Recombinant Xenopus tropicalis Transmembrane protein 173 (tmem173)
Description
Overview of Recombinant Xenopus tropicalis Transmembrane Protein 173 (TMEM173)
Recombinant Xenopus tropicalis Transmembrane protein 173 (TMEM173) is a synthetically produced version of the TMEM173 protein found in the Xenopus tropicalis frog species . TMEM173, also known as STING (Stimulator of Interferon Genes), is an endoplasmic reticulum transmembrane protein involved in innate immunity and inflammation .
Characteristics of TMEM173
-
TMEM173 is a transmembrane protein that resides in the endoplasmic reticulum (ER) .
-
It functions as a sensor for cytosolic double-stranded DNA (dsDNA) and cyclic dinucleotides (CDNs) .
-
TMEM173 directly binds to bacterial second messengers, such as cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and 3'3'-cGAMP .
-
It has four amino-terminal transmembrane domains spanning the first 136 amino acids, followed by the helix α1 at residues 153-177 .
-
Helix α1, or the dimerization domain, is essential for protein stability, intraprotein interactions, and ligand binding .
-
The CDN binding domain (residues 153-340) is part of the cytoplasmic carboxy-terminus having multiple phosphorylation and downstream signaling interaction sites .
Function and Mechanism of Action
The TMEM173 gene encodes STING, which plays a crucial role in the innate immune response . The protein recognizes nucleic acids or cyclic nucleotides, which then triggers the production of type I interferon (IFN) and other inflammatory cytokines, leading to nucleic-acid driven inflammation . TMEM173-induced type I IFN signaling requires the activation of TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) .
Role in Disease
Mutations in TMEM173 are associated with several diseases . Somatic mutations in human TMEM173 are rare in cancers .
-
Gain-of-function mutations in TMEM173 can lead to early-onset vasculitis, autoinflammation, and interstitial lung disease, defining the SAVI phenotype .
-
TMEM173 drives coagulation through ER stress-initiated activation of gasdermin D (GSDMD), leading to the release of coagulation factor III, an initiator of the coagulation cascade .
-
TMEM173-dependent systemic coagulation is implicated in septic death in mice and humans .
TMEM173 and Sepsis
TMEM173 plays an essential role in the dysregulated coagulation that leads to lethal sepsis . In a study, myeloid TMEM173 was found to be critical in this process, independent of a classical TMEM173-induced type I IFN response in monocytes and macrophages .
Conditional ablation of TMEM173 in mice revealed that TMEM173 expressed by myeloid cells plays a major role in mediating CLP-induced septic death and coagulation activation . The deletion of Tmem173 in myeloid cells prolonged animal survival and was associated with:
TMEM173 Mutations in Cancer
The Catalogue of Somatic Mutations in Cancer (COSMIC) database provides information on TMEM173 mutations in various cancers .
| Mutation (Amino Acid) | Mutation ID (COSF) | Count | Mutation Type |
|---|---|---|---|
Research Findings
-
A novel gain-of-function G207E STING mutation can cause a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of autoinflammation .
-
The common TMEM173 HAQ, AQ alleles can rescue CD4 T cellpenia .
-
HAQ, AQ, and Q293 splenocytes resist STING-mediated cell death ex vivo, establishing a critical role of STING residue 293 in cell death .
-
The N-terminal domain (NTD) of STING affects DNA responses via control of trafficking .
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized preparation.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires advance notification and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us; we will prioritize its development.
Target Background
A cytosolic DNA sensor from bacteria and viruses that promotes autophagy. It functions by recognizing and binding cyclic GMP-AMP (cGAMP), a messenger molecule produced by CGAS in response to cytosolic DNA. cGAMP binding triggers autophagosome formation, targeting cytosolic DNA for lysosomal degradation. This protein exhibits guanine base-specific ligand recognition, binding 3'-3' linked cGAMP, 2'-3' linked cGAMP, and 3'-3' linked c-di-GMP with significantly higher affinity than 3'-3' linked c-di-AMP. Unlike its vertebrate orthologs, it lacks the C-terminal tail (CTT) crucial for interferon signaling.
Q&A
What is the basic structure of Xenopus tropicalis TMEM173 and how does it differ from mammalian STING?
Xenopus tropicalis TMEM173 contains the conserved transmembrane domains and ligand-binding domain (LBD) found in other species but notably lacks the C-terminal tail (CTT) that is present in most vertebrates . The protein contains:
-
Transmembrane domains that anchor it to the endoplasmic reticulum membrane
-
A cytoplasmic domain for ligand binding
-
A conserved TMEM173 domain (amino acids 50-342 in the chicken homolog, which shares structural similarity)
While mammalian STING contains a functional CTT that recruits TBK1 and IRF3 to induce type I interferon signaling, the absence of this domain in Xenopus tropicalis STING makes it an interesting evolutionary case study .
Can Xenopus tropicalis TMEM173 bind cyclic dinucleotides despite lacking the CTT?
Yes, despite lacking the CTT, Xenopus tropicalis TMEM173 can still bind 2'-3' cGAMP as demonstrated by electron mobility shift assays . This suggests that the ligand-binding function is preserved even though downstream signaling may differ from species with intact CTT domains. The binding capacity reflects the conservation of key residues in the cGAMP binding pocket, although their binding affinity may be lower compared to mammalian counterparts .
What is the consequence of the missing CTT in Xenopus tropicalis TMEM173 for signaling?
