Recombinant Rat ORM1-like protein 3 (Ormdl3)

What is Ormdl3 and what is its cellular localization?

Ormdl3 (ORM1-like protein 3) is an endoplasmic reticulum (ER) trans-membrane protein that belongs to the ORM family. It has been identified as a candidate gene for susceptibility to asthma through genome-wide association studies. In mammalian cells, Ormdl3 is primarily localized in the endoplasmic reticulum, where it functions in regulating ER-mediated Ca²⁺ signaling and cellular stress responses by facilitating the unfolded-protein response .

When visualized through immunofluorescence techniques, Ormdl3 shows a characteristic reticular pattern consistent with ER localization. The protein is expressed in multiple tissues, including lungs, where it is found not only in airway epithelial cells and endothelial cells but also in inflammatory cells recruited to allergic airways .

How conserved is Ormdl3 across species?

Ormdl3 demonstrates remarkable evolutionary conservation across species. Human ORMDL3 shows approximately 96% homology with mouse Ormdl3, indicating strong evolutionary pressure to maintain its structure and function . This high degree of conservation suggests fundamental biological importance and allows for translational research between rodent models and human studies.

The human ORMDL family includes three members: ORMDL1 on chromosome 2, ORMDL2 on chromosome 12, and ORMDL3 on chromosome 17, all of which are expressed ubiquitously in adult and fetal tissues .

What are the known physiological functions of Ormdl3?

Ormdl3 serves multiple physiological functions:

• Regulation of sphingolipid homeostasis: Ormdl3 negatively regulates serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid biosynthesis .

• Calcium homeostasis: It modulates ER-mediated Ca²⁺ signaling, with dysregulation potentially contributing to cellular stress responses .

• Inflammatory cell regulation: In eosinophils, Ormdl3 plays a critical role in trafficking, adhesion, and activation. It regulates expression of adhesion molecules (CD49d and CD18) that are essential for eosinophil recruitment to inflammatory sites .

• Immune cell activation: Ormdl3 regulates cytoskeletal rearrangement and cellular polarization in response to inflammatory stimuli, which are essential for directed migration and function of immune cells .

• Cell signaling: Ormdl3 influences the ERK (extracellular signal-regulated kinase) pathway and nuclear translocation of NF-κB, affecting downstream gene expression in inflammatory cells .

What are the optimal methods for expressing recombinant rat Ormdl3?

Based on established protocols for human and mouse Ormdl3, the following approach can be adapted for rat Ormdl3:

• Vector selection: Clone full-length cDNA encoding rat Ormdl3 into an appropriate expression vector (e.g., pET-28a for His-tagged protein or pGEX-6p-2 for GST-tagged protein).

• Bacterial expression system: Transform the expression plasmid into E. coli Rosetta (DE3) pLysS cells, which are optimized for expression of eukaryotic proteins that contain rare codons.

• Induction conditions: Induce protein expression with 1 μM IPTG. Optimization of temperature (typically 16-30°C) and duration (4-16 hours) may be necessary for optimal expression.

• Protein extraction: Lyse the bacteria in RIPA buffer containing protease inhibitors and 1 mM DTT to maintain protein stability.

• Verification: Confirm expression using western blot analysis with antibodies specific to Ormdl3 or to the fusion tag .

For mammalian expression, transfection of Ormdl3 cDNA into an appropriate expression vector (such as pEGFP-N1 for GFP-tagged protein) can be performed using lipid-based transfection reagents like Trans IT-2020 .

What approaches are effective for Ormdl3 knockdown studies?

RNA interference techniques have been successfully employed for Ormdl3 knockdown:

• siRNA transfection: Use Ormdl3-specific siRNA with appropriate transfection reagents (e.g., INTERFERin). The efficiency of knockdown should be assessed at both mRNA level by RT-PCR and protein level by western blot analysis .

• Validation protocol:

  • Transfect cells with Ormdl3-specific siRNA or scrambled control siRNA

  • Incubate for 24-48 hours to allow for protein turnover

  • Confirm knockdown efficiency by RT-PCR (mRNA) and western blot or immunofluorescence (protein)

  • Assess cell viability using Trypan blue exclusion to ensure knockdown does not significantly affect cell survival

• Considerations: Optimize siRNA concentration to achieve maximum knockdown with minimal off-target effects. For studying long-term effects, stable knockdown using shRNA may be preferable.

How can Ormdl3 expression be induced in experimental systems?

Ormdl3 expression can be induced by specific cytokines and chemokines in a cell-type specific manner:

• In eosinophils:

  • IL-3 (100 ng/ml) and eotaxin-1 (100 nM) significantly induce Ormdl3 expression

  • IL-5 and RANTES do not induce Ormdl3 expression

• Protocol for induction:

  • Suspend cells (1 × 10⁶ per well) in culture medium

  • Add appropriate cytokine/chemokine (IL-3, eotaxin-1)

  • Incubate for 2 hours at 37°C and 5% CO₂ for mRNA expression

  • For protein expression analysis, extend incubation to 12 hours

  • Analyze Ormdl3 expression by RT-PCR, qPCR, or western blot

• Verification of induction: Quantify expression changes using qPCR for mRNA and western blot with densitometry (e.g., using ImageJ) for protein levels.

