Recombinant Human V-set domain-containing T-cell activation inhibitor 1 (VTCN1), partial

What is VTCN1 and what is its primary function in the immune system?

VTCN1, also known as B7-H4, is a negative co-stimulatory ligand that inhibits T cell activation and proliferation through receptors on activated T and B cells . As a member of the B7 family of cosignal molecules, it plays a crucial role in regulating both cellular and humoral immune responses . Studies demonstrate that VTCN1 signaling is impaired in type 1 diabetes (T1D) in both mouse models and human patients, and aberrant VTCN1 expression has been associated with certain ovarian and renal carcinomas .

What are the key structural domains of VTCN1 and how do they contribute to its function?

Structurally, VTCN1 is a 283 amino acid-long heavily glycosylated protein with several distinct domains:

• A very short intracellular tail

• A type 1 hydrophobic transmembrane domain

• A long extracellular part consisting of Ig-like V-set (IgV) and Ig-like C-set (IgC) domains

Research has demonstrated functional specialization between these domains:

• The IgV domain primarily constrains T cell proliferation

• The IgC domain predominantly inhibits cytokine production

Both domains retain inhibitory activities, though their mechanisms of action differ. This domain-specific functionality provides important insights for researchers developing targeted therapeutic approaches.

What is the relationship between membrane-bound VTCN1 and its soluble form (sVTCN1)?

Membrane-tethered VTCN1 can undergo proteolytic cleavage mediated by the metalloproteinase nardilysin, resulting in a soluble fragment called sVTCN1 . This process appears dysregulated in T1D, with sVTCN1 detected at high levels in the peripheral blood of 53% of T1D patients compared to only 9% of healthy subjects . In NOD mice (a T1D model), despite elevated VTCN1 mRNA levels, surface-associated VTCN1 protein is low due to extensive release of sVTCN1 . The elevated blood sVTCN1 levels appear early in disease progression and correlate with aggressive disease pace, highlighting sVTCN1's potential as a T1D biomarker .

How can researchers effectively design functional assays to evaluate the inhibitory capacity of recombinant VTCN1?

Effective functional assays for evaluating VTCN1's inhibitory capacity should include:

• Proliferation assays: Studies have shown that immobilized fusion protein human B7-H4.Ig (coated at 5 μg/ml) clearly inhibits the proliferation of activated CD4+ and CD8+ T cells from patients induced by anti-CD3 antibody in CFSE assays .

• Cell cycle analysis: B7-H4.Ig has been demonstrated to arrest cell cycle progression of T cells in G0/G1 phase as measured by BrdU-7-AAD flow cytometric analysis .

• Apoptosis assessment: Research confirms that B7-H4.Ig induces T cell apoptosis .

• Cytotoxicity assays: The expression of cell-associated B7-H4.Ig on human beta-cells inhibits the cytotoxicity of T-cell clones to targeted human beta-cells in 51Cr release cytotoxicity assays .

These methodologies provide comprehensive assessment of VTCN1's functional impact on T cell responses.

How does the glycosylation status of recombinant VTCN1 affect its binding affinity and inhibitory function?

Glycosylation of VTCN1 is crucial for:

• Membrane trafficking

• Proper protein folding

• Negative co-stimulatory functions

Research using mouse VTCN1 protein mutants with either deletions or point mutations of potential glycosylation sites has demonstrated that proper glycosylation is essential for VTCN1 to:

• Bind effectively to pre-activated T cells in culture

• Influence T cell proliferation

• Inhibit cytokine production

This highlights the critical importance of considering glycosylation when working with recombinant VTCN1, as improperly glycosylated protein may exhibit diminished or altered functionality.

How can researchers distinguish between the effects of IgV and IgC domains in VTCN1's inhibitory function?

To distinguish between domain-specific effects, researchers have successfully employed a reductionist approach using:

• Domain-specific mutant proteins: Generation of mouse VTCN1 mutants engineered to contain each domain (IgV or IgC) alone or in combination .

• Domain-specific functional assays:

  • Proliferation assays to evaluate the IgV domain's effect on T cell proliferation

  • Cytokine production assays to assess the IgC domain's impact

• Binding studies: Evaluation of each domain's ability to bind pre-activated T cells in culture

This methodological approach has revealed that while both domains retain inhibitory activities, the IgV domain primarily constrains T cell proliferation, while the IgC domain predominantly inhibits cytokine production .

What are the optimal experimental conditions for studying VTCN1's role in type 1 diabetes models?

When investigating VTCN1 in T1D contexts, researchers should consider:

• Cell types: Include both macrophages (which show elevated VTCN1 mRNA but low surface protein in NOD mice) and T cells from both diabetes-prone (NOD) and control mice .

• Analytical approaches:

  • FACS and immunofluorescence analyses for membrane-bound VTCN1

  • ELISA analysis for sVTCN1 in blood sera

• Timing considerations: Since elevated blood sVTCN1 levels appear early in disease progression and correlate with aggressive disease pace, timing sample collection relative to disease stage is critical .

• Comparative analysis: Include both T1D patients, their first-degree relatives, and healthy age-matched control subjects for human studies .

This multifaceted approach can provide comprehensive insights into the role of VTCN1 in T1D pathogenesis.

What are the key considerations when designing experiments to explore VTCN1's role in trophoblast development?

