UPB1 Antibody

What is UPB1 and what specific biological function does it perform?

UPB1 (Beta-ureidopropionase) belongs to the CN hydrolase family and catalyzes the final step in the pyrimidine degradation pathway. It converts N-carbamoyl-beta-alanine (3-ureidopropanoate) into beta-alanine, ammonia, and carbon dioxide. Similarly, it converts N-carbamoyl-beta-aminoisobutyrate (3-ureidoisobutyrate) into beta-aminoisobutyrate, ammonia, and carbon dioxide .

The pyrimidine bases uracil and thymine are degraded through sequential enzymatic processes involving:

• Dihydropyrimidine dehydrogenase (DHPDH)

• Dihydropyrimidinase (DHP)

• Beta-ureidopropionase (UPB1)

Deficiency in UPB1 is associated with N-carbamyl-beta-amino aciduria and can lead to neurological abnormalities .

Which tissue types show significant UPB1 expression and how should samples be prepared for antibody-based detection?

UPB1 is expressed in multiple tissues, with notable expression in:

• Liver (particularly fetal liver shows strong expression)

• Kidney

• Brain tissue (relevant for neurological studies)

For optimal sample preparation:

• For Western blot analysis: Extract proteins from tissue lysates and use 5-10 μg of protein. Human fetal liver lysate has been used successfully as a positive control .

• For immunohistochemistry: Use paraffin-embedded tissue sections with heat-mediated antigen retrieval using citrate buffer pH 6 before commencing with IHC staining protocols .

• For protein fractionation: Use NuPAGE® 4–12% Bis-Tris Mini Gels or similar systems, with transfer to nitrocellulose membranes .

How can UPB1 antibodies be incorporated into investigations of UPB1 gene mutations and their functional consequences?

UPB1 antibodies provide crucial tools for studying the effects of gene mutations on protein expression, stability, and function:

• Protein Expression Analysis: Western blot analysis using UPB1 antibodies can reveal whether specific mutations affect protein expression levels. Cell supernatants containing 5 μg protein can be fractionated on gradient gels and probed with anti-UPB1 antibodies (typically at 1:1000 dilution) .

• Oligomerization Studies: Blue native PAGE followed by Western blot analysis using polyclonal anti-β-ureidopropionase can determine whether specific mutations affect the oligomeric state of the protein. This is particularly valuable since certain point mutations have been shown to prevent proper subunit association to larger oligomers, affecting enzyme functionality .

• Mutant Protein Characterization: After site-directed mutagenesis to introduce UPB1 mutations (using tools like QuikChange™ Site-Directed Mutagenesis Kit), antibodies can be used to detect expression and stability of mutant proteins. This approach has been used to study how various missense mutations affect UPB1 protein structure and function .

• Correlation with Clinical Phenotypes: Combining antibody-based detection with clinical data can help establish genotype-phenotype correlations, as seen in studies of patients with β-ureidopropionase deficiency .

What methodological approaches are recommended for validating UPB1 antibody specificity in experimental systems?

For robust validation of UPB1 antibody specificity, researchers should implement multiple strategies:

• Multiple Antibody Approach: Use different antibodies targeting distinct UPB1 epitopes. For example, comparing results between rabbit monoclonal antibodies like EPR9132 and EPR9133(B), which recognize different epitopes .

• Knockout/Knockdown Controls: Include UPB1 knockout or knockdown samples as negative controls to confirm signal specificity.

• Recombinant Protein Controls: Use purified recombinant UPB1 protein as a positive control for antibody binding.

• Co-labeling Experiments: Combine UPB1 antibody with secondary detection antibodies that have minimal cross-reactivity:

  • IRDye800 conjugated goat anti-rabbit

  • IRDye680 conjugated donkey anti-mouse

• Blocking Experiments: Pre-incubate antibodies with immunogen peptides to demonstrate specific blocking of signal.

• Western Blot Validation: Confirm antibody detects a band of expected size (approximately 43 kDa for UPB1) .

How do researchers combine genetic screening and antibody-based methods for comprehensive UPB1 deficiency diagnosis?

