Recombinant Human ORM1-like protein 3 (ORMDL3)

What is ORMDL3 and what are its primary functions?

ORMDL3 (ORM1-like protein 3) is a member of the ORM family proteins that are conserved from yeast to humans. It functions as a crucial modulator in lipid metabolism, inflammation, and endoplasmic reticulum (ER) stress responses . ORMDL3 is predominantly expressed in immune cells, particularly CD4+ T lymphocytes, where its expression can be significantly upregulated in individuals carrying specific risk alleles .

The protein is involved in several cellular processes:

• Regulation of sphingolipid biosynthesis

• Modulation of ER stress responses

• Influence on calcium homeostasis

• Mediation of autophagy in various cell types, including endothelial cells

• Impact on T-cell function, including reduced IL-2 expression when overexpressed

Research examining ORMDL3's role in disease pathways has demonstrated its significance in immune regulation, cellular stress responses, and lipid metabolism, all of which contribute to its associations with inflammatory conditions like asthma and atherosclerosis.

How does ORMDL3 differ from other ORM family proteins?

While ORMDL3 shares structural similarities with other ORM family proteins (ORMDL1 and ORMDL2), it has distinct expression patterns and genetic associations that set it apart. Unlike its family members, ORMDL3 has been universally confirmed as a susceptibility gene for asthma and has emerging associations with atherosclerosis .

The key differences include:

• ORMDL3 expression is highly regulated by specific SNPs on chromosome 17q21, particularly rs7216389, rs9303277, and rs12603332, which have not been consistently linked to regulation of other ORM family members

• CD4+ T lymphocytes show a particularly pronounced (3-fold) increase in ORMDL3 expression in individuals with 17q12-21 risk SNPs, suggesting unique immune regulatory functions

• ORMDL3 responds to oxidized low-density lipoprotein (ox-LDL) with increased expression in endothelial cells, demonstrating a specific role in vascular pathology not clearly established for other family members

Understanding these distinctions is crucial for research targeting ORMDL3 without affecting other ORM family proteins.

What are the key SNPs associated with ORMDL3 expression and how do they function?

Several SNPs on chromosome 17q21 have been identified as significant regulators of ORMDL3 expression, with functional consequences for disease susceptibility. The most prominently studied include:

The functionality of these SNPs has been experimentally validated. For instance, rs12603332 has been proven functional through Cytosine Base Editor (CBE) technology, which demonstrated that editing the C risk allele to the T non-risk allele in both Jurkat cells and primary human CD4 T cells significantly reduced ORMDL3 expression .

The molecular mechanism underlying this regulation involves transcription factor binding: the non-risk allele rs12603332-T forms part of an E-box binding motif (CANNTG) that is recognized by the E47 transcription factor, while the risk allele rs12603332-C disrupts this binding site. E47 binding to the non-risk allele appears to suppress ORMDL3 expression, explaining the increased ORMDL3 levels associated with the risk allele .

How can researchers effectively genotype ORMDL3-related SNPs in clinical samples?

For researchers working with clinical samples to study ORMDL3-related polymorphisms, several methodological approaches can be employed:

• PCR-RFLP Analysis: Design primers flanking the SNP of interest (e.g., rs12603332), followed by restriction enzyme digestion that differentially cleaves risk versus non-risk alleles.

• TaqMan SNP Genotyping Assays: Use fluorescent probes specific to each allele variant. This allows for high-throughput screening of clinical samples with minimal DNA input.

• Next-Generation Sequencing (NGS): For comprehensive analysis of multiple ORMDL3-related SNPs, targeted sequencing of the 17q21 region can provide detailed haplotype information.

• Sanger Sequencing: For validation or small sample sets, direct sequencing remains a gold standard for SNP identification.

• Digital PCR: Offers absolute quantification and higher sensitivity for detecting allelic imbalances in heterozygous samples.

When implementing these techniques, researchers should consider:

• Including known control samples with verified genotypes

• Analyzing multiple SNPs within the 17q21 locus for comprehensive haplotype assessment

• Correlating genotype with ORMDL3 expression levels via RT-qPCR from the same samples

• Accounting for population-specific allele frequencies, as these may vary across ethnic groups

The choice of method should be guided by available resources, sample size, and the specific research questions being addressed.

How does ORMDL3 contribute to asthma pathophysiology?

