Recombinant Human Serpin H1 (SERPINH1)

What is the molecular structure and function of SERPINH1?

SERPINH1 (also known as HSP47) belongs to the serpin superfamily of serine proteinase inhibitors. It is a 47 kDa heat shock protein that functions as a collagen-specific molecular chaperone. The protein contains a signal sequence at the N-terminus, two N-glycosylation sites, and an endoplasmic reticulum (ER) retention signal (RDEL) at the C-terminus . SERPINH1 is localized to the ER lumen and plays a crucial role in collagen biosynthesis by specifically binding to the folded triple helix, thereby stabilizing the structure . It prevents lateral aggregation of procollagen triple helices in the ER and guards their transport from the ER to the cis-Golgi. In the Golgi, the pH drop releases bound HSP47, which is then recycled back to the ER by its C-terminal RDEL sequence .

How does SERPINH1 differ from other members of the serpin family?

Unlike many other serpins that function as protease inhibitors through conformational changes and irreversible "suicide" inhibition mechanisms , SERPINH1 primarily functions as a molecular chaperone. Most serpins inhibit target enzymes by undergoing a dramatic conformational change involving the insertion of the reactive center loop (RCL) into a β-sheet, which distorts the active site of the target protease . In contrast, SERPINH1 doesn't function primarily as a protease inhibitor but instead acts as a collagen-specific chaperone, binding specifically to correctly folded triple-helical collagen and aiding in its proper folding and transport . Additionally, unlike other serpins whose activity can be controlled by specific cofactors like heparin (e.g., SERPINC1/antithrombin) , SERPINH1's function is regulated by pH changes during subcellular transport between the ER and Golgi apparatus .

What are the typical expression patterns of SERPINH1 in normal human tissues?

In normal human tissues, SERPINH1 is expressed primarily in collagen-producing cells. Immunohistochemistry demonstrates that SERPINH1 is highly expressed throughout the coronary vasculature and in fibroblasts in the human heart, with weak staining also detected in cardiomyocytes . According to analyses using the EndoDB database, the expression of SERPINH1 is highly similar in both veins and arteries across different tissues including heart, lungs, and liver . In the Human Protein Atlas database, SERPINH1 shows variable expression patterns in normal tissues, with higher expression in tissues with active collagen synthesis . SERPINH1 is constitutively expressed in these tissues, reflecting its role in normal collagen biosynthesis, but its expression can be significantly upregulated during stress conditions, particularly those requiring increased collagen production .

How does overexpression of SERPINH1 induce mesenchymal features in endothelial cells, and what are the implications for cardiovascular disease research?

Overexpression of SERPINH1 in human endothelial cells (both HUVECs and HCAECs) significantly alters cellular morphology, characterized by impaired or discontinuous vascular endothelial cadherin junctions, increased stress fiber formation, and larger cell size . At the molecular level, SERPINH1 overexpression leads to significant repression of endothelial cell markers (CD31, CDH5, TIE1, NRARP, and ID1) and induction of mesenchymal/EndMT markers (TAGLN, αSMA, CD44, VIM, NOTCH3, ZEB2, SLUG, FN1, VCAM1) .

This transition has profound implications for cardiovascular disease (CVD) research, as cardiovascular risk factors (aging, obesity, hypertension) can induce SERPINH1 expression, while exercise training represses it . The upregulation of SERPINH1 appears to be a key molecular event in the pathogenesis of CVD, as it promotes endothelial dysfunction, inflammation, and fibrotic changes. These findings suggest that SERPINH1 could serve as both a biomarker and therapeutic target in CVD, potentially allowing for the development of exercise-mimetic treatments that could benefit patients unable to follow training programs to reduce their CVD risk .

For researchers, this indicates that SERPINH1 modulation could be used to develop experimental models of endothelial dysfunction and to screen potential CVD therapeutics targeting endothelial-to-mesenchymal transition processes.

What is the relationship between SERPINH1 expression and immune cell infiltration in cancer, and how might this impact immunotherapy approaches?

SERPINH1 expression exhibits strong correlations with immune cell infiltration across multiple cancer types. Studies using the TIMER2 database and "CIBERSOFT" method have demonstrated associations between SERPINH1 expression and infiltration of various immune cell types, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells .

