Recombinant Mouse V-set domain containing T-cell activation inhibitor 1 (Vtcn1)

What is the structure and functional significance of mouse VTCN1 protein domains?

VTCN1 (also known as B7-H4, B7S1, B7X) is a 283 amino acid-long heavily glycosylated protein with a distinctive structure consisting of:

• A short intracellular tail

• A type 1 hydrophobic transmembrane domain

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

Functional studies have revealed that both IgV and IgC VTCN1 domains retain inhibitory activities, though they control different aspects of T-cell function. The IgV domain primarily constrains T-cell proliferation, while the IgC domain predominantly inhibits cytokine production. Additionally, glycosylation of VTCN1 is crucial for its membrane trafficking, proper folding, and negative co-stimulatory functions .

Experimental approaches for domain analysis include:

• Generation of domain-specific mutants (IgV or IgC alone or in combination)

• Creation of glycosylation site mutants through deletion or point mutations

• Assessment of these mutants' binding to pre-activated T cells and their effects on T-cell proliferation and cytokine production

How does mouse VTCN1 inhibit T-cell activation and what methodologies best demonstrate this function?

VTCN1 negatively regulates T-cell-mediated immune responses through multiple mechanisms:

• Inhibition of T-cell activation and proliferation

• Reduction of cytokine production

• Inhibition of development of cytotoxicity

Research methodologies to assess VTCN1's inhibitory function include:

T-cell proliferation assays:

• Addition of recombinant mouse VTCN1 1 hour before activation beads

• Collection of medium aliquots 48 hours after activation for ELISA analysis

Important experimental considerations:

• The inhibitory effect is typically observed with plate-bound VTCN1 protein but not with soluble VTCN1, suggesting that crosslinking with putative T-cell receptors is required for inhibitory signal delivery

• Recombinant mouse VTCN1 protein construction typically includes the extracellular domain (Leu25-Ser256) expressed with a Fc tag at the C-terminus

• Purification by SDS-PAGE to >95% purity ensures reliable experimental results

What are the differential expression patterns of VTCN1 in mouse tissues and disease models?

VTCN1 exhibits varied expression patterns across different tissues and disease conditions:

Normal tissue expression:

• Expressed in various lymphoid and non-lymphoid tissues including kidney, liver, lung, pancreas, placenta, prostate, spleen, testis, and thymus

• In pancreatic islets, both α and β cells express VTCN1

Expression in disease models:

• In diabetes-susceptible NOD mice, there is a gradual loss of membrane-tethered VTCN1 from antigen-presenting cells (APCs) despite upregulation of the VTCN1 gene

• This loss correlates with disease progression in autoimmune diabetes development

Methodological approaches for expression analysis:

• Flow cytometry and immunofluorescence for membrane-bound VTCN1

• ELISA for detection of soluble VTCN1 (sVTCN1) in serum

• RT-PCR and RNA-seq for transcriptional analysis

• Immunohistochemistry for tissue localization

How does proteolytic processing regulate VTCN1 function in autoimmune disease models?

A key regulatory mechanism of VTCN1 involves proteolytic processing that significantly impacts its function:

Process and mechanism:

• Membrane-tethered VTCN1 undergoes proteolytic cleavage mediated by the metalloproteinase nardilysin (NRD1)

• This cleavage results in the release of soluble VTCN1 (sVTCN1) and diminishment of functional cell-surface VTCN1

• The proteolysis occurs in two synergistic modes: cell-intrinsic intracellular and cell-extrinsic systemic

Research findings in disease models:

• In diabetes-susceptible NOD mice, increased NRD1 expression correlates with enhanced VTCN1 proteolysis

• Inhibition of NRD1 activity stabilizes VTCN1 on cell surfaces and reduces anti-islet T-cell responses

• The loss of membrane-bound VTCN1 combined with increased sVTCN1 release occurs in parallel with natural T1D development

Experimental approaches:

• Western blotting to detect VTCN1 cleavage products

• ELISA to measure sVTCN1 levels in serum or culture supernatants

• NRD1 inhibition studies to assess VTCN1 stabilization effects

• Correlation of sVTCN1 levels with disease progression markers

What experimental systems are available to study VTCN1 function in vitro?

Several experimental systems have been developed to study VTCN1 function:

Recombinant protein systems:

• Mammalian expression systems producing recombinant mouse VTCN1 (typically encoding Leu25-Ser256 with a Fc tag)

• Stable cell lines expressing full-length or truncated VTCN1 through retroviral vector systems

• Domain-specific mutants for structure-function analyses

Cell culture models:

• 3T3 cells stably expressing recombinant VTCN1 proteins established after viral transduction and puromycin selection

• T-cell co-culture systems to assess inhibitory functions

Protein characteristics for experimental planning:

• Molecular mass: 52.7 kDa (theoretical); 90 kDa (apparent mass due to glycosylation)

• Optimal storage: Lyophilized proteins stable for up to 12 months at -20 to -80°C

• Working solutions: Reconstituted protein solution can be stored at 4-8°C for 2-7 days

• Long-term storage: Aliquots of reconstituted samples stable at < -20°C for 3 months

How can researchers effectively study VTCN1 in autoimmune disease models?