ORMDL3 contributes to asthma pathophysiology through multiple mechanisms affecting both immune and non-immune cells:

• T Cell Dysregulation: Enhanced ORMDL3 expression in CD4+ T cells leads to reduced interleukin-2 (IL-2) production, altering T cell function and immune responses . This modification in T cell behavior may contribute to the immune dysregulation characteristic of asthma.

• Increased Th2 Responses: Transgenic mice overexpressing ORMDL3 exhibit heightened Th2 responses when challenged with allergens . This skewing toward Th2 immunity promotes the production of cytokines that drive allergic inflammation in asthma.

• Airway Hyperreactivity: Global ORMDL3 overexpression in mouse models leads to increased baseline airway hyperreactivity and exacerbated responses in ovalbumin-induced asthma models . This directly connects ORMDL3 to one of the hallmark features of asthma.

• Effects on Airway Structural Cells: ORMDL3 affects the function of airway smooth muscle (ASM) cells and bronchial epithelial cells (BEC), potentially altering airway remodeling processes and epithelial barrier function .

• ER Stress Induction: ORMDL3 activates endoplasmic reticulum stress pathways, which can trigger inflammatory responses and alter cellular homeostasis in lung tissues.

• Sphingolipid Dysregulation: As a regulator of sphingolipid biosynthesis, abnormal ORMDL3 expression can disrupt sphingolipid balance, affecting membrane integrity and signaling processes relevant to asthma pathology.

The genetic evidence strongly supports these mechanisms, as individuals carrying the risk alleles (e.g., rs12603332-C) show significantly higher ORMDL3 expression, particularly in CD4+ T lymphocytes (up to 3-fold increase) .

What is the evidence linking ORMDL3 to atherosclerosis development?

The connection between ORMDL3 and atherosclerosis (AS) is supported by both genetic association studies and functional investigations:

• Genetic Association: Two single nucleotide polymorphisms regulating ORMDL3 expression (rs7216389 and rs9303277) have been significantly associated with atherosclerosis risk in Chinese Han populations . This provides statistical evidence for ORMDL3's involvement in AS pathogenesis.

• Differential Expression: Increased ORMDL3 expression has been documented in atherosclerosis cases compared to controls, suggesting a direct correlation between ORMDL3 levels and disease presence .

• Response to Oxidized LDL: In endothelial cells, oxidized low-density lipoprotein (ox-LDL)—a key driver of atherosclerosis—upregulates ORMDL3 expression. This indicates ORMDL3 is responsive to atherogenic stimuli .

• Autophagy Regulation: Knockdown of ORMDL3 alleviates both ox-LDL-induced and basal autophagy in endothelial cells. Since autophagy plays a complex role in atherosclerosis progression, this suggests a mechanistic link .

• BECN1 Regulation: Silencing ORMDL3 suppresses ox-LDL-induced as well as basal BECN1 expression, an essential protein for autophagy initiation .

• Cell Survival Impact: Deletion of ORMDL3 results in greater sensitivity to ox-LDL-induced cell death, suggesting ORMDL3 may protect vascular cells from lipid-induced damage .

These findings collectively suggest ORMDL3 might represent a causal gene mediating autophagy in endothelial cells during atherosclerosis development. The link between ORMDL3 and key atherogenic processes (lipid metabolism, inflammation, ER stress) provides a biological basis for its contribution to atherosclerosis pathogenesis.

How can CRISPR-based technologies be utilized to study ORMDL3 function?

CRISPR-based technologies offer powerful approaches for studying ORMDL3 function, as demonstrated by recent research using Cytosine Base Editors (CBE). Researchers can implement these methodologies through several targeted strategies:

• Single Base Editing of Regulatory SNPs:

  • Using CBE to convert the rs12603332 C risk allele to the T non-risk allele in human cell lines or primary cells

  • The CBE approach uses a catalytically impaired CRISPR-associated nuclease [nCas9(D10A)] complexed with a guide RNA for sequence-specific targeting, fused to cytosine deaminase enzyme rAPOBEC1 and Uracil glycosylase inhibitor (UGI)

  • This methodology avoids double-strand breaks while achieving precise single nucleotide modifications

• Complete ORMDL3 Knockout:

  • Traditional CRISPR-Cas9 with paired guide RNAs can create larger deletions for complete functional elimination

