Recombinant Mouse Transmembrane protein 165 (Tmem165)
Description
Introduction to Transmembrane Protein 165
Transmembrane protein 165 (Tmem165) is a Golgi-localized protein that plays essential roles in ion homeostasis and vesicular trafficking within the Golgi apparatus. The mouse variant of this protein shares significant homology with human TMEM165, making it a valuable model for studying the protein's functions across species. In mice, Tmem165 is also known by several alternative names including TPA-regulated locus protein, transmembrane protein PFT27, and transmembrane protein TPARL, with synonyms in research literature including AV026557, pFT27, Tpardl, and Tparl . This protein has gained scientific interest due to its critical roles in cellular physiology and its associations with certain pathological conditions, particularly after discoveries linking mutations in TMEM165 to congenital disorders of glycosylation, which represent a class of recessive autosomal metabolic diseases .
Recombinant mouse Tmem165 refers to artificially produced versions of this protein, typically generated using expression systems such as Escherichia coli. These recombinant forms serve as valuable research tools for investigating the protein's structure, function, and potential therapeutic applications. The availability of purified recombinant Tmem165 enables researchers to conduct detailed biochemical analyses, develop antibodies, and explore potential drug targets related to this protein's activity.
Genetic Information
Mouse Tmem165 is encoded by the Tmem165 gene, with its human ortholog identified by the gene symbol TMEM165 and NCBI Gene ID 55858 . The mouse protein is cataloged in UniProt under the accession number P52875. While the specific chromosomal location is not detailed in the provided search results, the gene's expression is known to be regulated through various tissue-specific mechanisms, with notable expression patterns in multiple tissues including the liver, where its dysregulation has been associated with pathological conditions .
Role in Golgi Apparatus
Tmem165 functions primarily within the Golgi apparatus, where it plays crucial roles in maintaining ion homeostasis, particularly for calcium and manganese ions. This ion regulation is essential for normal Golgi function, as these ions serve as cofactors for various enzymes involved in post-translational modifications of proteins . Additionally, Tmem165 participates in vesicular trafficking processes within the Golgi network, facilitating the transport of cargo molecules between different Golgi cisternae and to their final cellular destinations.
The protein's strategic localization within the Golgi membrane allows it to act as a potential ion channel or transporter, regulating the flux of specific ions across the membrane. This function is critical for maintaining the optimal ionic environment required for the numerous enzymatic reactions that occur within the Golgi compartment, particularly those involved in protein glycosylation and processing .
Role in Glycosylation
One of the most significant functions of Tmem165 relates to its impact on glycosylation processes. Glycosylation, the attachment of sugar moieties to proteins and lipids, is a fundamental post-translational modification that affects protein folding, stability, and function. Mutations in the TMEM165 gene in humans have been linked to congenital disorders of glycosylation, highlighting the protein's essential role in this process .
The specific mechanisms through which Tmem165 influences glycosylation remain under investigation, but current evidence suggests that the protein's ion transport functions are crucial for the activity of glycosyltransferases and other enzymes involved in glycosylation. By maintaining the appropriate concentrations of calcium and manganese ions within the Golgi lumen, Tmem165 enables these enzymes to function optimally, ensuring proper glycosylation of proteins and lipids destined for various cellular compartments or secretion.
Production Methods
Recombinant mouse Tmem165 is typically produced using bacterial expression systems, particularly Escherichia coli. The process involves introducing the Tmem165 gene sequence into expression vectors, transforming these vectors into bacterial cells, and inducing protein expression under controlled conditions. According to available product information, commercial recombinant mouse Tmem165 is produced in vitro using E. coli expression systems .
The recombinant protein is often engineered to include specific tags that facilitate purification and detection. For example, commercial preparations may include an N-terminal 10xHis-tag, which allows for efficient purification using affinity chromatography techniques . Following expression, the protein undergoes purification steps to remove bacterial contaminants and yield a product suitable for research applications.
Laboratory Techniques
Recombinant mouse Tmem165 and antibodies against this protein find application in various laboratory techniques essential for studying protein function and expression patterns. These applications include:
| Technique | Typical Dilution/Usage | Notes |
|---|---|---|
| Western Blot (WB) | 1:1000-1:6000 | For protein expression quantification |
| Immunoprecipitation (IP) | 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate | For protein interaction studies |
| Immunofluorescence (IF)/ICC | 1:400-1:1600 | For cellular localization studies |
| ELISA | Variable based on assay design | For quantitative protein detection |
These techniques enable researchers to investigate various aspects of Tmem165 biology, including expression levels in different tissues or cell types, subcellular localization, interaction partners, and responses to experimental manipulations or disease states .
