Recombinant Pongo abelii ORM1-like protein 3 (ORMDL3)

What is Pongo abelii ORM1-like protein 3 and what is its structure?

ORMDL3 is a protein originally identified as a candidate gene for susceptibility to asthma. The protein consists of 153 amino acids with a molecular sequence that includes multiple transmembrane domains. The recombinant form of Pongo abelii (Sumatran orangutan) ORMDL3 shares significant homology with human ORMDL3, making it valuable for comparative studies. The full amino acid sequence is: MNVGTAHSEVNPNTRVMNSRGIWLSYVLAIGLLHVVLLSIPFVSVPVVWTLTNLIHNMGMYIFLHTVKGTPFETPDQGKARLLTHWEQMDYGVQFTASRKFLTITPIVLYFLTSFYTKYDQIHFVLNTVSLMSVLIPKLPQLHGVRIFGINKY . The protein is primarily localized to the endoplasmic reticulum, where it appears to regulate various cellular functions including calcium homeostasis and protein folding.

How does ORMDL3 differ across species and what are the implications for research models?

While the search results focus primarily on ORMDL3 from Pongo abelii and mouse models, it's important to note that researchers can work with both human and mouse ORMDL3 for comparative studies. Different expression vectors have been used for each type: human ORMDL3 has been cloned into pET-28a vector containing a His tag, while mouse ORMDL3 has been cloned into pGEX-6p-2 vector containing a GST tag . These differences in tagging strategy allow for specific isolation and purification of the respective proteins. When designing experiments, researchers should consider that while there is high conservation of function across mammals, species-specific differences may affect certain pathway interactions or regulatory mechanisms. Cross-species validation is recommended when translating findings from animal models to human applications.

Which cytokines and chemokines regulate ORMDL3 expression in eosinophils?

Experimental data demonstrates that ORMDL3 expression in eosinophils is selectively regulated by specific cytokines and chemokines. RT-PCR and qPCR analyses have shown that IL-3 and eotaxin-1 significantly induce ORMDL3 expression, while IL-5 and RANTES (CCL5) have no effect on ORMDL3 expression levels . Western blot analysis confirms that IL-3 exposure increases ORMDL3 protein expression in eosinophil lysates . This selective regulation is particularly interesting as the receptors for IL-3 and IL-5 share a common β-chain, suggesting that the IL-3-induced upregulation of ORMDL3 likely occurs via an IL-3R α-chain-specific mechanism independent of common β-chain-mediated signaling . Similarly, despite both eotaxin-1 and RANTES binding to CCR3, only eotaxin-1 induces ORMDL3, indicating selective signaling mechanisms in the regulation of this protein in eosinophils.

How can ORMDL3 expression be experimentally induced in laboratory settings?

For researchers looking to induce ORMDL3 expression in eosinophils for experimental purposes, the following protocol has been validated: bone marrow-derived eosinophils (1 × 10^6/well) should be suspended in culture medium containing IL-3 (100 ng/ml) or eotaxin-1 (100 nM) and incubated for 2 hours at 37°C with 5% CO2 . For protein expression analysis, a longer incubation of 12 hours with IL-3 is recommended. Following treatment, cells can be analyzed for ORMDL3 mRNA expression using RT-PCR and qPCR, while protein expression can be evaluated by Western blot analysis with subsequent densitometry quantification using ImageJ software. This methodology provides a reliable approach for studying the regulation and function of ORMDL3 in immune cells under controlled laboratory conditions.

What are the optimal methods for recombinant ORMDL3 production and purification?

The most effective protocol for recombinant ORMDL3 production involves cloning the full-length cDNA encoding either human or mouse ORMDL3 into appropriate expression vectors. For human ORMDL3, the pET-28a vector with a His tag is recommended, while for mouse ORMDL3, the pGEX-6p-2 vector with a GST tag has shown good results . The expression plasmids should be transformed into E. coli Rosetta™ (DE3) pLysS cells for protein production. Protein expression is optimally induced with 1 μM IPTG. For extraction, bacteria should be lysed in RIPA buffer containing protease inhibitors and 1 mM DTT. Expression of tagged ORMDL3 can be confirmed by Western blot analysis using polyclonal antibodies against ORMDL3. This method provides high yields of functional recombinant protein suitable for a range of experimental applications including structural studies, antibody production, and functional assays.

What techniques are available for modulating ORMDL3 expression in eosinophils?