  • Target critical exons or regulatory regions to ensure complete loss of function

  • Compare phenotypes against wild-type or base-edited variants

• CRISPRi/CRISPRa for Expression Modulation:

  • Use CRISPR interference (CRISPRi) with dCas9-KRAB to repress ORMDL3 expression

  • Apply CRISPR activation (CRISPRa) with dCas9-VP64 to enhance expression

  • These approaches allow titratable expression changes without altering genetic sequence

• Prime Editing for Precise Modifications:

  • Utilize prime editing to make specific modifications to ORMDL3 coding sequences

  • Introduce specific amino acid changes to study structure-function relationships

  • Create mutations that mimic disease-associated variants

Implementation considerations include:

• Design guide RNAs with minimal off-target effects using computational prediction tools

• Validate editing efficiency via sequencing, TIDE analysis, or restriction fragment length polymorphism

• Confirm functional consequences through expression analysis (RT-qPCR, Western blot)

• Assess downstream effects on target pathways (sphingolipid metabolism, ER stress markers, autophagy)

The successful application of CBE technology to edit rs12603332 in Jurkat cells and primary human CD4 T cells, achieving 90 ± 3% editing efficiency, demonstrates the feasibility and power of these approaches for ORMDL3 research .

What are the optimal methods for measuring ORMDL3 protein expression and activity?

Accurate measurement of ORMDL3 protein expression and functional activity requires a multi-faceted approach:

For ORMDL3 Protein Expression:

• Western Blotting:

  • Use validated antibodies specific to human ORMDL3 (avoiding cross-reactivity with ORMDL1/2)

  • Include appropriate loading controls (β-actin, GAPDH)

  • Quantify band intensity using densitometry software for semi-quantitative analysis

• Immunohistochemistry/Immunofluorescence:

  • Particularly useful for tissue samples or cellular localization studies

  • Apply antigen retrieval protocols optimized for ORMDL3 detection

  • Include co-staining with ER markers to confirm subcellular localization

• Flow Cytometry:

  • Provides quantitative assessment in individual cells

  • Particularly valuable for immune cell populations (e.g., CD4+ T cells)

  • Allows simultaneous assessment of ORMDL3 with cell surface markers

• ELISA:

  • For serum or plasma samples if extracellular ORMDL3 is of interest

  • Sandwich ELISA with capture and detection antibodies offers high specificity

For ORMDL3 Functional Activity:

• Sphingolipid Profiling:

  • Liquid chromatography-mass spectrometry (LC-MS) to quantify ceramides and other sphingolipids

  • Measure baseline levels and changes after ORMDL3 modulation

• ER Stress Markers:

  • RT-qPCR for downstream genes (e.g., ATF6α) shown to be regulated by ORMDL3

  • Western blot for ER stress proteins (BiP/GRP78, CHOP, phospho-eIF2α)

• Autophagy Assessment:

  • LC3-II/LC3-I ratio by Western blot

  • Fluorescent LC3 puncta quantification by microscopy

  • p62/SQSTM1 accumulation as marker of autophagy inhibition

• BECN1 Expression Analysis:

  • RT-qPCR and Western blotting for BECN1 levels after ORMDL3 manipulation

  • Correlation analysis between ORMDL3 and BECN1 expression

• Cell Functional Assays:

  • Measure sensitivity to ox-LDL-induced cell death in ORMDL3-modified cells

  • Assess CD4+ T cell IL-2 production in cells with varying ORMDL3 expression

Validation strategies should include:

• Positive and negative controls (ORMDL3 overexpression or knockdown)

• Comparison across multiple cell types relevant to disease (T cells, airway cells, endothelial cells)

• Correlation of protein levels with mRNA expression

• Assessment under both basal and stimulated conditions (e.g., allergen exposure, ox-LDL)

How might targeting ORMDL3-regulated pathways provide novel therapeutic strategies for asthma?

