Recombinant Mouse Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D (Ssc5d), partial

What is the molecular structure of mouse SSC5D and how does it compare to its human homolog?

Mouse S5D-SRCRB (SSC5D) is a soluble member of the scavenger receptor cysteine-rich superfamily (SRCR-SF). The protein contains five SRCR domains at the N-terminus and a heavily glycosylated, mucin-like domain at the C-terminus. The s5d-srcrb gene maps to mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 amino acids, with a predicted molecular weight of 144.6 kDa .

Although the predicted molecular weight is 144.6 kDa, experimental evidence using recombinant mouse S5D-SRCRB-HA expressed in HEK 293-EBNA cells revealed that the secreted protein appears as a single broad band of approximately 200 kDa when analyzed by SDS-PAGE under reducing conditions. This difference between observed and predicted molecular weight suggests extensive post-translational modifications .

The human and mouse SSC5D proteins share high sequence homology, with the N-terminal SRCR domains showing 95% amino acid sequence identity . This high conservation suggests functional importance across species.

What expression systems are most effective for producing recombinant mouse SSC5D?

Based on published methodologies, mammalian expression systems appear to be most effective for producing properly folded and post-translationally modified recombinant mouse SSC5D. Specifically:

• Mammalian expression system: The full-length cDNA sequence of mouse s5d-srcrb has been successfully expressed using an episomal mammalian-expression system in HEK 293-EBNA cells, yielding a glycosylated soluble recombinant form >200 kDa .

• Expression vector strategy: Fusion of the full-length cDNA sequence with a C-terminal HA tag and cloning into the pCEP-Pu vector has proven effective .

• Protein collection: The recombinant protein is secreted into the culture medium, allowing collection from serum-free supernatants of stable transfectants .

Although commercial sources indicate that E. coli, yeast, and baculovirus expression systems can also be used , the complex glycosylation pattern and large size of SSC5D suggest that mammalian systems may provide more physiologically relevant protein forms.

What is the tissue expression pattern of SSC5D in mice?

Mouse SSC5D exhibits a restricted tissue-expression pattern, with significant expression in specific organs:

Immunohistochemistry findings:

• Strong expression in: serosal salivary gland, exocrine pancreas, testis

• Moderate expression throughout: gastrointestinal tract, genitourinary tract

• Selective expression in: kidney tubular structures (likely distal collecting tubules)

• Negative expression in: lung, heart

• Few scattered positive cells in: spleen

RT-qPCR analysis results:
The following table summarizes relative expression levels of s5d-srcrb in mouse tissues:

Interestingly, SSC5D expression was not detected in resting or LPS-stimulated bone marrow-derived monocytes, nor in various cell lines of monocytic, lymphocytic, or epithelial origin .

Has SSC5D expression been found to change in disease states?

Yes, recent research has identified significant changes in SSC5D expression in heart failure. Despite previous immunohistochemistry showing negative expression in normal heart tissue, heart failure appears to induce SSC5D expression:

• Using RNA sequencing data analysis, SSC5D levels were found to be significantly elevated in failing hearts compared to non-failing hearts .

• In murine models of myocardial infarction or transverse aortic constriction, Ssc5d mRNA levels were markedly increased compared to sham groups .

• Single-cell RNA sequencing data demonstrated that Ssc5d is predominantly expressed in cardiac fibroblasts in the context of heart failure .

• Serum SSC5D levels were considerably elevated in heart failure patients compared to control groups:

  • Heart failure group: 15,789.35 (10,745.32–23,110.65) pg/mL

  • Control group: 8,938.72 (6,154.97–12,778.81) pg/mL (p < 0.0001)

These findings suggest that SSC5D expression can be induced in tissues where it is normally absent in response to pathological conditions.

What molecular interactions does SSC5D participate in, and how can they be studied experimentally?

