id: Q9NR71
gene_symbol: ASAH2
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  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.
existing_annotations:
  - term:
      id: GO:0042759
      label: long-chain fatty acid biosynthetic process
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      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.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "Neutral ceramidase (nCDase) catalyzes conversion of the
            apoptosis-associated lipid ceramide to sphingosine, the precursor for
            the proliferative factor sphingosine-1-phosphate"
        - reference_id: file:human/ASAH2/ASAH2-deep-research-falcon.md
          supporting_text: 'model: Edison Scientific Literature'
  - term:
      id: GO:0046512
      label: sphingosine biosynthetic process
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      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.
      action: ACCEPT
      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].
      supported_by:
        - reference_id: PMID:10781606
          supporting_text: "the enzyme catalyzes the hydrolysis of ceramide in the
            neutral alkaline range"
        - reference_id: PMID:26190575
          supporting_text: "Neutral ceramidase (nCDase) catalyzes conversion of the
            apoptosis-associated lipid ceramide to sphingosine, the precursor for
            the proliferative factor sphingosine-1-phosphate"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        This is the precise molecular function term for ceramidase activity.
        IBA annotation well-supported by phylogenetic conservation and extensive
        experimental validation.
      action: ACCEPT
      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].
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "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"
        - reference_id: PMID:16229686
          supporting_text: "the enzyme exhibited classical Michaelis-Menten kinetics,
            with an optimum activity at pH 7.5"
  - term:
      id: GO:0005576
      label: extracellular region
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      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.
      action: ACCEPT
      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].
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "neutral ceramidase is expressed in human intestine, released
            in the intestinal lumen and plays a major role in ceramide metabolism
            in the human gut"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        Ceramide catabolism is the core biological process function of ASAH2.
        IBA annotation reflects conserved function across species.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "Sphingolipids are degraded by sphingomyelinase and ceramidase
            in the gut to ceramide and sphingosine"
        - reference_id: PMID:26190575
          supporting_text: "Neutral ceramidase (nCDase) catalyzes conversion of the
            apoptosis-associated lipid ceramide to sphingosine, the precursor for
            the proliferative factor sphingosine-1-phosphate"
  - term:
      id: GO:0000139
      label: Golgi membrane
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      summary: >-
        IEA annotation based on UniProt subcellular location mapping. Supported
        by experimental evidence showing ASAH2 localization to Golgi apparatus.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: "nCDase was found to be located in both the plasma membrane
            and in the Golgi apparatus"
  - term:
      id: GO:0005576
      label: extracellular region
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      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.
      action: ACCEPT
      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].
      supported_by:
        - reference_id: PMID:24798654
          supporting_text: "cytokines at a low concentration stimulated neutral ceramidase
            (NCDase) release via exosomes from INS-1 cells"
  - term:
      id: GO:0005737
      label: cytoplasm
    evidence_type: IEA
    original_reference_id: GO_REF:0000117
    review:
      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.
      action: KEEP_AS_NON_CORE
      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.
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      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].
      action: UNDECIDED
      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.
      additional_reference_ids:
        - PMID:15845354
  - term:
      id: GO:0005886
      label: plasma membrane
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        IEA annotation consistent with experimental IDA evidence. ASAH2 is a
        type II membrane protein localized to plasma membrane.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: "nCDase was found to be located in both the plasma membrane
            and in the Golgi apparatus"
  - term:
      id: GO:0005901
      label: caveola
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      summary: >-
        IEA annotation based on UniProt subcellular location. Caveolae are
        specialized plasma membrane microdomains enriched in sphingolipids.
      action: ACCEPT
      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.
  - term:
      id: GO:0006629
      label: lipid metabolic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      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.
      action: ACCEPT
      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.
  - term:
      id: GO:0006665
      label: sphingolipid metabolic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        IEA annotation from combined automated methods. Sphingolipid metabolism
        is a core function of ASAH2 as a ceramidase enzyme.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "Sphingolipids are degraded by sphingomyelinase and ceramidase
            in the gut"
  - term:
      id: GO:0006915
      label: apoptotic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      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.
      action: MARK_AS_OVER_ANNOTATED
      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.
      supported_by:
        - reference_id: PMID:15946935
          supporting_text: "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"
  - term:
      id: GO:0016787
      label: hydrolase activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        IEA annotation from UniProt keyword mapping. This is a very general
        parent term for the more specific ceramidase activity.
      action: ACCEPT
      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.
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        IEA annotation from combined automated methods including InterPro and
        RHEA mappings. This is the correct specific molecular function term
        for ceramidase activity.
      action: ACCEPT
      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.
  - term:
      id: GO:0045121
      label: membrane raft
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      summary: >-
        IEA annotation based on UniProt subcellular location. Membrane rafts
        (lipid rafts) are cholesterol and sphingolipid-enriched microdomains.
      action: ACCEPT
      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.
  - term:
      id: GO:0046512
      label: sphingosine biosynthetic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000117
    review:
      summary: >-
        IEA annotation from ARBA machine learning. Consistent with experimental
        evidence (IMP) showing ASAH2 involvement in sphingosine production.
      action: ACCEPT
      reason: >-
        ASAH2 generates sphingosine as the primary product of ceramide hydrolysis.
