Recombinant Human ORM1-like protein 1 (ORMDL1)

What is ORMDL1 and what biological functions does it perform?

ORMDL1 is a member of the mammalian orosomucoid-like gene family that acts as a critical factor in maintaining cellular sphingolipid homeostasis. It functions primarily as a regulatory subunit of serine palmitoyltransferase (SPT), the enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis pathway . ORMDL1 plays a crucial role in regulating SPT activity through a mixed inhibition mechanism that involves both competitive inhibition (by decreasing SPT's affinity for palmitoyl-CoA) and non-competitive inhibition (by altering enzyme kinetics) .

Structurally, ORMDL1 contains specific residues that can interact with ceramide molecules, allowing it to function as a sensor within the sphingolipid homeostatic network. This sensing mechanism enables cells to regulate sphingolipid production based on existing ceramide levels, creating a feedback loop that maintains appropriate sphingolipid balance .

How does ORMDL1 expression vary across different tissues and cancer types?

Analysis of ORMDL1 expression using the GEPIA database shows differential expression patterns across various cancer tissues and their corresponding normal tissues. ORMDL1 is significantly upregulated in certain cancer types, with notably higher expression in cholangiocarcinoma (CHOL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (LAML), and thymoma (THYM) compared to their normal tissue counterparts .

Using the Cancer Cell Line Encyclopedia (CCLE) database, researchers have verified that ORMDL1 mRNA is highly expressed in cell lines derived from LAML and DLBCL, with these cancer types ranking 1st and 11th respectively among 40 different cancer types analyzed . This expression pattern consistency between tissue samples and cell lines provides strong evidence for the relevance of ORMDL1 in these particular cancer types.

What methodologies are commonly used to study ORMDL1 function?

Several methodological approaches have been employed to investigate ORMDL1 function:

• Genetic manipulation: CRISPR-Cas9 genome editing to generate knockout cell lines lacking specific ORMDL isoforms. This approach involves targeting regions between exons (typically between exons 2 and 3) followed by clone selection and validation through Sanger sequencing .

• Enzymatic assays: In vitro measurement of SPT activity in the presence or absence of ORMDL1 and/or ceramide analogs. These assays typically use purified protein complexes and analyze enzyme kinetics to determine inhibitory mechanisms .

• Structural biology: Determination of protein structures through techniques like X-ray crystallography or cryo-electron microscopy to elucidate the molecular interactions between ORMDL1, SPT, and ceramide molecules .

• Bioinformatics analysis: Use of databases like GEPIA, CCLE, LinkedOmics, and cBioPortal to analyze expression patterns, genetic alterations, and prognostic implications across various cancer types .

• Metabolic labeling: Analysis of sphingolipid metabolism through incorporation of isotopically labeled precursors (like d2-labeled serine) followed by liquid chromatography-mass spectrometry (LC-MS) detection .

What is the relationship between ORMDL1 and ceramide binding?

ORMDL1 contains a functional ceramide binding site that allows it to sense cellular ceramide levels and modulate SPT activity accordingly. Structural analysis has revealed specific residues involved in this interaction:

• Polar head coordination: Residues like Asn17 in ORM1 form hydrogen bonds with the polar head of ceramide molecules .

• Aliphatic chain coordination: Hydrophobic residues including Trp20, Ser67, and Trp88 in ORM1 interact with the aliphatic sphingosine and acyl chains of ceramide .

Structure-guided mutational analyses have demonstrated that altering these ceramide-binding residues (creating variants like ORM1-N17A, ORM1-S67R, ORM1-W20R, and ORM1-W88R) results in significantly increased enzymatic activity of the SPT-ORM1 complex compared to the wild-type complex. This confirms that ceramide binding to these residues is essential for ORMDL1-dependent SPT repression .

The specificity of this interaction varies among ceramide species. For example, C6-phytoceramide demonstrates stronger inhibitory effects (IC50 of 0.32 μM) compared to C6-ceramide (IC50 of 3.5 μM), while C6-dihydroceramide shows no apparent effect on the SPT-ORM1 complex activity .

What is the prognostic significance of ORMDL1 expression in different cancer types?

Additionally, analysis of genetic alterations using the cBioPortal database showed that DLBCL patients with ORMDL1 genetic alterations (primarily increased gene copy numbers and some gene deletions) exhibited worse prognosis compared to those without alterations. This finding suggests that genetic changes in ORMDL1 may contribute to its role in DLBCL pathogenesis and patient outcomes .

What molecular mechanisms explain ORMDL1-mediated regulation of SPT activity?

ORMDL1 regulates SPT activity through a complex mechanism involving:

• Mixed inhibition model: ORMDL1 inhibits SPT activity through both competitive and non-competitive mechanisms. The competitive aspect involves decreasing SPT's affinity toward palmitoyl-CoA (increasing Khalf value), while the non-competitive aspect involves reducing the maximum enzymatic activity (Vmax) when ceramide is bound .