Specifically, SERPINH1 expression shows strong associations with immunoregulators and immune checkpoint markers in testicular germ cell tumors, brain lower grade glioma (LGG), pheochromocytoma and paraganglioma . In breast invasive carcinoma, LGG, and liver hepatocellular carcinoma, the relationship between SERPINH1 expression and immune cell infiltration is particularly pronounced .

The research implications are significant for immunotherapy approaches. SERPINH1 expression correlates with immune regulation, chemokines, and immune checkpoints , suggesting it may influence the tumor immune microenvironment and potentially affect response to immunotherapies. For researchers, this means:

• SERPINH1 could serve as a predictive biomarker for immunotherapy response

• Targeting SERPINH1 might enhance immunotherapy efficacy through modification of the tumor immune microenvironment

• Combined approaches targeting both SERPINH1 and immune checkpoints could be an effective strategy to explore

These findings point to a potential dual role for SERPINH1 as both a prognostic marker and a target for enhancing cancer immunotherapy strategies.

How do SERPINH1 mutations contribute to the pathogenesis of osteogenesis imperfecta, and what cellular mechanisms are involved?

SERPINH1 mutations have been implicated in recessive osteogenesis imperfecta (OI), a genetic disorder characterized by bone fragility and susceptibility to fractures. Research on OI dachshunds revealed that an HSP47(L326P) mutation affects post-translational modification, secretion, and cross-linking of type I collagen .

The cellular mechanisms involved include:

• Impaired collagen chaperone function: The mutated SERPINH1 fails to properly guide collagen folding and stabilization, resulting in misfolded collagen molecules that are unable to form proper triple helices.

• ER stress: The accumulation of misfolded collagen in the ER triggers the unfolded protein response (UPR), leading to ER stress and potentially apoptosis of collagen-producing cells.

• Aberrant bone collagen cross-linking: The mutation affects not only the structure of collagen molecules but also their post-translational modifications and cross-linking, which are essential for bone strength and integrity.

These mechanisms collectively contribute to the bone fragility phenotype observed in OI. For researchers, understanding these pathways provides potential therapeutic targets, such as chemical chaperones that might compensate for defective SERPINH1 function or approaches to reduce ER stress in collagen-producing cells. Additionally, this knowledge informs the development of animal models for OI and the interpretation of collagen biosynthesis defects in human patients with SERPINH1 mutations .

What are the optimal expression systems for producing recombinant SERPINH1, and how do they affect protein functionality?

Different expression systems produce recombinant SERPINH1 with varying characteristics, affecting both structural and functional properties:

E. coli Expression System:

• Produces non-glycosylated SERPINH1 with a molecular mass of approximately 48.9 kDa

• Advantages: Higher yield, cost-effectiveness, simpler purification process

• Limitations: Lacks post-translational modifications, particularly glycosylation, which may affect proper folding and function

• Best for: Structural studies, antibody production, assays not requiring full biological activity

HEK293 Cell Expression System:

• Produces glycosylated SERPINH1 with observed molecular weights of 48-55 kDa due to glycosylation

• Advantages: Contains proper mammalian post-translational modifications, more likely to retain native conformation and biological activity

• Limitations: Lower yield, higher cost, more complex purification

• Best for: Functional studies, cell-based assays, applications requiring full biological activity

Affinity Tags and Their Impact:
Most recombinant SERPINH1 proteins incorporate a polyhistidine tag (His-tag) at either the N-terminus or C-terminus to facilitate purification . For C-terminal tagging, care must be taken to ensure the tag doesn't interfere with the RDEL ER retention signal. N-terminal tags avoid this issue but may affect signal peptide function.

Recommended Approach for Functional Studies:
For studies investigating SERPINH1's collagen chaperone function, researchers should use HEK293-expressed SERPINH1 with proper glycosylation. The protein should be stored in a stabilizing buffer (typically PBS, pH 7.4 with 5-20% glycerol or trehalose) and maintained at -20°C to -80°C for long-term storage, avoiding repeated freeze-thaw cycles .

What are the key considerations when designing experiments to study SERPINH1's role in epithelial-mesenchymal transition (EMT)?

