TMEM230 Antibody

What is TMEM230 and what is its cellular localization pattern?

TMEM230 (Transmembrane Protein 230), also known as C20orf30, is a ubiquitously expressed transmembrane protein with no obvious sequence homology to other known proteins. It is primarily localized to several vesicular compartments in cells. Studies have demonstrated that TMEM230 is enriched in STX-6-positive trans-Golgi network (TGN), with additional localization to VMAT2-positive vesicles, VPS35-positive endosomes, Rab11-positive recycling endosomes, and Rab5-positive early endosomes . The protein does not show apparent co-localization with markers for mitochondria, lysosomes, or endoplasmic reticulum, though some partial co-localization with mitochondria has been observed in certain studies . TMEM230 is particularly abundant in neurons where it functions as a transmembrane protein of synaptic vesicles .

What are the known molecular weights and isoforms of TMEM230?

Based on antibody validation data, TMEM230 has the following characteristics:

The protein has been detected consistently in multiple cell lines including HeLa, COLO 320, Caco-2, HT-29, A549, MCF-7, HSC-T6, NIH/3T3, PC-12, and HEK-293 cells .

How is TMEM230 associated with Parkinson's disease pathogenesis?

TMEM230 was identified as a causative gene for familial Parkinson's disease (PD) through genetic studies. Mutations in TMEM230 have been linked to autosomal dominant, clinically typical and Lewy body-confirmed PD . The disease-causing mechanism involves impairment of synaptic vesicle trafficking, which represents a novel pathogenic pathway in PD etiology .

Mechanistically, TMEM230 loss or dysfunction disrupts:

• Retromer cargo CI-M6PR (cation-independent mannose 6-phosphate receptor) trafficking

• Autophagic cargo degradation rates

• Extracellular secretion of autophagic cargo p62 and immature lysosomal hydrolases

• Secretory autophagy (exophagy)

These disruptions can lead to accumulation of α-synuclein, a hallmark of PD pathology. Indeed, TMEM230 has been found in α-synuclein-positive Lewy bodies and Lewy neurites in midbrain and neocortex sections from patients with sporadic PD and Dementia with Lewy bodies (DLB) .

What are the confirmed pathogenic mutations in TMEM230?

Several TMEM230 mutations have been identified in Parkinson's disease patients, though there is some debate about their pathogenicity:

It's worth noting that some research has questioned whether TMEM230 is truly a PD gene. A study by Farrer et al. found that several TMEM230 variants (p.Met1Val, p.Arg62His, p.Ile125Met, p.Arg171Cys) have appreciable frequencies in ExAC database, suggesting they may be benign polymorphisms rather than pathogenic mutations .

Beyond Parkinson's disease, what other conditions has TMEM230 been implicated in?

In GBM, TMEM230 functions as a pleiotropic protein with both intracellular and extracellular effects:

• Promotes glial tumor cell migration and adhesion

• Enhances vascular mimicry-like behavior of tumor cells

• Induces endothelial cells to sprout and form tubule-like structures

• Drives tissue remodeling and hypervascularization

These findings suggest TMEM230 as a promising novel target for both antiangiogenic and antitumor therapies in GBM treatment.

How does TMEM230 regulate autophagy and vesicle trafficking?

TMEM230 plays a critical role in multiple aspects of vesicle trafficking and autophagy:

Retromer Trafficking Regulation:
TMEM230 is required for proper retromer function. Loss of TMEM230 reduces steady-state levels of CI-M6PR (cation-independent mannose 6-phosphate receptor), a key retromer cargo . This suggests TMEM230 normally regulates retromer trafficking, and PD-linked mutations (particularly R141L) lead to partial loss of this function.

Autophagy Regulation:
TMEM230 regulates both conventional autophagy and secretory autophagy (exophagy):

• In conventional autophagy: TMEM230 depletion disrupts CCCP-induced autophagic clearance of p62 and α-synuclein, while not affecting proteasomal degradation (e.g., of Mfn1) .