The absence of the CTT in Xenopus tropicalis TMEM173 significantly impacts its signaling capabilities:
| Signaling Capability | Species with CTT | Xenopus tropicalis |
|---|---|---|
| cGAMP binding | Yes | Yes |
| IRF3 activation | Strong | Weak or absent |
| Type I IFN induction | Robust | Limited |
| NF-κB activation | Present | Not well characterized |
While Xenopus tropicalis TMEM173 can bind cyclic dinucleotides, its signaling function and role in antiviral immunity remains to be fully characterized . This makes it an excellent model for understanding the evolutionary development of STING-mediated interferon signaling.
Why is the Xenopus tropicalis TMEM173 an important evolutionary model?
Xenopus tropicalis TMEM173 represents a critical transitional form in the evolution of STING signaling due to its lack of the CTT domain. Research indicates that the ability of STING to induce type I IFN signaling through IRF3 phosphorylation is an evolutionarily recent event . The study of Xenopus STING provides insight into how this pathway developed and became specialized in higher vertebrates.
Phylogenetic analyses place Xenopus tropicalis TMEM173 in a unique position among vertebrates:
-
It possesses the conserved ligand-binding domain found across species
-
It lacks the specialized CTT found in most vertebrates
-
It represents an intermediate evolutionary state between invertebrate and mammalian STING proteins
How does studying Xenopus tropicalis TMEM173 inform our understanding of STING pathway evolution?
Studying Xenopus tropicalis TMEM173 provides a unique window into how the STING pathway evolved modular functionality. The STING CTT functions as a linear signaling hub that acquired new motifs throughout evolution to adapt downstream signaling . By examining a species lacking CTT, researchers can understand:
-
The baseline functionality of STING without CTT-mediated signaling
-
How the addition of CTT modules enhanced and diversified immune responses
-
The evolutionary pressures that shaped vertebrate innate immunity
For instance, research has shown that ray-finned fishes acquired a CTT extension that dramatically enhances NF-κB activation through recruitment of TRAF6, demonstrating how CTT can acquire new motifs to adapt downstream signaling .
What expression systems are optimal for recombinant Xenopus tropicalis TMEM173 production?
Based on available research and commercial products, several expression systems have been successfully used for recombinant Xenopus tropicalis TMEM173:
| Expression System | Advantages | Considerations |
|---|---|---|
| E. coli | High yield, cost-effective | May form inclusion bodies requiring refolding |
| Cell-free expression | Rapid, avoids toxicity issues | Higher cost, potentially lower yield |
| Baculovirus | Better folding for complex proteins | More time-consuming, specialized equipment |
| Mammalian cells | Native-like post-translational modifications | Lower yield, higher cost |
Commercial preparations generally achieve ≥85% purity as determined by SDS-PAGE . For research requiring functional studies, mammalian or baculovirus systems may be preferred to ensure proper protein folding and modification.
What purification strategies yield the most active recombinant Xenopus tropicalis TMEM173?
For optimal purification of functional recombinant Xenopus tropicalis TMEM173, a multi-step approach is recommended:
-
Affinity chromatography: Using tags such as His, GST, or Myc/DDK for initial capture
-
Size exclusion chromatography: To separate properly folded protein from aggregates
-
Ion exchange chromatography: For further purification and removal of contaminants
Buffer considerations are crucial for stability, with recommended conditions including:
For functional studies, verification of proper folding using circular dichroism or thermal shift assays is advisable.
How can recombinant Xenopus tropicalis TMEM173 be used to study the evolution of innate immune signaling?
Recombinant Xenopus tropicalis TMEM173 serves as an excellent tool for comparative studies of innate immune evolution:
-
Chimeric protein studies: Creating fusion proteins with CTT domains from other species can reveal how these modules affect signaling. For example, chimeric STING proteins containing zebrafish-specific STING CTT modules demonstrate enhanced NF-κB activation, illustrating how these domains evolved specific functions .
-
Binding assays: Comparing the binding affinities of various cyclic dinucleotides to Xenopus tropicalis TMEM173 versus other species' STING helps map the evolution of ligand recognition.
-
Comparative signaling studies: Transfection experiments in reporter cell lines can quantify differences in IRF3 and NF-κB activation between Xenopus tropicalis TMEM173 and other vertebrate STING proteins .
What approaches are effective for studying the signaling capabilities of Xenopus tropicalis TMEM173?
To characterize the signaling properties of Xenopus tropicalis TMEM173:
-
Reporter assays: Transfection of cells with plasmids encoding Xenopus tropicalis TMEM173 along with IRF3 or NF-κB luciferase reporters can assess activation of these pathways .
-
Phosphorylation analysis: Western blotting with phospho-specific antibodies against TBK1, IRF3, and IKK can determine whether these downstream mediators are activated.
-
Viral infection models: Transfection of fish cell lines (such as EPC) with Xenopus tropicalis TMEM173 followed by viral challenge can assess protection against viral replication, as demonstrated with MAVS and other STING variants .
-
Co-immunoprecipitation studies: These can identify binding partners of Xenopus tropicalis TMEM173 compared to other species, revealing differences in protein-protein interactions that explain functional variations .
How can researchers assess whether recombinant Xenopus tropicalis TMEM173 is properly folded and functional?
The functional integrity of recombinant Xenopus tropicalis TMEM173 can be assessed through multiple approaches:
-
Cyclic dinucleotide binding assays:
-
Structural analysis:
-
Circular dichroism to verify secondary structure elements
-
Limited proteolysis to assess proper folding
-
Thermal shift assays to determine protein stability
-
-
Subcellular localization:
What are the common challenges when working with recombinant Xenopus tropicalis TMEM173 and how can they be addressed?
For activity studies specifically, researchers should consider:
-
Using cell-based assays rather than in vitro assays due to the requirement for membrane localization
-
Including positive controls (human or mouse STING) when testing signaling capabilities
-
Employing chimeric approaches with known functional domains when studying specific aspects of signaling
