Recombinant Mouse Transmembrane protein 189 (Tmem189)

What is mouse Tmem189 and what is its primary function?

Mouse Transmembrane protein 189 (Tmem189) encodes the enzyme plasmanylethanolamine desaturase (PEDS), which catalyzes the final step in the biosynthesis of plasmalogens. This enzyme is responsible for introducing the characteristic alk-1′-enyl ether bond in plasmalogen formation . Plasmalogens are an abundant class of glycerophospholipids that play crucial roles in membrane structure and function, and are notably depleted in certain diseases such as Alzheimer's disease . The enzymatic activity of Tmem189 was confirmed through multiple experimental approaches, including gene knockout studies in cell lines and animal models, which demonstrated that Tmem189 is essential for plasmalogen biosynthesis rather than merely being an accessory protein .

What is the cellular localization of mouse Tmem189?

Mouse Tmem189 is primarily localized to the endoplasmic reticulum (ER). This localization has been experimentally confirmed using TMEM189-GFP fusion proteins expressed in HEK293T cells, which show a characteristic ER distribution pattern when visualized by confocal microscopy. The localization can be verified by co-staining with ER-specific markers such as ER Tracker Red, which shows overlapping fluorescence signals . Interestingly, while mutations in the conserved histidine residues dramatically affect the enzymatic activity of Tmem189, they do not alter its subcellular localization to the ER, suggesting that protein localization and catalytic function are determined by different structural elements .

What structural motifs are important in mouse Tmem189 protein?

Mouse Tmem189 contains a highly conserved protein motif (pfam10520) characterized by eight conserved histidine residues. This motif is shared with an alternative type of plant desaturase called fatty acid desaturase type 4 (FAD4) . The structure differs somewhat from classical eight-histidine motifs found in membrane-bound desaturases like stearoyl CoA desaturase.

Site-directed mutagenesis experiments have demonstrated that each of these eight histidines is essential for PEDS enzymatic activity. When any of these histidines is mutated to alanine, the enzymatic activity is abolished, strongly suggesting that these residues are directly involved in the catalytic function of the protein . Based on structural analogies with other desaturases, these histidines likely coordinate a di-metal center that participates in the enzymatic reaction mechanism.

How can Tmem189-deficient mouse models be generated and characterized?

Tmem189-deficient mouse models can be generated using knockout-first allele strategies. As described in the literature, Tmem189tm1a(KOMP)Wtsi mice have been successfully created using a strategy that introduces an artificial splice site downstream of exon 2, resulting in the production of a truncated, inactive TMEM189 protein .

For proper characterization of these models:

• Genotyping: Confirm the genetic modification through PCR-based genotyping.

• Expression analysis: Verify the absence or reduction of Tmem189 mRNA using RT-PCR and protein levels using Western blotting.

• Enzymatic activity: Measure PEDS activity in tissue homogenates to confirm functional deficiency.

• Lipid analysis: Quantify plasmalogen levels in various tissues using techniques such as liquid chromatography tandem-mass spectrometry (LC-MS/MS).

• Developmental monitoring: Track body weight from 3 to 8 weeks of age to assess growth phenotypes.

• Tissue collection: Harvest and analyze relevant tissues (preferably after euthanasia by cervical dislocation) with immediate snap-freezing in liquid nitrogen and storage at -80°C until analysis .

What phenotypic characteristics do Tmem189-deficient mice exhibit?

Tmem189-deficient mice exhibit several notable phenotypic characteristics:

• Growth impairment: These mice show reduced body weight gain during development compared to wild-type littermates .

• Biochemical abnormalities: They completely lack plasmanylethanolamine desaturase activity in all tissues examined .

• Lipid profile alterations: Dramatic reduction in plasmalogen levels across various tissues, with a corresponding accumulation of plasmanylethanolamines (the substrate for PEDS) .

• Membrane properties: Likely alterations in membrane structure and dynamics due to the absence of plasmalogens, which affect membrane curvature and fusion properties.

These phenotypic changes provide valuable insights into the physiological importance of plasmalogens and can serve as models for studying conditions associated with plasmalogen deficiency, such as certain neurodegenerative disorders .

How can cell lines with altered Tmem189 expression be created?

Several approaches can be used to create cell lines with altered Tmem189 expression:

• CRISPR-Cas9 knockout: Generate complete knockout cell lines by targeting the TMEM189 gene, as has been done in human HAP1 cells . This approach results in a complete loss of PEDS activity and plasmalogen synthesis.

