ASAH2 encodes human neutral ceramidase (N-CDase, EC 3.5.1.23), a Zn2+-dependent type II membrane glycoprotein that catalyzes the hydrolysis of ceramide to sphingosine and free fatty acid at neutral pH. The enzyme is highly expressed at the intestinal brush border where it plays an essential role in digesting dietary sphingolipids. ASAH2 also catalyzes the reverse reaction, allowing synthesis of ceramides from fatty acids and sphingosine. The enzyme contains a ~20 Angstrom deep hydrophobic active site pocket that specifically recognizes ceramide's small hydroxyl headgroup while sterically excluding sphingolipids with bulky headgroups. While ASAH2's ceramide-degrading activity can shift the ceramide/S1P rheostat toward pro-survival signaling, this represents a downstream metabolic consequence rather than its evolved primary function.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0042759 long-chain fatty acid biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: The hydrolysis of ceramide by ASAH2 releases a free fatty acid as one of the products. IBA annotation based on phylogenetic analysis across multiple species orthologs indicates conserved involvement in fatty acid generation through ceramide catabolism. Reason: This annotation is well-supported. ASAH2 catalyzes ceramide + H2O -> sphingosine + fatty acid. The released fatty acid is a direct enzymatic product. This is a core metabolic outcome of the ceramidase reaction, not a downstream pleiotropic effect. Supporting Evidence: PMID:26190575 Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate file:human/ASAH2/ASAH2-deep-research-falcon.md model: Edison Scientific Literature |
| GO:0046512 sphingosine biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: ASAH2 generates sphingosine as a direct product of ceramide hydrolysis. IBA annotation based on phylogenetic conservation across multiple species. This is a core function of the neutral ceramidase. Reason: Sphingosine is the primary product of ASAH2's ceramidase activity. This is central to the enzyme's biochemical function. Well-supported by structural and biochemical studies [PMID:26190575, PMID:10781606, PMID:17475390]. Supporting Evidence: PMID:10781606 the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range PMID:26190575 Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IBA GO_REF:0000033 | ACCEPT | Summary: This is the precise molecular function term for ceramidase activity. IBA annotation well-supported by phylogenetic conservation and extensive experimental validation. Reason: N-acylsphingosine amidohydrolase activity (ceramidase activity) is the defining molecular function of ASAH2. Multiple experimental studies demonstrate this activity with defined kinetic parameters [PMID:26190575, PMID:10781606, PMID:16229686, PMID:17475390]. Supporting Evidence: PMID:26190575 Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases PMID:16229686 the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5 |
| GO:0005576 extracellular region | IBA GO_REF:0000033 | ACCEPT | Summary: ASAH2 is a type II membrane protein with a large extracellular/lumenal domain where the catalytic activity occurs. The enzyme is active in the intestinal lumen for digestion of dietary sphingolipids and can be released as a soluble form. Reason: ASAH2's catalytic domain faces the extracellular/lumenal side as a type II membrane protein. A soluble form can be generated by proteolytic cleavage. In the intestine, the enzyme functions at the brush border to digest dietary sphingolipids in the lumen [PMID:17475390]. Supporting Evidence: PMID:17475390 neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut |
| GO:0046514 ceramide catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: Ceramide catabolism is the core biological process function of ASAH2. IBA annotation reflects conserved function across species. Reason: Ceramide catabolism is the primary biological process carried out by ASAH2. The enzyme hydrolyzes ceramides to generate sphingosine and fatty acids. This is its defining physiological role, especially in intestinal digestion of dietary sphingolipids. Supporting Evidence: PMID:17475390 Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine PMID:26190575 Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate |
| GO:0000139 Golgi membrane | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation based on UniProt subcellular location mapping. Supported by experimental evidence showing ASAH2 localization to Golgi apparatus. Reason: Golgi localization has been experimentally demonstrated by IDA evidence [PMID:30154232]. The IEA annotation is consistent with this experimental finding. ASAH2 functions at both plasma membrane and Golgi to metabolize ceramide in different cellular compartments. Supporting Evidence: PMID:30154232 nCDase was found to be located in both the plasma membrane and in the Golgi apparatus |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation based on UniProt subcellular location mapping. Consistent with the enzyme's type II membrane topology with extracellular catalytic domain and its presence in exosomes. Reason: ASAH2 is a type II membrane protein with its catalytic domain facing the extracellular/lumenal space. It is also secreted via exosomes [PMID:24798654] and released into the intestinal lumen as a soluble form [PMID:17475390]. Supporting Evidence: PMID:24798654 cytokines at a low concentration stimulated neutral ceramidase (NCDase) release via exosomes from INS-1 cells |
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: IEA annotation from ARBA machine learning. This is a very broad localization term. ASAH2 has a short cytoplasmic N-terminal region (residues 1-12) but the bulk of the protein and catalytic domain is extracellular/lumenal. Reason: While ASAH2 has a small cytoplasmic tail, the term "cytoplasm" is too general and does not reflect the enzyme's primary localization at membranes (plasma membrane, Golgi). The catalytic domain is entirely extracellular/lumenal. This is not wrong but not particularly informative for this enzyme. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | UNDECIDED | Summary: IEA annotation based on UniProt subcellular location. The original PMID:10781606 reported mitochondrial localization using GFP-tagged constructs, but this could not be confirmed in subsequent studies [PMID:15845354]. Reason: Mitochondrial localization was proposed based on GFP-fusion experiments [PMID:10781606] but could not be confirmed in later studies. UniProt notes this caution. The IEA annotation derives from this disputed finding. The mouse and rat orthologs may show mitochondrial localization, but human ASAH2 localization to mitochondria remains uncertain. |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation consistent with experimental IDA evidence. ASAH2 is a type II membrane protein localized to plasma membrane. Reason: Plasma membrane localization is well-established experimentally [PMID:30154232, PMID:15845354]. As a type II membrane protein, ASAH2 is anchored in the plasma membrane with its catalytic domain facing extracellularly. This is a core localization. Supporting Evidence: PMID:30154232 nCDase was found to be located in both the plasma membrane and in the Golgi apparatus |
| GO:0005901 caveola | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation based on UniProt subcellular location. Caveolae are specialized plasma membrane microdomains enriched in sphingolipids. Reason: Caveolar localization is inferred from mouse ortholog studies (ISS evidence). As a plasma membrane ceramidase, localization to caveolae (which are enriched in sphingolipids) is biochemically sensible. Supported by ISS from mouse ortholog Q9JHE3. |
| GO:0006629 lipid metabolic process | IEA GO_REF:0000043 | ACCEPT | Summary: IEA annotation from UniProt keyword mapping. This is a high-level parent term; more specific terms (ceramide catabolism, sphingolipid metabolism) better describe ASAH2's function. Reason: ASAH2 is clearly involved in lipid metabolism as a ceramidase. While this term is broad, it is accurate. The more specific child terms (ceramide catabolic process, sphingolipid metabolic process) provide better annotation precision, but this parent term is not wrong. |
| GO:0006665 sphingolipid metabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation from combined automated methods. Sphingolipid metabolism is a core function of ASAH2 as a ceramidase enzyme. Reason: ASAH2 is central to sphingolipid metabolism - it hydrolyzes ceramides (sphingolipids) to sphingosine and can catalyze the reverse reaction. This is a core pathway annotation. Supporting Evidence: PMID:17475390 Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut |
