Recombinant Danio rerio ORM1-like protein 1 (ormdl1)

What is Recombinant Danio rerio ORM1-like protein 1 and how is it structurally characterized?

Recombinant Danio rerio ORM1-like protein 1 (ormdl1) is a member of the evolutionarily conserved ORMDL gene family, which encodes transmembrane proteins anchored in the endoplasmic reticulum (ER). The full-length protein consists of 153 amino acids and is typically expressed with an N-terminal His tag when produced recombinantly. The amino acid sequence of the protein is: MNVGVAHSEVNPNTRVMNSRGIWLTYALGVGMLHIVLLSIPFFSVPVVWTLTNVIHNFGMYVFMHAVKGTPFETPDQGKARLLTHWEQLDYGVQFTSSRKFFTISPIILYFLASFYTKYDTAHFVINTASLLSVLIPKLPQLHGVRIFGINKY . The protein belongs to a family that includes ORMDL1, ORMDL2, and ORMDL3 in humans, with homologs found across various organisms including yeast, plants, Drosophila, and vertebrates .

What expression systems are most effective for producing recombinant Danio rerio ormdl1 protein?

E. coli expression systems are effectively used for recombinant production of Danio rerio ormdl1 protein. The protein is commonly expressed as a full-length construct (amino acids 1-153) fused to an N-terminal His tag to facilitate purification . When establishing an expression protocol, researchers should consider that as a transmembrane protein, ormdl1 may require optimization of expression conditions to maintain proper folding and avoid aggregation. Protein yield and purity greater than 90% can be achieved as determined by SDS-PAGE analysis . Alternative expression systems like yeast could potentially be employed, especially since functional studies have demonstrated that human ORMDL homologs can rescue yeast mutants lacking their endogenous ORMDL genes, suggesting proper folding and functionality can be maintained across expression systems .

How should researchers properly store and reconstitute recombinant Danio rerio ormdl1 protein?

For optimal stability and activity preservation, recombinant Danio rerio ormdl1 protein should be stored at -20°C or -80°C upon receipt. The lyophilized powder should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To prevent protein degradation during storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being optimal for long-term storage) before aliquoting for storage at -20°C/-80°C . These aliquots help avoid repeated freeze-thaw cycles, which can compromise protein integrity. For short-term usage, working aliquots can be stored at 4°C for up to one week . Prior to opening, vials should be briefly centrifuged to ensure all content is at the bottom. The reconstituted protein is typically stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 .

What functional assays are appropriate for studying Danio rerio ormdl1 protein activity?

Based on ORMDL family functional studies, several assays can be implemented to investigate Danio rerio ormdl1 activity:

• Sphingolipid Regulation Assays: Since ORMDLs regulate sphingolipid synthesis, measuring sphingolipid levels (particularly ceramides and sphingosine-1-phosphate) via liquid chromatography-mass spectrometry (LC-MS) after ormdl1 manipulation can reveal functional activity .

• ER Stress Response Assays: As ER-resident proteins, ormdl1 function can be assessed through measurement of ER stress markers (such as XBP1 splicing, BiP/GRP78 upregulation) using RT-qPCR or Western blotting before and after ormdl1 overexpression or knockdown .

• Complementation Studies: Functional conservation can be tested through rescue experiments in yeast models lacking endogenous ORMDL homologs. Growth rate measurements and sensitivity to ER stressors like tunicamycin and dithiothreitol can determine if Danio rerio ormdl1 can functionally complement the yeast orthologs .

• Protein Interaction Assays: Co-immunoprecipitation or proximity ligation assays can identify protein-protein interactions, helping elucidate the molecular partners of ormdl1 in the ER membrane.

How can researchers effectively design knockdown or knockout experiments for Danio rerio ormdl1?