How does Ormdl3 regulate immune cell trafficking and recruitment?

Ormdl3 plays a complex role in immune cell trafficking through multiple mechanisms:

• Adhesion molecule regulation: Ormdl3 regulates the expression of critical adhesion molecules, including α4 (CD49d) and β2 (CD18) integrins. Knockdown of Ormdl3 decreases mRNA levels of these integrins, reducing cell adhesion to VCAM-1 and ICAM-1 .

• Cytoskeletal rearrangement: Ormdl3 influences cell polarization and cytoskeletal changes necessary for directed migration. Cells with Ormdl3 knockdown show:

  • Reduced cell spreading and polarization

  • Limited development of leading edges, uropodia, and filopodia

  • Diminished adhesion to substrate proteins

• Signaling pathway activation: Ormdl3 overexpression increases phosphorylated ERK (1/2) levels and promotes nuclear translocation of NF-κB, activating signaling cascades essential for cell migration and activation .

• In vivo trafficking: In mouse models, Ormdl3 knockdown significantly reduces inflammatory cell recruitment to sites of allergic inflammation, demonstrating its physiological relevance to inflammatory diseases .

The table below summarizes the effects of Ormdl3 manipulation on immune cell functions:

What is the relationship between Ormdl3 and inflammatory diseases, particularly asthma?

Ormdl3 has been strongly linked to asthma pathogenesis through several mechanisms:

• Genetic association: Genome-wide association studies have identified Ormdl3 as a candidate gene for susceptibility to both childhood and adult-onset asthma across ethnically diverse populations .

• Allergic inflammation: In allergen-challenged mice, Ormdl3 expression is increased in the airways, particularly in inflammatory cells recruited to allergic airways .

• Eosinophil function: Ormdl3 promotes eosinophil trafficking and activation, key processes in allergic asthma. It regulates:

  • Eosinophil rolling and adhesion

  • Expression of integrins (CD49d and CD18)

  • CD48-mediated eosinophil degranulation

• Neutrophilic inflammation: Recent research indicates that neutrophilia in severe asthma is reduced in Ormdl3 overexpressing mice, suggesting a complex role in different asthma phenotypes .

• Sphingolipid metabolism: As a negative regulator of serine palmitoyltransferase, Ormdl3 influences sphingolipid synthesis, which may contribute to asthma pathophysiology through effects on inflammatory cell function and airway hyperresponsiveness .

How does Ormdl3 influence cell signaling pathways?

Ormdl3 interacts with multiple signaling pathways that regulate immune cell function:

• ERK signaling: Overexpression of Ormdl3 in eosinophils results in increased levels of phosphorylated ERK (1/2), but not p38 MAPK, indicating selective pathway activation .

• NF-κB pathway: Ormdl3 overexpression leads to nuclear translocation of NF-κB, which can induce expression of multiple proteins including cytokines and adhesion molecules .

• Cytokine-specific signaling: IL-3, but not IL-5, induces Ormdl3 expression despite shared receptor components (common β-chain), suggesting Ormdl3 expression is regulated through the IL-3R α-chain-specific mechanism .

• Chemokine signaling: Eotaxin-1, but not RANTES, induces Ormdl3 expression despite both binding to CCR3, indicating selective signaling mechanisms .

• Calcium signaling: Ormdl3 binds to sarco-ER Ca²⁺ pump, regulating ER-mediated Ca²⁺ signaling and cellular stress responses .

What assays can be used to assess Ormdl3-mediated effects on cell function?

Several established assays can measure Ormdl3's impact on cellular functions:

• Cell adhesion assay:

  • Coat plates with recombinant adhesion proteins (VCAM-1, ICAM-1)

  • Add cells with manipulated Ormdl3 expression

  • Quantify adherent cells to assess adhesion capacity

• Cytoskeletal assessment:

  • Permeabilize cells with 4% paraformaldehyde containing 0.5% saponin

  • Stain with rhodamine-conjugated phalloidin (2.5 U/ml)

  • Evaluate by confocal microscopy to assess cytoskeletal rearrangement

• In vitro flow chamber assay:

  • Coat coverslips with recombinant adhesion molecules

  • Flow cells (1-2 × 10⁵/ml) over the substrate under controlled shear stress (1.0-2.0 dynes/cm²)

  • Record and analyze cell rolling behavior

• Degranulation assay:

  • Stimulate cells with appropriate activators (e.g., anti-CD48 antibodies)

  • Measure release of granule proteins (e.g., eosinophil peroxidase)

  • Compare degranulation in cells with normal vs. altered Ormdl3 expression

• Signaling pathway activation:

  • Assess ERK phosphorylation by western blot

  • Examine NF-κB nuclear translocation by immunofluorescence

  • Quantify changes in downstream target gene expression

How can researchers analyze the impact of Ormdl3 on sphingolipid metabolism?