VTCN1 plays a significant role in autoimmune conditions, particularly Type 1 Diabetes (T1D). Research approaches include:

Animal models:

• NOD (non-obese diabetic) mice as a model for T1D development

• Comparison with control strains (B6 g7, DBA) to identify VTCN1-related pathogenic mechanisms

Key parameters to measure:

• Membrane-associated VTCN1 levels on antigen-presenting cells (particularly macrophages and dendritic cells)

• Soluble VTCN1 levels in sera

• VTCN1 gene expression versus protein levels to identify post-translational regulation

• NRD1 (nardilysin) expression and activity

Therapeutic intervention studies:

• Treatment with VTCN1-Ig fusion protein to attenuate T1D in NOD mice

• Inhibition of NRD1 activity to stabilize VTCN1 and reduce anti-islet T-cell responses

• Assessment of T-cell proliferation and cytokine production in response to interventions

Translational approaches:

• Correlation of findings in mouse models with human T1D patient samples

• Analysis of soluble VTCN1 as a potential biomarker (elevated in 53% of T1D patients compared to 9% of healthy subjects)

What are the considerations for designing experiments to study VTCN1's role in cancer models?

VTCN1 has significant implications in cancer research, with distinct experimental considerations:

Expression analysis in cancer models:

• VTCN1 is frequently upregulated in multiple neoplasms and is associated with tumor-protective down-regulation of anti-tumor T cell responses

• High upregulation observed in several solid tumors including ovarian cancer

• Expression correlates with other checkpoint genes such as CTLA4, HAVCR2, LAG3, and TIGIT

Functional studies:

• VTCN1 expressed on tumor macrophages plays a role in suppressing tumor-associated antigen-specific T-cell immunity

• When studying VTCN1 in tumors, analyze both cancer cells and tumor-associated macrophages (TAMs)

Methodological approaches:

• Protein-protein interaction (PPI) network analysis to identify VTCN1-related pathways in cancer

• Gene Set Enrichment Analysis (GSEA) to determine VTCN1-associated signaling pathways

• Functional annotation of VTCN1-related module genes using tools like ClueGo

• Gene Ontology (GO) analysis to identify biological processes regulated by VTCN1

Pathway analysis findings:
VTCN1 in cancer is associated with multiple critical pathways:

• IL-2/STAT5 signaling

• p53 pathway

• mTORC1 signaling

• TNF-α signaling via NF-κB

• Inflammatory responses

• IFN-γ and IFN-α responses

• IL-6/JAK/STAT3 signaling

How can researchers differentiate between membrane-bound and soluble forms of VTCN1 in experimental systems?

Distinguishing between membrane-bound and soluble VTCN1 is crucial for understanding its biology:

Detection methods for membrane-bound VTCN1:

• Flow cytometry using anti-VTCN1 antibodies

• Immunofluorescence microscopy for visualization on cell surfaces

• Western blotting of membrane fractions

• Surface biotinylation followed by pull-down assays

Detection methods for soluble VTCN1 (sVTCN1):

• ELISA assays of serum or culture supernatants

• Western blotting of concentrated culture media

• Immunoprecipitation followed by mass spectrometry for detailed characterization

Key experimental findings:

• In diabetes-susceptible NOD mice, there is extensive release of sVTCN1 from macrophages compared to control strains

• sVTCN1 levels in NOD mice blood sera are significantly higher than in B6 g7 mice

• An inverse correlation (R² = 0.62, P = 0.0001) exists between surface VTCN1 levels on macrophages and sVTCN1 levels in sera of T1D patients versus healthy controls

• The cleaved sVTCN1 fragment appears early in disease progression and correlates with aggressive disease pace in T1D

What approaches are effective for studying the interaction between VTCN1 and its putative receptor(s)?

Although the specific receptor for VTCN1 has not been definitively identified, several methodologies can investigate these interactions:

Binding assays:

• Recombinant VTCN1-Fc fusion proteins for pull-down experiments

• Surface plasmon resonance (SPR) to measure binding kinetics

• Co-immunoprecipitation to identify interacting partners

• Cell-based binding assays with pre-activated T cells

Functional validation:

• T-cell proliferation assays with either plate-bound or soluble VTCN1

• Cytokine production measurements via ELISA

• Assessment of T-cell signaling pathway activation/inhibition

• Competition assays with potential receptor-blocking antibodies

Important considerations:

• The inhibitory effect of VTCN1 is typically observed only with plate-bound but not soluble VTCN1 protein, suggesting that crosslinking with a putative binding partner on T cells is required to deliver inhibitory signals

• Both IgV and IgC domains of VTCN1 retain inhibitory activities but may interact with different components of the receptor complex

• Glycosylation state of VTCN1 is critical for receptor binding and should be considered in experimental design

What advanced techniques can assess VTCN1 glycosylation and its impact on function?

Glycosylation of VTCN1 is crucial for its membrane trafficking, folding, and negative co-stimulatory functions:

Analysis techniques:

• Site-directed mutagenesis of N-linked glycosylation sites

• Enzymatic deglycosylation using PNGase F or Endo H

• Lectin blotting to characterize glycan structures

• Mass spectrometry for detailed glycan profiling

• Glycoproteomic approaches for site-specific glycan analysis

Functional assessment methods:

• Comparison of glycosylated vs. deglycosylated VTCN1 in T-cell inhibition assays

• Trafficking studies using fluorescently tagged glycosylation mutants

• Binding studies with putative receptors on T cells

• Structural analyses to determine how glycosylation affects protein conformation

Research findings:

• VTCN1 is a heavily glycosylated protein with N-linked modifications

• Glycosylation mutations significantly affect VTCN1's ability to bind pre-activated T cells

• Both proper membrane trafficking and functional activity require intact glycosylation

• Generation of glycosylation site mutants through deletion or point mutations provides valuable tools for studying glycosylation's role in VTCN1 function