HTRA1 Antibody

What is HTRA1 and what cell/tissue types express it?

HTRA1, also known as HTRA, PRSS11, and L56, belongs to the peptidase S1B family. It functions as a protease that regulates the availability of various proteins in cellular pathways. HTRA1 is expressed in multiple tissue types with significant expression observed in:

• Human tissues: Placenta shows strong expression as demonstrated in immunohistochemistry studies

• Cell lines: L02 cells, HAP1 cells, and HepG2 cells consistently show HTRA1 expression in Western blot analyses

• Animal tissues: Mouse brain tissue exhibits detectable levels of HTRA1 protein

• Vascular system: Vascular smooth muscle cells (VSMCs) express HTRA1, where it plays a crucial role in vascular maturation and homeostasis

• Renal system: HTRA1 serves as a podocyte antigen in the glomerular system

HTRA1 has a calculated molecular weight of 51 kDa, though observed molecular weights in experimental settings may vary (35 kDa, 42 kDa, and 50 kDa) depending on post-translational modifications and experimental conditions .

What applications are validated for HTRA1 antibodies?

HTRA1 antibodies have been validated for multiple research applications with specific dilution recommendations:

It is recommended to titrate the antibody in each testing system to obtain optimal results, as the effective concentration may be sample-dependent .

How should HTRA1 antibodies be stored for maximum stability?

For optimal preservation of antibody activity:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• HTRA1 antibodies in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) are typically stable for one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage

• Small volume formats (20μl) may contain 0.1% BSA as a stabilizing agent

What controls should be included when using HTRA1 antibodies?

To ensure experimental validity:

• Positive controls: Use L02 cells, HAP1 cells, HepG2 cells, or mouse brain tissue for Western blot applications

• Negative controls: HTRA1 knockout or knockdown samples provide optimal negative controls, as demonstrated in studies using HtrA1-knockout mice for antibody development

• Specificity verification: Compare HTRA1 wild-type samples with HtrA1 serine-to-alanine catalytically inactive mutants to confirm specificity in activity-based assays

• Cross-reactivity assessment: Test against human, mouse, and rat samples, as the antibody shows documented reactivity with these species

How are HTRA1 antibodies utilized in therapeutic development?

HTRA1 antibodies have shown significant potential in therapeutic development, particularly for age-related macular degeneration (AMD):

• Development approach: Anti-HtrA1 antibodies can be obtained by immunizing HtrA1-knockout mice with recombinant HtrA1 protease domain (HtrA1-PD)

• Optimization process: The therapeutic development pipeline typically includes:

  • Initial hybridoma screening (identifying clone 15H6 with strong inhibition of HtrA1 enzymatic activity)

  • Humanization through CDR grafting into human consensus frameworks

  • Modification of problematic residues (N94A for cleavage, D55 for isomerization)

  • Affinity maturation via Fab phage display with deep-sequencing analysis

• Clinical application: The development of HtrA1-blocking Fab fragments has enabled testing of the therapeutic hypothesis that HtrA1 protease activity contributes to AMD progression

• Significance: Genome-wide association studies have identified genetic variation at the ARMS2/HTRA1 locus as a risk factor for AMD development and progression, making HtrA1 a relevant therapeutic target

What methodologies can confirm target engagement of anti-HTRA1 antibodies in vivo?

Confirming target engagement is crucial for validating anti-HTRA1 antibody efficacy:

• Activity-based probes (ABPs):

  • Development of HtrA1-directed ABPs using diphenyl phosphonate as the reactive group targeting the nucleophilic active-site serine residue

  • Incorporation of Val and Leu at P1 and P2 positions to direct ABP reactivity against HtrA1

  • Addition of carboxytetramethylrhodamine (TAMRA) through a polyethylene glycol linker as a fluorescent reporter

• N-terminomic proteomic profiling:

  • Compare amino termini of proteins in biological samples (e.g., rat vitreous humor) upon incubation with HtrA1 versus catalytically inactive HtrA1

  • Identify signature cleavage patterns specific to HtrA1 activity

  • Monitor changes in substrate processing upon antibody administration

• Pharmacodynamic biomarkers:

  • Dickkopf-related protein 3 (DKK3) has been identified as a robust biomarker for anti-HtrA1 activity

  • This biomarker can be measured in both preclinical animal models and clinical samples

  • Successfully employed in a phase 1 study of geographic atrophy patients to demonstrate anti-HtrA1 Fab activity and duration

What role does HTRA1 play in cellular signaling pathways?