Given Ormdl3's role in regulating sphingolipid synthesis, the following approaches can be used:

• Sphingolipid profiling:

  • Extract cellular lipids using appropriate solvent systems

  • Perform liquid chromatography-mass spectrometry (LC-MS) to identify and quantify sphingolipid species

  • Compare profiles between cells with normal, overexpressed, or knocked-down Ormdl3

• SPT activity assay:

  • Measure the activity of serine palmitoyltransferase (the enzyme negatively regulated by Ormdl3)

  • Use radiolabeled serine incorporation into sphinganine as a readout

  • Compare activity levels in cells with manipulated Ormdl3 expression

• Gene expression analysis:

  • Assess expression of key sphingolipid metabolism enzymes using qPCR or RNA-seq

  • Focus on genes encoding SPT subunits and downstream enzymes in the sphingolipid pathway

  • Correlate changes with Ormdl3 expression levels

How do findings from rodent Ormdl3 models translate to human disease?

When interpreting results from rat Ormdl3 studies in the context of human disease:

• Sequence homology: The high conservation (96% homology) between human and mouse Ormdl3 suggests functional conservation across mammalian species, including rats .

• Expression patterns: Both human and rodent Ormdl3 are expressed in similar tissues, including the lung, and show increased expression in inflammatory cells during allergic responses .

• Genetic associations: Variations in the ORMDL3 locus contribute to asthma susceptibility in both humans and mouse models, suggesting conserved pathophysiological mechanisms .

• Cellular functions: Ormdl3 regulates similar cellular processes (adhesion, migration, degranulation) in both human and rodent immune cells .

• Limitations to consider:

  • Species differences in immune system development and composition

  • Variations in asthma phenotypes between humans and rodent models

  • Potential differences in regulatory mechanisms controlling Ormdl3 expression

How can Ormdl3 research contribute to understanding different asthma phenotypes?

Ormdl3 research provides insights into asthma heterogeneity:

• Eosinophilic vs. neutrophilic asthma: Ormdl3 has been linked to both eosinophil function in allergic asthma and neutrophilia in severe asthma , suggesting it may play different roles in distinct asthma phenotypes.

• T helper cell responses: The relationship between Ormdl3 and both Th2-driven eosinophilic inflammation and non-Th2 severe asthma with high IL-17 levels points to its involvement in multiple inflammatory pathways .

• Sphingolipid metabolism: As a regulator of sphingolipid synthesis, Ormdl3 may contribute to asthma heterogeneity through differential effects on sphingolipid profiles in different patient populations.

• Genetic associations: The strength of association between ORMDL3 gene variants and asthma varies across populations, potentially explaining some of the heterogeneity in disease presentation.

What are common challenges in working with recombinant Ormdl3 and how can they be addressed?

Researchers may encounter several challenges when working with recombinant Ormdl3:

• Protein solubility issues:

  • Challenge: As a transmembrane protein, Ormdl3 can be difficult to solubilize.

  • Solution: Use mild detergents (0.1% Triton X-100, 0.1% NP-40, or 0.5% CHAPS) during extraction, and consider adding 5-10% glycerol to stabilize the protein.

• Low expression levels:

  • Challenge: Membrane proteins often express poorly in bacterial systems.

  • Solution: Optimize codon usage for the expression system, reduce induction temperature to 16-20°C, and consider fusion tags that enhance solubility (MBP, SUMO).

• Antibody specificity:

  • Challenge: Cross-reactivity with other ORMDL family members.

  • Solution: Validate antibodies using positive controls (recombinant Ormdl3) and negative controls (tissues or cells with Ormdl3 knockdown).

• Functional assays:

  • Challenge: Maintaining physiologically relevant conditions.

  • Solution: Use primary cells or cell lines that naturally express Ormdl3, and validate findings across multiple assay systems.

What controls are essential for Ormdl3 knockdown and overexpression experiments?

Proper controls are critical for interpreting Ormdl3 manipulation experiments:

• For knockdown studies:

  • Negative control: Scrambled siRNA with the same nucleotide composition but randomized sequence

  • Transfection control: Cells treated with transfection reagent only

  • Positive control: siRNA targeting a housekeeping gene with known knockdown phenotype

  • Validation controls: RT-PCR and western blot to confirm knockdown efficiency

• For overexpression studies:

  • Empty vector control: Cells transfected with the same vector lacking the Ormdl3 insert

  • Tag-only control: If using a tagged construct, include a control expressing only the tag

  • Positive control: Known gene with established overexpression phenotype

  • Expression verification: Confirm overexpression by RT-PCR, western blot, or fluorescence (for GFP-tagged constructs)

• Cell viability assessment: For both approaches, monitor cell viability to ensure observed effects are not due to cytotoxicity .