TMEM2 Antibody, FITC conjugated

What is TMEM2 and why is it important in research?

TMEM2 (Transmembrane Protein 2) is a type II transmembrane protein that functions as a cell surface hyaluronidase. It mediates the initial cleavage of extracellular high-molecular-weight hyaluronan into intermediate-size hyaluronan fragments of approximately 5-10 kDa . TMEM2 plays crucial roles in regulating cell adhesion and migration via hyaluronan degradation at focal adhesion sites, and acts as a regulator of angiogenesis and heart morphogenesis by mediating degradation of extracellular hyaluronan, thereby regulating VEGF signaling . Recent research has implicated TMEM2 in various pathological conditions, including Graves' orbitopathy, where it inhibits inflammation, adipogenesis, and fibrosis .

What are the functional domains of TMEM2 important for its hyaluronidase activity?

TMEM2 contains several functional domains critical for its hyaluronidase activity. Studies using chimeric constructs have identified that the GG domain is particularly important for the hyaluronidase activity of mouse TMEM2. Research has shown that when the His248 and Ala303 residues in mouse TMEM2 are simultaneously replaced by the corresponding residues of human TMEM2 (Asn248 and Phe303), the HA-degrading activity is abolished . The protein also contains G8 domains and PbH1 repeats in the C-terminal extracellular domain (ECD), with experiments demonstrating that the ectodomain of TMEM2 exhibits robust hyaluronidase activity .

What are the advantages of using FITC-conjugated TMEM2 antibodies in research?

FITC-conjugated TMEM2 antibodies offer several advantages for research applications:

• Direct visualization without secondary antibodies, reducing background and cross-reactivity issues

• Compatible with multi-color immunofluorescence experiments due to FITC's distinct emission spectrum (peak ~520 nm)

• Enables live-cell imaging of TMEM2 localization and trafficking

• Allows for quantitative analysis through flow cytometry

• Provides higher sensitivity for detecting low-abundance TMEM2 expression compared to unconjugated antibodies in certain applications

What are the optimal protocols for using FITC-conjugated TMEM2 antibodies in immunofluorescence studies?

For optimal results with FITC-conjugated TMEM2 antibodies in immunofluorescence applications:

• Fixation method: A combination of paraformaldehyde (PFA) and Triton X-100 is recommended for optimal results

• Working dilution: Typically 1:50 to 1:200 for immunofluorescence applications

• Blocking: Use 5-10% normal serum (from the same species as the secondary antibody would be) in PBS with 0.1-0.3% Triton X-100

• Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Washing: Multiple PBS washes (3-5 times for 5 minutes each)

• Counterstaining: DAPI for nuclear visualization

• Mounting: Use anti-fade mounting medium to prevent photobleaching of FITC

• Storage: Protect slides from light and store at 4°C for short-term or -20°C for long-term storage

Research shows that TMEM2 localizes to the cell surface, cytosol, and potentially nucleoli in certain cell types .

How can researchers validate the specificity of TMEM2 antibody staining?

Validating TMEM2 antibody specificity is crucial for reliable research outcomes. Recommended validation approaches include:

• Positive controls: Use cell lines with confirmed TMEM2 expression (MG-63 and 293T cells have been demonstrated to express TMEM2 at the cell surface)

• Knock-down validation: Compare staining patterns between wild-type cells and cells with TMEM2 knockdown using siRNA

• Overexpression validation: Use cells transfected with tagged TMEM2 (such as FLAG-tagged or mCherry-tagged constructs) to confirm antibody co-localization

• Blocking peptide competition: Pre-incubation of antibody with the immunizing peptide should abolish specific staining

• Multiple antibody validation: Use different TMEM2 antibodies targeting distinct epitopes to confirm staining patterns

• Cross-species validation: Compare staining patterns in human versus mouse samples (noting species-specific differences in TMEM2 activity)

Studies have successfully validated TMEM2 antibodies using surface biotinylation assays and live immunostaining approaches .

What techniques can be used to quantify TMEM2 expression levels in different tissue samples?

Research has demonstrated that TMEM2 expression levels are significantly decreased in Graves' orbitopathy tissue samples compared to control samples, as measured by Western blot, qRT-PCR, and immunohistochemistry .

How can TMEM2 antibodies be used to study the hyaluronidase activity of TMEM2 in live cells?

To study TMEM2 hyaluronidase activity in live cells, researchers can employ several advanced techniques:

• In situ HA degradation assays: Cells expressing TMEM2 are cultured on fluorescein-labeled high-molecular-weight HA (FITC-HMW-HA1500) substrate, and degradation is visualized as the disappearance of fluorescence . This technique has revealed that TMEM2-expressing cells create conspicuous holes in the HA substrate at sites of cell-substratum contacts .

