L-proline cis-3-hydroxylase 1 Antibody

What is L-proline cis-3-hydroxylase 1 (P3H1) and what is its biological function?

P3H1 is an enzyme that catalyzes the hydroxylation of specific proline residues in collagen alpha chains to form (3S)-hydroxyproline (3-Hyp). The reaction requires 2-oxoglutarate, oxygen, and iron as cofactors. P3H1 specifically modifies a single proline residue in the α chains of type I, II, and III collagens . The systematic enzyme name is L-proline,2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating) .

The catalyzed reaction is:
L-proline + 2-oxoglutarate + O₂ → cis-3-hydroxy-L-proline + succinate + CO₂

P3H1 functions in the rough endoplasmic reticulum as part of a multiprotein complex with cartilage-associated protein (CRTAP) and cyclophilin B (CypB) in a 1:1:1 stoichiometry . This complex has dual functions:

• Hydroxylation of specific proline residues in collagen

• Acting as a molecular chaperone to facilitate collagen folding

How does P3H1 differ from other proline hydroxylases?

P3H1 differs from other proline hydroxylases in several key aspects:

P3H1 has a higher degree of substrate specificity compared to proline 4-hydroxylases, modifying only specific proline residues in collagen alpha chains . Bacterial proline 3-hydroxylases, in contrast, can hydroxylate free L-proline to form cis-3-hydroxy-L-proline .

What are the essential properties of antibodies against P3H1?

High-quality P3H1 antibodies should have the following properties:

• Specificity: Able to distinguish P3H1 from other proline hydroxylases, particularly P3H2 and P3H3 isoforms

• Recognition of native confirmation: Effective in applications requiring recognition of the native protein such as immunoprecipitation and ChIP

• Complex recognition: Some antibodies should be able to detect P3H1 within the P3H1-CRTAP-CypB complex, while others might be specifically designed to recognize only free P3H1

• Cross-reactivity: Well-characterized cross-reactivity across species (human, mouse, and other model organisms) should be documented

• Application versatility: Validated for multiple applications including Western blotting, immunohistochemistry, immunofluorescence, and ELISA

When selecting antibodies for P3H1 research, consider the specific epitope recognized, as P3H1 has distinct domains that may be differentially accessible in various experimental conditions .

How is P3H1 involved in disease pathology?

Defects in P3H1 have been associated with several connective tissue disorders:

• Recessive Osteogenesis Imperfecta: Mutations inhibiting 3-Hyp formation cause recessive osteogenesis imperfecta, a brittle bone disease .

• Collagen Maturation Disorders: Impaired P3H1 activity affects collagen triple helix stability and fibril assembly.

• Cancer Progression: P3H1 expression has been investigated as a prognostic marker in clear cell renal cell carcinoma (ccRCC) .

Research using P3H1 antibodies has helped elucidate these disease mechanisms by:

• Identifying altered P3H1 expression levels in patient tissues

• Localizing P3H1 in cellular compartments during disease states

• Monitoring changes in the P3H1-CRTAP-CypB complex formation

What are the challenges in differentiating between cis-3-hydroxy-L-proline and other hydroxyproline isomers in experiments?

Distinguishing between the various stereoisomers of hydroxyproline presents several challenges:

• Structural similarity: The cis-3-hydroxy, trans-3-hydroxy, and trans-4-hydroxy isomers of proline have similar chemical properties.

• Detection methods: HPLC analysis with postcolumn derivatization using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is required for accurate identification and quantification .

• Stereoisomer abundance: trans-4-hydroxyproline is much more abundant in biological samples, making detection of the less common cis-3-hydroxyproline challenging.

For accurate isomer identification, researchers should:

• Use authentic standards of each isomer (cis-3-hydroxy-L-proline, trans-3-hydroxy-L-proline, trans-4-hydroxy-L-proline) for comparison

• Employ LC-MS analysis to confirm molecular ions (typically detected at m/z 132.06 [M+H]⁺)

• Validate fragmentation patterns against known standards

Antibodies specifically recognizing 3-hydroxyproline-containing peptides can be valuable tools but must be extensively validated for isomer specificity.

How can I validate P3H1 antibody specificity for experimental applications?