When investigating SERPINH1's role in EMT, several critical experimental design considerations should be addressed:

Cell Model Selection:

• Endothelial cells (HUVECs, HCAECs) are optimal for studying endothelial-to-mesenchymal transition (EndMT)

• Cancer cell lines with epithelial characteristics are suitable for classical EMT studies

• Primary cells are preferred over immortalized lines to avoid confounding effects of immortalization on EMT programs

Genetic Manipulation Approaches:

• Overexpression: Use lentiviral vectors encoding myc-tagged hSERPINH1 for stable expression

• Silencing/Knockout: Apply multiple independent shRNA constructs (aim for ≥80% knockdown efficiency) or CRISPR-Cas9 for complete knockout

• Controls: Include proper vector controls and validate knockdown/overexpression at both mRNA and protein levels

Key Readouts to Measure:

• Morphological Changes: Document alterations in cell shape, size, and junction formation using phase-contrast and fluorescence microscopy

• Marker Expression: Assess both downregulation of epithelial/endothelial markers (e.g., CD31, CDH5, TIE1) and upregulation of mesenchymal markers (e.g., TAGLN, αSMA, VIM, FN1) using qRT-PCR, western blotting, and immunofluorescence

• Functional Assays: Measure changes in cell migration, invasion, and collagen deposition

• Time-Course Analysis: Examine both early (2 days) and late (10+ days) effects to capture the complete transition process

Validation in Multiple Systems:

• Confirm findings in at least two different cell types

• Verify with both in vitro and in vivo models when possible

• Use patient samples to establish clinical relevance

By addressing these considerations, researchers can design robust experiments to elucidate SERPINH1's mechanistic role in EMT, which has significant implications for understanding cardiovascular disease, cancer progression, and fibrotic disorders.

What techniques are most effective for analyzing SERPINH1's prognostic value in cancer, and how should contradictory findings be interpreted?

To effectively analyze SERPINH1's prognostic value in cancer, researchers should employ a multi-faceted approach combining various computational and experimental techniques:

Experimental Validation Approaches:

• Tissue Microarray Analysis: Develop and analyze tissue microarrays containing paired tumor/normal samples across multiple stages to validate expression patterns

• Correlation with Clinical Parameters: Compare SERPINH1 expression with T-stage progression, recurrence, and metastasis

• Functional Validation: Perform in vitro and in vivo experiments to establish causality between SERPINH1 expression and aggressive phenotypes

Interpreting Contradictory Findings:
When faced with contradictory findings regarding SERPINH1's prognostic value (e.g., favorable in CHOL, OV, and THCA but unfavorable in most other cancers) :

• Consider Tissue Context: SERPINH1 function may be tissue-specific due to differences in collagen dependency and microenvironment

• Analyze Genetic Background: VHL mutation status significantly impacts SERPINH1's prognostic value in clear cell renal cell carcinoma (ccRCC)

• Examine Splice Variants: Different SERPINH1 isoforms may have opposing functions

• Evaluate Immune Context: SERPINH1's relationship to immune infiltration varies across cancer types

• Account for Technical Variation: Methodology differences (antibodies, platforms, scoring methods) may contribute to discrepancies

What are the most effective assays for measuring SERPINH1's collagen chaperone activity in vitro?

Several specialized assays can be employed to measure SERPINH1's collagen chaperone activity effectively:

Collagen Fibril Formation Assay:
This assay directly measures SERPINH1's impact on collagen assembly by monitoring the kinetics of fibril formation. Type I collagen (0.5-1 mg/mL) is incubated with or without recombinant SERPINH1 at physiological temperature (37°C), and turbidity is measured at 313 nm over time. SERPINH1's chaperone activity is reflected by changes in the lag phase, growth rate, and final turbidity of collagen fibrils . This approach allows quantitative assessment of how SERPINH1 influences the rate and extent of collagen fibril assembly.

Collagen Thermal Stability Assay:
This technique evaluates SERPINH1's ability to stabilize collagen triple helices against thermal denaturation. Circular dichroism (CD) spectroscopy at 221 nm can monitor the helical content of collagen during controlled temperature increases (typically 25-50°C). The presence of functional SERPINH1 will increase the melting temperature (Tm) of collagen, indicating stabilization of the triple helix . Alternative approaches include differential scanning calorimetry or fluorescence-based thermal shift assays.

