SERPINA5 Human, Active

What is the molecular classification and primary function of SERPINA5?

SERPINA5 (Serpin Peptidase Inhibitor Clade A Member 5), also known as protein C inhibitor (PCI), alpha-1 antiproteinase, or antitrypsin, belongs to the serpin family of glycoproteins. It was first identified as an inhibitor of the anticoagulant protease activated protein C (APC) . As a secreted protein with extensive tissue distribution, SERPINA5 is found in various body fluids including blood plasma, seminal plasma, and cervicovaginal fluid .

Functionally, SERPINA5 serves as a broad-spectrum inhibitor of several serine proteases, plasminogen activators, and kallikreins . Beyond its canonical roles in hemostasis and thrombosis, recent research has uncovered novel functions in antiviral immunity, tumor suppression, and host defense mechanisms .

How is SERPINA5 expression regulated in different tissue types?

SERPINA5 exhibits a wide tissue distribution pattern in humans, with expression profiles that vary significantly across tissue types . In particular:

• In normal human tissues: SERPINA5 shows broad expression across multiple organ systems

• In immune cells: Expression is upregulated following stimulation with Toll-like receptor (TLR) agonists (LPS, PolyI:C, R848), interferon α (IFN α), and during viral infections in a time-dependent manner

• In mouse models: Expression is predominantly restricted to the reproductive tract, creating important species-specific differences in SERPINA5 biology

Expression regulation occurs through:

• IFN-dependent mechanisms, as demonstrated by abrogated upregulation in IFN α receptor-deficient BMM cells (IFNAR−/−)

• Transcriptional activation through STAT1 signaling pathways

• Tissue-specific regulatory elements that control baseline expression

What is known about the subcellular localization of SERPINA5?

SERPINA5 demonstrates dynamic subcellular localization patterns that contribute to its diverse functions:

• Primary localization: As a secreted protein, SERPINA5 is initially synthesized in the endoplasmic reticulum and processed through the secretory pathway

• Extracellular distribution: Present in multiple body fluids including plasma, seminal fluid, and cervicovaginal fluid

• Cellular internalization: SERPINA5 can be internalized by cells through a mechanism dependent on:

  • Phosphatidylethanolamine interaction

  • The intact N-terminus which functions as a cell-penetrating peptide

• Nuclear translocation: Following internalization, SERPINA5 can be transported to the nucleus, suggesting potential roles in transcriptional regulation

This complex localization pattern enables SERPINA5 to perform distinct functions in different cellular compartments and contributes to its multifaceted biological roles.

How does SERPINA5 contribute to antiviral immunity and what are the underlying mechanisms?

SERPINA5 functions as an IFN-stimulated gene (ISG) with significant antiviral properties through several distinct mechanisms:

• Transcriptional activation of antiviral pathways:

  • Promotes activation of IFN-β promoter and IFN-stimulated response element (ISRE) promoter

  • Upregulates expression of IFN-related genes including IFN-β, MX1, CXCL10, IL-1β, and IFN-λ

• Enhancement of JAK-STAT signaling:

  • Interacts directly with STAT1 but not with STAT2 or IRF9

  • Promotes STAT1 phosphorylation and nuclear translocation

  • Facilitates formation of transcriptionally active ISGF3 complexes

• Direct inhibition of viral replication:

  • Significantly suppresses HSV-1 replication in multiple cell types in a dose-dependent manner

  • Cells with SERPINA5 knockdown demonstrate increased susceptibility to HSV-1 infection

• Signaling pathway independence:

  • Functions independently of the cGAS-STING pathway, as SERPINA5 overexpression upregulates IFN-β and ISG56 expression even in the presence of STING inhibitor C-176

Transcriptomic analysis revealed that SERPINA5 treatment significantly alters the expression landscape with 1099 upregulated genes and 894 downregulated genes, with enrichment in pathways related to TNF signaling, viral protein interaction with cytokine receptors, and Toll-like receptor signaling .

What are the experimental approaches to studying SERPINA5's role in tumor suppression?

