GO:0017040 N-acylsphingosine amidohydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0017040 (N-acylsphingosine amidohydrolase activity) is a molecular function that catalyzes the hydrolysis of N-acylsphing-4-enine (ceramide) into a fatty acid and sphing-4-enine (sphingosine).
• This activity is mediated by ceramidases, including acid, neutral, and alkaline isoforms, which are critical for sphingolipid metabolism and signaling.
• Dysregulation of ceramidase activity is implicated in cancer, Alzheimer's disease, atherosclerosis, and abdominal aortic aneurysm [1,2,3,4,6].
• ASAH1 (acid ceramidase) promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production.
• ACER3 (alkaline ceramidase 3) attenuates cholestasis by mitigating bile acid overload via unsaturated ceramide-mediated LXRβ signaling.
• Targeting ceramidases, such as ASAH1, enhances antitumor immune responses in colorectal cancer, highlighting therapeutic potential.
Description
N-acylsphingosine amidohydrolase activity (GO:0017040) is a molecular function that catalyzes the hydrolysis of N-acylsphing-4-enine (ceramide) to a fatty acid and sphing-4-enine (sphingosine). This reaction is central to sphingolipid metabolism, regulating the balance between pro-apoptotic ceramides and pro-survival sphingosine-1-phosphate. Researchers study this activity because it influences diverse physiological and pathological processes, including cancer progression, neurodegeneration, and cardiovascular disease [1,2,3,4,6]. The enzymes responsible, ceramidases, are classified by their optimal pH as acid, neutral, and alkaline, and they exhibit distinct tissue distributions and substrate specificities. Understanding GO:0017040 is essential for developing therapies that modulate sphingolipid signaling [4,8].
N-acylsphingosine amidohydrolase activity At A Glance
| GO ID | GO:0017040 |
|---|---|
| GO term | N-acylsphingosine amidohydrolase activity |
| Ontology | molecular_function |
| Synonym | ceramidase activity; acylsphingosine deacylase activity; glycosphingolipid ceramide deacylase |
| Definition | Catalysis of the reaction: an N-acylsphing-4-enine + H2O = a fatty acid + sphing-4-enine. |
| Major function | Hydrolysis of ceramides to sphingosine and fatty acids |
| EC number | 3.5.1.23 |
| Related genes | ASAH1, ASAH2, ACER1, ACER2, ACER3 |
What Is GO:0017040?
In my own words, GO:0017040 describes the catalytic activity of enzymes that cleave the amide bond in N-acylsphing-4-enine (a ceramide), using water to release a fatty acid and sphing-4-enine (sphingosine). This activity is synonymous with ceramidase activity and is fundamental to sphingolipid catabolism.
Why Is N-acylsphingosine amidohydrolase activity Important in Cell Biology?
GO:0017040 is important because ceramide hydrolysis products, particularly sphingosine and its phosphorylated derivative sphingosine-1-phosphate, are potent signaling molecules that regulate cell survival, proliferation, migration, and immune responses. Dysregulation of this activity contributes to cancer, metabolic disorders, and inflammatory diseases, making it a promising therapeutic target [2,4,6,8].
• Regulates the balance between pro-apoptotic ceramides and pro-survival sphingosine-1-phosphate.
• Promotes melanoma growth and metastasis through ASAH1-mediated suppression of ROS.
• Modulates adipose ceramide catabolism and atherosclerosis suppression via HIF-2α.
• Enhances antitumor immune responses when acid ceramidase is targeted in colorectal cancer.
• Mitigates vascular inflammation and abdominal aortic aneurysm via ACER1-mediated platelet ceramide catabolism.
• Attenuates cholestasis by ACER3-mediated unsaturated ceramide signaling to LXRβ.
• Associated with mental activity and population genetics of the ASAH1 gene.
• Implicated in Alzheimer's disease pathology through ASAH2 and related proteins.
• Provides targets for pharmacological inhibitors of ceramidases.
• Essential for sphingolipid homeostasis in health and disease.