Collagen Secretion and Deposition Assays:
These cellular assays assess SERPINH1's role in collagen biosynthesis and secretion:

• Immunohistochemistry for type 1 collagen detection in SERPINH1-modified cells (overexpression or silencing)

• Pulse-chase experiments with radiolabeled proline to track collagen synthesis and secretion rates

• Hydroxyproline quantification assays to measure total collagen content

SERPINH1-Collagen Binding Assays:
Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can determine the binding affinity and kinetics between recombinant SERPINH1 and various collagen types under different pH conditions, mimicking the ER-to-Golgi transport pathway .

When conducting these assays, researchers should include appropriate controls such as heat-inactivated SERPINH1, other molecular chaperones, and pH variations to account for SERPINH1's pH-dependent binding properties.

How can researchers effectively investigate the relationship between SERPINH1 and TGF-β signaling in disease models?

Investigating the relationship between SERPINH1 and TGF-β signaling requires a comprehensive approach combining molecular, cellular, and in vivo methodologies:

Protein-Level Interaction Analysis:

• Co-immunoprecipitation (Co-IP): Determine whether SERPINH1 directly interacts with TGF-β receptors or downstream SMAD proteins

• Proximity Ligation Assay (PLA): Visualize and quantify interactions between SERPINH1 and TGF-β pathway components in intact cells

• Chromatin Immunoprecipitation (ChIP): Assess whether TGF-β-activated SMADs bind to the SERPINH1 promoter to regulate its expression

Signaling Pathway Analysis:

• SMAD Phosphorylation: Measure phosphorylation of SMAD2/3 following TGF-β treatment in cells with SERPINH1 overexpression or knockdown

• Transcriptional Reporter Assays: Use SMAD-responsive luciferase reporters to quantify TGF-β signaling activity in the presence or absence of SERPINH1

• RNA-Seq Analysis: Compare transcriptomic changes induced by TGF-β in control versus SERPINH1-modified cells to identify differentially regulated pathways

Functional Consequences:

• EMT Marker Expression: Analyze epithelial and mesenchymal markers (E-cadherin, N-cadherin, Vimentin) in response to TGF-β with or without SERPINH1 modulation

• Collagen Production: Quantify collagen synthesis and deposition following TGF-β treatment in SERPINH1-modified cells

• Cell Migration/Invasion Assays: Assess whether SERPINH1 affects TGF-β-induced changes in cell motility and invasiveness

In Vivo Validation:

• Conditional Knockout Models: Generate tissue-specific SERPINH1 knockout animals and assess their response to TGF-β administration or TGF-β-driven disease models

• TGF-β Antagonism in SERPINH1-Overexpressing Models: Test whether TGF-β inhibitors can rescue phenotypes in SERPINH1-overexpressing disease models

• Patient Sample Analysis: Evaluate correlation between SERPINH1 expression and TGF-β pathway activation markers in disease tissues

The findings from research on clear cell renal cell carcinoma suggest that antagonizing TGF-β can suppress tumorigenesis and regress established tumors, and SERPINH1/HSP47 may function as a driver gene in this process . This indicates a potential co-targeting strategy involving both SERPINH1 and TGF-β pathway inhibition for therapeutic development.

What are the current challenges in developing selective inhibitors of SERPINH1 for therapeutic applications?

Developing selective inhibitors of SERPINH1 faces several significant challenges that researchers must address:

Selectivity Challenges:

• Serpin Family Homology: SERPINH1 shares structural features with other serpins, creating potential off-target effects. Inhibitors must selectively target SERPINH1 without affecting related serpins that regulate critical proteolytic cascades .

• Tissue Distribution: SERPINH1 is expressed in multiple collagen-producing tissues, not just disease-affected tissues. Tissue-specific delivery approaches may be necessary to avoid disrupting normal collagen biosynthesis in healthy tissues.

Efficacy and Safety Concerns:

• Complete vs. Partial Inhibition: Total SERPINH1 inhibition might disrupt essential collagen biosynthesis, whereas partial inhibition might be insufficient for therapeutic effect. Determining the optimal degree of inhibition represents a significant challenge.

• Long-term Safety: Chronic inhibition of SERPINH1 might lead to connective tissue abnormalities similar to those observed in osteogenesis imperfecta caused by SERPINH1 mutations .