Designing effective knockdown or knockout experiments for Danio rerio ormdl1 requires careful consideration of functional redundancy within the ORMDL family:

• CRISPR/Cas9 Genome Editing: This approach has been successfully used to create ormdl1 knockouts in model organisms. Target guide RNAs should be designed to introduce frameshifts in early exons (e.g., exon 2, the first protein-coding exon) to ensure complete protein loss. Verification of knockout should include RT-qPCR with probes corresponding to the deleted coding sequences .

• Morpholino Oligonucleotides: For transient knockdown in zebrafish embryos, splice-blocking or translation-blocking morpholinos can be designed. Due to potential off-target effects, results should be validated with rescue experiments using morpholino-resistant mRNA.

• Redundancy Considerations: Research shows that Ormdl1 often functions redundantly with other family members, particularly Ormdl3. Single gene knockouts may not display overt phenotypes, while double knockouts (e.g., Ormdl1/3) have demonstrated significant phenotypes including elevated sphingolipid metabolites and myelination defects . Therefore, consider simultaneous targeting of multiple ORMDL family members.

• Validation Strategy: Knockout validation should include:

  • Genomic DNA sequencing to confirm mutations

  • RT-qPCR to verify absence of wild-type transcript

  • Western blotting to confirm protein loss

  • Functional assays to demonstrate altered sphingolipid metabolism

What are the optimal conditions for analyzing ormdl1 protein-protein interactions?

When investigating ormdl1 protein-protein interactions, researchers should consider the following methodological approaches:

• Co-immunoprecipitation (Co-IP): Use mild detergents (e.g., 1% Digitonin, 0.5% CHAPS, or 1% NP-40) that preserve membrane protein interactions while effectively solubilizing the ER membrane. Include protease inhibitors and perform at 4°C to prevent degradation. The N-terminal His tag on recombinant ormdl1 can be exploited for pull-down using anti-His antibodies or Ni-NTA resin .

• Proximity-based Labeling: Techniques like BioID or APEX2 proximity labeling, where ormdl1 is fused to a biotin ligase or peroxidase, can capture transient or weak interactions within the native ER environment.

• Membrane Yeast Two-Hybrid: Modified yeast two-hybrid systems designed for membrane proteins can identify interacting partners while maintaining the proper membrane context.

• Crosslinking Mass Spectrometry: Chemical crosslinking combined with mass spectrometry can capture native interactions before membrane disruption.

• Sample Preparation Considerations: Since ormdl1 is an ER membrane protein, standard buffers for cytosolic proteins are inadequate. Use buffers containing glycerol (10-15%) to stabilize the protein structure during isolation procedures. Consider using nitrogen cavitation or sonication for efficient membrane disruption while preserving protein-protein interactions.

How does Danio rerio ormdl1 function compare to its mammalian homologs?

Danio rerio ormdl1 shares significant functional conservation with its mammalian homologs despite evolutionary distance. Both zebrafish and mammalian ORMDL proteins function as regulators of sphingolipid biosynthesis and are localized to the endoplasmic reticulum as transmembrane proteins . Cross-species functional studies have demonstrated that human ORMDL homologs can rescue phenotypes in yeast models lacking endogenous ORMDL genes, suggesting fundamental mechanistic conservation .

Key similarities include:

• ER membrane localization

• Role in sphingolipid metabolism regulation

• Functional redundancy among family members

• Ubiquitous expression across tissues

• Tissue expression patterns during development

• Regulatory mechanisms controlling expression

• Specific binding partners and downstream effectors

• Relative importance of different ORMDL family members

Research indicates that functional redundancy exists between Ormdl1 and Ormdl3, as single knockout models often lack overt phenotypes, while double knockouts exhibit significant defects, particularly in myelination processes . This suggests that experimental designs should consider targeting multiple family members simultaneously when studying function.

What is the role of ormdl1 in sphingolipid metabolism and how can researchers measure its impact?