SERPINA5 demonstrates tumor suppressive activity, particularly in hepatocellular carcinoma (HCC), which can be investigated through these methodological approaches:

• Expression analysis in clinical samples:

  • Quantitative real-time PCR to assess DNA dosage and expression levels

  • Correlation of expression with clinical progression parameters and patient outcomes

• Functional assessment through gain/loss-of-function models:

  • Lentiviral expression systems for ectopic SERPINA5 expression

  • shRNA or siRNA-mediated knockdown to evaluate oncogenic phenotypes

  • CRISPR/Cas9 genome editing for complete gene knockout

• Migration and invasion assays:

  • Transwell migration assays with or without Matrigel coating

  • Wound healing assays to assess collective cell migration

  • 3D spheroid invasion assays in extracellular matrix

• In vivo metastasis models:

  • Orthotopic xenograft models with cells expressing different levels of SERPINA5

  • Tail vein injection models to assess lung colonization capacity

  • Intrasplenic injection models for liver metastasis studies

• Pathway analysis techniques:

  • Protein interaction studies (co-immunoprecipitation, proximity ligation assays)

  • Analysis of fibronectin–integrin β1 signaling pathway components

  • Phosphorylation status assessment of downstream effectors

Research has shown that SERPINA5 inhibits HCC metastasis through direct interaction with fibronectin, disrupting the fibronectin–integrin signaling pathway crucial for cell migration and invasion .

How do post-translational modifications affect SERPINA5 function and activity?

SERPINA5 undergoes several post-translational modifications that significantly impact its inhibitory activity, binding specificity, and cellular localization:

• Glycosylation:

  • As a glycoprotein, SERPINA5 contains N-linked glycosylation sites

  • Glycosylation patterns influence protein stability and half-life

  • May affect binding to proteases and other interaction partners

• Phosphorylation:

  • Potential phosphorylation sites may regulate activity

  • Phosphorylation status can influence subcellular localization

  • May create docking sites for signaling molecule interactions

• Binding-induced conformational changes:

  • Interaction with glycosaminoglycans modulates inhibitory activity and specificity

  • Phospholipid binding (particularly phosphatidylethanolamine) facilitates cellular internalization

  • Retinoic acid binding may regulate inhibitory functions

• Proteolytic processing:

  • Target protease interaction involves conformational change in the reactive center loop

  • Proteolytic cleavage may generate fragments with distinct biological activities

Experimental approaches to study these modifications include mass spectrometry-based proteomic analysis, site-directed mutagenesis of modification sites, and structure-function relationship studies using recombinant variants.

What are optimal expression systems for producing active recombinant human SERPINA5?

Producing functionally active recombinant human SERPINA5 requires careful consideration of expression systems to ensure proper folding, post-translational modifications, and biological activity:

• Mammalian expression systems:

  • HEK293 cells: Provide proper glycosylation and secretion

  • CHO cells: High protein yields with mammalian-like modifications

  • Methods: Stable transfection or transient expression using lipid-based transfection, electroporation, or viral vectors

  • Vectors: pCDNA3.1, pSecTag2, or lentiviral constructs with signal peptides for secretion

• Insect cell expression:

  • Sf9 or High Five cells with baculovirus expression systems

  • Provides higher yields than mammalian systems while maintaining core glycosylation

  • Bac-to-Bac or flashBAC systems with polyhedrin or p10 promoters

• Purification strategies:

  • Affinity tags: His-tag, FLAG-tag, or Strep-tag for initial capture

  • Ion-exchange chromatography for further purification

  • Size-exclusion chromatography as a polishing step

  • Importance of including protease inhibitors throughout purification

• Activity validation:

  • Protease inhibition assays using chromogenic or fluorogenic substrates

  • Target protease (e.g., activated protein C) inhibition kinetics determination

  • Binding assays with glycosaminoglycans, phospholipids, and retinoic acid

  • Cell-based functional assays (antiviral, anti-migration)

For antiviral studies, specialized activity assays include viral plaque reduction assays, TCID50 determination, and quantification of viral gene expression by RT-qPCR as demonstrated in previous studies with HSV-1 .

