Recombinant Chicken Prolyl 4-hydroxylase subunit alpha-2 (P4HA2), partial

What is Prolyl 4-hydroxylase subunit alpha-2 (P4HA2) and what is its primary function?

Prolyl 4-hydroxylase subunit alpha-2 (P4HA2) is a component of the prolyl 4-hydroxylase enzyme (EC 1.14.11.2), which plays a key role in collagen synthesis. The enzyme functions as a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2), where the alpha subunit provides the major part of the catalytic site. P4HA2 specifically catalyzes the post-translational hydroxylation of proline residues in -Xaa-Pro-Gly- sequences in collagens and related proteins. This hydroxylation is essential for the proper three-dimensional folding of newly synthesized procollagen chains, directly impacting the structural integrity of connective tissues .

What is the molecular structure of chicken P4HA2?

The mature chicken P4HA2 subunit consists of 516 amino acids with a calculated molecular mass of 59,373 Da. The protein contains two potential glycosylation sites, which aligns with previous demonstrations that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA has detected an mRNA of approximately 3.5 kilobases for P4HA2. The recombinant partial version typically maintains the catalytic region while potentially lacking some structural elements of the full-length protein .

How does recombinant chicken P4HA2 differ from endogenous P4HA2?

Recombinant chicken P4HA2 is typically produced in expression systems such as yeast, while endogenous P4HA2 is naturally expressed in chicken tissues that synthesize and secrete collagens. The recombinant version often includes a tag for purification purposes, which might not be present in the endogenous form. While the recombinant partial form preserves the key functional domains necessary for enzymatic activity, it may lack certain regions that are present in the full-length endogenous protein. Despite these differences, recombinant P4HA2 maintains greater than 85% purity as determined by SDS-PAGE analysis and retains the essential catalytic functions when properly reconstituted .

What are the optimal storage conditions for recombinant chicken P4HA2?

The stability of recombinant chicken P4HA2 depends on multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself. For liquid formulations, the shelf life is typically 6 months when stored at -20°C/-80°C. Lyophilized forms exhibit greater stability with a shelf life of 12 months at -20°C/-80°C. To maintain protein integrity during research, it is recommended to avoid repeated freeze-thaw cycles. Working aliquots may be stored at 4°C for up to one week. For long-term storage, adding glycerol to a final concentration of 5-50% (optimally 50%) before aliquoting and freezing is recommended to preserve enzymatic activity .

How should recombinant chicken P4HA2 be reconstituted for experimental use?

For optimal reconstitution of recombinant chicken P4HA2, the protein vial should first be briefly centrifuged to bring the contents to the bottom. The protein should then be reconstituted in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. For long-term storage, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation). After reconstitution, the solution should be aliquoted to minimize freeze-thaw cycles and stored at -20°C/-80°C. This methodological approach ensures maintained protein stability and enzymatic activity for subsequent experimental applications .

What detection methods are available for measuring P4HA2 expression and activity in research samples?

Several detection methods can be employed to measure P4HA2 expression and activity in research samples:

The Chicken P4HA2 ELISA Kit offers high sensitivity and specificity for quantitative detection in chicken serum, plasma, and cell lysates. For activity measurements, researchers typically assess the hydroxylation of proline residues in collagen peptide substrates. RNA blot hybridization can detect P4HA2 mRNA with a characteristic size of approximately 3.5 kilobases .

How is P4HA2 involved in cancer progression, and what methodologies can be used to study this relationship?

P4HA2 has been implicated in multiple cancer types, with its overexpression correlating with tumor aggressiveness and poor prognosis. In head and neck squamous cell carcinoma (HNSCC), elevated P4HA2 expression promotes cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) through activation of the PI3K/AKT signaling pathway. Similarly, in breast ductal carcinoma in situ (DCIS) and glioma, high P4HA2 expression serves as a predictor of disease progression and shorter recurrence-free intervals .