Integrating genetic and antibody-based methods provides a comprehensive approach to UPB1 deficiency diagnosis:

• Initial HRM-Based Screening: High Resolution Melting (HRM) analysis can rapidly screen for UPB1 gene mutations. This method has been established using DNA samples with known UPB1 mutations as controls:

  • Each mutation produces specific melting curve profiles that differ from wild-type

  • HRM clearly distinguishes heterozygote variants from homozygote variants

  • The method can correctly identify genetic mutations in patients with β-ureidopropinoase deficiency

• Confirmatory Sanger Sequencing: Following HRM analysis, suspicious fragments should be sequenced to confirm pathogenic mutations .

• Antibody-Based Protein Expression Analysis: Western blot analysis using UPB1 antibodies can evaluate protein expression levels in patient samples, complementing genetic findings.

• Biochemical Verification: Analysis of pyrimidine metabolites in patient samples (serum, urine, CSF) to detect elevated levels of N-carbamyl-β-alanine (NCβA) and N-carbamyl-β-aminoisobutyric acid (NCβAIBA), which strongly suggests UPB1 deficiency .

This integrated approach has successfully identified novel UPB1 mutations and confirmed diagnoses in patients with suspected β-ureidopropionase deficiency .

What are the technical challenges in detecting variant UPB1 proteins and how can they be overcome?

Detecting variant UPB1 proteins presents several technical challenges:

• Variant-Specific Epitope Changes: Missense mutations may alter epitope structure, potentially reducing antibody recognition. Solution: Use antibodies targeting conserved regions or multiple antibodies targeting different epitopes.

• Reduced Protein Stability: Some UPB1 variants show reduced stability, resulting in lower protein levels. Solution: Optimize protein extraction protocols and increase sample loading for Western blot detection.

• Alternative Splicing Effects: Splice-site mutations (e.g., c.[364+6T>G] and c.[916+1_916+2dup]) can lead to exon skipping and truncated proteins . Solution: Use antibodies that can detect N-terminal portions of the protein to identify truncated variants.

• Oligomerization Defects: Some mutations affect UPB1 protein oligomerization. Solution: Use blue native PAGE followed by Western blotting to analyze the oligomeric state of variant proteins .

• Antibody Specificity: Commercial antibodies may have varying specificity for different UPB1 variants. Solution: Validate antibody reactivity using recombinant proteins expressing the specific variants of interest.

How can researchers establish correlations between UPB1 protein detection and enzymatic activity in clinical samples?

To correlate UPB1 protein detection with enzymatic activity, researchers should implement:

• Parallel Analysis Approach:

  • Quantitative Western blot using calibrated UPB1 antibodies to measure protein levels

  • Enzymatic assays measuring conversion of N-carbamoyl-beta-alanine to beta-alanine

  • Correlation analysis between protein levels and enzyme activity

• Structure-Function Correlation:

  • Analyze how specific mutations affect both antibody detection signal and enzymatic function

  • Focus on mutations that affect substrate binding (e.g., p.Ser300Leu) or prevent proper subunit association

• Clinical Correlation:

  • Compare antibody-based protein detection with clinical biomarkers like elevated levels of N-carbamyl-β-amino acids in urine

  • For example, patients with homozygous p.R326Q and heterozygous p.G31S mutations show both protein expression changes and elevated metabolites

• Expression System Validation:

  • Express wild-type and variant UPB1 in cellular expression systems

  • Compare antibody detection with enzymatic activity measurements

  • Use site-directed mutagenesis to introduce specific mutations and assess their impact

What are the optimal blocking conditions and antibody dilutions for Western blot analysis of UPB1?

For optimal Western blot detection of UPB1, consider the following parameters:

Blocking Conditions:

• Use Odyssey blocking buffer (LI-COR) for minimal background

• Alternative blocking solution: 50% Odyssey blocking buffer, 50% PBS and 0.1% Tween

Antibody Dilutions and Conditions:

• Primary antibody:

  • Monoclonal anti-UPB1 (e.g., EPR9132, EPR9133(B)): 1:1000-1:10000 dilution

  • Polyclonal anti-UPB1: 1:1000 dilution

• Primary antibody incubation: 1 hour at room temperature or overnight at 4°C

• Secondary antibodies:

  • IRDye800 conjugated goat anti-rabbit: 1:10,000 dilution

  • IRDye680 conjugated donkey anti-mouse: 1:10,000 dilution

• Secondary antibody incubation: 1 hour at room temperature

Sample Preparation:

• Optimal protein loading: 5-10 μg per lane

• Positive control: Human fetal liver lysate consistently shows strong UPB1 expression

How can UPB1 antibodies be utilized in studying disease mechanisms in β-ureidopropionase deficiency?

