Recombinant Bovine UPF0197 transmembrane protein C11orf10 homolog

What are the functional roles of TMEM258 in cellular processes?

TMEM258 serves as a crucial component of the oligosaccharyltransferase (OST) complex, which is essential for N-linked protein glycosylation . This post-translational modification is vital for:

• Proper protein folding

• Maintaining endoplasmic reticulum (ER) quality control

• Cellular homeostasis, particularly in secretory cell types

Research has demonstrated that TMEM258 specifically interacts with RPN1, another OST complex component, and co-expression of both proteins elevates their steady-state levels . TMEM258 is required for proper N-linked glycosylation, as its depletion results in defective glycosylation, profound ER stress, and perturbed ER homeostasis .

What expression systems are most effective for producing Recombinant Bovine TMEM258?

Multiple expression systems have been utilized for recombinant TMEM258 production, each with distinct advantages depending on research needs:

For studies focused on protein-protein interactions within the OST complex, mammalian expression systems are preferred as they provide the most physiologically relevant post-translational modifications and protein folding environment .

How can researchers optimize purification of Recombinant Bovine TMEM258?

Purification of membrane proteins like TMEM258 presents unique challenges. A recommended stepwise approach includes:

• Membrane fraction isolation: Cell fractionation analyses indicate that TMEM258 is present in the membrane fraction but not in the soluble fraction .

• Detergent selection: Use mild detergents (such as DDM or CHAPS) for solubilization to maintain protein structure and function.

• Affinity chromatography: Utilize His-tag, FLAG-tag, or other affinity tags for initial purification.

• Size exclusion chromatography: Further purify based on molecular size to achieve >85% purity.

• Validation: Confirm purity by SDS-PAGE and identity by Western blotting using specific antibodies against TMEM258 or its tags.

For functional studies, it's crucial to verify that the purified protein maintains its native conformation and ability to interact with OST complex partners like RPN1 .

How can researchers study TMEM258's role in the oligosaccharyltransferase complex?

To investigate TMEM258's role in the OST complex, several complementary approaches are recommended:

• Co-immunoprecipitation assays: Use tagged TMEM258 (e.g., V5-tagged) as bait to identify interaction partners within the OST complex. This approach has successfully identified RPN1 as a key interactor with TMEM258 .

• Glycosylation assays: Monitor the glycosylation status of model glycoproteins (e.g., basigen/BSG) in the presence or absence of TMEM258 using methods like:

  • PNGaseF treatment followed by Western blotting

  • Lectin binding assays (e.g., concanavalin A)

  • Gel mobility shift assays

• Functional reconstitution: Reconstitute the OST complex in vitro with purified components to assess the direct contribution of TMEM258.

Research has shown that TMEM258 knockdown dramatically reduces glycoprotein surface content and prevents proper N-glycosylation of proteins like BSG, demonstrating its essential role in this process .

What methodologies are effective for studying TMEM258 subcellular localization?

For accurate characterization of TMEM258 subcellular localization, a multi-method approach is recommended:

• Cell fractionation: Separate cellular components (membranes, cytosol, nuclear fractions) followed by Western blot analysis to detect TMEM258 in different fractions .

• Immunofluorescence microscopy: Use specific antibodies against TMEM258 or epitope tags. Co-staining with markers such as Lamin B1 (nuclear envelope) can help define precise localization .

• Digitonin permeabilization: This selective permeabilization technique can distinguish between cytoplasmic and membrane-embedded portions of TMEM258 .

• Protease protection assays: After digitonin permeabilization, treat samples with proteinase K to assess which domains are protected within membranes .

Studies have revealed that TMEM258 is found in both the cytoplasm and at the nuclear envelope, where it colocalizes with nuclear lamina markers like Lamin B1 .

How is TMEM258 implicated in inflammatory bowel disease pathogenesis?

TMEM258 has been identified as a potential regulator of intestinal inflammation through several mechanisms:

• Genetic association: The gene-dense locus on chromosome 11 (61.5-61.65 Mb) containing TMEM258 has been associated with inflammatory bowel disease (IBD), rheumatoid arthritis, and coronary artery disease .

