ORM1 Human, HEK

What is ORM1 and what are its known biological functions?

ORM1 (Orosomucoid 1), also known as alpha-1-acid glycoprotein 1 (AGP1), is an acute phase plasma protein primarily synthesized by the liver. It is encoded by the ORM1 gene located on human chromosome 9 . This 21.56 kDa protein (calculated without glycosylation) consists of 183 amino acid residues and functions as a key mediator in the interaction between blood cells and endothelial cells .

ORM1 works alongside other acute phase proteins such as haptoglobin and C-reactive protein to regulate cell extravasation during infection and inflammation . While its precise function remains not fully characterized, ORM1 is believed to play important roles in immunosuppression and inflammatory responses . Expression of ORM1 is induced by acute-phase stimulatory mediators, including bacterial lipopolysaccharides, highlighting its involvement in immune responses .

At the molecular level, ORM1 has been shown to interact with Plasminogen activator inhibitor-1, suggesting potential roles in coagulation and fibrinolysis pathways . In plant systems, ORM1 homologs have been demonstrated to participate in sphingolipid homeostasis regulation through interaction with serine palmitoyltransferase (SPT), which may provide insights into potential mammalian functions .

How is ORM1 different from ORM2, and what are the implications for experimental design?

ORM1 and ORM2 are highly homologous genes that both encode orosomucoid proteins (alpha-1-acid glycoproteins), but they exhibit differential regulation patterns that researchers must consider when designing experiments . These differences manifest primarily in their promoter regions and responses to various stimuli.

Key differences that impact experimental design include:

• Promoter region functionality: ORM1 shows higher transcription-induction activity in its distal promoter region in the absence of C/EBPβ overexpression, while ORM2's distal promoter shows higher activity under C/EBPβ overexpression conditions .

• Response to inflammatory stimuli: When treated with IL-1β, ORM1 full-length promoter constructs show significantly higher activity than deletion constructs. In contrast, for ORM2, deletion constructs show significantly lower activity at basal levels and after dexamethasone treatments .

• Transcriptional regulation: Both genes contain putative glucocorticoid responsive elements and C/EBPβ binding sites in their proximal promoter regions, but their responses to these transcription factors differ .

When designing experiments involving ORM proteins, researchers should specifically identify which variant they are studying and consider using gene-specific primers or antibodies that can distinguish between these homologs. Failure to distinguish between ORM1 and ORM2 may lead to confounding results, especially in studies examining regulatory mechanisms or expression patterns during acute phase responses.

What are the optimal conditions for expressing human ORM1 in HEK293 cells?

Based on successful experimental protocols, the following conditions represent optimized approaches for human ORM1 expression in HEK293 cells:

Expression System Considerations:

• HEK293F cells have been successfully used for expressing ORM1-containing protein complexes .

• For functional studies of ORM1, standard HEK293 cells can be utilized as they maintain normal sphingolipid homeostasis regulatory machinery .

Expression Vector Requirements:

• Vectors containing strong promoters (CMV or EF1α) are recommended for high expression levels.

• For structural and interaction studies, expression vectors allowing C-terminal or N-terminal tagging (His, FLAG, or Strep tags) facilitate purification without disrupting function .

Transfection Protocol:

• Lipid-based transfection reagents (Lipofectamine or PEI) show good efficiency.

• For stable expression, selection with appropriate antibiotics (G418 or puromycin) for 2-3 weeks is advised.

• Transfection efficiency can be monitored using co-transfected GFP markers.

Culture and Induction Conditions:

• For optimal protein yield, cells should be grown in DMEM supplemented with 10% FBS.

• For induction studies examining ORM1 regulation, treatments with dexamethasone (DEX) or interleukin-1β (IL-1β) can induce significant changes in expression levels .

• C/EBPβ overexpression can be employed to study transcriptional regulation mechanisms .

For validation of expression, Western blotting with specific antibodies or RT-qPCR analysis should be performed. Purification from HEK cells can be achieved through affinity chromatography approaches using the introduced tags.

How can researchers effectively measure ORM1 gene expression changes in response to inflammatory stimuli?

