Periostin Human, HEK

What is human periostin and what are its key structural features?

Human periostin (gene name POSTN) is a secreted extracellular matrix protein composed of five globular domains arranged in an elongated structure with an extensive disordered C-terminal tail. Structurally, periostin contains 11 cysteine residues, with 10 forming five intramolecular disulfide bonds and one remaining unpaired . Recent research has revealed that periostin exists as a disulfide-bonded homodimer, challenging previous structural models . The protein shares homology with fasciclin I, a secreted cell adhesion molecule found in insects . Periostin functions primarily as a ligand for alpha-V/beta-3 and alpha-V/beta-5 integrins, supporting adhesion and migration of epithelial cells in various tissues .

How does periostin function in normal physiology versus pathological conditions?

In normal physiology, periostin plays critical roles in tissue development and homeostasis, particularly in:

• Development of heart valves and associated cardiac structures

• Tissue remodeling and wound healing responses

• Extracellular matrix organization and integrity

• Mesenchymal cell differentiation and function

In pathological conditions, periostin demonstrates altered expression and activity:

• Cancer progression: Periostin binds to integrins on cancer cells, activating Akt/PKB and FAK-mediated signaling pathways that promote cell survival, invasion, angiogenesis, and metastasis

• Inflammatory responses: Forms a self-amplifying loop with NF-κB, accelerating cellular senescence in conditions like intervertebral disc degeneration

• Fibrotic disorders: Prominent expression in desmoplastic stroma of aggressive tumors, contributing to fibrotic tissue remodeling

• Atopic diseases: Involved in driving inflammatory states in various atopic conditions

What post-translational modifications are known to occur in human periostin?

Despite previous annotations in UniProt suggesting vitamin K-dependent γ-carboxylation, comprehensive biochemical analysis has conclusively demonstrated that periostin does not undergo γ-carboxylation of glutamic acid residues . Research using monoclonal antibodies specific for γ-carboxyglutamic acid (Gla) modification showed no reactivity with periostin in either tissue extracts or recombinant protein produced in HEK293 cells optimized for γ-carboxylation . Mass spectrometry with over 67% coverage of recombinant periostin detected no γ-carboxylation modifications on any of the 19 examined glutamate residues (out of 24 total potential sites) .

Periostin does undergo significant glycosylation, with distinct molecular weight forms observed at approximately 40 kDa (less glycosylated) and 50 kDa (more heavily glycosylated) . The differential expression of these glycoforms appears to correlate with malignant progression, with the 40 kDa form associated with more aggressive phenotypes .

What are the optimal conditions for expressing recombinant human periostin in HEK293 cells?

For optimal expression of functional human periostin in HEK293 cells, researchers should consider the following methodological approach:

• Vector selection: Use mammalian expression vectors with strong constitutive promoters (CMV) or inducible systems (tetracycline-regulated) depending on experimental requirements

• Cell line optimization: Standard HEK293 cells produce periostin with normal mammalian post-translational modifications, but for specific applications:

  • HEK293 cells transfected with vitamin K 2,3-epoxide reductase C1 (VKORC1) can be used to test potential γ-carboxylation, though research indicates this modification does not occur naturally

  • Suspension-adapted HEK293F or ExpiHEK293 cells may provide higher yields for large-scale production

• Culture conditions:

  • Temperature: 37°C

  • CO₂: 5%

  • Media: DMEM supplemented with 10% FBS

  • Duration: 48-72 hours post-transfection for optimal protein secretion

• Purification approach:

  • Collect serum-free conditioned media

  • Concentrate using ultrafiltration

  • Purify using affinity chromatography with tagged constructs or immunoaffinity methods

• Quality control assessments:

  • SDS-PAGE analysis under reducing and non-reducing conditions to assess disulfide bonding

  • Western blot confirmation of homodimeric structure

  • Glycosylation profiling to characterize post-translational modifications

What analytical methods are most effective for characterizing recombinant periostin structure and function?

Comprehensive characterization of periostin requires multiple complementary analytical approaches:

• Structural Analysis:

  • Western blotting under reducing and non-reducing conditions to assess disulfide bond formation and homodimerization

  • Mass spectrometry (LC-MS/MS) with at least 67% sequence coverage to confirm protein identity and detect potential post-translational modifications

  • 2D gel electrophoresis to determine isoelectric points (pIs) of 7.0 to >8.0, confirming proper folding and absence of γ-carboxylation

• Functional Assessment:

  • Integrin binding assays using alpha-V/beta-3 and alpha-V/beta-5 integrin-expressing cells

  • Cell adhesion and migration assays to confirm biological activity

  • Co-immunoprecipitation studies to identify interaction partners, particularly fibronectin

• Conformational Analysis:

  • Circular dichroism spectroscopy to assess secondary structure

  • Size exclusion chromatography to confirm dimeric state

  • Surface plasmon resonance to measure binding kinetics with integrins and other partners

How can researchers distinguish between different splicing variants and post-translationally modified forms of periostin?