• Expression patterns: TMEM258 is expressed at significantly higher levels than other neighboring genes in ileum biopsies, with highest expression in secretory cell types such as goblet and Paneth cells .

• Experimental evidence: Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in models of colitis, with increased frequencies of epithelial cells positive for BiP/GRP78 (a marker of ER stress) and a greater frequency of apoptotic epithelial cells .

• Mechanistic insight: Complete deficiency of Tmem258 in colonic organoids results in unresolved ER stress culminating in apoptosis, suggesting that TMEM258 is essential for intestinal epithelial homeostasis .

The precise mechanism involves TMEM258's role in N-linked glycosylation, which is critical for proper folding of secretory proteins abundant in intestinal epithelial cells. Disruption of this process leads to ER stress and subsequent inflammation .

What genetic evidence links TMEM258 to metabolic and cardiovascular disorders?

Genetic studies have identified significant associations between the chromosomal region containing TMEM258 and various metabolic and cardiovascular traits:

• GWAS findings: The region containing TMEM258, FADS1, FADS2, and other genes on chromosome 11 has shown strong associations with:

  • Polyunsaturated fatty acid (PUFA) levels

  • Lipid traits including LDL-C, HDL-C, and triglycerides

  • Coronary artery disease

• Expression quantitative trait loci (eQTL): SNPs in this region affect TMEM258 expression levels, potentially influencing downstream metabolic processes .

• Network analysis: Integration of genetic variation and gene expression data has revealed connections between this locus and cardiovascular disease traits, suggesting TMEM258 may be part of regulatory networks affecting these conditions .

The strongest associations have been observed on chromosome 11 in the region containing FADS1/2, FEN1, C11orf9, and C11orf10/TMEM258, suggesting these genes may cooperatively influence metabolic traits .

How can CRISPR-Cas9 be effectively employed to study TMEM258 function?

CRISPR-Cas9 technology offers powerful approaches for investigating TMEM258 function, with specific considerations for this essential gene:

• Inducible knockout systems: Since complete TMEM258 deficiency is embryonically lethal, inducible Cas9 systems (such as stop-floxed Cas9-2A-GFP with Cre-mediated activation) allow temporal control of gene editing .

• Organoid models: Colonic organoids derived from Tmem258 heterozygous mice have been successfully used with CRISPR-Cas9 to target the remaining Tmem258 allele, enabling study of complete deficiency in a relevant tissue context .

• sgRNA design considerations:

  • Target exonic regions for gene disruption

  • Use multiple independent sgRNAs to confirm phenotypes

  • Include control sgRNAs targeting non-coding intronic regions

• Phenotypic readouts:

  • Monitor cell viability (loss of GFP-positive cells)

  • Assess morphological changes in organoids

  • Analyze ER stress markers (BiP/GRP78, XBP1 splicing)

  • Evaluate N-glycosylation using lectin binding or glycoprotein mobility

Research has shown that sgRNAs targeting Tmem258 exons in organoids result in fewer viable Cas9-GFP-positive cells compared to control sgRNAs, with morphological changes including small spheroids containing dead cells .

What are the considerations for studying TMEM258 homologs across different species?

Comparative studies of TMEM258 across species provide valuable insights into conserved functions and species-specific adaptations:

• Sequence conservation: The UPF0197 family proteins are evolutionarily conserved with high sequence similarity:

  • Human TMEM258 and Drosophila Kud (CG9669) share 65% identity

  • Functional conservation is demonstrated by the ability of human TMEM258 to rescue phenotypes in Drosophila Kud mutant cells

• Experimental approaches for cross-species studies:

  • Complementation assays to test functional conservation

  • Comparative subcellular localization studies

  • Interaction partner identification across species

• Model system selection:

  • Drosophila: Kud (CG9669) knockout flies show growth retardation and larval lethality

  • Zebrafish: tmem258 (zgc:73269) for developmental studies

  • Mouse: Tmem258 knockout is embryonically lethal; heterozygotes viable but susceptible to colitis

The conserved subcellular localization patterns and functional roles across species suggest that fundamental aspects of TMEM258 biology are evolutionarily ancient and likely central to eukaryotic cell biology .