Targeting ORMDL3-regulated pathways offers several promising therapeutic avenues for asthma treatment, given the established role of ORMDL3 in disease pathogenesis:

• E47 Transcription Factor Modulation:

  • The discovery that E47 binds to the non-risk allele rs12603332-T to suppress ORMDL3 expression suggests potential for therapeutic mimicry

  • Small molecules or peptides that enhance E47 binding to the ORMDL3 promoter region could reduce ORMDL3 expression

  • E47 agonists might preferentially benefit patients carrying the risk allele rs12603332-C

• Sphingolipid Metabolism Targeting:

  • Since ORMDL3 regulates sphingolipid biosynthesis, normalizing sphingolipid profiles could mitigate downstream effects

  • Sphingosine-1-phosphate receptor modulators (already in clinical use for multiple sclerosis) might be repurposed

  • Ceramide synthesis inhibitors could counteract ORMDL3-induced alterations

• ER Stress Pathway Intervention:

  • Chemical chaperones like 4-phenylbutyric acid (4-PBA) that reduce ER stress could counteract ORMDL3-mediated effects

  • ATF6α inhibitors might be particularly effective, as ATF6α is a downstream target of ORMDL3

  • Targeting the PERK-eIF2α arm of the unfolded protein response could alleviate inflammatory consequences

• T Cell Function Modulation:

  • Given that increased ORMDL3 expression reduces IL-2 production in T cells , IL-2 supplementation strategies might restore normal immune function

  • Therapies that rebalance Th1/Th2 responses could counteract the enhanced Th2 responses observed with ORMDL3 overexpression

• Precision Medicine Approaches:

  • Genotyping patients for ORMDL3-related SNPs (particularly rs12603332) could identify individuals most likely to benefit from targeted therapies

  • Different therapeutic strategies might be optimal for patients with different genetic profiles

Experimental approaches to develop and validate these therapeutic strategies should include:

• High-throughput screening for compounds that reduce ORMDL3 expression or activity

• Testing in both cell culture systems and transgenic mouse models overexpressing ORMDL3

• Validation in primary cells from asthmatic patients with known genotypes for ORMDL3-related SNPs

• Assessment of effects on multiple endpoints (airway hyperreactivity, inflammation, remodeling)

What is the interplay between ORMDL3, autophagy, and endoplasmic reticulum stress in different cell types?

The interplay between ORMDL3, autophagy, and endoplasmic reticulum (ER) stress represents a complex relationship that varies across cell types and has significant implications for disease pathogenesis:

Endothelial Cells and Atherosclerosis:

• ORMDL3 upregulation by ox-LDL induces autophagy in endothelial cells

• Knockdown of ORMDL3 alleviates both ox-LDL-induced and basal autophagy

• ORMDL3 silencing suppresses BECN1 expression, an essential initiator of autophagy

• This suggests ORMDL3 serves as a positive regulator of autophagy in endothelial cells, potentially as a cellular protective mechanism against lipid-induced stress

T Lymphocytes and Asthma:

• In CD4+ T cells, increased ORMDL3 expression correlates with altered immune function, including reduced IL-2 production

• ORMDL3 overexpression is linked to enhanced Th2 responses , though the exact autophagy-related mechanisms remain to be fully elucidated

• The specific role of autophagy in T cell regulation by ORMDL3 warrants further investigation, particularly regarding T cell differentiation and cytokine production

Airway Cells:

• ORMDL3 may influence ER stress responses in airway smooth muscle and epithelial cells

• Autophagy processes in these cells could affect airway remodeling and hyperresponsiveness

• The balance between pro-survival and pro-inflammatory autophagy functions might determine disease outcomes

Molecular Mechanisms Across Cell Types:

• ORMDL3 regulates ATF6α expression , a key transcription factor in the ER stress response

• ER stress can both induce and be regulated by autophagy, creating a feedback loop

• Sphingolipid alterations caused by ORMDL3 may affect membrane dynamics important for autophagosome formation

This complex interplay suggests several important research directions:

• Comparative studies of autophagy flux in different cell types with controlled ORMDL3 expression

• Investigation of cell-specific transcriptional networks connecting ORMDL3, ER stress, and autophagy

• Temporal analysis of these pathways to determine sequence of activation

• Assessment of how genetic variants in ORMDL3 differentially affect these pathways

• Exploration of crosstalk between these cellular processes and other pathways, such as inflammasome activation

Understanding these intricate relationships could reveal cell-specific therapeutic targets and explain the tissue-specific manifestations of ORMDL3-associated diseases.

What are the most promising future directions for ORMDL3 research?