Molecular Mechanism of N-acylsphingosine amidohydrolase activity
Substrate recognition and binding
In simple terms: The enzyme grabs a ceramide molecule and holds it in place.
Ceramidases recognize N-acylsphing-4-enine (ceramide) as substrate, binding the sphingoid base and fatty acyl chain in the active site. Different ceramidase isoforms exhibit preferences for ceramide species with varying acyl chain lengths and saturation.
Catalytic hydrolysis
In simple terms: Water is used to split the ceramide into two parts.
The enzyme catalyzes the hydrolysis of the amide bond between the fatty acid and sphingosine, releasing a free fatty acid and sphing-4-enine (sphingosine). This reaction is dependent on the presence of water and is optimal at specific pH ranges depending on the isoform.
Isoform-specific mechanisms
In simple terms: Different versions of the enzyme work best in different cellular environments.
Acid ceramidase (ASAH1) functions in lysosomes at acidic pH, neutral ceramidase (ASAH2) operates in the cytosol and plasma membrane at neutral pH, and alkaline ceramidases (ACER1-3) work in the endoplasmic reticulum and Golgi at alkaline pH. Each isoform contributes to distinct sphingolipid pools and signaling outcomes.
Regulation by inhibitors and activators
In simple terms: Small molecules can turn the enzyme on or off.
Ceramidase activity can be modulated by pharmacological inhibitors, such as those targeting acid ceramidase, which have been explored for cancer therapy [4,7]. Endogenous regulators include sphingosine-1-phosphate and other sphingolipid metabolites that feedback on enzyme activity.
Key Genes Involved in GO:0017040 N-acylsphingosine amidohydrolase activity
The following genes encode enzymes with N-acylsphingosine amidohydrolase activity or related proteins, each with distinct roles and research relevance.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ASAH1 | Acid ceramidase; hydrolyzes ceramides in lysosomes | Promotes melanoma growth and metastasis; target for antitumor immunity [2,4] |
| ASAH2 | Neutral ceramidase; hydrolyzes ceramides in cytosol and plasma membrane | Implicated in Alzheimer's disease pathology |
| ACER1 | Alkaline ceramidase 1; hydrolyzes ceramides in ER | Mitigates vascular inflammation and abdominal aortic aneurysm |
| ACER2 | Alkaline ceramidase 2; hydrolyzes ceramides in Golgi | Involved in sphingolipid metabolism and stress responses |
| ACER3 | Alkaline ceramidase 3; hydrolyzes unsaturated ceramides | Attenuates cholestasis via LXRβ signaling |
| HIF2A | Hypoxia-inducible factor 2α; regulates ceramide catabolism | Suppresses atherosclerosis by promoting adipose ceramide catabolism |
| LXRβ | Liver X receptor beta; mediates bile acid signaling | Transduces unsaturated ceramide signals in cholestasis |
| Peroxisome biogenesis proteins | Maintain peroxisome function and ROS balance | Suppressed by ASAH1 in melanoma |
| Sphingosine kinase | Phosphorylates sphingosine to S1P | Downstream of ceramidase activity |
| Ceramide synthase | Synthesizes ceramides from sphingosine | Opposes ceramidase activity |
| S1P receptors | Mediate sphingosine-1-phosphate signaling | Effectors of ceramidase downstream effects |
| Cytokines (e.g., TNF-α) | Inflammatory mediators | Modulate ceramidase expression in disease |
| Platelets | Cell type with high ACER1 activity | Regulate vascular inflammation |
| Adipocytes | Fat cells with ceramide catabolism | Linked to atherosclerosis suppression |
| Colorectal cancer cells | Malignant cells with acid ceramidase overexpression | Target for immune therapy |
| Melanoma cells | Malignant cells dependent on ASAH1 | Model for metastasis studies |
| Cholangiocytes | Bile duct cells | ACER3 regulates bile acid overload |
How Is N-acylsphingosine amidohydrolase activity Regulated?