To overcome these challenges, researchers might consider:

• Structure-based design approaches focusing on the collagen-binding interface

• Allosteric modulators that tune SERPINH1 activity rather than completely inhibiting it

• Targeted delivery systems to direct inhibitors specifically to disease-relevant tissues

• Combination approaches targeting both SERPINH1 and downstream effectors (e.g., TGF-β pathway)

Finding highly specific inhibitors of SERPINH1/HSP47, including small molecules with acceptable safety profiles, remains an active area of research with broad therapeutic applicability in conditions ranging from cardiovascular disease to cancer and fibrotic disorders .

How is SERPINH1 being investigated as a biomarker in liquid biopsies for cancer detection and monitoring?

SERPINH1 shows promise as a biomarker in liquid biopsies for cancer detection and monitoring, with several research approaches currently being explored:

Circulating Tumor Cell (CTC) Analysis:
SERPINH1 expression in CTCs may serve as a marker for cells undergoing epithelial-mesenchymal transition (EMT), a critical process in metastasis. Research indicates that SERPINH1 is significantly associated with metastasis in multiple cancer types, including gastric cancer and renal cell carcinoma . Detection methodologies focus on:

• Immunocytochemical staining of SERPINH1 in isolated CTCs

• Single-cell RNA sequencing of CTCs to detect SERPINH1 expression patterns

• Correlation of SERPINH1-positive CTCs with clinical outcomes and treatment responses

Cell-Free DNA Methylation Patterns:
Studies have demonstrated correlations between SERPINH1 expression and DNA methylation in various cancers . Current research investigates:

• SERPINH1 promoter methylation patterns in circulating cell-free DNA

• Development of sensitive PCR-based assays to detect SERPINH1 methylation signatures

• Integration of SERPINH1 methylation data with other cancer-specific methylation markers for improved sensitivity and specificity

Exosomal SERPINH1:
Tumor-derived exosomes may contain SERPINH1 protein or mRNA that can be detected in liquid biopsies. Research directions include:

• Development of exosome isolation protocols optimized for SERPINH1 detection

• Correlation between exosomal SERPINH1 levels and tumor burden or treatment response

• Functional studies on whether exosomal SERPINH1 contributes to pre-metastatic niche formation

Multi-Marker Panel Development:
Given the complex heterogeneity of cancer, SERPINH1 is being investigated as part of multi-marker panels. For example, in clear cell renal cell carcinoma, researchers are comparing SERPINH1 with other reported biomarkers including HADHA, DIABLO, PDZK1, LDHA, BIRC5, CA9, FSCN2, and IMP3 . SERPINH1 demonstrated superior capability in predicting recurrence compared to several of these markers.

The development of SERPINH1 as a liquid biopsy biomarker requires rigorous validation across large patient cohorts with diverse cancer types, stages, and treatments to establish its clinical utility for early detection, monitoring of minimal residual disease, and early prediction of recurrence or treatment resistance.

What are the implications of SERPINH1's role in collagen biosynthesis for tissue engineering and regenerative medicine?

SERPINH1's critical role in collagen biosynthesis has significant implications for tissue engineering and regenerative medicine applications:

Enhancing Scaffold Biomimicry and Integrity:
SERPINH1 manipulation could improve the production and quality of collagen-based scaffolds:

• Co-expression systems: Engineering cells to co-express SERPINH1 with specific collagen types could enhance the correct folding and assembly of collagen triple helices within bioengineered constructs.

• Quality control enhancement: SERPINH1 supplementation during scaffold fabrication might improve collagen fibril organization, leading to superior mechanical properties and stability.

• Cross-linking optimization: As SERPINH1 affects collagen cross-linking , modulating its activity could allow fine-tuning of scaffold degradation rates to match tissue regeneration timelines.

Cell-Based Therapeutic Approaches:
Manipulating SERPINH1 expression in therapeutic cells shows promise for several applications:

• Enhancing MSC therapy: Transient upregulation of SERPINH1 in mesenchymal stem cells could improve their collagen production capacity for cartilage or tendon repair.

• Fibrosis control: Controlled downregulation of SERPINH1 in fibroblasts might prevent excessive collagen deposition in wound healing applications, reducing scarring.

