Recombinant Papio anubis Serpin B10 (SERPINB10)

What is SERPINB10 and what is its fundamental function in Papio anubis?

SERPINB10 (also known as PI-10 or PI10) is a member of the serpin family B, which belongs to the superfamily of high molecular weight serine proteinase inhibitors. In Papio anubis (olive baboon), SERPINB10 is a protein-coding gene that functions similarly to other serpins by regulating proteolytic processes .
Serpins regulate diverse intracellular and extracellular processes including complement activation, fibrinolysis, coagulation, cellular differentiation, tumor suppression, apoptosis, and cell migration. SERPINB10 specifically belongs to the ov-serpin subfamily, which is characterized by:

• High degree of homology to chicken ovalbumin

• Lack of N- and C-terminal extensions

• Absence of a signal peptide

• Presence of serine rather than asparagine at the penultimate position

How does SERPINB10 structure compare between Papio anubis and human models?

While specific structural comparisons between human and Papio anubis SERPINB10 are not extensively documented in the provided materials, serpins across species share well-conserved tertiary structure consisting of:

• 3 beta sheets

• 8-9 alpha helices

• A reactive center loop connecting beta sheets A and C, which is critical for function
  Recombinant Papio anubis SERPINB10 protein is available commercially with His-tag conjugation, expressed in yeast expression systems, suggesting structural viability in heterologous expression systems .

What are the recommended protocols for recombinant expression of Papio anubis SERPINB10?

Based on commercial production methods, recombinant Papio anubis SERPINB10 can be successfully expressed in yeast expression systems with His-tag conjugation . Researchers should consider:

• Expression system selection: Yeast systems appear viable for functional SERPINB10 expression

• Affinity tag placement: His-tagging is demonstrated to be compatible with SERPINB10

• Codon optimization: Adapting the Papio anubis sequence for the chosen expression system

• Growth conditions: Optimizing temperature, induction timing, and media composition
  When designing expression constructs, researchers should consider the protein's tertiary structure and ensure the reactive center loop remains unimpeded by fusion tags.

What methods are effective for measuring SERPINB10 levels in biological samples?

Several validated methods have been successfully applied to measure SERPINB10:

• ELISA: Successfully used to detect SERPINB10 levels in induced sputum from asthmatic patients

• Quantitative PCR (qPCR): Effective for measuring SERPINB10 mRNA expression levels following UV irradiation

• Protein immunoblotting: Can be used to assess protein levels in various experimental conditions
  When examining SERPINB10 in the context of inflammation, correlations with other inflammatory markers should be considered, as research has demonstrated correlations between SERPINB10 levels and:

• FeNO levels (r = 0.4620, p < 0.0001)

• Eosinophils in peripheral blood (r = 0.2500, p = 0.0218)

• FEV1 (%predicted) (r = −0.4161, p < 0.0001)

• FEV1/FVC% (r = −0.4383, p < 0.0001)

How can researchers effectively validate SERPINB10 activity in experimental models?

Functional validation of SERPINB10 should address both expression and activity:

• Expression validation:

  • Western blotting with anti-SERPINB10 antibodies

  • Quantitative PCR for transcript levels

  • Immunofluorescence for cellular localization

• Activity assays:

  • Protease inhibition assays measuring inhibition of target serine proteases

  • Cell-based functional assays relevant to specific research questions (e.g., UV response assays or inflammation models)

• Knockdown validation:

  • siRNA approaches have been successfully used to silence SERPINB10 and study functional effects

  • Control experiments should include non-targeting scrambled siRNA (siSCR)

What is the role of SERPINB10 in the UV-induced cellular response?

Research has revealed that SERPINB10 is dramatically upregulated following UV irradiation, with this response observed across various cell types. Key findings include:

How does SERPINB10 contribute to inflammatory processes, particularly in airway inflammation?