        This IEA annotation is consistent with experimental IMP evidence from
        PMID:30154232.
  - term:
      id: GO:0046513
      label: ceramide biosynthetic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000117
    review:
      summary: >-
        IEA annotation from ARBA machine learning. ASAH2 can catalyze the reverse
        reaction to synthesize ceramide from sphingosine and fatty acids.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:11278489
          supporting_text: "the same enzyme is able to catalyze the reverse reaction
            of ceramide synthesis"
        - reference_id: PMID:17475390
          supporting_text: "The enzyme has neutral pH optimum and catalyses both hydrolysis
            and formation of ceramide"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        IEA annotation from combined automated methods. Ceramide catabolism is
        the core biological process of ASAH2.
      action: ACCEPT
      reason: >-
        Ceramide catabolism is the defining biological function of ASAH2. This
        IEA annotation is consistent with the IBA and experimental evidence.
  - term:
      id: GO:0046872
      label: metal ion binding
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        IEA annotation from UniProt keyword mapping. ASAH2 binds Zn2+ (essential
        for catalysis) and Ca2+ (structural/regulatory).
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "nCDase uses a new catalytic strategy for Zn(2+)-dependent
            amidases"
  - term:
      id: GO:0005886
      label: plasma membrane
    evidence_type: ISS
    original_reference_id: GO_REF:0000024
    review:
      summary: >-
        ISS annotation based on sequence similarity to mouse ortholog Q9JHE3.
        Consistent with IDA evidence from human studies.
      action: ACCEPT
      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].
  - term:
      id: GO:0005901
      label: caveola
    evidence_type: ISS
    original_reference_id: GO_REF:0000024
    review:
      summary: >-
        ISS annotation based on sequence similarity to mouse ortholog Q9JHE3.
        Caveolae are plasma membrane invaginations enriched in sphingolipids.
      action: ACCEPT
      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.
  - term:
      id: GO:0044241
      label: lipid digestion
    evidence_type: ISS
    original_reference_id: GO_REF:0000024
    review:
      summary: >-
        ISS annotation based on sequence similarity to mouse ortholog. ASAH2
        plays a key role in dietary sphingolipid digestion at the intestinal
        brush border.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "neutral ceramidase is expressed in human intestine, released
            in the intestinal lumen and plays a major role in ceramide metabolism
            in the human gut"
  - term:
      id: GO:0045121
      label: membrane raft
    evidence_type: ISS
    original_reference_id: GO_REF:0000024
    review:
      summary: >-
        ISS annotation based on sequence similarity to rat ortholog Q91XT9.
        Uses colocalizes_with qualifier indicating association rather than
        integral component.
      action: ACCEPT
      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.
  - term:
      id: GO:0070062
      label: extracellular exosome
    evidence_type: IDA
    original_reference_id: PMID:24798654
    review:
      summary: >-
        IDA annotation based on experimental detection of ASAH2 in exosomes
        secreted from INS-1 cells upon cytokine stimulation.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:24798654
          supporting_text: "cytokines at a low concentration stimulated neutral ceramidase
            (NCDase) release via exosomes from INS-1 cells"
  - term:
      id: GO:0071345
      label: cellular response to cytokine stimulus
    evidence_type: IDA
    original_reference_id: PMID:24798654
    review:
      summary: >-
        IDA annotation based on ASAH2 secretion being induced by cytokine
        treatment. The enzyme's release via exosomes is modulated by cytokine
        concentration.
      action: KEEP_AS_NON_CORE
      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.
      supported_by:
        - reference_id: PMID:24798654
          supporting_text: "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"
  - term:
      id: GO:0005794
      label: Golgi apparatus
    evidence_type: IDA
    original_reference_id: PMID:30154232
    review:
      summary: >-
        IDA annotation based on direct experimental localization of ASAH2 to
        Golgi apparatus in HCT116 colorectal cancer cells.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: "nCDase was found to be located in both the plasma membrane
            and in the Golgi apparatus"
  - term:
      id: GO:0005886
      label: plasma membrane
    evidence_type: IDA
    original_reference_id: PMID:30154232
    review:
      summary: >-
        IDA annotation based on direct experimental localization of ASAH2 to
        plasma membrane in HCT116 cells.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: "nCDase was found to be located in both the plasma membrane
            and in the Golgi apparatus"
  - term:
      id: GO:0007346
      label: regulation of mitotic cell cycle
    evidence_type: IMP
    original_reference_id: PMID:19345744
    review:
      summary: >-
        IMP annotation based on siRNA knockdown experiments showing that ASAH2
        depletion causes cell cycle arrest at G0/G1 phase.
      action: KEEP_AS_NON_CORE
      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.
      supported_by:
        - reference_id: PMID:19345744
          supporting_text: "NCDase siRNA transfection was sufficient to induce a cell
            cycle arrest at G(0)/G(1) and an increase in total ceramide levels"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IDA
    original_reference_id: PMID:30154232
    review:
      summary: >-
        IDA annotation based on direct enzymatic assay demonstrating ceramidase
        activity of ASAH2 in HCT116 cells and purified enzyme.
      action: ACCEPT
      reason: >-
        This is the core molecular function of ASAH2. Direct enzymatic
        characterization confirms the ceramidase activity.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: Aug 28. Functions of neutral ceramidase in the Golgi 
            apparatus.