• In secretory autophagy: TMEM230 is required for Baf-A1-induced secretion of p62 into extracellular media. TMEM230 knockdown dramatically impairs this secretion, suggesting that TMEM230 dysfunction results in p62 accumulation due to defective secretory autophagy .

Expression of PD-linked TMEM230 mutants increases intracellular levels of both p62 and LC3-II compared to wildtype TMEM230, indicating disruption of autophagic flux .

What is the relationship between TMEM230 and mitochondrial function?

TMEM230 has been implicated in mitochondrial function, particularly in the context of cell death pathways:

• Ectopic expression of TMEM230 variants increases mitochondrial reactive oxygen species (ROS), leading to mitochondrial dysfunction and triggering apoptotic cell death

• Expression of TMEM230 variants decreases cellular ATP levels compared to control cells

• These effects suggest TMEM230 plays a critical role in normal mitochondrial function

This mitochondrial dysfunction ultimately activates the caspase cascade, leading to apoptosis. Specifically, expression of TMEM230 variants significantly induces caspase 3/7 activation, which are execution caspases .

How does TMEM230 contribute to PARP1-linked cell death pathways?

TMEM230 mediates a poly(ADP-ribose) polymerase-1 (PARP1)-linked cell death pathway through the following mechanism:

• Expression of TMEM230 variants (especially PD-linked mutations) increases mitochondrial ROS

• Elevated ROS leads to mitochondrial dysfunction and decreased ATP levels

• This triggers caspase 3/7 activation

• Activated caspases cleave PARP1 to produce 89 and 24 kD fragments

• PARP1 cleavage results in induced apoptosis

This pathway can be interrupted by treatment with pan-caspase inhibitor Z-VAD-FMK or the ROS scavenger NAC, which attenuate PARP1 cleavage and protect against TMEM230-induced toxicity . This suggests an ROS/caspase/PARP pathway mediates TMEM230-induced apoptotic cell death.

What are the validated applications for TMEM230 antibodies?

Based on the search results, TMEM230 antibodies have been validated for multiple applications:

The antibodies have demonstrated reactivity with human, mouse, and rat samples, with some also showing cross-reactivity with bovine, canine, equine, guinea pig, rabbit, and zebrafish samples .

What is the optimal protocol for using TMEM230 antibodies in Western blot analysis?

For Western blot analysis of TMEM230, researchers should consider the following methodological details:

Sample Preparation:

• Multiple cell lines can be used as positive controls, including HeLa, COLO 320, HEK-293, and HepG2 cells

• Expected molecular weight is approximately 13-16 kDa (lower than the calculated 20 kDa)

Protocol Recommendations:

• Use dilutions between 1:1000-1:50000 depending on the specific antibody

• For maximum sensitivity, the 67247-1-Ig antibody can be used at higher dilutions (1:5000-1:50000)

• For the 21466-1-AP antibody, use at 1:1000-1:4000 dilution

• Storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Store antibodies at -20°C for long-term storage; they are stable for approximately one year after shipment

Detection Considerations:

• TMEM230 may run at a lower molecular weight (13-16 kDa) than predicted (20 kDa)

• Sample-dependent variations may occur; calibration within each testing system is recommended

What are effective methods for TMEM230 knockdown in experimental systems?