| GO:0006915 apoptotic process | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: IEA annotation from UniProt keyword (Apoptosis). This is a classic OVER-ANNOTATION case. ASAH2 is not directly involved in apoptotic machinery; rather, it metabolizes ceramide (which happens to be a pro-apoptotic signal). The anti-apoptotic effect is a downstream metabolic consequence, not a direct function. Reason: This annotation represents the "causal downstream effect" pattern of over-annotation. ASAH2's core function is ceramide hydrolysis for sphingolipid metabolism (especially dietary sphingolipid digestion in intestine). Ceramide happens to be a pro-apoptotic lipid, and by reducing ceramide levels while increasing sphingosine/S1P (pro-survival), ASAH2 can shift the balance away from apoptosis. However, ASAH2 did not evolve TO regulate apoptosis - it evolved to metabolize ceramides. The enzyme has no direct role in apoptotic machinery (caspases, Bcl-2 family, death receptors, etc.). The keyword "Apoptosis" in UniProt leads to this over-broad annotation. Supporting Evidence: PMID:15946935 Because the dynamic balance between the intracellular levels of ceramide and S1P (the "ceramide/S1P rheostat") may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-alpha-induced apoptosis of primary hepatocytes |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | ACCEPT | Summary: IEA annotation from UniProt keyword mapping. This is a very general parent term for the more specific ceramidase activity. Reason: ASAH2 is indeed a hydrolase - it hydrolyzes the amide bond in ceramide. While this is a general term, it is accurate. The more specific child term GO:0017040 (N-acylsphingosine amidohydrolase activity) provides better specificity. |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation from combined automated methods including InterPro and RHEA mappings. This is the correct specific molecular function term for ceramidase activity. Reason: This annotation is correct and represents the core molecular function of ASAH2. Supported by extensive experimental evidence with IDA and IMP codes as well. The IEA annotation is consistent with the experimental evidence. |
| GO:0045121 membrane raft | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation based on UniProt subcellular location. Membrane rafts (lipid rafts) are cholesterol and sphingolipid-enriched microdomains. Reason: Membrane raft localization is inferred from ortholog studies. As a sphingolipid-metabolizing enzyme, localization to lipid rafts (which are enriched in sphingolipids) is biochemically appropriate. |
| GO:0046512 sphingosine biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: IEA annotation from ARBA machine learning. Consistent with experimental evidence (IMP) showing ASAH2 involvement in sphingosine production. Reason: ASAH2 generates sphingosine as the primary product of ceramide hydrolysis. This IEA annotation is consistent with experimental IMP evidence from PMID:30154232. |
| GO:0046513 ceramide biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: IEA annotation from ARBA machine learning. ASAH2 can catalyze the reverse reaction to synthesize ceramide from sphingosine and fatty acids. Reason: The reverse (synthetic) activity of ASAH2 has been experimentally characterized [PMID:11278489, PMID:17475390]. The enzyme can synthesize ceramide in a CoA-independent manner. This is a documented activity, though the hydrolytic direction is the primary physiological function. Supporting Evidence: PMID:11278489 the same enzyme is able to catalyze the reverse reaction of ceramide synthesis PMID:17475390 The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide |
| GO:0046514 ceramide catabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation from combined automated methods. Ceramide catabolism is the core biological process of ASAH2. Reason: Ceramide catabolism is the defining biological function of ASAH2. This IEA annotation is consistent with the IBA and experimental evidence. |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | ACCEPT | Summary: IEA annotation from UniProt keyword mapping. ASAH2 binds Zn2+ (essential for catalysis) and Ca2+ (structural/regulatory). Reason: ASAH2 is a metalloenzyme requiring Zn2+ for catalysis. The crystal structure [PMID:26190575] shows Zn2+ coordination at the active site and Ca2+ binding. More specific terms (zinc ion binding, calcium ion binding) are annotated with IDA evidence. Supporting Evidence: PMID:26190575 nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation based on sequence similarity to mouse ortholog Q9JHE3. Consistent with IDA evidence from human studies. Reason: Plasma membrane localization is well-established for ASAH2. The ISS annotation from mouse ortholog is consistent with direct experimental evidence in human [PMID:30154232]. |
| GO:0005901 caveola | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation based on sequence similarity to mouse ortholog Q9JHE3. Caveolae are plasma membrane invaginations enriched in sphingolipids. Reason: Caveolar localization is inferred from well-characterized mouse ortholog. Given ASAH2's role in sphingolipid metabolism and plasma membrane localization, this is biochemically plausible. |