To study this relationship, researchers can employ multiple methodologies:

• Immunohistochemical analysis of P4HA2 expression in tumor tissues and adjacent normal tissues

• Genetic manipulation through knockdown (siRNA/shRNA) or overexpression systems in cancer cell lines

• Functional assays including colony formation, cell proliferation (CCK-8), apoptosis (flow cytometry), migration (wound healing), and invasion (transwell) assays

• Western blotting to assess EMT markers and signaling pathway components (PI3K/AKT)

• Xenograft models to evaluate tumor growth in vivo

• Analysis of collagen deposition and extracellular matrix remodeling using specialized staining techniques

These approaches collectively provide comprehensive insights into how P4HA2 contributes to cancer pathogenesis .

What is the relationship between P4HA2 expression and extracellular matrix remodeling in pathological conditions?

P4HA2 plays a crucial role in extracellular matrix (ECM) remodeling by catalyzing the hydroxylation of proline residues in collagen, which is essential for proper collagen folding, secretion, and fibril formation. In pathological conditions, dysregulated P4HA2 expression leads to abnormal collagen deposition and altered ECM properties, creating a favorable microenvironment for disease progression .

Research has demonstrated that P4HA2 mRNA positively correlates with the expression of multiple collagen genes. When P4HA2 is silenced or its enzymatic activity is inhibited, both collagen protein expression and downstream signaling pathways like PI3K/AKT are downregulated. This relationship is particularly significant in cancer progression, where altered ECM composition contributes to increased cell migration, invasion, and metastasis .

Methodologically, researchers can study this relationship through:

• Co-expression analysis of P4HA2 and collagen genes using RNA-seq or qRT-PCR

• Collagen deposition assessment using Masson's trichrome or picrosirius red staining

• Mechanical testing of ECM properties in tissues with varying P4HA2 expression

• Atomic force microscopy to evaluate nanoscale ECM changes

• Second harmonic generation microscopy for fibrillar collagen visualization

These approaches provide mechanistic insights into how P4HA2-mediated collagen modification influences disease progression through ECM remodeling .

How can researchers effectively target P4HA2 activity in experimental models to study its functional roles?

Researchers can effectively target P4HA2 activity through multiple complementary approaches:

For robust experimental designs, combining multiple approaches is recommended. For instance, genetic manipulation of P4HA2 expression can be complemented with pharmacological intervention targeting its enzymatic activity. Additionally, rescuing P4HA2 function after knockdown through expression of a modified, inhibitor-resistant version can help establish specificity. When studying signaling pathways, researchers should include positive controls such as PI3K/AKT activators or inhibitors to validate downstream effects attributed to P4HA2 modulation .

What are the challenges in differentiating between the roles of P4HA2 and other prolyl hydroxylase isoforms in collagen synthesis?

Differentiating between the roles of P4HA2 and other prolyl hydroxylase isoforms presents several methodological challenges:

• Structural Similarity: P4HA isoforms (P4HA1, P4HA2, P4HA3) share considerable sequence homology, making selective targeting difficult.

• Compensatory Mechanisms: Knockdown of one isoform often leads to compensatory upregulation of others, potentially masking phenotypic effects.

• Overlapping Functions: All P4HA isoforms catalyze similar reactions, complicating functional dissection.

• Tissue-Specific Expression: The isoforms exhibit differential tissue expression patterns, requiring context-specific experimental designs.

• Substrate Specificity: Subtle differences in substrate preferences exist but are challenging to leverage experimentally.

To overcome these challenges, researchers should employ:

• Isoform-specific antibodies validated for both Western blotting and immunohistochemistry

• Highly specific siRNA/shRNA sequences with minimal off-target effects

• Rescue experiments with isoform-specific constructs resistant to knockdown

• Tissue-specific conditional knockout models

• Mass spectrometry to analyze hydroxylation patterns on specific collagen types

• Computational modeling to predict isoform-specific substrate interactions

These strategies collectively enable more precise characterization of P4HA2's unique contributions to collagen synthesis and pathological processes .

What methodological approaches can resolve the molecular mechanisms connecting P4HA2 activity to signaling pathway activation in disease models?