Reactivity and Specificity
Commercial antibodies against Tmem165 have demonstrated reactivity with samples from multiple species, including human, mouse, and rat, making them versatile tools for comparative studies across species . The cross-reactivity profile is particularly valuable for translational research that aims to connect findings from mouse models to human health and disease.
The specificity of these research tools has been validated in various cell lines, with positive Western blot detection reported in Jurkat and HeLa cells, positive immunoprecipitation in HeLa cells, and positive immunofluorescence/immunocytochemistry in both HeLa and Jurkat cells . This established specificity provides confidence in experimental results and facilitates accurate interpretation of findings related to Tmem165 function and expression.
Role in Hepatocellular Carcinoma
Recent research has revealed significant associations between Tmem165 and hepatocellular carcinoma (HCC), a primary form of liver cancer. Studies have demonstrated that TMEM165 is overexpressed in HCC tissues compared to adjacent non-tumor tissues and normal liver samples . This overexpression suggests a potential role for Tmem165 in cancer development or progression.
Quantitative real-time RT-PCR analysis of HCC tissues (n=88) has revealed associations between TMEM165 overexpression and several clinicopathological features, including more frequent macroscopic vascular invasion, microscopic serosal invasion, and higher α-fetoprotein levels . These associations suggest that Tmem165 may contribute to the aggressive behavior of HCC cells, potentially influencing their invasive and metastatic capabilities.
Potential as a Biomarker
The consistent overexpression of Tmem165 in HCC tissues compared to normal liver tissues suggests its potential utility as a biomarker for this malignancy. Research has shown significant elevation of TMEM165 expression in HCC compared to adjacent non-tumor liver tissues (P<0.001) and normal liver tissues (P=0.034) . This differential expression pattern provides a potential basis for diagnostic applications.
Similar to other Golgi proteins like GP73 (a 73-kDa Golgi transmembrane protein), which has been documented as a useful serum biomarker for HCC, Tmem165 may offer complementary or independent diagnostic value . Further research is needed to evaluate its specificity, sensitivity, and predictive value in clinical settings, particularly in combination with established biomarkers.
Therapeutic Potential
Beyond its diagnostic potential, Tmem165 has emerged as a possible therapeutic target for HCC treatment. Experimental studies have demonstrated that depletion of TMEM165 leads to a marked decrease in the invasive activity of HCC cell lines, including Huh7 and SNU475 . This reduced invasiveness is accompanied by downregulation of matrix metalloproteinase-2 (MMP-2), a key enzyme involved in degrading extracellular matrix components and facilitating cancer cell invasion.
These findings suggest that targeting Tmem165 could represent a novel strategy for inhibiting HCC progression, particularly by limiting the invasive and metastatic capabilities of cancer cells. The development of specific inhibitors or other therapeutic approaches directed against Tmem165 could potentially complement existing treatments for HCC, addressing the current challenges in managing this aggressive malignancy.
Product Specs
Note: We will prioritize shipping the format that is currently in stock. However, if you have specific format requirements, please indicate them in your order notes. We will accommodate your request to the best of our ability.
Note: All protein shipments are standardly packaged with blue ice packs. If you require dry ice packaging, please inform us in advance as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
- The timing, magnitude, and Golgi location of TMEM165 expression support its role as a contributor to mammary Golgi calcium transport needs, alongside the well-established roles of SPCA1 and 2. PMID: 24530912
Q&A
What is the primary molecular function of TMEM165 in cellular systems?
TMEM165 is a Golgi transmembrane protein critical for maintaining ion homeostasis, particularly manganese (Mn²⁺) and calcium (Ca²⁺), which are essential cofactors for glycosylation enzymes . Its deficiency disrupts the elongation of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycan (GAG) chains on proteoglycans (PGs), leading to skeletal dysplasia and dwarfism in model systems . Methodologically, TMEM165’s role in Mn²⁺ transport has been validated using yeast complementation assays (Saccharomyces cerevisiae gdt1Δ pmr1Δ strains), where codon-optimized TMEM165 variants restored growth under high Ca²⁺ stress . Immunofluorescence colocalization with GM130 (a Golgi marker) and radiolabeled GAG chain assays in tmem165-knockout ATDC5 cells further confirmed its Golgi-specific activity .
How does TMEM165 deficiency affect proteoglycan synthesis in chondrocytes?
