm specificity

• Cell-based activity assays:

  • Express fluorescent protein-tagged substrates in cells with variable ASPRV1 expression

  • Monitor substrate cleavage through change in localization or FRET signal

  • Compare activity in wild-type versus ASPRV1 knockout cells

• Mass spectrometry approaches:

  • Perform proteomic analysis to identify cleaved peptides in biological samples

  • Compare peptide profiles between wild-type and ASPRV1-deficient samples

  • Use terminal amine isotopic labeling of substrates (TAILS) to identify ASPRV1-specific cleavage products

• In vitro reconstitution:

  • Express and purify recombinant ASPRV1 (both zymogen and catalytic domain)

  • Test processing of candidate substrates under controlled conditions

  • Characterize kinetic parameters and substrate specificity

• Activity-based probes:

  • Develop chemical probes that covalently bind to active ASPRV1

  • Label active enzyme in complex biological samples

  • Visualize activity patterns in tissues or cell populations

When designing activity assays, researchers should consider:

• The pH-dependence of ASPRV1 activation and activity

• Potential contributions from other aspartic proteases

• The cell type-specific context of ASPRV1 function

• Different substrate preferences in epithelial versus inflammatory settings

These approaches complement antibody-based detection methods and provide crucial information about ASPRV1's functional role in different biological contexts.