• Live-cell imaging with dual labeling: Cells expressing fluorescently-tagged TMEM2 (e.g., mCherry-mTMEM2) can be combined with fluorescently-labeled HA to visualize real-time degradation events. This approach has demonstrated colocalization of mCherry-TMEM2 signals with vinculin-positive puncta and sites of HA removal .

• FRET-based assays: By using HA substrates with FRET pairs that separate upon degradation, researchers can quantify TMEM2 activity in real-time.

• Size-exclusion chromatography of collected media: Media from TMEM2-expressing cells can be analyzed to detect degradation products of defined sizes (typically 5-10 kDa fragments) .

These techniques have revealed that mouse TMEM2 exhibits stronger hyaluronidase activity than human TMEM2, with activity differences attributed to specific amino acid residues .

What are the current controversies surrounding human TMEM2's hyaluronidase activity, and how can antibodies help resolve them?

There is significant debate regarding whether human TMEM2 possesses intrinsic hyaluronidase activity or primarily functions as a regulator of hyaluronan metabolism:

Supporting intrinsic hyaluronidase activity:

• Studies demonstrate that purified human TMEM2 ectodomain (TMEM2 ECD) can degrade fluorescein-labeled HA into 5-10 kDa fragments

• Both N-terminally and C-terminally tagged human TMEM2 ECD exhibit HA-degrading activity

• Membrane fractions from TMEM2-transfected cells show hyaluronidase activity

Supporting regulatory role without catalytic activity:

• Research indicates human TMEM2 lacks catalytic hyaluronidase activity but instead regulates hyaluronan metabolism by promoting HAS2-dependent HA production and reducing HYBID-dependent HA depolymerization

• Chimeric studies suggest amino acid differences at positions 248 and 303 between mouse and human TMEM2 account for the difference in activity

Antibodies can help resolve this controversy through:

• Immunoprecipitation of TMEM2 followed by activity assays to test native enzyme function

• Immunofluorescence co-localization studies with hyaluronan and other hyaluronidases

• Proximity labeling approaches (BioID or APEX) to identify TMEM2 interaction partners

• Super-resolution microscopy to precisely localize TMEM2 at sites of HA degradation

How does TMEM2 expression correlate with JAK/STAT signaling, and what techniques can demonstrate this relationship?

TMEM2 has been shown to activate the JAK/STAT signaling pathway, which is implicated in mediating the progression of many autoimmune and inflammatory diseases. Research techniques to investigate this relationship include:

• Phospho-specific antibody analysis: Studies have shown that overexpression of TMEM2 leads to increased phosphorylation of Tyk2, JAK1, Stat1, and Stat2, without affecting levels of non-phosphorylated forms of these proteins .

• Gene set enrichment analysis (GSEA): GSEA analysis of the GSE116959 dataset revealed a positive correlation between TMEM2 expression and the JAK/STAT signaling pathway .

• Pharmacological inhibition: Treatment with JAK inhibitors like CYT387 (5 μM) has been shown to reverse the increase in downstream STAT pathway phosphorylation caused by TMEM2 overexpression .

• Co-immunoprecipitation: To detect physical interactions between TMEM2 and JAK/STAT pathway components.

• Chromatin immunoprecipitation (ChIP): To identify STAT binding to promoter regions of TMEM2-regulated genes.

This relationship is particularly relevant in Graves' orbitopathy, where TMEM2 has been shown to inhibit inflammation, adipogenesis, and fibrosis through JAK/STAT signaling .

What are common issues with FITC-conjugated TMEM2 antibodies and how can they be resolved?

Research has shown that for optimal TMEM2 detection, a combination of PFA fixation and Triton X-100 permeabilization is recommended .

How can researchers optimize TMEM2 antibody selection for specific applications?

When selecting TMEM2 antibodies for specific applications, researchers should consider:

• Epitope location: Antibodies targeting different domains may have different utilities

  • Antibodies against the extracellular domain are useful for live cell staining and flow cytometry

  • Antibodies against intracellular domains may be better for fixed cell applications

• Species reactivity: Consider cross-reactivity with TMEM2 from different species

  • Human and mouse TMEM2 share approximately 87% amino acid homology

  • Some antibodies are species-specific while others recognize conserved epitopes

• Validation for specific applications: Check if the antibody has been validated for your specific application

  • Some TMEM2 antibodies work well for Western blot but poorly for IHC

  • Applications like ChIP-seq require specific validation

• Clonality consideration:

  • Monoclonal antibodies provide high specificity for a single epitope

  • Polyclonal antibodies may provide higher sensitivity but with potential for cross-reactivity

• Testing multiple antibodies: When establishing a new system, test multiple antibodies targeting different epitopes

Research has demonstrated that antibodies recognizing the GG domain of TMEM2 are particularly useful for studying its hyaluronidase activity .