• siRNA knockdown: Reduce expression through RNA interference using siGenome Smart pools (as demonstrated in A431 cells), which allows for transient reduction in gene expression and subsequent analysis of PEDS activity .

• Overexpression systems: Transfect cells with expression plasmids containing the Tmem189 cDNA to increase protein levels and enzymatic activity. This approach has been demonstrated in HEK293T cells using transfection reagents like TurboFect .

• Fusion protein expression: Generate cell lines expressing TMEM189-GFP or TMEM189-myc fusion proteins for localization studies and protein detection, respectively .

• Site-directed mutagenesis: Create cell lines expressing Tmem189 variants with specific mutations (particularly in the conserved histidine residues) to study structure-function relationships .

Each of these approaches has specific applications depending on the research question, with CRISPR knockouts providing the most definitive loss-of-function model.

What techniques are used to measure PEDS activity in Tmem189 research?

PEDS (plasmanylethanolamine desaturase) activity can be measured through several complementary techniques:

• Enzymatic assays: Direct measurement of enzyme activity in cell homogenates or tissue extracts using radiolabeled substrates (such as [1-14C]-labeled plasmanylethanolamines) followed by product analysis.

• Lipid extraction and analysis: Extract total lipids from cells or tissues, followed by separation of lipid classes and analysis of plasmalogen content vs. plasmanylethanolamines.

• Mass spectrometry: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) to quantify glycerophosphoethanolamines and glycerophosphocholines, allowing detection of both substrate accumulation and product formation . This technique enables detailed characterization of lipid species with specific side chain patterns.

• Metabolic labeling: Incubation of cells with labeled alkylglycerols followed by analysis of their conversion to plasmalogens, which is absent in Tmem189-deficient cells .

• Complementation assays: Transfection of Tmem189-deficient cells with expression plasmids, followed by measurement of restored PEDS activity as a functional confirmation .

These approaches can be used individually or in combination to provide comprehensive assessment of PEDS activity in different experimental contexts.

How should Tmem189 protein expression and localization be analyzed?

Analysis of Tmem189 protein expression and localization requires multiple complementary approaches:

• Western blot analysis:

  • Use standard techniques with antibodies against Tmem189 or epitope tags (e.g., myc tag for recombinant proteins)

  • Include loading controls such as β-actin (MAB1501; Merck Millipore)

  • Visualize with fluorescently labeled secondary antibodies (Cy3/Cy5) and analyze using multi-wavelength laser scanners

• Confocal microscopy for localization:

  • Generate fusion proteins (e.g., TMEM189-GFP) by cloning Tmem189 cDNA into vectors like pEGF-N1

  • Express in appropriate cell lines (e.g., HEK293T)

  • Perform real-time confocal imaging using spinning-disk confocal systems

  • Co-stain with organelle markers (e.g., ER Tracker Red for endoplasmic reticulum)

  • Analyze co-localization patterns to determine subcellular distribution

• Flow cytometry:

  • For quantitative analysis of protein expression levels in cell populations

  • Particularly useful when comparing wild-type and mutant proteins

• Immunohistochemistry:

  • For tissue sections to analyze expression patterns in vivo

  • Requires specific antibodies against Tmem189 or epitope tags

The choice of method depends on the specific research question, with Western blotting providing quantitative expression data and microscopy offering spatial information about protein localization.

What expression systems are suitable for recombinant mouse Tmem189 production?

Several expression systems can be used for recombinant mouse Tmem189 production, each with specific advantages:

• Mammalian cell expression:

  • HEK293T cells have been successfully used for Tmem189 expression

  • Transfection with expression plasmids using reagents like TurboFect

  • Allows proper folding and post-translational modifications

  • Can include epitope tags (myc, FLAG, etc.) for detection and purification

  • Suitable for functional studies as the protein retains enzymatic activity

• Cell-free systems:

  • For small-scale protein production

  • Useful for initial characterization studies

  • May have limitations for membrane proteins like Tmem189

• Bacterial expression:

  • Challenging for multi-pass membrane proteins like Tmem189

  • May require fusion partners or solubilization tags

  • Less likely to produce functional protein due to lack of proper folding machinery

• Insect cell expression:

  • Baculovirus expression systems can be used for larger-scale production

  • Better suited for complex membrane proteins than bacterial systems

  • Can provide higher yields than mammalian systems

For functional studies of mouse Tmem189, mammalian expression systems are generally preferred as they provide the appropriate cellular environment for proper folding, membrane insertion, and enzymatic activity of this multi-pass membrane protein.