How can Recombinant Bovine TMEM258 be utilized as a research tool?

Recombinant Bovine TMEM258 has several applications as a research tool:

• Antibody production: As an immunogen for generating specific antibodies against TMEM258, useful for:

  • Western blotting

  • Immunofluorescence

  • Immunoprecipitation

  • ELISA

• Protein interaction studies: Purified TMEM258 can be used to:

  • Identify novel interaction partners

  • Characterize binding kinetics

  • Map interaction domains

• Structural biology: Purified protein for:

  • X-ray crystallography

  • Cryo-EM studies of the OST complex

  • NMR analysis of membrane topology

• Functional reconstitution: Component for in vitro reconstitution of N-linked glycosylation systems .

What methods can resolve contradictory data about TMEM258 subcellular localization?

Contradictory reports exist regarding TMEM258 subcellular localization, with some studies reporting cytoplasmic localization and others finding nuclear envelope association . To resolve these discrepancies:

• Expression level considerations: Different expression levels may affect localization patterns. Use:

  • Endogenous protein detection with validated antibodies

  • Inducible expression systems with titratable levels

  • Comparison of tagged and untagged versions

• Cell type-specific variations: Examine localization across:

  • Different cell types (epithelial, immune, neuronal)

  • Primary cells vs. cell lines

  • Normal vs. stressed conditions

• Technical approaches:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging to track dynamic localization

  • Subcellular fractionation with multiple markers

  • Electron microscopy for ultrastructural localization

• Protein modification status: Investigate how post-translational modifications affect localization:

  • Glycosylation

  • Phosphorylation

  • Ubiquitination

Research indicates that discrepancies in localization patterns may be due to differences in expression levels or detection methods .

What are promising approaches for investigating the therapeutic potential of targeting TMEM258?

Given TMEM258's role in N-linked glycosylation and association with inflammatory bowel disease, several approaches show promise for therapeutic development:

• Modulation strategies:

  • Small molecule stabilizers to enhance TMEM258 function

  • Peptide mimetics that simulate TMEM258-RPN1 interaction

  • Gene therapy approaches to restore optimal TMEM258 levels

• Disease models for assessment:

  • Tmem258 haploinsufficient mice with DSS-induced colitis

  • Patient-derived intestinal organoids

  • Inflammatory bowel disease tissue explants

• Readouts for therapeutic efficacy:

  • ER stress markers (BiP/GRP78, spliced XBP1)

  • Epithelial barrier function

  • Apoptosis markers

  • N-glycosylation efficiency

  • Inflammatory cytokine profiles

• Combination approaches:

  • ER stress modulators plus TMEM258-targeting agents

  • Anti-inflammatory agents with TMEM258 stabilizers

The high expression of TMEM258 in secretory intestinal epithelial cells makes it a particularly relevant target for inflammatory bowel disease therapies .

How can systems biology approaches advance our understanding of TMEM258's role in disease networks?

Systems biology offers powerful frameworks for contextualizing TMEM258 within broader disease networks:

• Integrative genomics approaches:

  • Combine GWAS, eQTL, and epigenomic data to understand TMEM258 regulation

  • Network analysis incorporating protein-protein interactions

  • Pathway enrichment analysis to identify biological processes

• Multi-omics integration:

  • Transcriptomics to identify genes co-regulated with TMEM258

  • Proteomics to map the extended interactome

  • Glycomics to assess systemic impacts on protein glycosylation

  • Metabolomics to identify downstream metabolic effects

• Mathematical modeling:

  • Dynamical modeling of ER stress responses

  • Prediction of network perturbations from TMEM258 dysregulation

  • Simulation of therapeutic interventions

Studies have already begun to incorporate TMEM258 into disease networks, revealing connections to inflammatory and metabolic pathways that may explain its associations with multiple diseases .