Ceramidase activity is regulated at multiple levels. Transcriptional regulation includes HIF-2α-mediated induction of ceramide catabolism genes in adipocytes. Post-translational modifications and protein-protein interactions also modulate enzyme activity. Pharmacological inhibitors can block ceramidase function, and feedback loops involving sphingosine-1-phosphate and other sphingolipids fine-tune activity. In disease contexts, inflammatory cytokines and metabolic stress alter ceramidase expression and activity [6,8].
N-acylsphingosine amidohydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ASAH1 | Melanoma growth and metastasis | ASAH1 knockout melanoma cell lines and mouse xenografts |
| ASAH1 | Colorectal cancer antitumor immunity | Acid ceramidase inhibitors in syngeneic mouse models |
| ASAH2 | Alzheimer's disease | ASAH2 knockout neurons and AD mouse models |
| ACER1 | Abdominal aortic aneurysm | Platelet-specific ACER1 knockout mice |
| ACER3 | Cholestasis | ACER3 knockout or inhibitor-treated mice |
Cancer
Acid ceramidase (ASAH1) promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production. Targeting acid ceramidase enhances antitumor immune responses in colorectal cancer, suggesting that inhibition of this activity could boost immunotherapy efficacy.
Neurodegeneration
N-acylsphingosine amidohydrolase 2 (ASAH2) and related proteins have been investigated for pathological relevance in Alzheimer's disease, where sphingolipid dysregulation contributes to neuronal dysfunction. Population genetic analysis of the ASAH1 gene has associated variants with mental activity in humans.
Cardiovascular disease
Adipocyte HIF-2α suppresses atherosclerosis by promoting adipose ceramide catabolism, linking ceramidase activity to vascular health. Alkaline ceramidase 1 (ACER1)-mediated platelet ceramide catabolism mitigates vascular inflammation and abdominal aortic aneurysm formation.
Cholestasis
Targeting the ceramidase ACER3 attenuates cholestasis in mice by mitigating bile acid overload via unsaturated ceramide-mediated LXRβ signaling transduction.
From N-acylsphingosine amidohydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ASAH1 affect melanoma growth? | ASAH1 knockout melanoma cell lines and xenografts |
| Can acid ceramidase inhibition enhance antitumor immunity? | Colorectal cancer mouse models with ASAH1 inhibitors |
| What is the role of ACER1 in platelet ceramide catabolism? | Platelet-specific ACER1 knockout mice |
| How does ACER3 regulate bile acid signaling? | ACER3 knockout mice and cholestasis models |
| Is ASAH2 involved in Alzheimer's pathology? | ASAH2 knockout neurons and AD mouse models |
| Does HIF-2α regulate adipose ceramide catabolism? | Adipocyte-specific HIF-2α knockout mice |
How to Study the N-acylsphingosine amidohydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Fluorescent ceramidase assay | Enzymatic hydrolysis of ceramide | In vitro inhibitor screening |
| LC-MS/MS sphingolipidomics | Ceramide, sphingosine, S1P levels | Metabolic profiling in disease models [3,8] |
| CRISPR knockout | Loss-of-function phenotypes | Target validation in cancer cells [2,4] |
| RNA interference | Gene knockdown effects | Transient silencing in cell lines |
| Immunohistochemistry | Protein expression and localization | Tissue analysis in patient samples |
| Mouse xenografts | Tumor growth and metastasis | Preclinical drug testing |
| Platelet aggregation assays | Platelet function | Vascular inflammation studies |
| Bile acid quantification | Cholestasis severity | Liver disease models |
Enzymatic activity assays
Ceramidase activity can be measured using fluorescent or radioactive ceramide substrates, followed by HPLC or mass spectrometry to quantify product formation. These assays are used to characterize isoform-specific activity and inhibitor efficacy.
Sphingolipid profiling
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables comprehensive quantification of ceramides, sphingosine, and sphingosine-1-phosphate in cells and tissues, revealing changes in sphingolipid metabolism upon genetic or pharmacological manipulation [3,8].
Genetic knockout and knockdown
CRISPR-Cas9 knockout or RNA interference knockdown of ASAH1, ASAH2, ACER1-3 allows researchers to study loss-of-function phenotypes in cancer, neurodegeneration, and cardiovascular models [2,4,6].