How is TMEM2 implicated in disease pathogenesis, and how can antibodies help investigate these connections?

TMEM2 has been implicated in several disease processes, with antibody-based research providing critical insights:

• Graves' orbitopathy (GO): TMEM2 expression is decreased in GO tissues compared to controls. Overexpression of TMEM2 reduces inflammation, adipogenesis, and fibrosis through the JAK/STAT pathway, identifying it as a potential therapeutic target .

• Cancer biology: TMEM2 expression varies across cancer types:

  • Lower expression in invasive bladder cancer cells compared to non-invasive cells

  • Altered expression during epithelial-mesenchymal transition (EMT)

  • Associated with bladder, breast, and pancreatic cancer progression

• Developmental disorders: Conditional knockout of TMEM2 in neural crest cells leads to severe craniofacial abnormalities, demonstrating its essential role in neural crest development .

• Inflammatory conditions: TMEM2 influences hyaluronan metabolism in response to inflammatory cytokines like IL-1β and TGF-β .

Antibody-based approaches to study these connections include:

• Immunohistochemical analysis of TMEM2 expression in patient tissues

• Correlation of TMEM2 levels with disease progression and outcomes

• Mechanistic studies using cell models with TMEM2 manipulation

• In vivo imaging of TMEM2 expression in disease models

What are emerging research areas for TMEM2 that would benefit from advanced antibody applications?

• Single-cell analysis of TMEM2 expression:

  • Single-cell RNA sequencing combined with antibody-based protein validation

  • Mass cytometry (CyTOF) with TMEM2 antibodies to correlate with other markers

  • Spatial transcriptomics combined with TMEM2 immunofluorescence

• TMEM2 in tissue engineering and regenerative medicine:

  • Role in extracellular matrix remodeling during tissue regeneration

  • Potential applications in hyaluronan-based biomaterials

  • Engineering of TMEM2-expressing cells for controlled HA degradation

• Therapeutic targeting of TMEM2:

  • Development of agonistic or antagonistic antibodies

  • Monitoring of TMEM2 expression as a biomarker for treatment response

  • CAR-T approaches targeting TMEM2 in cancer contexts

• TMEM2 in immune regulation:

  • Interactions with immune cells in hyaluronan-rich environments

  • Role in regulating inflammation through JAK/STAT signaling

  • Potential involvement in autoimmune disease mechanisms

• Structural biology of TMEM2:

  • Epitope mapping with diverse antibodies

  • Conformational changes during substrate binding and catalysis

  • Structure-function relationships of different domains

Recent research has demonstrated that TMEM2 plays roles beyond simple hyaluronidase activity, including regulation of gene expression and signal transduction pathways , opening new avenues for investigation.

How do mouse and human TMEM2 differ in their functions, and what methodological approaches can distinguish these differences?

Significant functional differences exist between mouse and human TMEM2:

• Hyaluronidase activity: Mouse TMEM2 exhibits stronger HA-degrading activity than human TMEM2 .

• Key residue differences: Critical amino acid differences at positions 248 and 303 (His248 and Ala303 in mouse vs. Asn248 and Phe303 in human) account for differences in catalytic activity .

• Functional roles:

  • Human TMEM2 appears to function more as a regulator of hyaluronan metabolism

  • Mouse TMEM2 demonstrates direct, robust hyaluronidase activity

• Response to stimuli: Different responses to inflammatory cytokines between species

Methodological approaches to distinguish these differences include:

• Chimeric protein analysis: Studies using human-mouse chimeric TMEM2 constructs have identified domains critical for activity. Chimera 1, containing human N-terminal and G8 domains with mouse GG domain and C-terminal tail, degraded extracellular HA in a dose-dependent manner .

• Site-directed mutagenesis: Replacement of His248 and Ala303 in mouse TMEM2 with human counterparts abolishes activity .

• Comparative activity assays: Using both fluorescein-labeled and native high-molecular-weight HA to compare degradation kinetics between species .

• Species-specific antibodies: Using antibodies that specifically recognize either human or mouse TMEM2 for comparative studies.

• Cross-species complementation: Testing whether mouse TMEM2 can rescue phenotypes in human cells with TMEM2 knockdown, and vice versa. Studies have shown that expression of mouse TMEM2 in human TMEM2-depleted cells fully restores their ability to migrate on high-molecular weight HA substrates .