Recombinant Danio rerio UPF0197 transmembrane protein C11orf10 homolog (zgc:73269)

What is the functional role of Recombinant Danio rerio UPF0197 transmembrane protein C11orf10 homolog?

This protein functions as a critical component of the oligosaccharyltransferase (OST) complex, which catalyzes the transfer of defined glycan structures (Glc3Man9GlcNAc2 in eukaryotes) from dolichol-pyrophosphate to asparagine residues within Asn-X-Ser/Thr consensus motifs in nascent polypeptide chains. This cotranslational process represents the initial step in N-glycosylation, which occurs as proteins are being synthesized and translocated across the endoplasmic reticulum membrane. The OST complex associates with the Sec61 complex at the translocon, facilitating protein translocation across the ER membrane while simultaneously enabling glycosylation. This protein's evolutionary conservation across species highlights its fundamental importance in eukaryotic cellular processes.

What are the optimal storage and handling conditions for this recombinant protein?

For optimal preservation of structural integrity and biological activity, the recombinant protein should be stored in a Tris-based buffer containing 50% glycerol at pH 8.0 . The recommended storage temperature is -20°C for routine storage, while -80°C is preferable for extended storage periods . It is critically important to avoid repeated freeze-thaw cycles, as these can significantly compromise protein stability and functionality . For ongoing experiments requiring regular access to the protein, working aliquots can be maintained at 4°C for up to one week . This aliquoting strategy minimizes the need for repeated freezing and thawing of the main stock, thereby preserving long-term protein integrity.

How does the zebrafish model complement research on this protein's human homolog?

Zebrafish (Danio rerio) serve as an excellent model organism for studying this protein due to the high degree of genetic conservation between zebrafish and humans, with approximately 69% of zebrafish genes having clear human orthologs . The human homolog TMEM258 (transmembrane protein 258, also known as C11orf10) shares significant sequence and functional similarity with the zebrafish protein.

Zebrafish offer several experimental advantages that make them particularly valuable for this research:

• Rapid development - their digestive organs are fully mature in larvae by 5 days of age

• Optical clarity - enables direct visualization of developmental and disease processes

• High fecundity - produces large clutches of embryos for experimental analysis

• Amenability to genetic manipulation - facilitates gene editing and screening approaches

These characteristics enable researchers to investigate the protein's function in vivo, establishing connections between molecular mechanisms and physiological outcomes that may be relevant to human disease states associated with TMEM258, including Spinocerebellar Ataxia 20.

How does this protein function mechanistically within the oligosaccharyltransferase complex?

This protein functions as an essential subunit of the oligosaccharyltransferase (OST) complex, which catalyzes the transfer of preassembled oligosaccharide moieties to nascent polypeptide chains. Mechanistically, the OST complex recognizes the Asn-X-Ser/Thr consensus sequence (where X can be any amino acid except proline) in newly synthesized proteins and transfers the Glc3Man9GlcNAc2 glycan from dolichol-pyrophosphate to the asparagine residue.

The zebrafish UPF0197 transmembrane protein integrates into the ER membrane through its transmembrane domains, positioning its catalytic regions appropriately for interaction with both the lipid-linked oligosaccharide donor and the nascent polypeptide acceptor. The protein's association with the Sec61 translocon complex ensures that glycosylation occurs cotranslationally as proteins are being synthesized and inserted into the ER. This spatial and temporal coordination is critical for proper protein folding, quality control, and subsequent trafficking through the secretory pathway.

Disruption of this protein's function can lead to hypoglycosylation of proteins, ER stress, and activation of the unfolded protein response (UPR), potentially contributing to disease pathogenesis .

What experimental approaches are most effective for studying the protein's role in glycosylation defects?

Several complementary experimental approaches can be employed to investigate this protein's role in glycosylation processes:

These methodologies can be integrated with the unique advantages of the zebrafish model system, including the ability to perform high-throughput chemical and genetic screens to identify modifiers of glycosylation phenotypes . Forward genetic screens have generated zebrafish mutants with hereditary liver diseases and biliary defects that can serve as models for human conditions associated with glycosylation abnormalities .

What expression systems provide optimal yield and functionality for this recombinant protein?

The selection of an appropriate expression system is critical for obtaining functional recombinant Danio rerio UPF0197 transmembrane protein with high yield and purity. Several expression platforms have been evaluated:

Experience with similar zebrafish proteins has demonstrated that careful selection of the host/vector combination is essential. For instance, the pIVEX vector system carrying zebrafish proteins has been reported to cause growth inhibition in BL21(DE3)pLysS cells even before expression induction . The KRX strain has shown superior results for proteins that exhibit toxicity in standard expression hosts .

For the Danio rerio UPF0197 transmembrane protein specifically, cell-free systems are preferred for initial production, while optimized bacterial or yeast systems may be more suitable for scaled-up production once expression conditions have been established.