| GO:0044241 lipid digestion | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation based on sequence similarity to mouse ortholog. ASAH2 plays a key role in dietary sphingolipid digestion at the intestinal brush border. Reason: Lipid digestion is a primary physiological function of ASAH2 in the intestine. The enzyme is highly expressed at the brush border where it degrades dietary sphingolipids [PMID:17475390]. Mouse knockout studies confirm this role. Supporting Evidence: PMID:17475390 neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut |
| GO:0045121 membrane raft | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation based on sequence similarity to rat ortholog Q91XT9. Uses colocalizes_with qualifier indicating association rather than integral component. Reason: Membrane raft association is biochemically sensible for a sphingolipid- metabolizing enzyme. The colocalizes_with qualifier appropriately indicates co-localization rather than being an integral component. |
| GO:0070062 extracellular exosome | IDA PMID:24798654 Low-dose cytokine-induced neutral ceramidase secretion from ... | ACCEPT | Summary: IDA annotation based on experimental detection of ASAH2 in exosomes secreted from INS-1 cells upon cytokine stimulation. Reason: The study [PMID:24798654] directly demonstrated that neutral ceramidase is secreted via exosomes upon low-dose cytokine treatment. This represents a mechanism for extracellular release of the enzyme. Supporting Evidence: PMID:24798654 cytokines at a low concentration stimulated neutral ceramidase (NCDase) release via exosomes from INS-1 cells |
| GO:0071345 cellular response to cytokine stimulus | IDA PMID:24798654 Low-dose cytokine-induced neutral ceramidase secretion from ... | KEEP AS NON CORE | Summary: IDA annotation based on ASAH2 secretion being induced by cytokine treatment. The enzyme's release via exosomes is modulated by cytokine concentration. Reason: While this is a valid observation from PMID:24798654, it represents a regulatory response in a specific cell type (INS-1 cells) rather than a core function of ASAH2. The cytokine-induced secretion is a context- specific phenomenon that may be relevant in beta cell biology but not the enzyme's primary physiological role. Supporting Evidence: PMID:24798654 We also found that cytokines at a low concentration stimulated neutral ceramidase (NCDase) release via exosomes from INS-1 cells, whereas cytokines at a high concentration inhibited NCDase release |
| GO:0005794 Golgi apparatus | IDA PMID:30154232 Functions of neutral ceramidase in the Golgi apparatus. | ACCEPT | Summary: IDA annotation based on direct experimental localization of ASAH2 to Golgi apparatus in HCT116 colorectal cancer cells. Reason: The study [PMID:30154232] directly demonstrated ASAH2 localization to the Golgi apparatus where it metabolizes ceramide. This is a well- characterized localization site for the enzyme. Supporting Evidence: PMID:30154232 nCDase was found to be located in both the plasma membrane and in the Golgi apparatus |
| GO:0005886 plasma membrane | IDA PMID:30154232 Functions of neutral ceramidase in the Golgi apparatus. | ACCEPT | Summary: IDA annotation based on direct experimental localization of ASAH2 to plasma membrane in HCT116 cells. Reason: Plasma membrane localization is well-documented. ASAH2 is a type II membrane protein anchored at the plasma membrane with its catalytic domain facing the extracellular space. Supporting Evidence: PMID:30154232 nCDase was found to be located in both the plasma membrane and in the Golgi apparatus |
| GO:0007346 regulation of mitotic cell cycle | IMP PMID:19345744 Downregulation of neutral ceramidase by gemcitabine: Implica... | KEEP AS NON CORE | Summary: IMP annotation based on siRNA knockdown experiments showing that ASAH2 depletion causes cell cycle arrest at G0/G1 phase. Reason: This represents a downstream effect of ceramide accumulation when ASAH2 is depleted, rather than a direct role in cell cycle machinery. The enzyme does not directly regulate cyclins, CDKs, or checkpoint proteins. Rather, ceramide elevation (due to reduced catabolism) leads to Rb dephosphorylation and cell cycle arrest. This is a metabolic consequence rather than a core cell cycle regulatory function. Supporting Evidence: PMID:19345744 NCDase siRNA transfection was sufficient to induce a cell cycle arrest at G(0)/G(1) and an increase in total ceramide levels |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IDA PMID:30154232 Functions of neutral ceramidase in the Golgi apparatus. | ACCEPT | Summary: IDA annotation based on direct enzymatic assay demonstrating ceramidase activity of ASAH2 in HCT116 cells and purified enzyme. Reason: This is the core molecular function of ASAH2. Direct enzymatic characterization confirms the ceramidase activity. Supporting Evidence: PMID:30154232 Aug 28. Functions of neutral ceramidase in the Golgi apparatus. |