UPB1 antibodies are valuable tools for investigating disease mechanisms in β-ureidopropionase deficiency:

• Characterizing Novel Mutations:

  • Western blot analysis can reveal how mutations affect protein expression

  • Blue native PAGE can detect oligomerization defects in variant proteins

  • Immunohistochemistry can assess tissue-specific expression patterns

• Investigating Structural Consequences:

  • Antibodies can help characterize how point mutations affect substrate binding

  • Detected changes can be correlated with 3D structural models of UPB1

• Studying Splicing Defects:

  • Combine minigene approaches with antibody detection to understand how intronic variants (e.g., c.[364+6T>G] and c.[916+1_916+2dup]) affect UPB1 pre-mRNA splicing and subsequent protein expression

• Genotype-Phenotype Correlations:

  • Use antibody-based detection to measure UPB1 protein levels in patients with different mutations

  • Correlate protein expression with clinical severity

  • Example: The c.[899C>T] (p.Ser300Leu) variant identified in Swedish patients shows specific effects on UPB1 function

• Carrier Screening Applications:

  • Combined with HRM genetic screening, antibody-based methods can help validate carrier status in population screening studies

  • This approach successfully identified both known and novel UPB1 mutations in a screening of 50 healthy individuals

What methodological differences exist between detecting wild-type versus mutant UPB1 proteins?

Detecting wild-type versus mutant UPB1 proteins requires consideration of several methodological differences:

• Antibody Selection Considerations:

  • Wild-type detection: Standard commercial antibodies targeting conserved epitopes work effectively

  • Mutant detection: May require antibodies targeting specific regions not affected by mutations

  • For mutations affecting protein folding, use antibodies targeting different epitopes to ensure detection

• Expression Level Adjustments:

  • Mutant UPB1 proteins often show reduced expression levels

  • Increase protein loading (10-15 μg vs. standard 5 μg) when detecting mutant proteins

  • Extend exposure times when imaging Western blots of mutant proteins

• Detection of Truncated Proteins:

  • For splice site mutations causing exon skipping:

    • Use antibodies targeting N-terminal regions to detect truncated proteins

    • Consider using gradient gels (4-16%) to better resolve smaller protein fragments

• Oligomerization Analysis:

  • Wild-type UPB1 forms specific oligomeric structures

  • Some mutations prevent proper subunit association

  • Use blue native PAGE (4-16%) followed by Western blot analysis to detect differences in oligomeric states

• Expression System Selection:

  • Wild-type protein: Standard expression systems work well

  • Mutant proteins: May require optimization of expression conditions, including lower temperature expression or use of chaperone co-expression systems

How can researchers develop an integrated workflow for UPB1 genetic and protein analysis in clinical research?

An integrated workflow for comprehensive UPB1 analysis should include:

• Initial Clinical Assessment and Biomarker Screening:

  • Measure pyrimidine degradation products in urine samples using reversed-phase HPLC with electrospray tandem mass spectrometry

  • Look for elevated levels of N-carbamyl-β-alanine (NCβA) and N-carbamyl-β-aminoisobutyric acid (NCβAIBA)

• Genetic Analysis Pipeline:

  • DNA extraction from whole blood using QIAamp DNA Micro kit or similar

  • HRM-based rapid screening for UPB1 mutations covering exons 1-10

  • Confirmation of suspected mutations by Sanger sequencing

• Protein Expression and Function Analysis:

  • Western blot analysis using validated UPB1 antibodies (dilution 1:1000-1:10000)

  • Immunohistochemistry of relevant tissues (kidney, liver) using optimized protocols

  • Functional enzyme assays to correlate genotype with biochemical phenotype

• Mutation Characterization:

  • For novel mutations, use site-directed mutagenesis to introduce changes into expression vectors

  • Express recombinant proteins and analyze using Western blot

  • For intronic variants affecting splicing, implement minigene approach to study pre-mRNA processing

• Data Integration and Reporting:

  • Correlate genetic findings with protein expression data and clinical phenotype

  • Document all findings in standardized formats for contribution to mutation databases

  • Develop patient-specific therapeutic strategies based on integrated analysis

This workflow has been successfully implemented in multiple studies, leading to the identification of novel UPB1 mutations and improved understanding of β-ureidopropionase deficiency .