In vivo disease models
Mouse models of melanoma, colorectal cancer, atherosclerosis, abdominal aortic aneurysm, and cholestasis are used to evaluate the therapeutic potential of targeting ceramidase activity [2,3,4,6,8].
How CRISPR Can Be Used to Study GO:0017040 N-acylsphingosine amidohydrolase activity
Knockout
CRISPR-Cas9 knockout of ASAH1, ASAH2, or ACER genes is used to abolish ceramidase activity and study consequent changes in sphingolipid metabolism, cell survival, and disease progression. For example, ASAH1 knockout reduces melanoma growth and metastasis.
Point Mutation
Point mutations can be introduced into the catalytic domain of ceramidases to dissect the contribution of enzymatic activity versus non-enzymatic functions. Such models help identify residues critical for substrate binding and catalysis.
Knock-in
Knock-in of tagged ceramidase alleles (e.g., FLAG or GFP) enables visualization and immunoprecipitation of endogenous enzymes, facilitating studies of localization and interactome in disease models.
Overexpression
Overexpression of wild-type or mutant ceramidases in cell lines allows gain-of-function studies to assess effects on proliferation, migration, and signaling. For instance, ASAH1 overexpression promotes melanoma growth.
How EDITGENE Supports N-acylsphingosine amidohydrolase activity Research
Researchers studying N-acylsphingosine amidohydrolase activity-related genes often need to determine whether a candidate gene is causally involved in a specific disease or cellular process. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell and animal models, enabling rigorous functional validation of ceramidase genes and their variants.
Contact EDITGENE today to design your custom CRISPR model for N-acylsphingosine amidohydrolase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| ACER2 Knockout HEK293 Cell Line | EDJ-KQ1093 | Human | 340485 | Details Get a Quote |
| ASAH2 Knockout HEK293 Cell Line | EDJ-KQ1744 | Human | 56624 | Details Get a Quote |
| ACER1 Knockout HEK293 Cell Line | EDJ-KQ1745 | Human | 125981 | Details Get a Quote |
| ACER3 Knockout HEK293 Cell Line | EDJ-KQ2859 | Human | 55331 | Details Get a Quote |
| NAAA Knockout HEK293 Cell Line | EDJ-KQ8703 | Human | 27163 | Details Get a Quote |
| ASAH2B Knockout HEK293 Cell Line | EDJ-KQ12449 | Human | 653308 | Details Get a Quote |
| ASAH1 Knockout HEK293 Cell Line | EDJ-KQ17855 | Human | 427 | Details Get a Quote |
| NAAA Knockout HCT 116 Cell Line | EDJ-KQ34927 | Human | 27163 | Details Get a Quote |
| NAAA Knockout HeLa Cell Line | EDJ-KQ34928 | Human | 27163 | Details Get a Quote |
| ASAH2B Knockout A-549 Cell Line | EDJ-KQ41381 | Human | 653308 | Details Get a Quote |
| ASAH2B Knockout HCT 116 Cell Line | EDJ-KQ41382 | Human | 653308 | Details Get a Quote |
| ASAH2B Knockout HeLa Cell Line | EDJ-KQ41383 | Human | 653308 | Details Get a Quote |
| ASAH1 Knockout A-549 Cell Line | EDJ-KQ21602 | Human | 427 | Details Get a Quote |
| ASAH1 Knockout HCT 116 Cell Line | EDJ-KQ21603 | Human | 427 | Details Get a Quote |
| ASAH1 Knockout HeLa Cell Line | EDJ-KQ21604 | Human | 427 | Details Get a Quote |
Displaying Records 1 To 15 Of 28 Records
Frequently Asked Questions About N-acylsphingosine amidohydrolase activity
What is N-acylsphingosine amidohydrolase activity?
It is a molecular function (GO:0017040) that catalyzes the hydrolysis of ceramide to sphingosine and a fatty acid, also known as ceramidase activity.