Distinguishing between periostin variants requires:

• Splicing Variant Analysis:

  • RT-PCR with primers targeting the C-terminal region where alternative splicing occurs

  • Western blot analysis using antibodies targeting specific isoform regions

  • Mass spectrometry proteomics to identify unique peptides from alternatively spliced regions

• Post-translational Modification Assessment:

  • Western blot analysis under reducing conditions can differentiate between the ~40 kDa (less glycosylated) and ~50 kDa (more glycosylated) forms of periostin

  • Glycosidase treatments (PNGase F, Endo H) to assess N-linked glycosylation patterns

  • Lectin binding assays to characterize glycan structures

• Quantitative Analysis:

  • Densitometric analysis of Western blots to determine the ratio of 40 kDa to 50 kDa periostin forms, which correlates with malignant progression

  • ELISA-based methods for quantifying specific variants in biological samples

How does the homodimeric structure of periostin influence its biological function and experimental approaches?

The recent discovery that periostin exists as a disulfide-bonded homodimer fundamentally changes our understanding of its biological mechanisms and experimental considerations :

Functional Implications:

• Enhanced binding avidity to integrin receptors through multivalent interactions

• Formation of more complex extracellular matrix networks through simultaneous binding of multiple partners

• Potential for spatially regulated signaling through clustering of integrin receptors

Experimental Approach Modifications:

• Protein Production:

  • Expression systems must maintain native disulfide bond formation capability

  • Purification under non-reducing conditions is critical to preserve dimeric structure

  • Quality control should include analysis under both reducing and non-reducing conditions

• Interaction Studies:

  • Co-immunoprecipitation protocols should be adapted to preserve native disulfide bonds

  • Binding assays should account for potential avidity effects from bivalent binding

  • Cell-based assays may require different interpretations considering receptor clustering effects

• Structural Analysis:

  • Techniques like analytical ultracentrifugation or size exclusion chromatography coupled with multi-angle light scattering are recommended to confirm dimeric state

  • Cryo-electron microscopy may be more suitable than X-ray crystallography for structural determination

What are the molecular mechanisms by which periostin participates in disease progression?

Periostin contributes to disease progression through multiple interrelated mechanisms:

• In Cancer Progression:

  • Binds to integrins (α-V/β-3, α-V/β-5) on cancer cells, activating Akt/PKB and FAK-mediated signaling

  • Promotes cell survival, invasion, angiogenesis, and metastasis

  • Undergoes alternative splicing in the C-terminal region, producing specific isoforms associated with various cancers (pancreatic, colon, breast)

  • Expression of the less glycosylated 40 kDa form over the 50 kDa form correlates with increased malignant progression

• In Inflammatory Conditions:

  • Forms a self-amplifying loop with NF-κB, initiated by PIEZO1 mechanosensor activation

  • Accelerates cellular senescence and promotes senescence-associated secretory phenotype (SASP)

  • Interacts with integrin αVβ3 to activate NF-κB p65 and promote inflammatory responses

• In Tissue Remodeling and Fibrosis:

  • Acts as a mediator between appropriate and inappropriate responses to tissue damage

  • Forms complexes with fibronectin both in vitro and in vivo, potentially modulating extracellular matrix architecture

  • Promotes extracellular matrix restructuring and epithelial-mesenchymal transition

How can researchers reconcile contradictory findings about periostin's vitamin K-dependent γ-carboxylation?

The discrepancy between UniProt annotations indicating vitamin K-dependent γ-carboxylation of periostin and experimental evidence showing absence of this modification presents an instructive case study in resolving contradictory research findings :

Methodological Approach to Resolution:

• Multiple Independent Analytical Methods:

  • Western blotting with anti-Gla antibodies showed no reactivity with periostin

  • 2D gel electrophoresis demonstrated pIs consistent with unmodified protein

  • Mass spectrometry with 67% coverage found no modified glutamate residues

  • Comparative analysis with known γ-carboxylated proteins (Factor VII) showed clear differences

• Optimized Experimental Systems:

  • Use of HEK293 cells transfected with vitamin K 2,3-epoxide reductase C1 to create ideal conditions for γ-carboxylation

  • Parallel expression of known γ-carboxylated proteins as positive controls

  • Testing of both recombinant and tissue-derived protein sources

• Critical Evaluation of Prediction Algorithms:

  • Recognition that the presence of putative recognition sequences for γ-glutamyl carboxylase does not guarantee modification

  • Importance of experimental validation over in silico predictions

  • Need for database curation and correction of annotation errors

How can periostin serve as a therapeutic target or biomarker in disease?