HTRA1 functions as a critical regulator of multiple signaling pathways:

• TGFβ signaling regulation:

  • Loss of HTRA1 increases TGFβ1 availability and signaling

  • HTRA1 can cleave either pro-TGFβ1 or GFD6

  • HTRA1 silencing leads to elevated phosphorylated SMAD2/3 proteins

  • Increased SMAD-dependent luciferase activity is observed in co-culture experiments with HTRA1-silenced vascular smooth muscle cells

• Notch signaling modulation:

  • The Notch ligand JAG1 is a substrate for HTRA1

  • HTRA1 cleaves JAG1 in the cytosol, leading to rapid degradation of the remaining JAG1 protein

  • JAG1/NOTCH3 signaling is crucial for differentiation, maintenance, and contractility of vascular smooth muscle cells

  • Loss of HTRA1 leads to over-activation of NOTCH3 signaling

• Synergistic effects:

  • TGFβ and Notch pathways synergistically stimulate expression of HES and HEY transcriptional repressors

  • Moderate overexpression of NOTCH3-ICD increases SM22α protein levels

  • Higher NOTCH3-ICD expression decreases SM22α expression, indicating hyper-activation of a negative feedback loop

  • Combined overexpression of HES1 and HEYL represses transcription of α-SMA and SM22α in human umbilical artery smooth muscle cells

How can HTRA1 antibodies be used in studying pathological conditions?

HTRA1 antibodies serve as valuable tools in investigating various disease states:

• Age-related macular degeneration (AMD):

  • Anti-HtrA1 Fab fragments help test the hypothesis that HtrA1 protease activity contributes to AMD progression

  • DKK3 serves as a pharmacodynamic biomarker in aqueous humor samples from geographic atrophy patients, providing evidence of anti-HtrA1 Fab activity and duration in phase 1 clinical studies

• Membranous nephropathy (MN):

  • HTRA1 has been identified as a novel podocyte antigen in a subset of patients with primary MN

  • Patient sera react by immunoblotting with a 51-kD protein within glomerular extracts

  • Anti-HTRA1 antibody titers appear to correlate with disease course

  • Serial monitoring of anti-HTRA1 antibodies could facilitate diagnostic and therapeutic decisions in this demographic group (mean age 67.3 years)

• Vascular disorders:

  • Loss of HTRA1 in vascular smooth muscle cells (VSMCs) impairs their maturation and function

  • HTRA1 is essential for vascular homeostasis through its regulation of Notch and TGFβ signaling

  • HTRA1 antibodies can help investigate the role of this protease in familial small vessel disease, where blood vessel functions are impaired

What are the methodological considerations when designing experiments to identify HTRA1 substrates?

When investigating HTRA1 substrates, researchers should consider:

• N-terminomics approach:

  • Compare amino termini of proteins in biological samples with and without active HTRA1

  • Incubate samples with HtrA1 or catalytically inactive HtrA1 (HtrA1-SA mutant)

  • Use mass spectrometry to identify cleaved peptides

• Activity-based profiling:

  • Develop HtrA1-directed activity-based probes

  • Design probes based on consensus cleavage sites determined by N-terminomics

  • Include appropriate reactive groups (e.g., diphenyl phosphonate) and reporter tags (e.g., TAMRA)

• Cross-species validation:

  • Perform experiments in multiple species to identify conserved substrates

  • This approach has successfully identified ocular substrates that were consistent across independent cross-species studies

• Biomarker identification:

  • Test potential substrates as pharmacodynamic biomarkers

  • Assess correlation between substrate cleavage and HtrA1 activity

  • Validate biomarker utility in both preclinical models and clinical samples

  • Example: Dickkopf-related protein 3 was identified as a robust biomarker for anti-HtrA1 activity

What are the optimal antigen retrieval methods for HTRA1 immunohistochemistry?

For effective immunohistochemical detection of HTRA1:

• Primary recommendation: Antigen retrieval with TE buffer at pH 9.0

• Alternative method: Antigen retrieval with citrate buffer at pH 6.0

• Validated tissue: Human placenta tissue shows positive IHC results

• Recommended dilution: 1:50-1:500, with optimization for specific tissue types

How can researchers differentiate between HTRA1 isoforms?

When analyzing HTRA1 expression:

• Expected molecular weights:

  • Calculated molecular weight: 51 kDa

  • Observed molecular weights: 35 kDa, 42 kDa, and 50 kDa

  • Variations likely represent alternative splicing, post-translational modifications, or proteolytic processing

• Verification strategy:

  • Use HTRA1 knockout/knockdown controls to confirm specificity

  • Compare with recombinant HTRA1 proteins of known molecular weight

  • Perform immunoprecipitation followed by mass spectrometry to confirm identity of bands

How should experimental design be modified when studying HTRA1 in different species?