A comprehensive validation strategy for P3H1 antibodies should include:

• Western blot analysis:

  • Using recombinant P3H1 as a positive control

  • Including P3H1 knockout/knockdown samples as negative controls

  • Testing cross-reactivity with P3H2 and P3H3 proteins

• Immunoprecipitation validation:

  • Co-IP experiments to verify capture of known binding partners (CRTAP and CypB)

  • Mass spectrometry analysis of immunoprecipitated proteins

• Immunohistochemistry controls:

  • Tissues from P3H1-deficient models

  • Peptide competition assays to confirm epitope specificity

  • Comparison with mRNA expression (in situ hybridization)

• Functional validation:

  • Confirming antibody ability to detect changes in P3H1 levels after relevant experimental manipulations

  • Assessing antibody interference with enzymatic activity

• Cross-species reactivity:

  • Testing antibody performance across relevant model organisms

  • Aligning epitope sequences across species to predict cross-reactivity

What are the optimal experimental conditions for studying P3H1 enzymatic activity?

Based on studies of purified P3H1, the optimal conditions for enzymatic activity are:

Key considerations for studying P3H1 activity:

• Maintain anaerobic conditions: Oxygen is a substrate, but excess can lead to oxidative damage to the enzyme

• Fresh preparation of iron solution: Fe(II) oxidizes readily in solution

• Include protease inhibitors: Especially when working with tissue-derived enzyme

• Consider the entire complex: When studying collagen substrate hydroxylation, the P3H1-CRTAP-CypB complex shows higher activity than P3H1 alone

How do fungal and bacterial P3H1 enzymes differ from mammalian P3H1?

The P3H1 enzymes from different kingdoms show significant differences:

In Streptomyces sp. strain TH1, proline 3-hydroxylase has been purified and characterized as a 35 kDa protein with an isoelectric point of 4.3 . This bacterial enzyme can efficiently hydroxylate free L-proline, unlike mammalian P3H1 which primarily targets peptidyl-proline in collagen chains .

Fungal P3H1 enzymes, particularly those involved in echinocandin biosynthesis, can hydroxylate both free L-proline and specific proline residues in peptide contexts. Some fungal enzymes (HtyE) can produce both trans-4-hydroxyproline and trans-3-hydroxyproline from L-proline, with varying ratios depending on the specific enzyme variant .

What is the best protocol for immunoprecipitation of P3H1 from tissue samples?

For successful immunoprecipitation of P3H1 from tissue samples:

Materials:

• Anti-P3H1 antibody (monoclonal preferred for specificity)

• Protein A/G magnetic or agarose beads

• Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors

• Collagenase inhibitors (to prevent degradation of P3H1-associated collagens)

Protocol:

• Tissue preparation:

  • Homogenize tissue in ice-cold lysis buffer (10 ml per gram of tissue)

  • Include protease inhibitors and phosphatase inhibitors

  • For collagen-rich tissues, consider including specific collagenase inhibitors

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation (10,000g for 10 min)

• Immunoprecipitation:

  • Add 2-5 μg of anti-P3H1 antibody to 1 ml of pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 50 μl of protein A/G beads, incubate for 2-4 hours at 4°C

  • Wash beads 5 times with wash buffer (lysis buffer with reduced detergent)

• Elution:

  • For Western blot: Boil beads in SDS sample buffer

  • For activity assays: Use competitive elution with excess epitope peptide

Based on studies of the P3H1-CRTAP-CypB complex, using gelatin-Sepharose columns can enhance purification, as this complex shows affinity for collagen .

How can I establish a quantitative assay for measuring 3-hydroxyproline in biological samples?

For accurate quantification of 3-hydroxyproline in biological samples:

HPLC-Based Method:

• Sample preparation:

  • For tissue samples: Acid hydrolysis (6N HCl, 110°C, 24h) followed by neutralization

  • For cell culture: TCA precipitation followed by hydrolysis

  • Derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole or other suitable agents

• HPLC conditions:

  • Column: C18 reversed-phase

  • Mobile phase: Gradient of acetonitrile in water with 0.1% TFA

  • Detection: Fluorescence detection for derivatized samples

• Quantification:

  • Use authentic standards of cis-3-hydroxy-L-proline for calibration curves

  • Include internal standards to account for sample loss during preparation

LC-MS/MS Method:

• Preparation: Similar to HPLC but may require different derivatization

• Analysis:

  • Multiple reaction monitoring (MRM) for specific transitions

  • Molecular ion typically detected at m/z 132.06 [M+H]⁺

  • Fragment ions specific to 3-hydroxyproline isomers

Considerations:

• Account for potential conversion between isomers during sample preparation

• Include standards for all hydroxyproline isomers to ensure proper separation

• Consider acid hydrolysis conditions carefully, as they can affect recovery

• For collagen-derived 3-hydroxyproline, enzymatic digestion may better preserve the stereochemistry

What expression systems are most effective for producing recombinant P3H1 for antibody validation?