• N-terminal inhibitory conformation: The N-terminal region of ORM1 (particularly Met14 and Asn15) adopts a conformation that physically clashes with the CoA head of the acyl-CoA substrate. This structural impediment explains how ORMDL1 decreases SPT's affinity for palmitoyl-CoA .

• Hybrid β-sheet structure: A hybrid β-sheet formation between ORM1 and the LCB2a subunit of SPT stabilizes the ORM1 N-terminus in an inhibitory conformation. Experiments with ORM1-ΔN14 variant (lacking the first 14 N-terminal residues) showed significantly lower Khalf value toward palmitoyl-CoA compared to wild-type SPT-ORM1 complex, confirming the importance of this region for inhibition .

• Ceramide-dependent potentiation: Binding of ceramide to ORMDL1 enhances its inhibitory effect on SPT, creating a feedback mechanism whereby increased ceramide levels (downstream products in the sphingolipid pathway) suppress further sphingolipid synthesis through ORMDL1-mediated SPT inhibition .

How does ORMDL1 differ functionally from other ORMDL isoforms (ORMDL2 and ORMDL3)?

Research on the functional differences between ORMDL isoforms has utilized CRISPR-Cas9 technology to generate cell lines expressing only one of the three ORMDL isoforms (by deleting the other two) or lacking all three isoforms .

These studies suggest that:

• Regulatory potency: While all three isoforms can regulate SPT activity, they may do so with different potencies. For instance, ORMDL1 appears to regulate SPT activity in a manner that responds to ceramide levels .

• Ceramide responsiveness: SPT regulatory activity of ORMDL1 can be modulated by ceramide levels, with exogenous C-8 ceramide influencing its inhibitory effect on SPT .

• Physiological specialization: Despite structural similarities, the three ORMDL isoforms may have evolved to serve tissue-specific or context-dependent roles in sphingolipid homeostasis. Evidence for this comes from the differential expression patterns observed across tissues and cell types .

• Disease associations: While ORMDL3 has been extensively studied for its associations with asthma and inflammatory conditions, ORMDL1 shows stronger associations with certain cancer types, particularly DLBCL .

Complete understanding of the isoform-specific functions requires further research, as the evolutionary conservation of all three isoforms suggests non-redundant physiological roles that may become apparent only under specific conditions or in particular tissues.

What is the relationship between ORMDL1 expression and immune cell infiltration in cancers?

Analysis of the relationship between ORMDL1 expression and immune signatures in DLBCL revealed that ORMDL1 expression levels are significantly correlated with the infiltrating level of B cells, but not with other immune cell types such as CD8+ T cells, CD4+ T cells, macrophages, neutrophils, or dendritic cells .

Further investigation showed that gene gain mutations of ORMDL1 promote B cell infiltration in DLBCL, suggesting a potential mechanism by which ORMDL1 genetic alterations might influence the tumor microenvironment . This specific association with B cell infiltration is particularly relevant in DLBCL, which is a malignancy of B lymphocytes.

The mechanism behind this correlation remains to be fully elucidated, but it may involve ORMDL1-mediated changes in sphingolipid metabolism affecting B cell trafficking, proliferation, or survival within the tumor microenvironment. This finding opens potential avenues for investigating ORMDL1 as a target for immunotherapeutic approaches in DLBCL.

What coexpressed genes and pathways are associated with ORMDL1 in cancer contexts?

Correlation analysis performed via LinkedOmics has identified numerous genes that are positively or negatively correlated with ORMDL1 expression in DLBCL. Among the top positively correlated genes are SORBS1 (correlation coefficient: 4.469e-01) and PPWD1 (correlation coefficient: 4.299e-01) .

Gene Ontology (GO) analysis of ORMDL1 co-expressed genes in DLBCL revealed enrichment in several biological processes:

• DNA damage response

• Nucleus localization

• rRNA metabolic processes

• Cell cycle checkpoint regulation

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further identified enrichment in:

• Cell cycle

• ABC transporters

• Oxidative phosphorylation

• DNA replication

These findings suggest that ORMDL1 may be involved in fundamental cellular processes beyond sphingolipid metabolism, potentially explaining its role in cancer progression through effects on cell cycle regulation, DNA damage responses, and energy metabolism.

What are the critical parameters for successful purification and analysis of recombinant ORMDL1?

Successful purification and analysis of recombinant ORMDL1 requires attention to several critical parameters:

How can researchers accurately measure and interpret ORMDL1-mediated effects on SPT activity?

Accurate measurement and interpretation of ORMDL1-mediated effects on SPT activity involve:

• Enzyme kinetic analysis: Determining both Vmax and Khalf (or Km) values to distinguish between competitive and non-competitive inhibition mechanisms. This approach revealed that ORMDL1 exhibits mixed inhibition of SPT activity .

• Dose-response experiments: Testing the effects of varying concentrations of ceramide analogs (such as C6-ceramide or C6-phytoceramide) on SPT-ORM1 complex activity to determine IC50 values and maximum inhibition levels .