SERPINB10 has been implicated in airway inflammation, particularly in asthma. Key research findings include:

• Elevated levels in asthma: SERPINB10 levels in induced sputum were significantly higher in asthmatic patients compared to healthy controls

• Correlation with type 2 inflammation markers: SERPINB10 levels positively correlate with several markers of type 2 airway inflammation:

  • FeNO: r = 0.4620, p < 0.0001

  • Eosinophils in peripheral blood: r = 0.2500, p = 0.0218

  • Th2 cytokines:

    • IL-4: r = 0.6274, p < 0.0001

    • IL-5: r = 0.5166, p < 0.0001

    • IL-13: r = 0.5212, p = 0.0003

• Negative correlation with lung function: SERPINB10 levels showed significant negative correlations with:

  • FEV1 (%predicted): r = −0.4161, p < 0.0001

  • FEV1/FVC%: r = −0.4383, p < 0.0001

• Potential biomarker: Induced sputum SERPINB10 may serve as a signature protein for type 2 high asthma and could represent a potential target for addressing airway eosinophilic inflammation

What protein interactions are critical for SERPINB10 function?

Several protein interactions appear important for SERPINB10 function:

• Histone H3 interaction: Research has highlighted an interaction between SERPINB10 and histone H3, suggesting potential roles in chromatin-related processes

• PCNA interactions: SERPINB10 appears to influence chromatin-bound PCNA levels following UV irradiation, suggesting direct or indirect interactions with replication and repair machinery

• Protease targets: As a member of the serpin family, SERPINB10 likely interacts with specific target proteases, though the specific proteases targeted by Papio anubis SERPINB10 are not explicitly identified in the provided materials

What experimental controls should be included when studying SERPINB10 in UV response studies?

When investigating SERPINB10's role in UV response, researchers should include:

• Time-course controls:

  • Measure SERPINB10 expression at multiple time points (e.g., 2, 4, 6, and 24 hours) after UV irradiation

  • Include both early and late timepoints to capture immediate responses and recovery phases

• RNA interference controls:

  • Non-targeting scrambled siRNA (siSCR) as a negative control

  • Verify knockdown efficiency using qPCR and/or Western blotting

• Cell viability measurements:

  • Trypan blue staining to determine the ratio of live and dead cells

  • Monitor at multiple timepoints post-irradiation

• DNA repair assays:

  • Post-replication repair (PRR) comet assay under physiological conditions

  • Compare results between control and SERPINB10-silenced cells

What considerations are important when designing experiments to measure SERPINB10 in inflammatory conditions?

When investigating SERPINB10 in inflammation, researchers should consider:

• Sample collection:

  • Induced sputum collection has been validated for SERPINB10 research in asthma

  • Standardize collection protocols to minimize variability

• Correlation analysis:

  • Measure multiple inflammatory markers simultaneously (FeNO, eosinophil counts, Th2 cytokines)

  • Perform correlation analyses to establish relationships between SERPINB10 and established markers

• Clinical parameters:

  • Include lung function measurements (FEV1, FEV1/FVC%)

  • Record relevant clinical data for proper patient stratification

• Control groups:

  • Include appropriate healthy controls

  • Consider including disease controls with non-type 2 inflammation for specificity assessment

What approaches can be used to investigate the evolutionary conservation of SERPINB10 function?

To study evolutionary conservation of SERPINB10:

• Comparative genomics:

  • Leverage existing genomic data from Papio anubis and other species

  • Analyze sequence conservation, particularly in functional domains and the reactive center loop

• Cross-species functional assays:

  • Express recombinant SERPINB10 from multiple species

  • Compare biochemical properties and functional activities

• Phylogenetic analysis:

  • Construct phylogenetic trees of SERPINB10 across primates and other mammals

  • Identify patterns of selection and conservation

• Structural modeling:

  • Generate structural models based on the conserved serpin fold

  • Identify conserved and divergent structural elements that may influence function
    Papio anubis represents an important model for comparative studies due to its evolutionary relationship to humans and the availability of genomic resources, including draft assemblies with N50 scaffold sizes of 887 kbp .