What emerging technologies are being developed for high-throughput screening of UPB1 mutations?

Several emerging technologies show promise for high-throughput UPB1 mutation screening:

• Advanced HRM Analysis:

  • Current HRM methods can successfully detect known UPB1 mutations with specific melting curve profiles

  • The method can identify both heterozygous and homozygous variants

  • Future developments include automated analysis systems for large-scale population screening

• Next-Generation Sequencing Panels:

  • Gene panel testing including UPB1 has proven effective for diagnosing conditions like microcephaly

  • A Korean case of β-ureidopropionase deficiency was diagnosed using Illumina next-generation sequencing in a panel gene test for microcephaly and lissencephaly

• Integrated Bioinformatic Pipelines:

  • Combining HRM screening with automated sequence analysis

  • Development of databases specific for UPB1 mutations and their functional consequences

  • Algorithmic prediction of mutation effects on protein structure and function

• Metabolomic Profiling:

  • High-throughput screening of pyrimidine metabolites in urine or dried blood spots

  • Development of mass spectrometry methods that can screen for multiple metabolic disorders simultaneously

  • Integration with genetic data for comprehensive phenotype-genotype correlation

How can researchers validate novel UPB1 mutations using antibody-based approaches combined with functional assays?

A comprehensive validation approach for novel UPB1 mutations includes:

• Expression of Mutant Proteins:

  • Clone wild-type UPB1 into expression vectors (e.g., pcDNA3.1Zeo)

  • Introduce mutations using site-directed mutagenesis

  • Express in appropriate cell systems (e.g., HEK293, COS-7)

• Antibody-Based Detection and Quantification:

  • Western blot analysis using validated anti-UPB1 antibodies

  • Compare expression levels of wild-type and mutant proteins

  • Analyze protein stability over time using cycloheximide chase experiments

• Structural Analysis:

  • Use blue native PAGE followed by Western blotting to analyze oligomerization

  • Assess if mutations affect substrate binding or prevent proper subunit association

  • Correlate findings with 3D structural models of UPB1

• Splicing Analysis for Intronic Variants:

  • For intronic variants, implement minigene approach

  • Analyze effects on pre-mRNA splicing (e.g., exon skipping)

  • Use antibodies to detect resulting protein products

• Functional Enzymatic Assays:

  • Measure conversion of N-carbamoyl-beta-alanine to beta-alanine

  • Compare enzymatic activity of wild-type and mutant proteins

  • Correlate structural findings with functional consequences

This integrated approach has successfully validated several novel UPB1 mutations, including missense variants (c.[53C>T], c.[358G>T], c.[386C>T], c.[899C>T], c.[1034A>T]) and splice-site variants (c.[364+6]T>G, c.[916+1_916+2dup]) .

What role might UPB1 antibodies play in developing potential therapeutic approaches for β-ureidopropionase deficiency?

UPB1 antibodies could contribute to therapeutic development in several ways:

• Patient Stratification for Clinical Trials:

  • Antibody-based assays can help categorize patients based on UPB1 protein expression levels

  • Patients with residual protein expression might respond differently to therapies compared to those with complete absence of protein

• Therapeutic Screening Platforms:

  • UPB1 antibodies can be used in high-throughput screening assays to identify compounds that:

    • Stabilize mutant UPB1 proteins

    • Enhance residual enzymatic activity

    • Correct misfolding or improper oligomerization

• Monitoring Therapeutic Efficacy:

  • Antibody-based assays can track changes in UPB1 protein levels during treatment

  • Western blot or immunohistochemistry can assess if therapies restore protein expression in relevant tissues

• Enzyme Replacement Therapy Development:

  • Antibodies can help characterize recombinant UPB1 proteins for enzyme replacement therapy

  • Quality control of therapeutic enzyme preparations

  • Tracking biodistribution of administered enzyme

• Gene Therapy Monitoring:

  • For gene therapy approaches, antibodies can verify successful expression of the therapeutic UPB1 protein

  • Quantitative analysis of expression levels in different tissues