How should gene expression data for Tmem189 be analyzed?

Analysis of gene expression data for Tmem189 should follow rigorous statistical approaches:

• Quality control and normalization:

  • Assess data normality before applying parametric statistical tests

  • Apply appropriate normalization methods for the specific platform used (microarray or RNA-seq)

  • Check for batch effects and technical variations

• Differential expression analysis:

  • For comparing expression between experimental groups (e.g., normal vs. disease states), use:

    • Student's t-test for pairwise comparisons between two groups

    • ANOVA for comparisons across multiple groups

    • Apply appropriate multiple testing corrections (e.g., False Discovery Rate) with threshold of 5%

  • Consider both statistical significance (p-value < 0.05) and biological significance (fold change)

• Correlation analysis:

  • When correlating Tmem189 expression with enzyme activity or other parameters:

    • Calculate Pearson's or Spearman's correlation coefficients

    • Generate scatter plots to visualize relationships

    • The correlation approach was successfully used to identify Tmem189 as the top gene correlating with PEDS activity across multiple cell lines and tissues

• Pathway and network analysis:

  • Place Tmem189 in the context of biological pathways

  • Identify co-regulated genes that may function in related processes

  • Consider gene set enrichment analysis (GSEA) to identify affected pathways

• Visualization:

  • Use heat maps, volcano plots, and other visualization tools to effectively communicate results

  • Include error bars and indicators of statistical significance in all figures

Following these analytical approaches ensures robust interpretation of Tmem189 expression data across different experimental contexts.

What experimental controls are critical in Tmem189 functional studies?

Critical experimental controls for Tmem189 functional studies include:

• Genetic controls:

  • Wild-type (WT) cell lines or animals alongside Tmem189-deficient models

  • Heterozygous animals to assess gene dosage effects

  • Littermate controls to minimize background genetic variation effects

• Expression controls:

  • Empty vector controls for transfection experiments

  • Irrelevant protein expression (e.g., GFP alone) to control for non-specific effects of protein overexpression

  • Western blot verification of expression levels for all constructs tested

• Functional controls:

  • Other desaturase enzymes as negative controls (they should not rescue PEDS activity)

  • Positive controls with known PEDS activity

  • Site-directed mutants (e.g., histidine to alanine mutations) as catalytically inactive variants

• Methodological controls:

  • Non-targeting siRNA for knockdown experiments

  • Scrambled CRISPR guide RNAs for knockout experiments

  • Vehicle controls for any treatments

  • Internal standards for mass spectrometry analysis of lipids

• Validation across systems:

  • Confirmation of key findings in multiple cell types

  • Cross-validation between in vitro and in vivo models

  • Use of complementary methodological approaches

Implementation of these controls ensures robust and reproducible results in Tmem189 functional studies and allows confident interpretation of experimental findings.

How can conflicting data about Tmem189 function be resolved?

Resolving conflicting data about Tmem189 function requires a systematic approach:

• Methodological reconciliation:

  • Compare experimental protocols in detail to identify variations that might explain discrepancies

  • Consider differences in cell types, animal backgrounds, or assay conditions

  • Standardize measurement techniques when possible

• Independent replication:

  • Have conflicting results reproduced by independent laboratories

  • Use multiple complementary approaches to address the same question

  • Employ different methodologies to cross-validate findings

• Genetic approach:

  • Generate knockout models using different strategies

  • Compare phenotypes between different knockout lines

  • Use rescue experiments with wild-type protein to confirm specificity

• Statistical analysis:

  • Ensure adequate sample sizes for robust statistical power

  • Apply appropriate statistical tests and multiple comparison corrections

  • Consider meta-analysis when multiple datasets are available

• Contextual factors:

  • Investigate cell type-specific or tissue-specific differences in Tmem189 function

  • Consider developmental timing or physiological state as sources of variability

  • Examine potential compensatory mechanisms in chronic vs. acute loss of function

• Protein interactions and modifications:

  • Investigate potential binding partners or post-translational modifications

  • Consider that Tmem189 may have context-dependent functions

By systematically addressing these aspects, researchers can resolve conflicting data and develop a more comprehensive understanding of Tmem189 function in different biological contexts.