| GO:0046512 sphingosine biosynthetic process | IMP PMID:30154232 Functions of neutral ceramidase in the Golgi apparatus. | ACCEPT | Summary: IMP annotation based on showing that ASAH2 overexpression increases sphingosine levels when cells are treated with ceramide. Reason: Sphingosine production from ceramide is the direct enzymatic function of ASAH2. The IMP evidence shows functional consequence of the enzyme's activity in cells. Supporting Evidence: PMID:30154232 Cells overexpressing nCDase... showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine |
| GO:0046514 ceramide catabolic process | IMP PMID:19345744 Downregulation of neutral ceramidase by gemcitabine: Implica... | ACCEPT | Summary: IMP annotation based on showing that ASAH2 knockdown leads to increased ceramide levels, demonstrating its role in ceramide degradation. Reason: This IMP evidence demonstrates the functional consequence of ASAH2 loss - ceramide accumulation - confirming its role in ceramide catabolism. Supporting Evidence: PMID:19345744 NCDase siRNA transfection was sufficient to induce... an increase in total ceramide levels |
| GO:0046514 ceramide catabolic process | IMP PMID:30154232 Functions of neutral ceramidase in the Golgi apparatus. | ACCEPT | Summary: IMP annotation based on showing that ASAH2 overexpression reduces C6-ceramide levels in HCT116 cells. Reason: Demonstrates ASAH2's ability to catabolize ceramide in a cellular context. Core biological process function. Supporting Evidence: PMID:30154232 Cells overexpressing nCDase... showed reduced levels of C6-ceramide |
| GO:0005739 mitochondrion | IDA PMID:10781606 Molecular cloning and characterization of a human mitochondr... | UNDECIDED | Summary: IDA annotation based on GFP-ceramidase fusion protein showing mitochondrial localization pattern. However, this was later disputed. Reason: The original study [PMID:10781606] reported mitochondrial localization using a GFP-tagged construct. However, subsequent work [PMID:15845354] could not confirm this localization for human ASAH2. UniProt notes this discrepancy. The GFP tag may have artifactually altered localization. Mouse and rat orthologs may have genuine mitochondrial localization. Supporting Evidence: PMID:10781606 the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern |
| GO:0006670 sphingosine metabolic process | IMP PMID:10781606 Molecular cloning and characterization of a human mitochondr... | ACCEPT | Summary: IMP annotation based on overexpression increasing ceramidase activity and demonstrating sphingosine production from ceramide hydrolysis. Reason: ASAH2 is central to sphingosine metabolism as it produces sphingosine from ceramide. This is a core function. Supporting Evidence: PMID:10781606 the enzyme catalyzes the hydrolysis of ceramide |
| GO:0006670 sphingosine metabolic process | IMP PMID:15946935 Roles for C16-ceramide and sphingosine 1-phosphate in regula... | ACCEPT | Summary: IMP annotation based on showing that ASAH2 overexpression affects sphingolipid metabolite levels in hepatocytes. Reason: The study demonstrates ASAH2's role in sphingosine/S1P generation from ceramide in hepatocytes. Supporting Evidence: PMID:15946935 the survival effect of NCDase is due to not only C16-ceramide reduction but also S1P formation |
| GO:0006670 sphingosine metabolic process | IDA PMID:16229686 Identification of a novel amidase motif in neutral ceramidas... | ACCEPT | Summary: IDA annotation based on direct enzymatic characterization showing sphingosine production from ceramide hydrolysis. Reason: Direct enzymatic assay demonstrating sphingosine production. Core metabolic function. Supporting Evidence: PMID:16229686 Identification of a novel amidase motif in neutral ceramidase. |