The expanding understanding of ORMDL3 biology opens several promising research avenues that could significantly advance both basic science knowledge and therapeutic development:

• Multi-Omics Integration:

  • Combining transcriptomics, proteomics, and metabolomics data from ORMDL3-modulated systems

  • Integrating genotype information (particularly rs12603332, rs7216389, and rs9303277) with molecular phenotypes

  • Developing network biology approaches to understand ORMDL3's position in cellular signaling networks

• Tissue and Cell-Specific Functions:

  • Employing conditional and tissue-specific ORMDL3 transgenic or knockout models

  • Investigating cell-specific contributions to disease phenotypes

  • Exploring potential differential functions of ORMDL3 across immune cells, endothelial cells, and structural cells

• Therapeutic Development:

  • Advancing E47-based strategies to regulate ORMDL3 expression

  • Developing small molecule modulators of ORMDL3 activity

  • Exploring RNA-based therapeutics for precise ORMDL3 targeting

• Advanced Genetic Approaches:

  • Utilizing CRISPR-based epigenome editing to modulate ORMDL3 expression

  • Investigating long-range chromatin interactions affecting ORMDL3 expression

  • Exploring the complete haplotype structure of the 17q21 locus and its functional implications

• Disease Expansion Beyond Asthma and Atherosclerosis:

  • Investigating ORMDL3's role in other inflammatory and autoimmune conditions

  • Exploring potential connections to metabolic disorders given ORMDL3's role in lipid metabolism

  • Assessing ORMDL3 in neurodegenerative diseases where ER stress and autophagy play crucial roles

• Precision Medicine Applications:

  • Developing diagnostic assays based on ORMDL3 genotype and expression

  • Stratifying patients based on ORMDL3-related biomarkers for targeted therapies

  • Creating predictive models for disease risk and progression incorporating ORMDL3 status

• Translational Research:

  • Validating findings from model systems in human clinical samples

  • Investigating pharmacological modulators of ORMDL3 function

  • Developing biomarkers of ORMDL3 activity for clinical monitoring

The convergence of advanced genetic editing technologies, systems biology approaches, and increasing clinical data makes ORMDL3 research particularly poised for significant discoveries that could transform understanding of inflammatory disease mechanisms and lead to novel therapeutic strategies.

How can researchers effectively design experiments to resolve contradictory findings in ORMDL3 research?

Resolving contradictory findings in ORMDL3 research requires careful experimental design and methodological considerations:

• Standardized Expression Systems:

  • Establish consensus cell lines and expression vectors for ORMDL3 studies

  • Quantify expression levels precisely to ensure comparability across studies

  • Consider both transient and stable expression systems to distinguish acute versus chronic effects

• Genetic Background Considerations:

  • Account for genetic background in model organisms (e.g., mouse strain differences)

  • Document complete genotype at the 17q21 locus when using human samples

  • Consider potential compensatory mechanisms involving ORMDL1/2 in knockout studies

• Methodological Harmonization:

  • Develop standard operating procedures for key ORMDL3 assays

  • Establish reference materials and positive controls

  • Create a repository of validated reagents (antibodies, vectors, guide RNAs)

• Comprehensive Phenotyping:

  • Assess multiple endpoints across studies (e.g., not only asthma but also related phenotypes)

  • Document environmental conditions thoroughly (e.g., exposure to allergens, stress)

  • Consider temporal dynamics of ORMDL3 effects

• Statistical and Reporting Rigor:

  • Pre-register experimental designs when possible

  • Report negative and contradictory findings

  • Provide complete methodological details to facilitate replication

• Addressing Specific Contradictions:

  • For contradictory findings between Ormdl3 transgenic mouse models , design studies that:

    • Use the same allergen challenge protocols

    • Employ identical background strains

    • Measure expression levels consistently

    • Assess multiple endpoints (physiological, cellular, molecular)

  • For inconsistencies in SNP functionality:

    • Use single base editing as demonstrated with rs12603332

    • Analyze allele-specific effects in various cell types

    • Measure transcription factor binding directly (ChIP assays)

• Meta-analysis and Systematic Reviews:

  • Periodically conduct systematic reviews of ORMDL3 literature

  • Perform meta-analyses of genetic association studies

  • Identify sources of heterogeneity across studies

• Collaborative Approaches:

  • Establish multi-center studies with standardized protocols

  • Create ORMDL3 research consortia to share resources and data

  • Implement round-robin testing of key findings across laboratories