How does the function of this protein relate to disease models and comparative biology?

The study of this zebrafish protein provides valuable insights into disease mechanisms through comparative biology approaches. The human homolog TMEM258 is associated with Spinocerebellar Ataxia 20 and plays a role in colonic epithelial ER homeostasis, making the zebrafish protein an important model for understanding these conditions.

Zebrafish models offer several advantages for disease-related research:

• The rapid development and optical clarity of zebrafish embryos allow for real-time visualization of disease progression and response to interventions .

• Zebrafish embryos can develop hepatobiliary diseases caused by developmental defects or toxin/ethanol-induced injury, with premalignant changes manifesting within weeks .

• The conservation of cellular composition, function, signaling pathways, and response to injury between zebrafish and human livers makes them excellent models for liver disease research .

• Chemical screens using zebrafish can identify compounds that modulate disease processes or enhance recovery after organ injury .

A notable example from biliary disease research found that knocking down gpc1 (glypican 1) in zebrafish caused structural biliary defects and decreased bile secretion, providing functional validation of genome-wide association data that had identified GPC1 as a gene associated with biliary atresia in humans . This demonstrates how the zebrafish model can bridge genetic associations with functional mechanisms in disease processes.

What detection and quantification methods are most reliable for analyzing this protein in experimental settings?

Reliable detection and quantification of the Danio rerio UPF0197 transmembrane protein requires careful selection of analytical methods based on experimental objectives:

For purity assessment during recombinant protein production, SDS-PAGE analysis with a minimum threshold of ≥85% purity is standard, while His-tagged variants can achieve >90% purity through affinity chromatography. When designing detection strategies, researchers should consider the transmembrane nature of this protein, which may require specialized solubilization approaches for optimal extraction and analysis.

What are the most effective approaches for conducting cross-species comparative studies with this protein?

Cross-species comparative studies with this protein can provide valuable evolutionary and functional insights. Effective approaches include:

• Sequence alignment and phylogenetic analysis: Compare the zebrafish UPF0197 transmembrane protein sequence with orthologs from other species, including human TMEM258 and Xenopus tmem258 homologs. This identifies conserved domains that likely have crucial functional roles.

• Heterologous expression studies: Express the zebrafish protein in mammalian cell lines alongside its human counterpart to assess functional conservation and species-specific differences in localization, interaction partners, and contribution to glycosylation processes.

• Complementation assays: Test whether the zebrafish protein can rescue phenotypes in systems where the endogenous ortholog has been knocked out or down. This approach directly assesses functional conservation across species.

• Domain swapping experiments: Create chimeric proteins containing domains from zebrafish and human orthologs to identify which regions confer species-specific functions versus conserved activities.

• Comparative glycoproteomics: Analyze glycosylation profiles in zebrafish vs. mammalian systems to identify conserved and divergent N-glycosylation targets affected by manipulation of this protein.

When conducting these cross-species studies, it's essential to consider that while the core function in N-glycosylation is likely conserved, species-specific differences may exist in regulatory mechanisms, interaction partners, and downstream effects that could influence experimental interpretation .

How should researchers design functional studies to investigate the protein's role in ER stress and the unfolded protein response?

Designing functional studies to investigate this protein's role in ER stress and the unfolded protein response (UPR) requires a multi-faceted approach:

• Gain and loss of function studies: Employ CRISPR-Cas9 or morpholino technologies to create zebrafish with altered expression levels of the UPF0197 transmembrane protein. Monitor subsequent effects on ER morphology and function using fluorescent reporters for the ER network .

• UPR activation monitoring: Measure expression levels of key UPR markers (BiP/GRP78, CHOP, XBP1 splicing, ATF6 cleavage) following manipulation of the protein using qPCR, Western blotting, and reporter constructs. This reveals which arms of the UPR are specifically activated by disruption of this protein's function .

• Glycosylation status assessment: Employ enzymatic deglycosylation (PNGase F, Endo H) combined with Western blotting to evaluate how alterations in this protein affect the glycosylation status of known N-glycoproteins in zebrafish.

• ER stress inducer challenges: Expose zebrafish with modified expression of this protein to established ER stress inducers (tunicamycin, thapsigargin, DTT) and compare their response to wild-type counterparts, assessing survival, tissue-specific effects, and UPR dynamics .

• Chemical modifier screens: Conduct small molecule screens to identify compounds that can modulate the phenotypes associated with dysregulation of this protein, potentially revealing intervention points in ER stress pathways .

• Temporal dynamics analysis: Implement time-course studies to distinguish between primary effects of protein manipulation and secondary consequences of chronic ER stress, which is critical for establishing causal relationships.

The transparency of zebrafish embryos provides a unique advantage for these studies, allowing real-time visualization of ER stress responses in living organisms, particularly when combined with tissue-specific fluorescent reporters .

What strategies can researchers employ to address proteolysis and stability issues with this recombinant protein?