To effectively measure ORM1 gene expression changes in response to inflammatory stimuli, researchers should implement a comprehensive approach combining multiple techniques:

RT-qPCR Analysis:

• Design ORM1-specific primers that don't cross-react with the highly homologous ORM2 gene.

• Include appropriate housekeeping genes (GAPDH, β-actin) for normalization.

• Time-course experiments (0, 3, 6, 12, 24 hours) provide insights into the dynamic regulation.

Luciferase Reporter Assays:

• Compare full promoter constructs (~1.1 kbp) with deletion constructs (containing only ~188 bp of proximal promoter region) .

• Test responses to inflammatory stimuli like IL-1β, which has been shown to significantly increase ORM1 promoter activity .

• Include dexamethasone (DEX) treatments to assess glucocorticoid response elements function.

Inflammatory Stimuli Protocol:

• IL-1β treatment: 1-10 ng/ml is typically effective.

• Dexamethasone: 100 nM-1 μM concentration range.

• Combined treatments can reveal synergistic effects .

Transcription Factor Analysis:

• C/EBPβ overexpression experiments can provide insights into transcriptional regulation mechanisms.

• Chromatin immunoprecipitation (ChIP) assays can identify specific transcription factor binding sites in the ORM1 promoter.

Example Data Analysis Table:

When reporting results, it's important to distinguish between transcriptional activation (mRNA levels) and protein expression changes, as post-transcriptional mechanisms might affect the correlation between these measurements.

What techniques can be used to study ORM1 interactions with other proteins in HEK cell systems?

Several complementary techniques can be employed to comprehensively characterize ORM1 interactions with other proteins in HEK cell systems:

Co-Immunoprecipitation (Co-IP):

• Express tagged ORM1 (FLAG, HA, or Myc-tagged) in HEK293 cells.

• Lyse cells under non-denaturing conditions to preserve protein-protein interactions.

• Capture ORM1 complexes using tag-specific antibodies conjugated to beads.

• Identify interacting partners through Western blotting or mass spectrometry.

• This technique successfully identified interactions between ORM1 and plasminogen activator inhibitor-1 .

Proximity Labeling Approaches:

• BioID or TurboID systems can be used, where ORM1 is fused to a biotin ligase.

• Proximal proteins become biotinylated and can be captured using streptavidin pulldown.

• This approach is particularly useful for capturing transient or weak interactions.

Fluorescence Resonance Energy Transfer (FRET):

• Create fluorescent protein fusions (e.g., ORM1-CFP and potential interactor-YFP).

• Analyze energy transfer between fluorophores when proteins interact.

• Live-cell imaging enables visualization of interaction dynamics.

Structural Analysis:

• For detailed interaction interfaces, cryo-electron microscopy (cryo-EM) can be employed.

• The SPT-ORM1 complex structure was successfully determined at 3.2 Å resolution using this approach .

• Single-particle reconstruction can reveal binding interfaces and conformational changes.

Functional Validation Through Mutagenesis:

• Structure-guided mutational analysis of key residues can confirm interaction interfaces.

• For example, mutations of ceramide-binding residues in ORM1 (N17A, S67R, W20R, and W88R) significantly affected ORM1's ability to repress SPT activity .

Example Interaction Validation Table:

These techniques, when used in combination, provide comprehensive insights into ORM1's interactome and the functional significance of these interactions.

How can researchers effectively study the role of ORM1 in sphingolipid homeostasis in human cell lines?

To investigate ORM1's role in sphingolipid homeostasis in human cell lines, researchers should implement a multifaceted approach that combines genetic manipulation, biochemical assays, and lipidomic analyses:

Genetic Manipulation Strategies:

• CRISPR/Cas9-mediated knockout of ORM1 in HEK293 cells to establish loss-of-function models.

• Overexpression of wild-type and mutant ORM1 variants to assess gain-of-function effects.

• Expression of ORM1 mutants defective in ceramide binding (W20R, W88R, N17A, S67R) to dissect specific functions .

Serine Palmitoyltransferase (SPT) Activity Assays:

• Measure SPT activity using isotope-labeled serine incorporation into long-chain bases.

• Implement d2-labeled serine incorporation assays to quantify de novo sphingolipid synthesis .

• Compare SPT activity in cells expressing wild-type ORM1 versus ceramide-binding mutants.