The choice of expression system significantly impacts recombinant P3H1 quality:

Recommended approach for P3H1:

• For antibody production and validation:

  • Express the C-terminal dioxygenase domain in E. coli for antibody generation

  • Express full-length protein in mammalian cells (HEK293 or CHO) for validation

  • Consider co-expression with CRTAP and CypB to enhance stability

• For functional studies:

  • Mammalian expression in HEK293 cells provides the most native-like enzyme

  • Co-expression with chaperones enhances proper folding

  • Tag placement should avoid interfering with catalytic domains

Based on bacterial P3H1 studies, recombinant expression in E. coli can yield active enzyme with hydroxylating activity up to five times higher than in the original bacterium , but this applies to bacterial P3H1, not the mammalian enzyme.

How can I design experiments to distinguish between the enzymatic and chaperone functions of P3H1?

P3H1 has dual functions as both an enzyme and a chaperone as part of the P3H1-CRTAP-CypB complex . To experimentally distinguish between these functions:

Enzymatic Activity Assays:

• Direct measurement of hydroxylation:

  • Incubate purified P3H1 or the complex with synthetic collagen peptides

  • Measure 3-hydroxyproline formation by mass spectrometry or HPLC

  • Include controls with catalytically inactive P3H1 mutants

• Coupled enzyme assays:

  • Monitor oxygen consumption or 2-oxoglutarate decarboxylation

  • Measure succinate formation as a co-product

Chaperone Function Assays:

• Thermal aggregation protection:

  • Use citrate synthase thermal aggregation assay (the P3H1-CRTAP-CypB complex inhibits thermal aggregation of citrate synthase)

  • Monitor aggregation by light scattering at 320-360 nm

  • Compare with known chaperones like protein-disulfide isomerase

• Rhodanese refolding assay:

  • The complex is active in preventing aggregation during refolding of denatured rhodanese

  • Measure recovery of rhodanese activity with/without P3H1 complex

• Collagen folding kinetics:

  • Monitor the rate of collagen triple helix formation

  • Compare wild-type P3H1 complex with enzymatically inactive mutants

Separation of Functions:

• Generate point mutations that specifically disrupt the catalytic domain without affecting complex formation

• Create truncated variants retaining only the chaperone function

• Use specific inhibitors of prolyl hydroxylase activity to block enzymatic function while preserving protein-protein interactions

The chaperone activity of the P3H1-CRTAP-CypB complex has been shown to be higher than that of protein-disulfide isomerase, a well-characterized chaperone .

What are the most common issues when using P3H1 antibodies and how can they be resolved?

Common issues with P3H1 antibodies and their solutions:

For antibodies targeting P3H1 within the P3H1-CRTAP-CypB complex, consider that the native complex structure might mask certain epitopes. The complex can be isolated using gelatin-Sepharose columns followed by antibody columns with monoclonal antibodies against P3H1 .

How do different storage and handling conditions affect P3H1 enzymatic activity?

Optimal storage and handling conditions for preserving P3H1 activity:

Based on bacterial P3H1 studies, the enzyme shows relatively stable activity in the pH range of 6.5-8.0 and temperature range of 25-40°C, with optimal conditions at pH 7.0 and 35°C .

For mammalian P3H1, the complex with CRTAP and CypB provides significant stabilization. When purifying and storing the complex, maintaining the integrity of all three components is crucial for optimal activity.

What factors affect the specificity of hydroxyproline position in P3H1-catalyzed reactions?

Several factors influence the regio- and stereoselectivity of proline hydroxylation:

• Enzyme structure:

  • Specific amino acid residues in the active site determine positioning of the proline substrate

  • In fungal HtyE enzymes, Leu182 has been identified as a key residue determining regioselectivity between 3- and 4-hydroxylation

• Substrate conformation:

  • The orientation of proline within the active site affects accessibility of C-3 vs. C-4

  • For mammalian P3H1, the context of proline within the collagen chain is crucial

• Reaction conditions:

  • Iron concentration can affect the ratio of different hydroxylated products

  • Temperature and pH may influence enzyme conformation and thus specificity

• Evolutionary adaptations:

  • Different P3H1 enzymes across species show varying specificities

  • Fungal enzymes often show mixed 3- and 4-hydroxylation activities with different ratios

• Substrate modifications:

  • Modified prolines (e.g., 4R-methyl-proline) may show altered hydroxylation patterns

  • L-proline vs. D-proline stereochemistry significantly affects enzyme recognition

Some fungal P3H1 variants (HtyE) can produce both trans-4-hydroxyproline and trans-3-hydroxyproline with varying ratios (trans-4-Hyp/trans-3-Hyp ranging from 2.5:1 to 7.2:1) , demonstrating how enzyme structural differences affect position specificity.