Ormdl1 plays a crucial regulatory role in sphingolipid metabolism by functioning as a negative regulator of sphingolipid synthesis. Research indicates that Ormdl1, along with Ormdl3, functions redundantly to suppress the levels of bioactive sphingolipid metabolites, particularly during high-demand processes like myelination of the sciatic nerve .

To measure ormdl1's impact on sphingolipid metabolism, researchers can employ the following methodological approaches:

• Lipidomic Analysis: Liquid chromatography-mass spectrometry (LC-MS) to quantify changes in sphingolipid metabolites (ceramides, sphingosine, sphingosine-1-phosphate) in ormdl1 knockout or overexpression models compared to controls.

• Enzyme Activity Assays: Measuring the activity of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid biosynthesis that is regulated by ORMDL proteins. This can be done using isotope-labeled substrates and tracking their incorporation into sphingolipid products.

• Gene Expression Analysis: RT-qPCR or RNA-seq to analyze changes in expression of other sphingolipid metabolism genes following ormdl1 manipulation, revealing potential compensatory mechanisms.

• Functional Phenotyping: In zebrafish models, assessing myelination (through electron microscopy or myelin-specific staining) can provide functional readouts of disturbed sphingolipid homeostasis resulting from ormdl1 alteration .

• Stress Response Testing: Evaluating cellular responses to sphingolipid metabolism disruptors (e.g., myriocin, fumonisin B1) in the presence or absence of normal ormdl1 function.

What are the implications of ormdl1 in disease models and potential therapeutic applications?

Research on the ORMDL family has revealed significant implications for disease mechanisms and potential therapeutic targets, with insights that may extend to zebrafish ormdl1:

What experimental controls are essential when studying recombinant Danio rerio ormdl1 function?

When designing rigorous experiments to study recombinant Danio rerio ormdl1 function, researchers should implement the following essential controls:

• Expression System Controls:

  • Empty vector control: Cells expressing only the vector backbone without ormdl1 insert

  • Unrelated protein control: Expression of an unrelated protein with the same tag to control for tag-specific effects

  • Wild-type vs. mutant ormdl1: Include both functional and non-functional (mutated) versions of ormdl1 to confirm specificity of observed effects

• Functional Redundancy Controls:

  • Single vs. combined knockdown/overexpression: Since Ormdl1 often functions redundantly with Ormdl3, single manipulations should be compared with simultaneous manipulation of multiple family members

  • Rescue experiments: Reintroduction of wild-type ormdl1 should reverse phenotypes observed in knockout/knockdown models

• Biochemical Assay Controls:

  • Positive control for sphingolipid metabolism: Include samples treated with known sphingolipid synthesis inhibitors (e.g., myriocin)

  • Negative control for protein-protein interaction studies: Use a known non-interacting protein pair

  • Antibody specificity control: Validate antibody specificity using knockout/knockdown samples

• Cellular Localization Controls:

  • ER marker co-localization: Confirm proper localization using established ER markers

  • Mislocalization construct: Create a mutant lacking ER retention signals as a negative control

• Physiological Relevance Controls:

  • Dose-dependency: Demonstrate that effects scale with ormdl1 expression levels

  • Temporal controls: Analyze effects at multiple timepoints to distinguish primary from secondary effects

How can researchers address challenges in detecting and quantifying membrane-bound ormdl1 protein?

Detection and quantification of membrane-bound proteins like ormdl1 present several technical challenges. Researchers can implement these methodological solutions:

• Sample Preparation Optimization:

  • Use specialized membrane protein extraction buffers containing mild detergents (CHAPS, DDM, or digitonin) that maintain protein structure while effectively solubilizing membranes

  • Employ sequential extraction protocols to enrich ER membrane fractions before analysis

  • Consider on-membrane digestion protocols for mass spectrometry analysis to improve coverage of hydrophobic regions

• Western Blotting Enhancements:

  • Transfer optimization: Use specialized transfer conditions for hydrophobic proteins (lower methanol concentration, addition of SDS in transfer buffer)