SSC5D engages in multiple types of molecular interactions that can be studied through various experimental approaches:

1. Pathogen-Associated Molecular Pattern (PAMP) Recognition:
SSC5D binds to PAMPs present on the cell walls of Gram-positive and Gram-negative bacteria and fungi, functioning as a pattern recognition receptor . These interactions can be studied using:

• Conventional protein-bacteria binding assays

• Surface plasmon resonance (SPR)-based assays

• Bacterial aggregation assays

2. Extracellular Matrix Protein Binding:
SSC5D interacts with host extracellular matrix components:

• Laminin binding: Demonstrated using ELISA-based assays showing dose-dependent binding

• Fibronectin: Shows low binding

• Collagen I and IV: Shows negative binding

3. Lectin Interactions:
SSC5D displays sugar-dependent interaction with galectin-1:

• Method: GST-Gal1 Sepharose bead pull-down assays with competition by lactose (3-30 mM)

• Findings: Specific binding is competed in a dose-dependent manner by lactose but not by sucrose

These diverse molecular interactions suggest that SSC5D contributes to both innate immune defense and epithelial homeostasis through recognition of both pathogenic and endogenous elements.

What bacteria species are known to interact with SSC5D, and how strong are these interactions?

SSC5D has been demonstrated to interact with various bacterial species, with differential binding strength:

Experimental approaches used to assess bacterial interactions:

• Western blot detection of protein-bacteria interactions

• Surface plasmon resonance (SPR) quantification

Known bacterial interactions:

• E. coli RS218 (neuropathogenic strain): Shows strong interaction with N-terminal SRCR-containing moiety of SSC5D (N-SSC5D)

• E. coli IHE3034 (meningitis-causing pathogen): Shows moderate interaction, with variable binding detected between conventional assays and SPR experiments

• Listeria monocytogenes EGD-e: Shows subtle interaction detectable by SPR but not by conventional assays

Relative binding strength:
When compared to other SRCR family proteins like Spα, the interaction levels of N-SSC5D with bacteria were generally lower (between 15-40% across several experiments) but still distinctly positive compared to non-binding controls like sCD5 .

Methodological considerations:
SPR offers advantages over conventional binding assays as it:

• Allows real-time detection of bacteria

• Better mimics protein-bacteria interaction under physiological conditions with shear forces

• Enables simultaneous measurement of different protein-bacteria interactions within the same experiment

The reproducibility of these interaction measurements has been reported to be >82% for N-SSC5D binding across independent experiments .

What functional assays can demonstrate the biological activity of recombinant mouse SSC5D?

Several functional assays can be employed to demonstrate the biological activity of recombinant mouse SSC5D:

1. Bacterial Aggregation Assays:

• SSC5D binding to PAMPs induces microbial aggregation

• This can be visualized microscopically or quantified using flow cytometry or spectrophotometric methods

2. Cytokine Release Inhibition Assays:

• SSC5D has been shown to inhibit PAMP-induced cytokine release

• Experimental approach: Measure cytokine production (e.g., TNF-α, IL-6) from immune cells stimulated with PAMPs in the presence vs. absence of recombinant SSC5D

3. E. coli Bioparticle Binding:

• Recombinant Human SSC5D has been shown to bind fluorescein-conjugated E. coli bioparticles

• The ED50 for this effect is 0.2-1 μg/mL

• This assay can be adapted for mouse SSC5D

4. Matrix Protein Binding ELISAs:

• ELISA-based assays to measure dose-dependent binding to extracellular matrix proteins such as laminin

5. Galectin-1 Binding:

• Pull-down assays using GST-Gal1 Sepharose beads with competitive inhibition by lactose

6. SPR-Based Binding Assays:

• Real-time, label-free detection of SSC5D binding to immobilized bacteria or host proteins

• Provides quantitative binding kinetics and affinity constants

Researchers should select the most appropriate assay based on their specific research question and the aspect of SSC5D function they wish to investigate.

How does recombinant mouse SSC5D compare to the native protein?