For TMEM230 knockdown studies, lentiviral-mediated RNA interference has been successfully employed:

Protocol for Lentivirus-Mediated TMEM230 Knockdown:

• Transfect HEK293FT cells in a 15 cm tissue culture dish (70% confluent) with:

  • 10 μg of transfer vector (TMEM230-RNAi in pLKO.1 - recommended clone: TRCN0000131231)

  • 9 μg psPAX2

  • 1 μg VSVG

  • Use Lipofectamine2000 according to manufacturer's protocol for 16-18 hours

• Replace medium with DMEM containing 10% fetal bovine serum and 1% Pen/Strep

• Harvest virus 2 days post-transfection

• Centrifuge supernatant at 1000 rpm and filter through a 0.45 μm filter

• Concentrate virus 80X with LentiX-concentrator reagent

• Resuspend viral pellet in DMEM medium and freeze for future use

• For stable cell line generation, infect target cells with 1.5 μl of viral resuspension

• Select transduced cells using puromycin (2 μg/mL)

This method has been validated for creating stable TMEM230 knockdown cell lines for functional studies.

How do TMEM230 and LRRK2 pathways converge in Parkinson's disease pathogenesis?

An important finding in TMEM230 research is the convergence of TMEM230 and LRRK2 pathways in Parkinson's disease:

• Both TMEM230 and LRRK2 are PD-associated genes

• LRRK2 is known to phosphorylate Rab8a, a small GTPase

• Loss of TMEM230 function inhibits extracellular secretion mediated specifically by loss of Rab8a

• Knockdown of LRRK2 similarly impairs:

  • Retromer trafficking

  • Secretory autophagy

  • Golgi-derived vesicle secretion

This demonstrates converging roles of these two PD genes on Rab8a function, suggesting that retromer and secretory dysfunction are common mechanisms in PD pathogenesis mediated by different genetic causes. This convergence provides a potential explanation for the similar clinical manifestations observed in patients with mutations in these different genes.

What experimental approaches can distinguish the effects of different TMEM230 mutations?

To investigate the differential effects of TMEM230 mutations, researchers have employed several experimental approaches:

Comparative Mutant Expression Studies:

• Expression of wildtype vs. mutant TMEM230 (R141L, Y92C, 184Wext5, 184PGext5) in cellular models

• Measurement of protein levels of autophagic markers (p62, LC3-II)

• Quantification of α-synuclein accumulation

• Assessment of retromer cargo (CI-M6PR) trafficking

Cell Death and Mitochondrial Function Assays:

• Measurement of mitochondrial ROS after expression of different TMEM230 variants

• Quantification of cellular ATP levels

• Caspase 3/7 activation assays

• PARP1 cleavage detection via Western blot

• Cell viability assessments with treatments (Z-VAD-FMK, NAC)

Patient-Derived Cell Studies:

• Use of lymphoblastoid cell lines from PD patients with TMEM230 mutations

• Comparison with control lymphoblastoid cells

• Assessment of retromer and autophagic dysfunction

These approaches allow researchers to compare the severity and specific mechanisms affected by different TMEM230 mutations, providing insights into structure-function relationships and potential therapeutic targets.

How can researchers effectively study TMEM230's role in glioblastoma progression?

For researchers interested in TMEM230's role in glioblastoma, several methodological approaches have proven informative:

Cell-Based Functional Assays:

• U87-MG cell model (human GBM model)

• Assessment of migration capacity with TMEM230 knockdown

• Substratum adhesion assays

• Cell re-passaging capacity tests

• Vascular mimicry assays to evaluate vessel-like structure formation

Angiogenesis Assessment:

• Collection of conditioned media from U87 cells expressing endogenous TMEM230

• Application to HUVECs (Human Umbilical Vein Endothelial Cells)

• Quantification of endothelial sprouting and tubule-like structure formation

• Comparison with media from TMEM230-downregulated cells

Gene Expression Analysis:

• Transcriptomic analysis of patient glioma datasets (702 patients)

• Correlation of TMEM230 expression levels with glioma grade

• Survival analysis based on TMEM230 expression levels

• Pathway analysis to identify molecular mechanisms (e.g., ATP-dependent microtubule kinesin motor activity)

These approaches have revealed that TMEM230 is necessary for growth, migration, and adhesion of GBM cells, and promotes abnormal vascularization, making it a promising therapeutic target for both anti-angiogenic and anti-tumor strategies in GBM.