| GO:0006670 sphingosine metabolic process | IDA PMID:17475390 Purification and characterization of human intestinal neutra... | ACCEPT | Summary: IDA annotation based on purification and characterization of human intestinal neutral ceramidase and its sphingosine-producing activity. Reason: The study purified human intestinal ASAH2 and characterized its sphingosine-producing activity. Core enzymatic function. Supporting Evidence: PMID:17475390 Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IMP PMID:10781606 Molecular cloning and characterization of a human mitochondr... | ACCEPT | Summary: IMP annotation based on overexpression showing increased ceramidase activity in transfected cells. Reason: Functional demonstration of ceramidase activity upon overexpression. Core molecular function. Supporting Evidence: PMID:10781606 ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IMP PMID:15946935 Roles for C16-ceramide and sphingosine 1-phosphate in regula... | ACCEPT | Summary: IMP annotation based on demonstrating that ASAH2 overexpression leads to ceramide hydrolysis in hepatocytes. Reason: Functional demonstration of ceramidase activity in cellular context. Supporting Evidence: PMID:15946935 Overexpression of neutral CDase (NCDase) inhibited the TNF-alpha-induced increase of C16-ceramide |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IDA PMID:17475390 Purification and characterization of human intestinal neutra... | ACCEPT | Summary: IDA annotation based on purification and direct enzymatic characterization of human intestinal neutral ceramidase. Reason: Direct biochemical characterization of the purified enzyme with defined kinetic parameters. Core molecular function. Supporting Evidence: PMID:17475390 The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide |
| GO:0046513 ceramide biosynthetic process | IDA PMID:17475390 Purification and characterization of human intestinal neutra... | ACCEPT | Summary: IDA annotation based on demonstrating the reverse (synthetic) activity of purified human intestinal neutral ceramidase. Reason: The enzyme can catalyze ceramide synthesis in addition to hydrolysis. This reverse activity has been biochemically characterized. Supporting Evidence: PMID:17475390 The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide |
| GO:0046514 ceramide catabolic process | IMP PMID:10781606 Molecular cloning and characterization of a human mitochondr... | ACCEPT | Summary: IMP annotation based on demonstrating ceramide hydrolysis by overexpressed ASAH2. Reason: Functional demonstration of ceramide catabolism. Core biological process function. Supporting Evidence: PMID:10781606 the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range |
| GO:0046514 ceramide catabolic process | IMP PMID:15946935 Roles for C16-ceramide and sphingosine 1-phosphate in regula... | ACCEPT | Summary: IMP annotation based on showing reduced ceramide levels upon ASAH2 overexpression in hepatocytes. Reason: Demonstrates functional ceramide catabolism in cellular context. Supporting Evidence: PMID:15946935 Overexpression of neutral CDase (NCDase) inhibited the TNF-alpha-induced increase of C16-ceramide |
| GO:0046514 ceramide catabolic process | IDA PMID:16229686 Identification of a novel amidase motif in neutral ceramidas... | ACCEPT | Summary: IDA annotation based on direct biochemical characterization of ceramide hydrolysis by purified/recombinant enzyme. Reason: Direct demonstration of ceramide catabolism with defined kinetics. Supporting Evidence: PMID:16229686 the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5 |
| GO:0046514 ceramide catabolic process | IDA PMID:17475390 Purification and characterization of human intestinal neutra... | ACCEPT | Summary: IDA annotation based on characterization of purified human intestinal neutral ceramidase. Reason: Direct biochemical demonstration of ceramide catabolism. Supporting Evidence: PMID:17475390 Mar 19. Purification and characterization of human intestinal neutral ceramidase. |
| GO:2001234 negative regulation of apoptotic signaling pathway | IMP PMID:15946935 Roles for C16-ceramide and sphingosine 1-phosphate in regula... | KEEP AS NON CORE | Summary: IMP annotation based on showing that ASAH2 overexpression protects hepatocytes from TNF-alpha-induced apoptosis. Reason: While this effect is experimentally demonstrated, it represents a downstream consequence of ASAH2's metabolic activity rather than a direct role in apoptotic signaling. ASAH2 reduces pro-apoptotic ceramide and increases pro-survival S1P through its ceramidase activity - this shifts the ceramide/S1P rheostat. The enzyme does not directly inhibit apoptotic signaling components. This is a metabolic consequence, not a core function. However, it is kept (as non-core) because the effect is real and relevant in certain cellular contexts like hepatocyte stress responses. Supporting Evidence: PMID:15946935 NCDase prevented apoptosis both by reducing C16-ceramide and by activation of AKT through S1P formation |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IMP PMID:11278489 Biochemical characterization of the reverse activity of rat ... | ACCEPT | Summary: IMP annotation based on biochemical characterization of the reverse (ceramide synthase) activity, which demonstrates the bidirectional nature of the ceramidase/synthase activity. Reason: The study characterizes the reverse reaction but this demonstrates the bidirectional catalytic capability of the enzyme at the same active site. Supporting Evidence: PMID:11278489 the same enzyme is able to catalyze the reverse reaction of ceramide synthesis |