How does Tmem189 dysfunction relate to disease states?

Tmem189 dysfunction has significant implications for several disease states:

• Neurodegenerative disorders:

  • Plasmalogen deficiency is associated with Alzheimer's disease

  • Tmem189 dysfunction could contribute to lipid composition alterations in brain tissue

  • The neuroprotective functions of plasmalogens may be compromised in conditions of Tmem189 deficiency

• Metabolic disorders:

  • Alterations in membrane composition affect cellular signaling and metabolism

  • Defects in plasmalogen synthesis may impact peroxisomal function

  • Potential implications for disorders with aberrant lipid metabolism

• Growth and developmental disorders:

  • Tmem189-deficient mice show impaired growth

  • Suggests potential role in developmental processes

  • May contribute to rare inherited disorders of lipid metabolism

• Oxidative stress-related conditions:

  • Plasmalogens function as antioxidants in cellular membranes

  • Tmem189 dysfunction may increase vulnerability to oxidative damage

  • Potential implications for ischemia-reperfusion injury and inflammation

• Cancer biology:

  • Altered lipid metabolism is a hallmark of cancer cells

  • Changes in membrane composition affect proliferation and metastatic potential

  • Potential role in cancer cell adaptation to metabolic stress

Future research should focus on establishing causal relationships between Tmem189 dysfunction and these disease states, potentially identifying new therapeutic targets or biomarkers.

What are the most effective strategies for studying Tmem189 in different tissue contexts?

Studying Tmem189 across different tissue contexts requires tailored experimental approaches:

• Tissue-specific knockout models:

  • Generate conditional knockout mice using Cre-loxP systems

  • Target Tmem189 deletion to specific tissues of interest

  • Compare phenotypes with global knockout to identify tissue-specific roles

• Ex vivo tissue culture systems:

  • Organotypic cultures that maintain tissue architecture

  • Primary cell cultures from different tissues

  • Addition of labeled substrates to track plasmalogen synthesis

• Human patient samples:

  • Analysis of tissues from individuals with suspected plasmalogen disorders

  • Correlation of Tmem189 expression with plasmalogen levels

  • Genetic analysis for potential mutations or polymorphisms

• Imaging approaches:

  • Tissue-specific localization using immunohistochemistry

  • Multiplex imaging to correlate Tmem189 with other markers

  • Lipid imaging techniques to visualize plasmalogen distribution

• Transcriptomic and proteomic profiling:

  • Tissue-specific expression patterns of Tmem189

  • Co-expression network analysis to identify tissue-specific interaction partners

  • Correlation of expression with enzymatic activity across tissues

• Functional readouts:

  • Tissue-specific measurement of PEDS activity

  • Assessment of membrane properties in different tissues

  • Evaluation of tissue-specific phenotypes in knockout models

These complementary approaches provide a comprehensive understanding of Tmem189 function across different physiological contexts and disease states.

How can recombinant Tmem189 be used as a research tool?

Recombinant Tmem189 offers several valuable applications as a research tool:

• Enzyme activity standardization:

  • Purified recombinant protein can serve as a standard for PEDS activity assays

  • Enables quantitative comparison across different experimental systems

  • Provides positive control for enzymatic assays

• Structure-function studies:

  • Site-directed mutagenesis to probe the role of specific residues

  • The eight conserved histidines provide targets for systematic analysis

  • Chimeric proteins with plant FAD4 desaturases to identify functional domains

• High-throughput screening:

  • Identification of inhibitors or activators of Tmem189

  • Drug discovery for conditions with aberrant plasmalogen metabolism

  • Development of assay systems for monitoring enzyme activity

• Protein-protein interaction studies:

  • Identification of binding partners through co-immunoprecipitation

  • Characterization of potential regulatory proteins

  • Understanding the integration of Tmem189 in broader metabolic networks

• Educational and training tools:

  • Model system for teaching concepts of membrane biochemistry

  • Example of gene-to-function discovery approaches

  • Demonstration of structure-function relationships in membrane proteins

• Biosensor development:

  • Creation of fluorescence-based reporters of PEDS activity

  • Real-time monitoring of plasmalogen synthesis

  • Potential applications in live cell imaging

The versatility of recombinant Tmem189 as a research tool continues to expand as our understanding of its function and regulation improves, offering new opportunities for both basic and translational research in lipid biochemistry.