• Vascular tissue engineering: Carefully balancing SERPINH1 expression in endothelial cells could prevent unwanted EndMT while maintaining appropriate collagen production for vessel integrity .

Disease Modeling and Drug Screening:
SERPINH1 manipulation enables creation of more accurate disease models:

• Fibrotic disease modeling: Overexpression of SERPINH1 in tissue-specific organoids could recapitulate fibrotic pathologies for drug screening.

• Osteogenesis imperfecta models: Introduction of SERPINH1 mutations found in OI patients into stem cells allows development of personalized bone tissue models for therapy testing .

• Cardiovascular disease models: Modulation of SERPINH1 in cardiac organoids could mimic age-related or obesity-induced cardiovascular pathologies .

Challenges and Future Directions:
Several technical challenges remain to be addressed:

• Temporal regulation: Developing systems for precise temporal control of SERPINH1 expression during tissue maturation processes.

• Spatial patterning: Creating gradients of SERPINH1 activity to mimic tissue-specific collagen distribution patterns.

• Balance with other ECM components: Ensuring SERPINH1 manipulation doesn't disrupt the balance between collagen and other extracellular matrix components.

Research into these applications is advancing our understanding of how to leverage SERPINH1's functions for creating more biomimetic engineered tissues with appropriate collagen content, organization, and mechanical properties, potentially revolutionizing approaches to tissue replacement and regeneration.

How are genetic variations in SERPINH1 being studied in relation to diverse pathological conditions beyond osteogenesis imperfecta?

Genetic variations in SERPINH1 are being investigated across multiple pathological conditions, revealing its broader significance beyond osteogenesis imperfecta:

Cardiovascular Disorders:
SERPINH1 polymorphisms are being examined for associations with cardiovascular disease risk and progression:

• Coronary artery disease: Studies are analyzing whether SERPINH1 variants correlate with collagen content in atherosclerotic plaques and plaque stability.

• Aortic aneurysms: Research is investigating SERPINH1 variations in patients with thoracic and abdominal aortic aneurysms, particularly focusing on variants that may affect collagen quality in vascular walls .

• Heart failure: Genetic association studies are examining links between SERPINH1 SNPs and heart failure with preserved ejection fraction, where fibrosis plays a key role.

Fibrotic Disorders:
SERPINH1 variants are being studied in conditions characterized by excessive fibrosis:

• Pulmonary fibrosis: Case-control studies are investigating whether SERPINH1 polymorphisms confer susceptibility to idiopathic pulmonary fibrosis or affect disease progression rates.

• Liver fibrosis/cirrhosis: Research is examining associations between SERPINH1 variants and progression of liver fibrosis in various chronic liver diseases.

• Systemic sclerosis: Studies are exploring whether SERPINH1 polymorphisms contribute to the extensive fibrosis characteristic of this autoimmune condition.

Pregnancy Complications:
SERPINH1 variations have been associated with pregnancy-related complications:

• Preterm premature rupture of membranes (PPROM): Multiple studies indicate that nucleotide polymorphisms in SERPINH1 may be associated with PPROM, which can lead to preterm birth .

• Placental abnormalities: Research is investigating whether SERPINH1 variants affect collagen remodeling in the placenta, potentially influencing placental development and function.

Cancer Susceptibility and Progression:
The role of SERPINH1 variants in cancer is an emerging area of research:

• Tumor susceptibility: Studies are examining whether germline SERPINH1 polymorphisms correlate with cancer risk in specific populations.

• Progression markers: Research is analyzing whether somatic SERPINH1 mutations or specific variants correlate with cancer aggressiveness, metastatic potential, or drug resistance .

• Predictive biomarkers: Investigations are determining whether SERPINH1 genetic variations can predict treatment response, particularly to therapies targeting tumor microenvironment or fibrosis.

Methodological Approaches:
Researchers are employing multiple techniques to study these associations:

• Genome-wide association studies (GWAS) to identify SERPINH1 SNPs correlated with disease phenotypes

• Functional genomics to determine how specific variants affect SERPINH1 expression or function

• Gene-environment interaction studies to examine how environmental factors (stress, diet, etc.) interact with SERPINH1 variants to influence disease susceptibility

• Animal models with knocked-in human SERPINH1 variants to study phenotypic consequences in controlled systems