Investigating VTCN1's role in trophoblast (TB) development requires careful experimental design:

• Knockdown approach: siRNA-mediated VTCN1 knockdown has proven effective, with validation via qPCR, western blotting, and immunofluorescence histochemistry .

• Developmental assessment:

  • Direct phase contrast imaging and crystal violet staining to assess syncytialization

  • Fluorescence immunohistochemistry to evaluate protein markers of TB cell syncytialization (CGB, CGA)

  • Time-course experiments to document effects on hCG secretion (normalized to DNA content)

• Functional evaluation: Invasion assays to assess TB cell invasive capacity, which increases after VTCN1 knockdown .

• Molecular analysis: RNA-seq at multiple time points (24h, 48h, 72h post-knockdown) to define pathways regulated by VTCN1 .

This comprehensive experimental approach has revealed VTCN1's role in guiding trophoblast lineage development and anti-viral responses.

How does VTCN1 knockdown affect signaling pathways and what are the best methods to detect these changes?

VTCN1 knockdown impacts multiple signaling pathways, which can be analyzed through:

• RNA-sequencing: This approach has identified:

  • 61 upregulated and 155 downregulated genes 24h after VTCN1 knockdown

  • 21 enriched upregulated pathways (p<0.05) at 24h

  • Upregulated pathways associated with herpes simplex infection, influenza A, and cytokine receptor interaction

• Pathway analysis: Using tools like QIAGEN Ingenuity Pathway Analysis software to identify:

  • Affected functional categories

  • Potential upstream regulators of differentially expressed genes

• Protein expression analysis: Western blotting to examine:

  • MAPK and phospho-MAPK (pMAPK) levels

  • Phospho-Stat1 (pSTAT1) levels, which increase upon VTCN1 knockdown

• Expression verification: Correlation of transcriptional changes with protein-level changes for key molecules like IFITM1, which increases as syncytialization decreases when VTCN1 is knocked down .

These methodological approaches provide comprehensive insights into the molecular mechanisms through which VTCN1 regulates cellular processes.

How can VTCN1 be targeted for potential therapeutic applications in autoimmune diseases?

Research suggests several promising therapeutic approaches targeting VTCN1:

• Activation of the B7-H4 pathway: Studies indicate this may represent a novel immunotherapeutic approach to inhibit T-cell responses for the prevention of beta-cell destruction in T1D .

• Cell-associated B7-H4.Ig expression: Transfection of human beta-cell lines and islet cells with B7-H4.Ig plasmid results in inhibition of T-cell cytotoxicity against these cells .

• Nardilysin inhibition: Since nardilysin mediates the proteolytic cleavage of membrane-tethered VTCN1, its inhibition could potentially preserve surface VTCN1 expression and function, suggesting nardilysin as a potential therapeutic target .

The consistent finding that VTCN1 can inhibit T-cell proliferation, induce apoptosis, and prevent cytotoxicity highlights its significant therapeutic potential in autoimmune conditions like T1D.

What methods are most effective for measuring sVTCN1 as a potential biomarker in clinical samples?

For clinical application of sVTCN1 as a biomarker, particularly in T1D, researchers have employed:

• ELISA analysis: Successfully used to detect elevated sVTCN1 levels in NOD mouse blood sera compared to B6 g7 controls .

• Human sample processing: Processing of patient blood to obtain:

  • Sera for sVTCN1 quantification

  • PBMCs differentiated into PBMC-MΦs for FACS and immunofluorescence analyses of membrane-bound VTCN1

• Comparative analytics: Analyzing sVTCN1 levels in:

  • T1D patients (found in 53%)

  • First-degree relatives

  • Healthy age-matched control subjects (found in only 9%)

The correlation between elevated blood sVTCN1 levels, early disease progression, and aggressive disease pace highlights sVTCN1's potential value as a clinical biomarker for T1D .

What are the unresolved questions regarding VTCN1's receptor and signaling mechanism?

Despite significant progress in VTCN1 research, several critical questions remain:

• Receptor identification: "At present, neither the receptor, nor the mechanism of VTCN1 action is known" . Identifying VTCN1's receptor would significantly advance understanding of its inhibitory mechanism.

• Domain-specific signaling: While research shows the IgV domain primarily constrains T cell proliferation and the IgC domain predominantly inhibits cytokine production, the intracellular signaling pathways mediating these effects require further elucidation .

• Soluble VTCN1 function: The functional significance of elevated sVTCN1 in T1D pathogenesis remains to be fully characterized - whether it acts as an inhibitor by sequestering the receptor or has independent signaling capabilities .

Addressing these questions would significantly advance understanding of VTCN1 biology and potentially lead to novel therapeutic approaches.

How can advanced glycoproteomic approaches enhance recombinant VTCN1 production and analysis?

Given that glycosylation of VTCN1 is crucial for its membrane trafficking, folding, and negative co-stimulatory functions , advanced glycoproteomic approaches could:

• Map critical glycosylation sites: Building on existing research with mutant proteins to identify which specific glycosylation modifications are essential for function.

• Optimize expression systems: Developing expression platforms that produce recombinant VTCN1 with glycosylation patterns matching the native protein.

• Develop glycoform-specific analysis: Creating methods to distinguish between different glycoforms of VTCN1 and their respective activities.

• Engineer optimized variants: Designing VTCN1 variants with enhanced stability or activity through targeted glycoengineering.

These approaches would address the challenges in producing fully functional recombinant VTCN1 with proper glycosylation patterns for both research and potential therapeutic applications.