Lipidomic Analysis:

• Employ liquid chromatography-mass spectrometry (LC-MS/MS) to quantify sphingolipid species.

• Monitor changes in ceramides, sphingosines, sphingosine-1-phosphates, and complex sphingolipids.

• Perform time-course analyses following manipulation of ORM1 expression.

Ceramide Supplementation Experiments:

• Test the effects of exogenous ceramides (C6-ceramide, C6-phytoceramide, C6-dihydroceramide) on SPT activity.

• Short-chain ceramide analogs can be added to cell culture to assess ORM1-dependent SPT inhibition .

• Determine whether ceramide supplementation can rescue phenotypes in ORM1-deficient cells.

Protein Complex Analysis:

• Isolate and characterize SPT-ORM1 complexes from HEK cells using affinity purification.

• Assess the stoichiometry and composition of complexes under different conditions.

• Analyze conformational changes using limited proteolysis or hydrogen-deuterium exchange mass spectrometry.

Example Data: Impact of ORM1 Variants on Sphingolipid Levels

The SPT-ORM1 complex functions as a ceramide sensor in the endoplasmic reticulum, controlling cellular sphingolipid homeostasis. When ceramide levels rise, ceramide binding to ORM1 enhances its inhibitory effect on SPT, creating a negative feedback loop that prevents excessive sphingolipid production .

How can researchers design experiments to differentiate between direct and indirect effects of ORM1 on cellular processes?

Distinguishing between direct and indirect effects of ORM1 on cellular processes requires sophisticated experimental designs that isolate specific pathways and interactions:

Reconstitution in Purified Systems:

• Express and purify ORM1 and its potential interaction partners.

• Reconstitute functional complexes in vitro to test direct biochemical activities.

• The SPT-ORM1 complex has been successfully purified and shown to maintain catalytic activity in vitro, confirming direct interaction and functional effects .

Structure-Guided Mutagenesis:

Inducible Expression Systems:

• Utilize tetracycline-inducible or other tightly controlled expression systems.

• Time-course experiments following induction can distinguish immediate (likely direct) effects from delayed (likely indirect) effects.

• Monitor primary outcomes (e.g., SPT activity) versus secondary outcomes (e.g., cellular sphingolipid profiles).

Proximity-Dependent Labeling:

• Employ BioID or APEX2 fusion proteins to identify proteins in close proximity to ORM1.

• This approach helps distinguish between direct binding partners and proteins that are simply part of the same larger complex.

Domain Swapping Experiments:

• Create chimeric proteins exchanging domains between ORM1 and ORM2 or other related proteins.

• These experiments can identify which domains are responsible for specific functions.

• For example, swapping the N-terminal regions can help determine their importance in forming regulatory β-sheets with partner proteins .

Temporal Analysis of Signaling Events:

• Use phosphoproteomics or other signaling assays at multiple time points after ORM1 perturbation.

• Immediate changes in protein modifications likely represent direct effects.

Example Decision Matrix for Interpreting ORM1 Effects:

Through careful experimental design and interpretation, researchers can build a comprehensive understanding of ORM1's direct mechanistic roles versus its broader influences on cellular homeostasis.

What are the key considerations when using HEK cell models to study ORM1 functions that may differ between cell types?

When using HEK cells as a model system to study ORM1 functions, researchers must carefully consider several factors that might affect the translation of findings to other cell types:

Baseline Expression Profiles:

• HEK293 cells may have different baseline expression levels of ORM1, ORM2, and their interacting partners compared to primary cells or tissues.

• Quantitative proteomics and transcriptomics comparing HEK cells with target tissues should be performed to identify potential differences.

• Creating HEK cells with tissue-specific expression profiles through genetic engineering might be necessary.

Sphingolipid Metabolism Differences:

• Sphingolipid profiles and metabolic rates can vary significantly between cell types.

• Comprehensive lipidomic analysis should compare HEK cells with the cell type of interest (e.g., hepatocytes, which naturally produce ORM1).

• Adjustments to culture conditions might help align sphingolipid metabolism between model and target cells.

Signaling Pathway Completeness:

• Verify that HEK cells possess the complete signaling machinery relevant to ORM1 function.

• For acute phase response studies, confirm that HEK cells express relevant cytokine receptors and downstream signaling components.