Researchers have two primary approaches for experimental manipulation of ORMDL3 levels in eosinophils:

Overexpression Method:

• Clone full-length ORMDL3 cDNA into pEGFP-N1 vector using standard cloning procedures

• Transfect bone marrow-derived eosinophils with either ORMDL3-GFP or empty vector (Control-GFP) using Trans IT-2020 DNA transfection reagent

• Confirm transfection by confocal microscopy of cytocentrifuged, fixed, DAPI-mounted cells and by RT-PCR

Knockdown Method:

• Transfect bone marrow-derived eosinophils with ORMDL3-specific siRNA or scrambled control siRNA

• Use INTERFERin™ siRNA transfection reagent according to manufacturer's protocol

• Assess knockdown efficiency by RT-PCR and Western blot

• Verify cell viability post-transfection using Trypan blue exclusion

These complementary approaches allow for comprehensive investigation of ORMDL3 function through both gain-of-function and loss-of-function studies.

Which signaling pathways are activated by ORMDL3 in eosinophils?

ORMDL3 activation in eosinophils triggers specific intracellular signaling cascades that contribute to cellular activation and inflammatory responses. Research demonstrates that overexpression of ORMDL3 in eosinophils results in selective activation of the ERK (1/2) pathway but not the p38 MAPK pathway . This is evidenced by markedly increased levels of phosphorylated ERK (1/2) in ORMDL3-GFP-transfected eosinophils compared to control-GFP-transfected or non-transfected cells. Activated ERK subsequently induces nuclear translocation of NF-κB through activation of its cytoplasmic target IKK-Alpha (I-κB Kinase alpha) . This nuclear translocation of NF-κB is significant as it leads to the expression of multiple proteins, including cytokines and adhesion molecules, that further contribute to eosinophil activation and inflammatory responses. The selectivity of ERK activation by ORMDL3 suggests a specific role in the regulation of eosinophil function that may be distinct from other inflammatory mediators.

How does ORMDL3 influence cytoskeletal rearrangement in eosinophils?

ORMDL3 plays a critical role in regulating eosinophil cytoskeletal dynamics, which are essential for cell trafficking and migration. Confocal microscopy studies with phalloidin staining have revealed that eosinophils with normal ORMDL3 expression (either untreated or control-siRNA-treated) demonstrate pronounced cytoskeletal changes when adherent to VCAM-1 and ICAM-1, including cell spreading with distinct polarization, leading edge formation, and development of uropodia and filopodia . In contrast, knockdown of ORMDL3 using siRNA significantly impairs these cytoskeletal rearrangements, resulting in cells that maintain a compact round morphology without leading edges. Quantitative analysis shows that a significantly larger percentage of ORMDL3-silenced eosinophils fail to spread when adhered to adhesion molecules . These findings demonstrate that ORMDL3 is essential for the cytoskeletal reorganization that enables eosinophil trafficking, particularly during the post-adhesion phase when cells prepare for transmigration through the endothelium to inflammatory sites.

How does ORMDL3 regulate eosinophil adhesion and trafficking?

ORMDL3 regulates multiple aspects of eosinophil trafficking through its effects on adhesion molecule expression and cellular activation. Research has demonstrated that knockdown of ORMDL3 in eosinophils significantly reduces their adhesion to recombinant mouse (rm) VCAM-1 and rm ICAM-1 compared to control siRNA-treated cells . This impaired adhesion correlates with decreased mRNA levels of α4 and β2 integrins, which are counter receptors for VCAM-1 and ICAM-1 respectively. Immunostaining confirms reduced surface expression of α4 and Mac-1 (αM) in ORMDL3-silenced eosinophils . Conversely, eosinophils overexpressing ORMDL3 show increased expression of α4 and β2 integrins. Interestingly, while ORMDL3 knockdown significantly impairs adhesion, it does not affect eosinophil rolling, possibly due to redundant adhesion molecules like galectin-3 that can mediate this process . These findings establish ORMDL3 as a key regulator of eosinophil trafficking, primarily through modulation of integrin expression and subsequent adhesion-dependent processes.

What is the relationship between ORMDL3 and eosinophil degranulation?

ORMDL3 plays a critical role in regulating eosinophil degranulation, a key effector function in allergic inflammation. Research indicates that ORMDL3 mediates this process through regulation of CD48 expression. IL-3, which induces ORMDL3 expression, also upregulates CD48 on eosinophils . CD48 is a glycosylphosphatidylinositol-anchored protein that functions as a receptor for CD2 and facilitates eosinophil degranulation when engaged. Studies show that knockdown of ORMDL3 significantly reduces CD48 expression on eosinophils, while overexpression of ORMDL3 increases CD48 levels . This ORMDL3-dependent regulation of CD48 expression directly impacts the capacity of eosinophils to release granule proteins in response to stimulation, establishing a molecular link between ORMDL3 and the effector functions of eosinophils in allergic inflammation. This mechanism may be particularly relevant in the context of asthma, where eosinophil degranulation contributes significantly to airway damage and remodeling.