  • Blocking optimization: Test milk vs. BSA blocking solutions as milk proteins can interfere with detection of some membrane proteins

  • Loading control selection: Use ER-specific proteins (e.g., calnexin) rather than cytosolic proteins as loading controls

• Alternative Detection Methods:

  • Flow cytometry: For cell surface proteins or permeabilized cells to detect internal membrane proteins

  • ELISA: Development of sandwich ELISA using antibodies against different epitopes

  • Mass spectrometry: Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) targeting specific ormdl1 peptides

• Quantification Strategies:

  • Absolute quantification: Use isotope-labeled peptide standards matching ormdl1 tryptic peptides

  • Relative quantification: Compare to a standard curve of purified recombinant ormdl1 protein

  • Image analysis: Employ automated quantification software with appropriate background correction for immunofluorescence

• Expression Tag Utilization:

  • Leverage the N-terminal His tag commonly used in recombinant Danio rerio ormdl1 for detection and quantification when antibodies against the native protein are unavailable or lack specificity

What are the critical parameters to consider when investigating ormdl1 involvement in cellular stress responses?

When investigating ormdl1's role in cellular stress responses, researchers should consider these critical experimental parameters:

• Stress Induction Protocols:

  • ER stress: Titrate tunicamycin (0.1-10 μg/ml) or dithiothreitol (0.1-2 mM) concentrations and exposure times to induce different levels of ER stress

  • Sphingolipid stress: Use myriocin (0.5-5 μM) to inhibit sphingolipid synthesis or exogenous ceramides (5-50 μM) to increase sphingolipid levels

  • Oxidative stress: Hydrogen peroxide (50-500 μM) or paraquat treatments with appropriate dose-response curves

• Temporal Analysis:

  • Immediate early response: Examine changes 0.5-4 hours post-stress induction

  • Intermediate response: 4-24 hours post-stress

  • Adaptive response: 24-72 hours post-stress

  • Include multiple timepoints to distinguish primary effects from secondary adaptations

• Cell Type Considerations:

  • Cell types with high sphingolipid demands (e.g., oligodendrocytes, Schwann cells) versus those with lower requirements

  • Primary cells versus cell lines (which may have altered stress response pathways)

  • Developmental stage of cells, as ormdl1 significance may vary during development

• Readout Selection:

  • Transcriptional changes: RT-qPCR panel of ER stress genes (BiP, CHOP, XBP1s)

  • Protein-level changes: Western blot for stress markers (phospho-eIF2α, BiP, CHOP)

  • Morphological changes: ER structure (electron microscopy or fluorescent ER trackers)

  • Functional outcomes: Cell viability, proliferation, and differentiation assays

• Pathway Integration Analysis:

  • Pharmacological inhibitors of specific stress response pathways to delineate which arm of the stress response involves ormdl1

  • Combined genetic approaches (e.g., ormdl1 knockout in the context of XBP1 or PERK pathway manipulation)

  • Systems biology approaches to map stress response networks

How does ormdl1 expression vary across different tissues in Danio rerio?

While specific data on zebrafish ormdl1 tissue distribution is limited in the provided search results, information about the ORMDL family suggests patterns that likely apply to Danio rerio. ORMDL genes, including ormdl1, are generally expressed ubiquitously across adult and fetal tissues . In Drosophila, the ORMDL homolog is expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues , suggesting potential developmental regulation that may also occur in zebrafish.

For comprehensive analysis of ormdl1 expression across zebrafish tissues, researchers should employ:

• RT-qPCR Tissue Panel: Quantitative analysis of ormdl1 mRNA expression across multiple tissue types (brain, heart, liver, kidney, muscle, gill, gut, etc.) at different developmental stages from embryo to adult.

• In Situ Hybridization: Spatial localization of ormdl1 mRNA expression in whole embryos and tissue sections to reveal cell type-specific expression patterns during development.

• Immunohistochemistry: Using antibodies against ormdl1 to visualize protein localization in tissue sections, with special attention to ER distribution within different cell types.