Proteolysis and stability challenges have been reported with in vitro expression of this protein . Researchers can implement several strategies to mitigate these issues:

• Optimized expression systems: Bacterial overexpression, particularly in KRX cells rather than BL21(DE3)pLysS, has demonstrated reduced leaky expression and improved stability for similar zebrafish proteins . This approach minimizes toxicity while maximizing yield.

• Protease inhibitor cocktails: Incorporate comprehensive protease inhibitor cocktails during all purification steps, including both serine and cysteine protease inhibitors, to prevent degradation during extraction and processing.

• Buffer optimization: The recommended Tris-based buffer with 50% glycerol at pH 8.0 provides optimal stability . Further stability can be achieved by testing additives such as reducing agents (DTT, β-mercaptoethanol), salt concentrations, and stabilizing agents like glycerol or sucrose.

• Temperature management: Maintain strict temperature control during purification, keeping samples consistently cold (4°C) to minimize protease activity. For long-term storage, -80°C is preferable to -20°C when possible .

• Strategic aliquoting: Prepare single-use aliquots immediately after purification to eliminate freeze-thaw cycles, which significantly contribute to protein degradation .

• Fusion tags selection: Consider testing different fusion tags (His, GST, MBP) which may enhance solubility and stability. The MBP tag in particular can improve the solubility of transmembrane proteins.

• Co-expression with chaperones: For bacterial expression systems, co-express with molecular chaperones (GroEL/ES, DnaK/J) to facilitate proper folding and reduce aggregation of the recombinant protein.

By systematically implementing these strategies, researchers can significantly improve the stability and functional integrity of the recombinant Danio rerio UPF0197 transmembrane protein for downstream applications.

How can researchers validate experimental findings when working with this protein in zebrafish models?

Rigorous validation of experimental findings is essential when working with this protein in zebrafish models. Researchers should implement a multi-layered validation approach:

• Genetic validation: Employ multiple independent methods for gene manipulation, including CRISPR-Cas9 gene editing, morpholino knockdown, and overexpression approaches. Consistent phenotypes across these different methodologies strengthen confidence in findings .

• Rescue experiments: Perform rescue experiments by reintroducing the wild-type protein into knockdown or knockout models. Successful rescue confirms that observed phenotypes are specifically due to the protein's absence rather than off-target effects .

• Dose-response relationships: Establish dose-response relationships between the level of protein manipulation and observed phenotypes. This quantitative approach helps distinguish specific effects from general toxicity or developmental delays.

• Cross-platform validation: Verify key findings in complementary model systems (cell culture, other organisms) to ensure the observed phenomena are not zebrafish-specific artifacts .

• Positive and negative controls: Include appropriate controls in all experiments, including unrelated proteins with similar characteristics (negative controls) and known glycosylation pathway components (positive controls).

• Functional readouts: Employ multiple independent assays to measure glycosylation function, such as fluorescent glycoprotein reporters, glycan-specific lectins, and mass spectrometry-based glycoprotein profiling .

• Temporal analysis: Conduct detailed temporal analysis to distinguish between primary defects and secondary consequences, establishing clear cause-effect relationships in complex developmental contexts.

By implementing this comprehensive validation strategy, researchers can generate robust, reproducible findings that advance our understanding of this protein's function in zebrafish and its relevance to human biology and disease.

What common technical challenges arise in experimental design when studying transmembrane proteins like this one?

Studying transmembrane proteins like the Danio rerio UPF0197 protein presents several technical challenges that require specialized approaches:

• Protein solubilization and extraction: The hydrophobic nature of transmembrane domains makes extraction from membranes challenging. Researchers should optimize detergent selection (e.g., CHAPS, DDM, or Triton X-100) for efficient solubilization while maintaining protein structure and function.

• Expression toxicity: Expression of transmembrane proteins often causes toxicity in host cells, as observed with similar zebrafish proteins in BL21(DE3)pLysS cells . Alternative expression hosts like KRX cells may mitigate this issue by reducing leaky expression .

• Proper folding and membrane insertion: Ensuring correct folding and membrane insertion is crucial for functional studies. Specialized expression systems that mimic the native membrane environment may be required for optimal results.

• Antibody generation and validation: The limited extracellular/cytoplasmic domains of transmembrane proteins can make generating specific antibodies challenging. Careful epitope selection and rigorous validation are essential for reliable detection.

• Functional assays: Designing functional assays for transmembrane proteins requires consideration of their membrane context. Cell-based assays that preserve the native membrane environment are often more informative than solution-based biochemical assays.

• Structural analysis: Traditional structural biology techniques may be challenging to apply to transmembrane proteins. Newer approaches like cryo-electron microscopy or advanced NMR techniques may be more suitable for structural characterization.

• Live imaging challenges: When performing live imaging of this protein in zebrafish, the membrane localization may require specialized probes or tags that don't disrupt protein function or localization.