• Mutational analysis: Creating structure-guided mutations in ceramide-binding residues or N-terminal regions to dissect the contribution of specific domains to ORMDL1's inhibitory function .

• Control experiments: Performing parallel experiments with SPT alone (without ORMDL1) to distinguish ORMDL1-dependent effects from intrinsic variations in SPT activity .

• Cellular validation: Complementing in vitro findings with cellular assays, such as measuring de novo sphingolipid synthesis in cells expressing wild-type or mutant ORMDL1 variants .

• Sphingolipid profiling: Using liquid chromatography-mass spectrometry techniques to analyze changes in sphingolipid profiles resulting from ORMDL1 manipulation. Typical analyses include the separation of sphingolipid species using a combination of solvent systems (like water/methanol/formic acid gradients) followed by mass spectrometric detection .

What are the optimal experimental designs for studying ORMDL1 in cancer models?

Optimal experimental designs for studying ORMDL1 in cancer models include:

• Multiple cancer cell line comparison: Given the differential expression of ORMDL1 across cancer types, using a panel of cell lines representing different cancers (with emphasis on DLBCL and LAML where ORMDL1 is highly expressed) provides more comprehensive insights .

• Genetic manipulation approaches:

  • CRISPR-Cas9 knockout of ORMDL1 using guide RNAs targeting regions between exons 2 and 3

  • Generation of single-isoform expressing cell lines by knocking out the other two ORMDL isoforms

  • Creation of ceramide-binding mutants (N17A, W20R, W88R) to assess the role of ceramide sensing in cancer phenotypes

• Patient-derived samples: Correlating ORMDL1 expression or genetic alterations with clinical outcomes in patient samples, particularly for DLBCL where prognostic significance has been established .

• Immune microenvironment analysis: Investigating the relationship between ORMDL1 expression/mutation and immune cell infiltration, with specific focus on B cell populations in lymphomas .

• Pathway analysis: Examining the effects of ORMDL1 manipulation on key cancer-associated pathways identified through coexpression analysis, such as cell cycle regulation, DNA damage response, and oxidative phosphorylation .

• In vivo models: Developing xenograft or genetically engineered mouse models with ORMDL1 alterations to assess its role in tumor initiation, progression, and response to therapies.

How can contradictory findings regarding ORMDL1 function be reconciled in research?

Contradictory findings regarding ORMDL1 function can be reconciled through several approaches:

• Context-dependent effects: Recognize that ORMDL1 may have different functions depending on:

  • Cell or tissue type

  • Cancer subtype

  • Expression levels of other ORMDL isoforms

  • Sphingolipid metabolism status of the cell

• Methodological differences: Carefully examine differences in experimental approaches:

  • In vitro vs. cellular vs. in vivo systems

  • Overexpression vs. knockout methodologies

  • Short-chain vs. long-chain ceramide analogs used in experiments (as shown by the differential effects of C6-ceramide vs. native long-chain ceramides)

• Multifaceted regulation: Acknowledge that ORMDL1 may exert regulatory effects through multiple mechanisms:

  • Direct SPT enzyme inhibition

  • Altered gene expression programs

  • Immune cell infiltration modulation

  • Effects on other signaling pathways

• Isoform compensation: Consider the possibility that other ORMDL isoforms may compensate for ORMDL1 in knockout models, necessitating the generation of single-isoform expressing cells to truly isolate ORMDL1-specific functions .

• Technical validation: Employ multiple techniques to verify findings, such as combining structural studies, in vitro enzymatic assays, cellular sphingolipid profiling, and in vivo phenotypic analysis .

What are the most promising therapeutic applications targeting ORMDL1?

Based on current research, the most promising therapeutic applications targeting ORMDL1 include:

Development of specific inhibitors of the ORMDL1-ceramide interaction or molecules that disrupt the SPT-ORMDL1 complex formation represents a logical next step in translating the basic research findings into therapeutic applications.

What critical knowledge gaps remain in our understanding of ORMDL1 biology?

Despite significant advances, several critical knowledge gaps remain in our understanding of ORMDL1 biology:

• Isoform-specific functions: While studies have begun to explore the differences between ORMDL isoforms, a comprehensive understanding of why evolution has maintained three distinct isoforms remains incomplete .

• Tissue-specific roles: The physiological significance of differential ORMDL1 expression across tissues and its implications for tissue-specific sphingolipid homeostasis require further investigation.

• Upstream regulation: The mechanisms controlling ORMDL1 expression, localization, and activity beyond ceramide sensing are poorly understood.

• Cancer-promoting mechanisms: While associations between ORMDL1 and cancer prognosis have been established, the precise mechanisms by which ORMDL1 contributes to cancer progression, particularly in DLBCL, remain to be fully elucidated .

• Interaction network: The complete interaction network of ORMDL1 beyond SPT, including potential roles in regulating other proteins or cellular processes, warrants further exploration.

• Structural dynamics: How the conformational changes in the ORMDL1-SPT complex occur upon ceramide binding and how these changes translate to altered enzymatic activity require more detailed structural and biochemical analysis .