What methodologies are most effective for studying SERPINA5-protein interactions?

Investigating SERPINA5's interactions with binding partners requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Effective for detecting stable protein-protein interactions

  • Can be performed with endogenous proteins or overexpressed tagged variants

  • Western blot analysis of immunoprecipitated complexes

  • Useful for confirming interactions with STAT1 and other signaling molecules

• Pull-down assays:

  • GST-fusion or His-tagged SERPINA5 as bait protein

  • Useful for direct binding studies with purified partner proteins

  • Can identify direct vs. indirect interactions

• Surface Plasmon Resonance (SPR):

  • Determines binding kinetics and affinity constants

  • Particularly valuable for SERPINA5 interactions with:

    • Proteases (activated protein C, plasminogen activators)

    • Extracellular matrix proteins (fibronectin)

    • Glycosaminoglycans

• Proximity-based methods:

  • Proximity Ligation Assay (PLA) for detecting interactions in situ

  • FRET/BRET for dynamic interaction studies in living cells

  • BioID or APEX2 proximity labeling for interaction networks

• Analytical techniques for complex formation:

  • Size-exclusion chromatography coupled with multi-angle light scattering

  • Native PAGE and blue native PAGE

  • Analytical ultracentrifugation

• Structural approaches:

  • X-ray crystallography of SERPINA5-protein complexes

  • Cryo-EM for larger complexes

  • Hydrogen-deuterium exchange mass spectrometry for mapping interaction interfaces

Relevant experimental examples include co-immunoprecipitation assays demonstrating SERPINA5's interaction with STAT1 but not with STAT2 or IRF9, and biochemical analyses showing direct binding to fibronectin that disrupts integrin signaling .

How can researchers effectively design knockdown and overexpression systems for SERPINA5 functional studies?

Designing effective genetic manipulation systems for SERPINA5 requires consideration of several technical factors:

• SERPINA5 knockdown strategies:

  • siRNA approaches:

    • Validated siRNA sequences targeting SERPINA5 (e.g., siRNA-820 shown to have high knockdown efficiency)

    • Transfection methods: lipid-based reagents for transient knockdown

    • Concentration optimization to minimize off-target effects

  • shRNA approaches:

    • Lentiviral vectors for stable knockdown

    • Inclusion of appropriate selection markers

    • Verification of knockdown by RT-qPCR and Western blotting

  • CRISPR/Cas9 genome editing:

    • gRNA design targeting early exons

    • Screening methods for knockout validation

    • Single-cell cloning and genotyping

• SERPINA5 overexpression systems:

  • Expression constructs:

    • Full-length SERPINA5 cDNA with intact signal peptide

    • Epitope tags (FLAG, HA, V5) for detection and immunoprecipitation

    • Inducible expression systems (Tet-On/Off) for dose-dependent studies

  • Delivery methods:

    • Transient transfection for short-term studies

    • Viral vectors (lentivirus, adenovirus) for difficult-to-transfect cells

    • Generation of stable cell lines with controlled expression levels

  • Functional domain variants:

    • Site-directed mutagenesis of key functional residues

    • Domain deletion constructs to map functional regions

    • Secretion-deficient variants for studying intracellular functions

• Validation approaches:

  • mRNA level verification by RT-qPCR

  • Protein level confirmation by Western blotting

  • Secretion analysis by ELISA of culture media

  • Functional assays specific to research question (e.g., antiviral assays, migration assays)

Experimental research has successfully utilized these approaches to demonstrate SERPINA5's biological functions, including the creation of 293T cell lines stably expressing SERPINA5 (293T-SERPINA5 cells) for antiviral studies and lentiviral expression systems for analyzing anti-metastatic effects in HCC .

How might SERPINA5's antiviral properties be leveraged for therapeutic development?