What evidence supports ORMDL3's role in allergic asthma pathogenesis?

The role of ORMDL3 in asthma pathogenesis is supported by several lines of evidence spanning genetic associations and functional studies. ORMDL3 has been identified as a candidate gene for asthma susceptibility through genome-wide association studies . Functionally, research demonstrates that ORMDL3 regulates key eosinophil processes implicated in asthma pathophysiology:

• Eosinophil recruitment: ORMDL3 is expressed in eosinophils recruited to airways of allergen-challenged mice

• Trafficking regulation: ORMDL3 mediates eosinophil rolling, adhesion, and migration through modulation of integrin expression

• Signal transduction: ORMDL3 activates ERK and NF-κB pathways, leading to proinflammatory responses

• Degranulation control: ORMDL3 regulates CD48 expression and CD48-mediated eosinophil degranulation

This multifaceted involvement in eosinophil biology provides a mechanistic link between ORMDL3 expression and allergic airway inflammation, suggesting that targeting ORMDL3 or its downstream pathways may offer therapeutic potential for asthma and other eosinophilic disorders.

What are the recommended protocols for studying ORMDL3-mediated eosinophil trafficking in vitro?

For researchers investigating ORMDL3's role in eosinophil trafficking, the following validated in vitro protocols are recommended:

Eosinophil Rolling Assay:

• Coat cover-slips with recombinant mouse VCAM-1

• Suspend bone marrow-derived eosinophils (1-2 × 10^5/ml) in appropriate buffer

• Infuse cells through a parallel plate flow chamber at 1 ml/min (wall shear stress ~1.0-2.0 dynes/cm²)

• Observe cell-substrate interactions using an inverted microscope

• Record images for offline analysis

• Quantify rolling cells as those demonstrating multiple discrete interruptions and slow flow relative to non-interacting cells

• Express results as number of rolling cells per minute

Adhesion and Cell Morphology Analysis:

• Prepare ORMDL3-siRNA-treated, control-siRNA-treated, and untreated eosinophils

• Allow cells to adhere to VCAM-1 or ICAM-1 coated surfaces

• Perform phalloidin staining to visualize F-actin

• Examine cell spreading and polarization using confocal microscopy

• Quantify morphological changes by calculating percentage of cells showing compact morphology versus spread/polarized phenotype

These protocols enable detailed functional assessment of ORMDL3's role in distinct phases of eosinophil trafficking.

How can researchers quantitatively assess ORMDL3-dependent signaling in eosinophils?

To effectively measure ORMDL3-dependent signaling pathways in eosinophils, researchers should implement a multi-modal approach combining both protein phosphorylation analysis and nuclear translocation studies:

ERK Activation Assessment:

• Prepare eosinophils with varied ORMDL3 expression levels (overexpression via ORMDL3-GFP transfection or knockdown via siRNA)

• Lyse cells in buffer containing phosphatase inhibitors

• Perform Western blot analysis using antibodies against phospho-ERK (1/2) and total ERK

• Normalize phospho-ERK levels to total ERK expression

• Compare activation levels between ORMDL3-modulated and control cells

NF-κB Nuclear Translocation Analysis:

• Prepare ORMDL3-GFP transfected and control eosinophils

• Fix and permeabilize cells

• Perform immunofluorescence staining for NF-κB

• Counterstain nuclei with DAPI

• Analyze using confocal microscopy

• Quantify the percentage of cells showing nuclear NF-κB localization

• Compare nuclear translocation between ORMDL3-overexpressing and control cells

These complementary approaches provide robust quantitative data on the signaling consequences of ORMDL3 modulation in eosinophils.

What are the most promising areas for further investigation of ORMDL3 function?

Several high-priority research directions for ORMDL3 warrant investigation:

• Structural biology approaches: Determining the three-dimensional structure of ORMDL3 would provide insights into its functional domains and potential for targeted drug development. Given the recombinant expression systems now available for both human and Pongo abelii ORMDL3, X-ray crystallography or cryo-EM studies are feasible next steps .

• Tissue-specific functions: While the current research focuses on ORMDL3 in eosinophils, expanding studies to examine its role in other immune and structural cells involved in asthma pathogenesis would provide a more comprehensive understanding of its contribution to disease.

• Translational studies: Developing small molecule modulators of ORMDL3 or its downstream pathways could provide new therapeutic approaches for asthma and other eosinophilic disorders. The established overexpression and knockdown models provide excellent platforms for screening such compounds .

• Systems biology integration: Integrating ORMDL3 function into larger networks of inflammatory signaling in asthma could reveal synergistic therapeutic targets and improve understanding of disease heterogeneity.

Each of these directions builds upon the established methodologies from the current literature while addressing critical gaps in knowledge that limit clinical translation.