When evaluating recombinant mouse SSC5D compared to the native protein, several important considerations emerge:

1. Molecular Weight and Glycosylation:

• Native SSC5D undergoes extensive post-translational modifications

• Recombinant mouse SSC5D-HA produced in HEK 293-EBNA cells shows a molecular weight of ∼200 kDa, which is significantly higher than the predicted size of 144.6 kDa

• The intracellular form of rmSSC5D-HA displays a smaller molecular weight (∼150 kDa) than the secreted form (>200 kDa), suggesting that additional processing occurs during secretion

2. Expression Systems Impact:
Different expression systems produce proteins with varying characteristics:

• Mammalian systems (HEK cells) produce heavily glycosylated proteins similar to native SSC5D

• E. coli-derived recombinant proteins lack mammalian glycosylation patterns

• Commercial sources offer recombinant SSC5D from various expression systems including E. coli, yeast, baculovirus, and mammalian cells

3. Functional Domains:

• For bacterial binding studies, the N-terminal SRCR-containing moiety (N-SSC5D) has been expressed separately, excluding the mucin-like domain

• This approach allows investigation of SRCR domain-specific functions without potential confounding effects from the mucin-like domain, which by nature may also bind microorganisms

4. Purity Considerations:

• Commercial recombinant proteins typically have ≥85% purity as determined by SDS-PAGE

• For functional studies, higher purity may be required

When designing experiments, researchers should consider whether their specific research questions require native glycosylation patterns and post-translational modifications, or whether bacterial-derived recombinant protein would be sufficient.

What evidence suggests SSC5D could serve as a biomarker for heart failure?

Recent research has provided compelling evidence for SSC5D as a potential biomarker for heart failure:

1. Serum Level Elevation in Heart Failure:

• Serum SSC5D levels are significantly elevated in heart failure patients compared to controls:

  • Heart failure group: 15,789.35 (10,745.32–23,110.65) pg/mL

  • Control group: 8,938.72 (6,154.97–12,778.81) pg/mL (p < 0.0001)

2. Correlation with Established Heart Failure Markers:

• Serum SSC5D levels show significant correlation with:

  • N-terminal pro-B-type natriuretic peptide (NT-proBNP): R = 0.4, p = 7.9e-12

  • Left ventricular ejection fraction (LVEF): R = −0.46, p = 9.8e-16

3. Diagnostic Performance:

• Receiver operating characteristic (ROC) curve analysis showed:

  • AUC value of SSC5D: 0.831 (improved from 0.768 without SSC5D)

  • Specificity: 0.723

  • Sensitivity: 0.843

  • Optimal cut-off value: 10,853.98 pg/mL

4. Association with Heart Failure Risk:

• Logistic regression analysis demonstrated that log-transformed serum SSC5D levels were strongly associated with heart failure prevalence (OR: 3.23, 95% CI: 2.32–4.50, p < 0.001)

• The highest SSC5D tertile was associated with significantly higher risk of heart failure (OR: 11.02, 95% CI: 5.53–21.97, p < 0.001)

• This association remained significant after adjusting for multiple covariates (OR: 3.40, 95% CI: 2.10–5.51, p < 0.001)

5. Mechanistic Rationale:

• Single-cell RNA sequencing data demonstrates that Ssc5d is predominantly expressed in cardiac fibroblasts during heart failure

• This suggests a potential role in cardiac remodeling processes

These findings collectively suggest that SSC5D represents a promising new biomarker for heart failure diagnosis and potentially for monitoring disease progression.

What experimental models are suitable for studying SSC5D function in cardiovascular disease?

Several experimental models have proven effective for studying SSC5D function in cardiovascular disease:

1. Animal Models of Heart Failure:

• Myocardial infarction (MI) model: Ssc5d mRNA levels were shown to be markedly increased compared to sham-operated controls

• Transverse aortic constriction (TAC) model: Induces pressure overload and heart failure, with significant upregulation of Ssc5d expression

2. In Vitro Cellular Models:

• Cardiac fibroblast culture: Single-cell RNA sequencing data indicates that SSC5D is predominantly expressed in cardiac fibroblasts during heart failure

• Cardiomyocyte/fibroblast co-culture systems: May help elucidate paracrine effects

3. Human Samples:

• Serum biomarker studies: Comparing SSC5D levels between heart failure patients and controls

• Tissue expression analysis: Using cardiac tissue samples from heart failure patients and non-failing hearts

4. Gene Expression Manipulation:

• RNA interference: To knock down SSC5D expression in relevant cell types

• CRISPR/Cas9 gene editing: For creating knockout or knock-in models

5. RNA Sequencing Approaches:

• Bulk RNA sequencing: Was used to demonstrate elevated SSC5D levels in failing hearts compared to non-failing hearts

• Single-cell RNA sequencing: Identified cardiac fibroblasts as the main source of SSC5D in heart failure

Methodological Considerations:
When studying SSC5D in cardiovascular disease models, researchers should consider:

• The temporal dynamics of SSC5D expression following cardiac injury

• The relationship between tissue expression and serum levels

• Potential functional differences between mouse and human SSC5D

• The interaction between SSC5D and other cardiac stress response pathways

These models provide complementary approaches to understand SSC5D's role in cardiovascular pathophysiology, from molecular mechanisms to potential clinical applications.