Periostin shows significant potential as both a therapeutic target and biomarker across multiple diseases:

As a Therapeutic Target:

• In Intervertebral Disc Degeneration:

  • Knockdown of periostin gene expression via siRNA delivered by AAV2 attenuated disc degeneration in rat models

  • Periostin neutralizing antibodies significantly attenuated disc degeneration when injected locally into rat tails during mechanical stress

  • Targeting periostin disrupts the self-amplifying loop with NF-κB, reducing cellular senescence and inflammatory responses

• In Cancer:

  • Targeting the periostin-integrin interaction could potentially reduce cancer cell survival, invasion, and metastasis

  • Specific targeting of the 40 kDa form might selectively affect more aggressive cancer phenotypes

As a Biomarker:

• In Cholangiocarcinoma:

  • Increased tumor and serum periostin levels strongly correlate with liver tumor mass and peritoneal metastases

  • The ratio of 40 kDa to 50 kDa periostin forms predicts increased malignant progression

• In Inflammatory Conditions:

  • Elevated periostin levels in fibrotic tissue indicate active tissue remodeling and potential disease progression

  • Analysis of periostin in bronchial epithelial cells and lung fibroblasts may predict TH-2 cytokine activity in respiratory conditions

What considerations are important when developing therapeutic approaches targeting periostin?

When developing therapeutic strategies targeting periostin, researchers should consider:

• Specificity Considerations:

  • Target specificity must account for periostin's dimeric structure and binding partners

  • Approaches should distinguish between different splicing variants and post-translationally modified forms

  • Potential for differential targeting of tissue-specific expression patterns

• Delivery Challenges:

  • For genetic approaches (siRNA/shRNA), appropriate delivery vehicles like AAV2 have proven effective in animal models

  • For antibody-based approaches, tissue penetration into extracellular matrix-rich environments must be considered

  • Local vs. systemic administration depending on disease context and target tissue

• Efficacy Assessment:

  • Monitor both periostin levels and downstream effects (NF-κB activation, senescence markers)

  • Evaluate effects on both cellular phenotypes and tissue architecture

  • Consider combination approaches targeting multiple points in periostin-related pathways

• Safety Considerations:

  • Potential physiological roles of periostin in normal tissue repair and homeostasis

  • Possible compensatory upregulation of related proteins

  • Timing of intervention relative to disease progression

What are the most promising areas for future periostin research using HEK expression systems?

Future research should focus on:

• Structural Biology:

  • Cryo-EM or crystallographic studies of the full-length homodimeric periostin structure

  • Structural analysis of periostin-fibronectin complexes

  • Investigation of structural differences between splice variants

• Mechanistic Studies:

  • Detailed mapping of the self-amplifying loop between periostin and NF-κB in different cell types

  • Investigation of mechanosensing pathways like PIEZO1 in regulating periostin expression

  • Effects of different glycosylation patterns on periostin function

• Development of Research Tools:

  • Engineering of HEK293 cell lines with inducible periostin expression

  • Development of reporter systems for monitoring periostin-integrin interactions

  • Creation of domain-specific antibodies for detecting various periostin forms

• Therapeutic Development:

  • Design and testing of periostin-targeting antibodies or small molecules

  • Development of splice variant-specific inhibitors

  • Exploration of gene therapy approaches based on successful siRNA studies

How might multiomics approaches enhance our understanding of periostin biology?

Integrated multiomics approaches offer powerful strategies for elucidating periostin biology:

• Genomics/Transcriptomics:

  • Single-cell RNA sequencing to identify cell-specific expression patterns and regulatory networks

  • Alternative splicing analysis across different tissues and disease states

  • Genetic association studies linking POSTN variants to disease susceptibility

• Proteomics:

  • Advanced mass spectrometry to characterize post-translational modifications beyond glycosylation

  • Interaction proteomics to identify the complete "periostinome"

  • Spatial proteomics to map periostin distribution in tissue contexts

• Structural Biology:

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics

  • Cross-linking mass spectrometry to identify interaction interfaces

  • AlphaFold or similar AI approaches to predict structures of different splice variants

• Systems Biology Integration:

  • Network analysis of periostin-related signaling pathways

  • Mathematical modeling of the periostin-NF-κB feedback loop

  • Integration of omics data with clinical outcomes for biomarker development