| GO:0046513 ceramide biosynthetic process | IMP PMID:11278489 Biochemical characterization of the reverse activity of rat ... | ACCEPT | Summary: IMP annotation based on demonstrating that ASAH2 can synthesize ceramide from sphingosine and fatty acids (reverse activity). Reason: The reverse ceramide synthase activity of ASAH2 is well-characterized. This is a CoA-independent ceramide synthase activity distinct from classical ceramide synthases. Supporting Evidence: PMID:11278489 A CoA-independent and fumonisin B1-insensitive ceramide synthase |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IDA PMID:16229686 Identification of a novel amidase motif in neutral ceramidas... | ACCEPT | Summary: IDA annotation based on detailed biochemical characterization including identification of the novel amidase motif and catalytic residues. Reason: Definitive enzymatic characterization with identification of catalytic mechanism and key residues. Supporting Evidence: PMID:16229686 a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide |
| GO:0005509 calcium ion binding | IDA PMID:26190575 Structural Basis for Ceramide Recognition and Hydrolysis by ... | ACCEPT | Summary: IDA annotation based on crystal structure showing Ca2+ bound to ASAH2 at defined coordination sites. Reason: The crystal structure at 2.58 Angstrom resolution clearly shows Ca2+ bound to ASAH2. Ca2+ mildly stimulates enzyme activity but is not essential for catalysis (Zn2+ is the essential catalytic metal). Supporting Evidence: PMID:26190575 Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases |
| GO:0006670 sphingosine metabolic process | IDA PMID:26190575 Structural Basis for Ceramide Recognition and Hydrolysis by ... | ACCEPT | Summary: IDA annotation based on structural characterization showing how the enzyme binds and hydrolyzes ceramide to produce sphingosine. Reason: The structural study provides mechanistic insight into sphingosine production from ceramide. Supporting Evidence: PMID:26190575 Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate |
| GO:0006672 ceramide metabolic process | IDA PMID:26190575 Structural Basis for Ceramide Recognition and Hydrolysis by ... | ACCEPT | Summary: IDA annotation based on structural characterization of ceramide binding and hydrolysis mechanism. Reason: The structural study reveals how ASAH2 recognizes and metabolizes ceramide through its hydrophobic active site pocket. Supporting Evidence: PMID:26190575 generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl head group of ceramide |
| GO:0008270 zinc ion binding | IDA PMID:26190575 Structural Basis for Ceramide Recognition and Hydrolysis by ... | ACCEPT | Summary: IDA annotation based on crystal structure showing Zn2+ coordination at the active site, essential for catalysis. Reason: The crystal structure definitively shows Zn2+ at the active site, coordinated by His194, His196, His303, and Glu540. Zn2+ is essential for the amidase catalytic mechanism. Supporting Evidence: PMID:26190575 nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IDA PMID:26190575 Structural Basis for Ceramide Recognition and Hydrolysis by ... | ACCEPT | Summary: IDA annotation based on definitive structural and biochemical characterization of the ceramidase activity mechanism. Reason: The crystal structure provides molecular-level understanding of the ceramidase catalytic mechanism. Supporting Evidence: PMID:26190575 Structural Basis for Ceramide Recognition and Hydrolysis by Human Neutral Ceramidase |
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Download this section (compressed HTML)Q: What is the relative contribution of ASAH2 vs. ASAH1 (acid ceramidase) to cellular ceramide homeostasis in different tissues?
Q: Does ASAH2 have physiologically relevant mitochondrial localization in humans, or is this specific to rodent orthologs?
Q: What are the specific ceramide species (chain lengths) preferred by human ASAH2 in intestinal vs. other tissue contexts?
Experiment: Endogenous localization studies of ASAH2 in human intestinal tissue using validated antibodies without overexpression
Experiment: Lipidomics analysis comparing ceramide/sphingosine profiles in ASAH2 knockout vs. wild-type human intestinal organoids
Experiment: In vivo studies of dietary sphingolipid absorption in tissue-specific ASAH2 knockout models
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