For cross-species HTRA1 research:

• Validated reactivity: HTRA1 antibodies have been tested and confirmed to react with human, mouse, and rat samples

• Cited reactivity: Published literature reports additional reactivity with zebrafish samples

• Cross-species validation: Important substrates of HTRA1 have been identified through cross-species studies, suggesting conservation of function

• Knockout models: HtrA1-knockout mice have been generated by traditional homologous recombination methods and serve as valuable tools for specificity controls and comparative studies

What controls should be included when measuring HTRA1 activity inhibition?

To properly assess HTRA1 inhibition by antibodies:

• Enzymatic activity controls:

  • Positive control: Active recombinant HtrA1 protease domain

  • Negative control: Catalytically inactive version (HtrA1-SA mutant)

  • Dose-response measurements with varying antibody concentrations

• Substrate processing:

  • Monitor cleavage of known HTRA1 substrates (e.g., DKK3)

  • Use activity-based probes to directly measure active HTRA1

  • Employ reporter systems like SMAD-dependent luciferase assays to measure downstream signaling effects

• Signaling pathway analysis:

  • Measure phosphorylated SMAD2/3 levels to assess TGFβ pathway inhibition

  • Use gamma-secretase inhibitor DAPT as a control for Notch signaling inhibition

  • Use ALK5 inhibitor CAS 446859-33-2 as a control for TGFβ signaling inhibition

How can researchers enhance specificity when working with HTRA1 antibodies?

To improve specificity:

• Antibody selection: Use antibodies derived from HtrA1-knockout animals for immunization, which reduces background reactivity

• Purification method: Antigen affinity-purified antibodies offer higher specificity

• Validation approach: Confirm antibody specificity using multiple techniques (WB, IP, IHC, IF/ICC) and positive/negative controls

• Buffer optimization: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 provides optimal stability and specificity

What factors might influence HTRA1 antibody performance in different applications?

Performance variations may stem from:

• Sample preparation: Different lysis buffers, fixation methods, or antigen retrieval techniques can affect epitope accessibility

• Protein conformation: Native versus denatured conditions influence epitope exposure

• Background reduction: Increasing blocking agent concentration or adjusting antibody dilution can improve signal-to-noise ratio

• Application-specific considerations:

  • WB: Optimize transfer conditions and blocking agents

  • IHC: Test alternative antigen retrieval methods (TE buffer pH 9.0 versus citrate buffer pH 6.0)

  • IP: Adjust antibody-to-lysate ratio (0.5-4.0 μg antibody for 1.0-3.0 mg total protein)

  • IF/ICC: Consider permeabilization methods and fixation protocols

How can inconsistent results with HTRA1 antibodies be addressed?

When encountering variability:

• Antibody titration: Test multiple dilutions to determine optimal concentration for each application and sample type

• Controls expansion: Include both positive controls (L02 cells, HAP1 cells, HepG2 cells, mouse brain tissue) and negative controls (HTRA1 knockdown/knockout samples)

• Cross-validation: Confirm results using alternative detection methods or antibody clones

• Activity verification: For functional studies, use activity-based probes to confirm that observed effects correlate with HTRA1 enzymatic activity

How are HTRA1 antibodies advancing our understanding of disease mechanisms?

HTRA1 antibodies have facilitated significant discoveries:

• AMD pathogenesis: Anti-HtrA1 Fab inhibitors have demonstrated that HtrA1 proteolytic activity contributes to AMD progression

• Membranous nephropathy: Identification of HTRA1 as a novel podocyte antigen has expanded our understanding of this glomerular disease

• Vascular disorders: HTRA1 inhibition studies have revealed its role in vascular maturation and homeostasis through Notch and TGFβ signaling regulation

What are the future prospects for HTRA1 antibodies in clinical applications?

The translational potential includes:

• Diagnostic biomarkers: Anti-HTRA1 antibody titers may serve as biomarkers in membranous nephropathy, facilitating diagnosis and treatment decisions

• Therapeutic development: HtrA1-blocking antibodies show promise for treating AMD, with pharmacodynamic biomarkers like DKK3 enabling monitoring of therapeutic efficacy

• Personalized medicine: Genetic variation at the ARMS2/HTRA1 locus may inform patient selection for anti-HTRA1 therapies in AMD