• Research has shown that HEK293 cells can recapitulate Arabidopsis SPT regulation by ORM1, but complete validation is necessary for human pathways .

Genetic Background Considerations:

• Create isogenic cell lines through CRISPR/Cas9 to study ORM1 variants.

• When comparing results between cell types, consider genetic background differences that might influence outcomes.

• Patient-derived induced pluripotent stem cells (iPSCs) differentiated into relevant cell types can provide genetically matched comparisons.

Physiological Relevance Assessment:

• Validate key findings in more physiologically relevant systems.

• Primary human hepatocytes would provide a more natural context for ORM1 function.

• Organoid models might better recapitulate the complex cellular environment.

Example Validation Strategy Table:

HEK cells provide an experimentally tractable system that successfully demonstrated ORM1's role in SPT regulation , but findings should be validated in physiologically relevant systems to ensure biological accuracy.

What are common challenges in purifying active ORM1 protein from expression systems, and how can they be overcome?

Purifying active ORM1 protein presents several challenges due to its biochemical properties and functional requirements. Here are the common issues and recommended solutions:

Challenge: Maintaining Proper Protein Folding

• Problem: ORM1 contains multiple disulfide bonds crucial for its native structure.

• Solution:

  • Express in mammalian systems like HEK293F cells that provide appropriate post-translational modifications .

  • Include oxidized and reduced glutathione (3:1 ratio) in purification buffers to maintain disulfide bonds.

  • Purify under non-denaturing conditions to preserve native structure.

Challenge: Preserving Protein-Protein Interactions

• Problem: ORM1 functions as part of multi-protein complexes (e.g., with SPT).

• Solution:

  • Consider co-expression and co-purification strategies when studying functional complexes.

  • The SPT-ORM1 complex has been successfully purified from HEK293F cells with catalytic activity intact .

  • Use mild detergents (0.01-0.05% DDM or LMNG) for membrane-associated complexes.

Challenge: Low Solubility

• Problem: Membrane-associated ORM1 complexes may have solubility issues.

• Solution:

  • Add solubility tags (SUMO, MBP, or GST) that can be cleaved after purification.

  • Optimize buffer conditions (pH 7.4-7.8, 150-300 mM NaCl).

  • Human ORM1 has been successfully formulated in 20mM TRIS and 50mM NaCl at pH 7.5 .

Challenge: Heterogeneous Glycosylation

• Problem: Variable glycosylation can cause heterogeneity in purified protein.

• Solution:

  • Express in GnTI- HEK293 cells for more homogeneous glycosylation.

  • Consider enzymatic deglycosylation with PNGase F for structural studies.

  • For functional studies, maintain native glycosylation as it may be important for activity.

Challenge: Maintaining Ceramide Binding Capability

• Problem: Purification can strip away bound lipids necessary for function.

• Solution:

  • Add specific lipids (e.g., ceramides) during or after purification.

  • Screen different detergents that preserve lipid-protein interactions.

  • Validate lipid binding using thin-layer chromatography or mass spectrometry.

Purification Protocol Optimization Table:

When validating purified ORM1, assess both purity (>95% by SDS-PAGE) and functional activity (ceramide binding capacity and SPT inhibition potential).

What are promising unexplored research directions for understanding ORM1's role in disease processes?

Based on current knowledge and technological capabilities, several promising research directions could significantly advance our understanding of ORM1's role in disease processes:

Investigating ORM1 in Chronic Inflammatory Conditions:

• Examine ORM1 expression patterns and polymorphisms in chronic inflammatory diseases.

• Develop mouse models with tissue-specific ORM1 deletion or overexpression to assess inflammation progression.

• Study how altered ORM1 glycosylation patterns might contribute to inflammatory disease phenotypes.

ORM1's Role in Sphingolipid-Related Disorders:

• Investigate whether ORM1 dysregulation contributes to sphingolipid metabolism disorders.

• The established role of ORM1 in sphingolipid homeostasis through SPT regulation suggests it may impact conditions like Gaucher disease or Niemann-Pick disease.

• Explore therapeutic approaches targeting ORM1-SPT interaction to modulate sphingolipid levels in disease states.