SERPINA5's newly discovered antiviral properties open several avenues for therapeutic development:

• Direct antiviral applications:

  • Recombinant SERPINA5 as a biological therapeutic against susceptible viruses

  • Development of SERPINA5-derived peptides with enhanced stability and cell penetration

  • Combination approaches with existing antivirals for synergistic effects

• Mechanistic targets for drug development:

  • Small molecules enhancing SERPINA5-STAT1 interaction

  • Compounds promoting SERPINA5 nuclear translocation

  • Agents increasing endogenous SERPINA5 expression

• Delivery strategies:

  • Viral vector-mediated gene therapy for localized SERPINA5 expression

  • Nanoparticle-based delivery of recombinant protein or expression constructs

  • Cell-penetrating peptide conjugates based on SERPINA5's N-terminal domain

• Experimental validation requirements:

  • Efficacy testing in cellular infection models beyond HSV-1

  • Animal model validation in immunocompetent and immunocompromised settings

  • Safety and immunogenicity assessments

  • Pharmacokinetic/pharmacodynamic studies

Evidence supporting these approaches includes data showing SERPINA5 significantly suppresses HSV-1 replication in a dose-dependent manner, with viral titers markedly decreased as measured by TCID50 assays . Additional research demonstrated that cells become more susceptible to viral infection when SERPINA5 is knocked down .

What experimental approaches can resolve contradictions in SERPINA5 research findings?

Research on SERPINA5 has revealed apparently contradictory findings that require specialized experimental approaches to resolve:

• Species-specific differences:

  • Comparative studies between human SERPINA5 and mouse serpinA5

  • Development of humanized mouse models expressing human SERPINA5

  • Cross-species functional complementation experiments

  • Analysis reconciling restricted expression in mice (reproductive tract) versus broad distribution in humans

• Context-dependent activities:

  • Controlled expression studies in diverse cell types

  • Investigation of tissue-specific binding partners

  • Identification of cell-specific post-translational modifications

  • Systematic analysis of microenvironmental factors affecting function

• Contradictions in signaling mechanisms:

  • Temporal analysis of signaling pathway activation

  • Single-cell approaches to detect heterogeneous responses

  • Pathway inhibitor studies with precise timing controls

  • Systems biology modeling of interconnected pathways

• Conflicting clinical correlations:

  • Meta-analysis of expression data across multiple cancer types

  • Stratification by molecular subtypes within disease categories

  • Correlation with specific genetic alterations

  • Integration of genomic, transcriptomic, and proteomic data

For example, comparative studies could help understand why serpinA5-knockout mice show fertility defects in males but no other obvious phenotypes , while human SERPINA5 has broader biological functions including tumor suppression and antiviral activity .

What are the most promising research directions for understanding SERPINA5's role in cancer biology?

Current evidence suggests several high-priority research directions for elucidating SERPINA5's role in cancer:

• Mechanistic investigations of tumor suppression:

  • Comprehensive mapping of SERPINA5 interactions with:

    • Cell adhesion molecules (particularly fibronectin)

    • Integrin signaling components

    • Growth factor receptors and downstream effectors

  • Analysis of SERPINA5's impact on epithelial-mesenchymal transition

  • Evaluation of effects on cancer stem cell properties

• Clinical correlation studies:

  • Multi-cancer analysis of SERPINA5 expression patterns

  • Association with specific genomic alterations beyond HCC

  • Prognostic value assessment in various cancer types

  • Potential as a biomarker for metastatic risk

• Therapeutic exploitation approaches:

  • Development of SERPINA5 mimetics for anti-metastatic therapy

  • Combination strategies with immune checkpoint inhibitors

  • Targeted delivery to tumor microenvironment

  • Methods to restore expression in SERPINA5-deficient tumors

• Integration with immune response:

  • Investigation of dual roles in antiviral immunity and tumor biology

  • Effects on tumor-infiltrating immune cells

  • Potential impact on immunotherapy responses

  • Role in virus-associated cancers

Experimental data supporting these directions includes findings that SERPINA5 expression negatively correlates with malignant progression of HCC, and that it directly interacts with fibronectin to disrupt fibronectin-integrin signaling pathways critical for tumor cell migration and invasion .