How are recent advances in cryo-EM and structural biology contributing to our understanding of P3H1?

Recent structural biology approaches are revealing crucial insights about P3H1:

• Complex architecture: Cryo-EM studies are beginning to elucidate the precise arrangement of the P3H1-CRTAP-CypB complex, showing how these proteins interact in a 1:1:1 stoichiometry

• Substrate binding: Crystal structures of P3H1 complexed with substrates and inhibitors provide information about the enzyme's selectivity for L-stereoisomers. For example, studies of ALDH4A1 (another proline-metabolizing enzyme) show that hydrogen bonding interactions contribute to stereoisomer preference

• Conformational changes: Time-resolved structural studies can capture the enzyme during different stages of the catalytic cycle, revealing how substrate binding, hydroxylation, and product release are coordinated

• Integration with computational approaches: Molecular dynamics simulations based on structural data help predict how mutations might affect enzyme function

• Structure-guided antibody development: Detailed structural information enables the design of antibodies targeting specific epitopes that are accessible in the native protein configuration

The integration of structural data with biochemical characterization is essential for a complete understanding of P3H1 function and for developing tools like specific antibodies and inhibitors.

What are the applications of P3H1 antibodies in cancer research?

P3H1 antibodies are increasingly valuable tools in cancer research:

• Biomarker studies:

  • P3H1 expression has been investigated as a prognostic marker in clear cell renal cell carcinoma (ccRCC)

  • Antibodies enable detection of altered P3H1 expression levels in tumor tissues

• Extracellular matrix remodeling:

  • Cancer progression involves extensive collagen remodeling

  • P3H1 antibodies help assess changes in collagen hydroxylation patterns during carcinogenesis

• Drug sensitivity correlation:

  • Multi-omics analyses have revealed correlations between P3H1 expression and drug sensitivity

  • Antibodies facilitate validation of these findings at the protein level

• Immune checkpoint relationships:

  • Studies have investigated correlations between P3H1 and immune checkpoints

  • Antibodies enable co-localization studies with checkpoint proteins

• Therapeutic target validation:

  • For cancers where P3H1 may be a therapeutic target, antibodies provide essential tools for target validation

  • Both inhibition and activation strategies may be relevant depending on the cancer type

Recent research has employed a multidimensional approach integrating in vitro assays and multi-omics bioinformatics analyses to investigate P3H1's impact on ccRCC prognosis, immune modulation, and therapeutic responses .

How can CRISPR-Cas9 gene editing be used to enhance P3H1 antibody validation?

CRISPR-Cas9 technology provides powerful approaches for P3H1 antibody validation:

• Generation of knockout cell lines:

  • Create complete P3H1 knockout cells as negative controls

  • These provide the gold standard for antibody specificity testing

  • Compare antibody signals between wild-type and knockout cells across applications

• Epitope tagging:

  • Introduce epitope tags (FLAG, HA, etc.) at the P3H1 locus

  • Enables parallel detection with both P3H1 antibodies and tag-specific antibodies

  • Confirms antibody recognition of the correct protein

• Domain-specific mutations:

  • Engineer cells with specific mutations in different P3H1 domains

  • Test domain-specific antibodies against these variants

  • Helps map the precise epitope recognition regions

• Humanized animal models:

  • Create models expressing human P3H1 for better testing of human-specific antibodies

  • Particularly valuable for therapeutic antibody development

• Inducible expression systems:

  • Develop CRISPR-engineered cell lines with inducible P3H1 expression

  • Allows titration of P3H1 levels to test antibody sensitivity and dynamic range

• Partner protein knockouts:

  • Create CRTAP or CypB knockout lines to test antibody recognition of P3H1 in different complex states

  • Helps identify antibodies that recognize complex-specific conformations

When using these CRISPR-engineered systems, it's important to validate the genetic modifications through sequencing and to confirm the expected changes in P3H1 mRNA expression through RT-PCR before testing antibody specificity.