How does the bacterial binding specificity of SSC5D compare with other SRCR family members?

The bacterial binding properties of SSC5D can be compared with other SRCR family members using both conventional binding assays and surface plasmon resonance (SPR) techniques:

Comparative binding profiles:

*There are conflicting reports about CD6 bacterial binding properties across different studies.

Key differences in binding mechanisms:

• Binding domains: The N-terminal SRCR-containing domain of SSC5D is responsible for bacterial interactions, similar to other SRCR family members .

• Relative binding strength: SPR experiments demonstrate that N-SSC5D has lower bacterial binding capacity compared to Spα, with interaction levels between 15-40% of those observed for Spα .

• Bacteria specificity patterns:

  • N-SSC5D shows stronger binding to E. coli strains than to Listeria monocytogenes

  • Spα binds strongly to both E. coli and Listeria monocytogenes

  • sCD5 shows negligible binding to both bacterial species

• Methodological considerations:

  • SPR offers advantages over conventional binding assays, allowing real-time detection under conditions that better mimic physiological shear forces

  • This may explain some discrepancies between binding results obtained with different methods

These differential binding profiles suggest that despite structural similarities in their SRCR domains, there is functional specialization among SRCR family members in their pattern recognition properties, potentially reflecting evolutionary adaptations to different pathogenic challenges.

What experimental challenges exist when working with recombinant SSC5D, and how can they be addressed?

Researchers working with recombinant SSC5D face several experimental challenges that require specific methodological approaches:

1. Protein Size and Structural Complexity:

• Challenge: Large molecular weight (>200 kDa for glycosylated form) and complex domain structure

• Solution:

  • Express individual domains separately for domain-specific studies

  • Use mammalian expression systems for full-length protein

  • Optimize gel electrophoresis conditions for high molecular weight proteins

2. Post-translational Modifications:

• Challenge: Extensive glycosylation affects protein behavior and function

• Solution:

  • Select appropriate expression system (mammalian preferred for native glycosylation)

  • Characterize glycosylation pattern using lectin binding assays or mass spectrometry

  • Consider enzymatic deglycosylation for specific experiments

3. Protein Stability and Storage:

• Challenge: Large, heavily glycosylated proteins may have stability issues

• Solution:

  • Avoid repeated freeze-thaw cycles

  • Use carrier proteins (BSA) to enhance stability except when carrier-free preparations are required

  • Store lyophilized and reconstitute at recommended concentrations (e.g., 500 μg/mL in PBS)

4. Functional Assay Standardization:

• Challenge: Variable results between assay types (e.g., conventional binding vs. SPR)

• Solution:

  • Include appropriate positive and negative controls (Spα and sCD5, respectively)

  • Standardize bacterial preparations and concentrations

  • Consider flow conditions when studying pathogen interactions

5. Species-Specific Differences:

• Challenge: Potential functional differences between mouse and human SSC5D

• Solution:

  • Direct experimental comparison of both orthologs

  • Focus on conserved domains (95% sequence identity in SRCR domains)

  • Be cautious about extrapolating findings across species

6. Background Signal in Binding Assays:

• Challenge: SRCR proteins may have intrinsic unspecific binding properties

• Solution:

  • Use genetically and structurally related molecules (like sCD5) as reference controls

  • Design experiments with proper compensation for binding of non-target molecules

7. Protein Yield:

• Challenge: Large, complex proteins often express at lower yields

• Solution:

  • Optimize expression conditions (temperature, time, media additives)

  • Consider episomal expression systems that can maintain stable expression over extended periods

Addressing these challenges through careful experimental design and appropriate methodological approaches is crucial for generating reliable and reproducible results when working with recombinant SSC5D.