Integration of ORM1 in Multi-omics Disease Models:

• Implement integrated proteomics, lipidomics, and transcriptomics approaches to map ORM1's effect on global cellular networks.

• Develop computational models predicting how ORM1 variants might influence disease progression.

• Use single-cell multi-omics to understand cell-type-specific functions of ORM1 in complex tissues.

ORM1 in Cell-Cell Communication:

• Investigate whether secreted ORM1 functions as a signaling molecule between different tissues.

• Examine how ORM1 in circulation affects distant organs during inflammatory responses.

• Study potential receptor-mediated actions of ORM1 in various cell types.

Therapeutic Targeting Opportunities:

• Develop small molecules that can modulate ORM1-ceramide interaction for potential therapeutic applications.

• Structure-based drug design targeting the ORM1-SPT interface could provide new approaches for modulating sphingolipid metabolism .

• Investigate whether recombinant ORM1 administration could have therapeutic value in conditions with dysregulated inflammation.

Potential Research Questions Table:

These research directions could significantly expand our understanding of ORM1 beyond its established roles in acute phase response and sphingolipid homeostasis, potentially revealing new therapeutic targets for various disease processes.

What emerging technologies could significantly advance our understanding of ORM1 function in human systems?

Several cutting-edge technologies hold particular promise for advancing our understanding of ORM1 function in human systems:

Cryo-Electron Tomography:

• Application: Visualize ORM1-containing complexes in their native cellular environment at near-atomic resolution.

• Advancement: Recent improvements in cryo-ET sample preparation and image processing now enable visualization of macromolecular complexes in situ.

• Potential Insights: Could reveal how ORM1-SPT complexes are organized in the endoplasmic reticulum membrane and how their distribution changes in response to ceramide levels .

CRISPR-Based Functional Genomics:

• Application: Genome-wide screens to identify genes that synthetically interact with ORM1.

• Advancement: Base editing and prime editing technologies allow precise introduction of disease-associated ORM1 variants.

• Potential Insights: Could uncover unknown functional interactions and regulatory networks involving ORM1.

Spatial Multi-omics:

• Application: Map ORM1 expression, protein interactions, and downstream effects with spatial resolution in tissues.

• Advancement: Technologies like Slide-seq, Visium, and MERFISH can provide unprecedented spatial information about gene expression and protein localization.

• Potential Insights: Could reveal tissue-specific and cell-type-specific functions of ORM1 in complex organs.

Artificial Intelligence for Structure-Function Prediction:

• Application: Predict functional impacts of ORM1 variants and identify potential binding sites for drug development.

• Advancement: AlphaFold and RoseTTAFold have revolutionized protein structure prediction, while graph neural networks can predict protein-protein interactions.

• Potential Insights: Could help design therapeutics targeting specific ORM1 interfaces or predict the impact of genetic variants.

Advanced Sphingolipidomics:

• Application: Comprehensive characterization of sphingolipid species with high sensitivity and specificity.

• Advancement: Ion mobility-mass spectrometry and novel derivatization strategies have dramatically improved sphingolipid detection limits and structural characterization.

• Potential Insights: Could reveal how ORM1 specifically affects different sphingolipid species in various cellular compartments.

Organoid and Microphysiological Systems:

• Application: Study ORM1 function in more physiologically relevant 3D tissue models.

• Advancement: Liver organoids and multi-organ-on-chip systems can better recapitulate the complex environment in which ORM1 naturally functions.

• Potential Insights: Could reveal how ORM1 participates in inter-organ communication and tissue-specific responses.

Technology Implementation Roadmap:

By strategically implementing these emerging technologies, researchers can develop a more comprehensive understanding of ORM1's functions in human health and disease, potentially leading to novel therapeutic approaches targeting this important acute phase protein.

How can researchers integrate findings from plant and mammalian ORM1 studies to gain broader insights into conserved mechanisms?

The integration of insights from plant and mammalian ORM1 studies represents a powerful approach to understanding evolutionarily conserved mechanisms of sphingolipid regulation. Despite significant evolutionary distance, striking functional parallels exist that can inform research in both systems:

Comparative Structural Analysis:

• The structure of the Arabidopsis SPT-ORM1 complex reveals a ceramide-sensing mechanism that may be conserved in mammals .