What genes are involved in N-acylsphingosine amidohydrolase activity?
The main genes are ASAH1, ASAH2, ACER1, ACER2, and ACER3, which encode acid, neutral, and alkaline ceramidases.
What diseases are associated with ceramidase activity?
Ceramidase dysregulation is linked to melanoma, colorectal cancer, Alzheimer's disease, atherosclerosis, abdominal aortic aneurysm, and cholestasis [1,2,3,4,6,8].
How is N-acylsphingosine amidohydrolase activity measured?
It is measured using fluorescent or radioactive ceramide substrates, often coupled with LC-MS/MS for sphingolipid quantification.
What is the role of ASAH1 in cancer?
ASAH1 promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production, and its inhibition enhances antitumor immunity in colorectal cancer [2,4].
Can ceramidase activity be targeted therapeutically?
Yes, inhibitors of acid ceramidase are being explored for cancer therapy, and targeting ACER3 shows promise for cholestasis [4,8].
What is the difference between acid, neutral, and alkaline ceramidases?
They differ in optimal pH, subcellular localization, and tissue distribution, with ASAH1 being lysosomal acid, ASAH2 neutral, and ACER1-3 alkaline.
How does ceramide catabolism affect atherosclerosis?
Adipocyte HIF-2α promotes ceramide catabolism, which suppresses atherosclerosis, and ACER1-mediated platelet ceramide catabolism mitigates vascular inflammation [3,6].
Is there a link between ceramidase activity and Alzheimer's disease?
ASAH2 and related proteins have been investigated for pathological relevance in Alzheimer's disease, suggesting a role in neurodegeneration.
What CRISPR models are available for studying ceramidase genes?
EDITGENE provides knockout, point mutation, knock-in, and overexpression models for ASAH1, ASAH2, ACER1-3, and related genes, as well as CRISPR library screening and bioinformatics support.
Conclusion
N-acylsphingosine amidohydrolase activity (GO:0017040) is a critical molecular function in sphingolipid metabolism with far-reaching implications for cancer, neurodegeneration, and cardiovascular disease. The enzymes catalyzing this reaction, including ASAH1, ASAH2, and ACER1-3, are promising therapeutic targets. Leveraging CRISPR-based models and advanced bioinformatics, researchers can dissect the precise roles of these genes and develop novel interventions.
References
- 1. Khabazeh A et al.. 2025. Investigating the Pathological Relevance of N-acylsphingosine Amidohydrolase 2 (ASAH2) and Related Proteins in Alzheimer's Disease.. Cureus 17(7):e87463 PMID: 40772191
- 2. Malvi P et al.. 2021. N-acylsphingosine amidohydrolase 1 promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production.. Mol Metab 48:101217 PMID: 33766731
- 3. Zhang X et al.. 2019. Adipocyte Hypoxia-Inducible Factor 2α Suppresses Atherosclerosis by Promoting Adipose Ceramide Catabolism.. Cell Metab 30(5):937-951.e5 PMID: 31668872
- 4. Vijayan Y et al.. 2024. Targeting acid ceramidase enhances antitumor immune response in colorectal cancer.. J Adv Res 65:73-87 PMID: 38142035
- 5. Kim HL et al.. 2008. Population genetic analysis of the N-acylsphingosine amidohydrolase gene associated with mental activity in humans.. Genetics 178(3):1505-15 PMID: 18245333
- 6. Zhang X et al.. 2023. Alkaline ceramidase 1-mediated platelet ceramide catabolism mitigates vascular inflammation and abdominal aortic aneurysm formation.. Nat Cardiovasc Res 2(12):1173-1189 PMID: 39196139
- 7. Saied EM et al.. 2016. Inhibitors of Ceramidases.. Chem Phys Lipids 197:60-8 PMID: 26220616
- 8. Liao L et al.. 2025. Targeting the ceramidase ACER3 attenuates cholestasis in mice by mitigating bile acid overload via unsaturated ceramide-mediated LXRβ signaling transduction.. Nat Commun 16(1):2112 PMID: 40025008