Resolving the molecular mechanisms connecting P4HA2 activity to signaling pathway activation requires sophisticated methodological approaches:

• Temporal Analysis: Implementing time-course studies following P4HA2 modulation can distinguish between direct and indirect effects on signaling pathways. This involves collecting samples at multiple time points (minutes to hours) after intervention and analyzing the sequential activation of signaling components.

• Proximity-Based Interaction Studies:

  • Proximity ligation assays (PLA) to visualize protein-protein interactions in situ

  • Co-immunoprecipitation followed by mass spectrometry to identify interaction partners

  • FRET/BRET to detect real-time interactions in living cells

• Domain-Specific Mutational Analysis: Creating P4HA2 constructs with mutations in key domains (catalytic, binding, structural) to dissect which regions are essential for signaling activation.

• Pathway Dissection:

  • Pharmacological inhibitors at different levels of the signaling cascade

  • Genetic knockdown of intermediate signaling components

  • Phosphoproteomic analysis to identify directly affected phosphorylation sites

• Extracellular Matrix Analysis:

  • Decellularization techniques to isolate ECM components influenced by P4HA2

  • Mechanical testing of matrix properties

  • Integrin blocking antibodies to disrupt cell-ECM interactions

Research has demonstrated that P4HA2 influences PI3K/AKT pathway activation, but interestingly, P4HA2 mRNA correlates positively with collagen gene expression rather than with PI3K or AKT1/2 mRNA. This suggests an indirect mechanism potentially involving ECM-receptor interactions. When P4HA2 is silenced or its enzymatic activity is inhibited, both collagen protein expression and PI3K/AKT phosphorylation are downregulated, providing evidence for a mechanistic link between collagen modification and intracellular signaling activation .

How might novel recombinant P4HA2 variants contribute to understanding post-translational modifications in collagen-related disorders?

Engineered recombinant P4HA2 variants could significantly advance our understanding of collagen-related disorders through several innovative approaches:

• Activity-Modulated Variants: Creating recombinant P4HA2 with enhanced or reduced catalytic activity would enable dose-dependent studies of hydroxylation effects on collagen structure and disease progression. By precisely controlling the degree of proline hydroxylation, researchers could establish threshold levels required for normal versus pathological collagen assembly.

• Domain-Specific Variants: Recombinant P4HA2 constructs with modifications in specific functional domains would help delineate the roles of different protein regions in substrate recognition, catalytic activity, and interaction with other cellular components. This approach could identify critical regions for targeted drug development.

• Substrate-Specific Variants: Engineering P4HA2 variants with altered substrate specificity could reveal how hydroxylation of different collagen types contributes to tissue-specific pathologies. This would be particularly valuable for understanding disorders affecting multiple collagen-rich tissues.

• Labeled Variants: Fluorescently tagged or isotopically labeled recombinant P4HA2 would facilitate real-time tracking of enzyme localization, turnover, and interaction with collagen precursors in live cells and tissues.

• Inducible Systems: Developing recombinant P4HA2 variants that can be conditionally activated would allow temporal control over hydroxylation activity, enabling studies of acute versus chronic effects on collagen modification.

These advanced recombinant variants would provide unprecedented tools for investigating how aberrant post-translational modifications contribute to collagen-related disorders ranging from fibrosis to cancer progression .

What are the methodological considerations for investigating P4HA2 as a therapeutic target in cancer and fibrotic diseases?

Investigating P4HA2 as a therapeutic target requires rigorous methodological approaches to address several critical considerations:

Effective investigation should include parallel assessment of direct P4HA2 inhibition versus targeting downstream pathways like PI3K/AKT. Since P4HA2 expression correlates with poor prognosis in multiple cancers including HNSCC, breast DCIS, and glioma, stratifying patients based on P4HA2 expression levels would be crucial for clinical studies .

Additionally, researchers must consider the dual nature of collagen in disease—while excessive collagen deposition contributes to fibrosis and tumor progression, baseline collagen is essential for tissue integrity. This necessitates careful dose-finding studies and long-term safety assessments to balance therapeutic efficacy with potential adverse effects on normal collagen homeostasis .