Loss of TMEM165 reduces PG synthesis by 70% in pre-chondrogenic ATDC5 cells, as shown by [³⁵S]-sulfate metabolic labeling . The defect arises from truncated CS/HS-GAG chains, evidenced by SDS-PAGE migration patterns and chondroitinase ABC sensitivity assays . Importantly, mRNA levels of GAG-polymerizing enzymes (e.g., CHSY1, EXT1) remain unchanged, ruling out transcriptional dysregulation . Instead, the impairment stems from Mn²⁺ deficiency in the Golgi, which is rescued by Mn²⁺ supplementation . Researchers should combine radiolabeling with enzymatic digestion (e.g., chondroitinase ABC for CS chains) and Western blotting for core proteins (e.g., decorin) to distinguish GAG chain defects from core protein synthesis issues.
What experimental models are used to study TMEM165 function?
Key models include:
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ATDC5 cells: A pre-chondrogenic mouse cell line used to study TMEM165’s role in GAG synthesis and chondrocyte differentiation. CRISPR-Cas9 knockout clones show accelerated chondrocyte hypertrophy via dysregulated Indian hedgehog (Ihh) expression .
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Patient-derived fibroblasts: Cells from TMEM165-deficient patients exhibit impaired TGFβ/BMP signaling, providing a human pathophysiological model .
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Yeast complementation assays: Codon-optimized TMEM165 variants (e.g., Δ78TMEM165) in gdt1Δ pmr1Δ yeast restore Mn²⁺/Ca²⁺ homeostasis, validated via inductively coupled plasma atomic emission spectrometry (ICP-AES) .
How can researchers resolve contradictions between TMEM165’s role in glycosylation defects versus cancer metastasis?
While TMEM165 deficiency impairs GAG synthesis in chondrocytes , its overexpression in hepatocellular carcinoma (HCC) promotes invasiveness via MMP-2 upregulation . To reconcile these findings, researchers should consider tissue-specific roles:
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Skeletal systems: TMEM165 maintains Mn²⁺ for Golgi glycosyltransferases. Deficiency reduces GAG chain length, disrupting extracellular matrix (ECM) integrity and growth factor signaling (e.g., TGFβ) .
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Cancer cells: TMEM165 overexpression may alter Golgi pH or ion gradients, activating metalloproteinases like MMP-2 to enhance ECM degradation and metastasis .
Methodological strategy: Compare ion homeostasis (via ICP-AES) and glycosylation profiles (via LC-MS/MS GAG analysis) in HCC versus chondrocyte models under TMEM165 modulation.
What strategies are employed to study TMEM165’s dual role in Mn²⁺ and Ca²⁺ homeostasis?
TMEM165’s ion transport activity has been dissected using:
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Fluorescent probes: Fura-2-loaded Lactococcus lactis DML1 strains quantified Ca²⁺/Mn²⁺ efflux rates after heterologous TMEM165 expression .
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Truncation mutants: Δ55TMEM165 and Δ78TMEM165 variants revealed that the N-terminal domain autoinhibits ion transport, as truncations enhanced Mn²⁺ efflux in yeast .
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Therapeutic supplementation: Mn²⁺ rescue experiments in ATDC5 cells restored GAG chain elongation, confirming TMEM165’s dependence on Mn²⁺ availability .
How does TMEM165 regulate TGFβ/BMP signaling in a cell type-specific manner?
In chondrocytes, TMEM165 deficiency reduces TGFβ/BMP pathway activity due to shorter HS-GAG chains on proteoglycans like syndecan-4, which are required for growth factor receptor binding . Conversely, in HCC cells, TMEM165 overexpression may enhance MMP-2 secretion independently of TGFβ/BMP . To investigate this dichotomy:
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Approach 1: Use HS-GAG mimetics (e.g., heparin) to rescue TGFβ signaling in TMEM165-deficient chondrocytes.
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Approach 2: Perform RNA-seq on HCC cells after TMEM165 knockdown to identify MMP-2 regulators unrelated to TGFβ/BMP.
Confirming TMEM165’s Ion Transport Activity
Challenge: Direct measurement of Golgi Mn²⁺/Ca²⁺ fluxes is technically challenging.
Solution:
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Use organelle-specific fluorescent dyes (e.g., Golgi-targeted Fura-2).
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Employ yeast mutants (gdt1Δ pmr1Δ) complemented with TMEM165 variants to quantify ion sensitivity .
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Validate findings in mammalian cells via CRISPR interference (CRISPRi) and ion chelators (e.g., BAPTA-AM).
Addressing Conflicting Roles in Cancer vs. Developmental Disorders
Strategy:
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Comparative proteomics: Analyze TMEM165 interactomes in HCC versus chondrocytes to identify context-dependent binding partners.
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In vivo models: Generate conditional Tmem165 knockout mice to study tissue-specific phenotypes.