• Researchers should conduct detailed structural comparisons between plant and mammalian ORM1-containing complexes to identify conserved binding interfaces and regulatory motifs.

• Hybrid β-sheet structures formed between ORM1 and its binding partners appear to be functionally significant in plants and may represent a conserved regulatory mechanism .

Conservation of Regulatory Mechanisms:

• Both plant and mammalian ORM1 proteins participate in sphingolipid homeostasis through regulation of serine palmitoyltransferase (SPT) .

• Ceramide binding enhances ORM1's inhibitory effect on SPT in plants; researchers should investigate whether similar lipid-sensing mechanisms exist in mammalian systems .

• Cross-species complementation experiments (e.g., expressing mammalian ORM1 in plant systems and vice versa) can identify functionally conserved domains.

Evolutionary Analysis of ORM Protein Families:

• Comprehensive phylogenetic analysis of ORM protein sequences across diverse organisms can reveal conserved motifs under selective pressure.

• Key residues involved in ceramide binding (e.g., W20, W88, N17 in Arabidopsis ORM1) should be analyzed for conservation across species .

• Divergent regions may indicate species-specific adaptations in sphingolipid metabolism regulation.

Integrated Experimental Approaches:

• Develop standardized assays that can be applied across both plant and mammalian systems.

• Create chimeric proteins combining domains from plant and mammalian ORM1 to test functional conservation.

• Implement parallel genetic screens in both systems to identify conserved genetic interactions.

Translational Research Potential:

By systematically integrating findings from both plant and mammalian systems, researchers can accelerate discovery of fundamental mechanisms in sphingolipid homeostasis and potentially identify novel therapeutic approaches for diseases involving dysregulated sphingolipid metabolism.

What methodological advances are needed to resolve current contradictions in ORM1 research literature?

Several methodological advances could help resolve existing contradictions and knowledge gaps in the ORM1 research literature:

Standardized Experimental Systems:

• Current Issue: Studies use diverse cell types, expression systems, and experimental conditions, making direct comparisons difficult.

• Needed Advance: Establish consensus cell models, standardized purification protocols, and agreed-upon assay conditions for ORM1 functional studies.

• Implementation: Develop detailed standard operating procedures (SOPs) for ORM1 expression, purification, and functional characterization to be adopted across research groups.

Improved Specificity in Detection Methods:

• Current Issue: Cross-reactivity between ORM1 and ORM2 in antibody-based detection and functional attribution.

• Needed Advance: Development of highly specific antibodies or alternative detection methods that can distinguish between ORM1 and ORM2.

• Implementation: Validate antibody specificity using ORM1/ORM2 knockout cells and recombinant proteins; consider epitope tagging strategies or targeted mass spectrometry approaches for increased specificity.

Comprehensive Genetic Models:

• Current Issue: Limited understanding of ORM1-specific functions due to potential redundancy with ORM2.

• Needed Advance: Generation of clean ORM1 knockout, knockin, and conditional knockout models in relevant cell types and organisms.

• Implementation: Apply CRISPR/Cas9 technology to create isogenic cell lines differing only in ORM1 status; develop conditional knockout mouse models for tissue-specific ORM1 deletion.

Integrated Multi-omics Approaches:

• Current Issue: Isolated findings focusing on limited aspects of ORM1 function without system-level understanding.

• Needed Advance: Integration of transcriptomics, proteomics, lipidomics, and metabolomics data to build comprehensive models of ORM1 function.

• Implementation: Apply multi-omics approaches to the same experimental system under identical conditions to enable direct correlation of changes across different molecular levels.

Advanced Imaging Techniques:

• Current Issue: Limited understanding of ORM1 subcellular localization and dynamic changes during cellular responses.

• Needed Advance: Implementation of super-resolution microscopy and live-cell imaging to track ORM1 spatiotemporal dynamics.

• Implementation: Develop fluorescent protein fusions or specific labels for ORM1 that don't disrupt function; validate localization with complementary techniques.

Methodological Contradictions Resolution Table:

By implementing these methodological advances, the field can work toward resolving current contradictions in ORM1 research, ultimately building a more coherent and comprehensive understanding of this important acute phase protein and its diverse biological functions.