• Transgenic Reporter Lines: Generation of transgenic zebrafish expressing fluorescent reporters under the control of the ormdl1 promoter to visualize dynamic expression patterns in live animals.

• Single-cell RNA Sequencing: To identify cell populations with enriched ormdl1 expression and correlate with cell type-specific functions.

What transcriptional and post-translational mechanisms regulate ormdl1 function?

Regulation of ormdl1 likely occurs at multiple levels, though specific mechanisms for Danio rerio ormdl1 are not fully detailed in the provided search results. Based on ORMDL family research, the following regulatory mechanisms may apply:

• Transcriptional Regulation:

  • Promoter analysis to identify transcription factor binding sites that regulate ormdl1 expression

  • Epigenetic regulation through DNA methylation and histone modifications at the ormdl1 locus

  • Alternative splicing events that may generate different ormdl1 isoforms with altered functions

  • miRNA-mediated regulation of ormdl1 mRNA stability and translation

• Post-translational Modifications:

  • Phosphorylation sites that may alter protein activity or interactions

  • Glycosylation patterns that could affect protein stability or localization

  • Ubiquitination pathways controlling protein turnover and degradation

  • Conformational changes induced by sphingolipid binding or protein-protein interactions

• Protein-Protein Interactions:

  • Formation of homo- or hetero-oligomeric complexes with other ORMDL family members

  • Interactions with sphingolipid synthesis enzymes, particularly serine palmitoyltransferase

  • Association with ER stress response proteins or calcium handling machinery

• Feedback Mechanisms:

  • Sphingolipid-dependent feedback loops that adjust ormdl1 expression or activity based on cellular sphingolipid levels

  • ER stress-induced changes in ormdl1 expression as part of adaptive responses

Experimental approaches to investigate these regulatory mechanisms include site-directed mutagenesis of potential modification sites, co-immunoprecipitation studies to identify interacting partners, and reporter assays to analyze promoter activity under various conditions.

How can researchers integrate ormdl1 studies with broader sphingolipid metabolism networks?

To effectively integrate ormdl1 research within the broader context of sphingolipid metabolism networks, researchers should employ these methodological approaches:

• Systems Biology Frameworks:

  • Develop computational models of sphingolipid metabolism incorporating ormdl1 regulatory nodes

  • Use flux analysis to quantify changes in sphingolipid pathway activity upon ormdl1 manipulation

  • Perform network analysis to identify key nodes where ormdl1 exerts the strongest influence

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and lipidomics data to create comprehensive maps of cellular responses to ormdl1 perturbation

  • Temporal profiling to distinguish primary effects from secondary adaptations

  • Correlation analysis between ormdl1 expression levels and specific sphingolipid species across conditions

• Functional Genomics Approaches:

  • Genetic interaction screens to identify synthetic lethal or suppressor relationships between ormdl1 and other sphingolipid metabolism genes

  • CRISPR screens targeting multiple pathway components simultaneously

  • Epistasis analysis to place ormdl1 within pathway hierarchies

• Physiological Context Implementation:

  • Study ormdl1 function during processes with high sphingolipid demand (e.g., myelination, membrane remodeling)

  • Analyze ormdl1 contribution to sphingolipid homeostasis under stress conditions

  • Investigate tissue-specific requirements for ormdl1 regulation of sphingolipid metabolism

• Translational Applications:

  • Connect altered ormdl1 function to disease-relevant sphingolipid profiles

  • Identify pharmacological modulators that act on ormdl1 to influence sphingolipid metabolism

  • Develop biomarkers based on ormdl1 activity that predict sphingolipid dysregulation

A comprehensive approach might include creating a conditional knockout zebrafish model of ormdl1, performing lipidomic analysis across multiple tissues, and correlating sphingolipid changes with phenotypic outcomes, particularly focusing on processes known to require precise sphingolipid regulation such as myelination .