  - term:
      id: GO:0046512
      label: sphingosine biosynthetic process
    evidence_type: IMP
    original_reference_id: PMID:30154232
    review:
      summary: >-
        IMP annotation based on showing that ASAH2 overexpression increases
        sphingosine levels when cells are treated with ceramide.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: "Cells overexpressing nCDase... showed reduced levels of
            C6-ceramide and higher levels of S1P and sphingosine"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IMP
    original_reference_id: PMID:19345744
    review:
      summary: >-
        IMP annotation based on showing that ASAH2 knockdown leads to increased
        ceramide levels, demonstrating its role in ceramide degradation.
      action: ACCEPT
      reason: >-
        This IMP evidence demonstrates the functional consequence of ASAH2
        loss - ceramide accumulation - confirming its role in ceramide catabolism.
      supported_by:
        - reference_id: PMID:19345744
          supporting_text: "NCDase siRNA transfection was sufficient to induce...
            an increase in total ceramide levels"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IMP
    original_reference_id: PMID:30154232
    review:
      summary: >-
        IMP annotation based on showing that ASAH2 overexpression reduces
        C6-ceramide levels in HCT116 cells.
      action: ACCEPT
      reason: >-
        Demonstrates ASAH2's ability to catabolize ceramide in a cellular
        context. Core biological process function.
      supported_by:
        - reference_id: PMID:30154232
          supporting_text: "Cells overexpressing nCDase... showed reduced levels of
            C6-ceramide"
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IDA
    original_reference_id: PMID:10781606
    review:
      summary: >-
        IDA annotation based on GFP-ceramidase fusion protein showing
        mitochondrial localization pattern. However, this was later disputed.
      action: UNDECIDED
      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.
      additional_reference_ids:
        - PMID:15845354
      supported_by:
        - reference_id: PMID:10781606
          supporting_text: "the green fluorescent protein-ceramidase fusion protein
            presented a mitochondrial localization pattern"
  - term:
      id: GO:0006670
      label: sphingosine metabolic process
    evidence_type: IMP
    original_reference_id: PMID:10781606
    review:
      summary: >-
        IMP annotation based on overexpression increasing ceramidase activity
        and demonstrating sphingosine production from ceramide hydrolysis.
      action: ACCEPT
      reason: >-
        ASAH2 is central to sphingosine metabolism as it produces sphingosine
        from ceramide. This is a core function.
      supported_by:
        - reference_id: PMID:10781606
          supporting_text: "the enzyme catalyzes the hydrolysis of ceramide"
  - term:
      id: GO:0006670
      label: sphingosine metabolic process
    evidence_type: IMP
    original_reference_id: PMID:15946935
    review:
      summary: >-
        IMP annotation based on showing that ASAH2 overexpression affects
        sphingolipid metabolite levels in hepatocytes.
      action: ACCEPT
      reason: >-
        The study demonstrates ASAH2's role in sphingosine/S1P generation
        from ceramide in hepatocytes.
      supported_by:
        - reference_id: PMID:15946935
          supporting_text: "the survival effect of NCDase is due to not only C16-ceramide
            reduction but also S1P formation"
  - term:
      id: GO:0006670
      label: sphingosine metabolic process
    evidence_type: IDA
    original_reference_id: PMID:16229686
    review:
      summary: >-
        IDA annotation based on direct enzymatic characterization showing
        sphingosine production from ceramide hydrolysis.
      action: ACCEPT
      reason: >-
        Direct enzymatic assay demonstrating sphingosine production. Core
        metabolic function.
      supported_by:
        - reference_id: PMID:16229686
          supporting_text: Identification of a novel amidase motif in neutral 
            ceramidase.
  - term:
      id: GO:0006670
      label: sphingosine metabolic process
    evidence_type: IDA
    original_reference_id: PMID:17475390
    review:
      summary: >-
        IDA annotation based on purification and characterization of human
        intestinal neutral ceramidase and its sphingosine-producing activity.
      action: ACCEPT
      reason: >-
        The study purified human intestinal ASAH2 and characterized its
        sphingosine-producing activity. Core enzymatic function.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "Sphingolipids are degraded by sphingomyelinase and ceramidase
            in the gut to ceramide and sphingosine"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IMP
    original_reference_id: PMID:10781606
    review:
      summary: >-
        IMP annotation based on overexpression showing increased ceramidase
        activity in transfected cells.
      action: ACCEPT
      reason: >-
        Functional demonstration of ceramidase activity upon overexpression.
        Core molecular function.
      supported_by:
        - reference_id: PMID:10781606
          supporting_text: "ceramidase activity (at pH 9.5) increased by 50- and 12-fold,
            respectively"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IMP
    original_reference_id: PMID:15946935
    review:
      summary: >-
        IMP annotation based on demonstrating that ASAH2 overexpression leads
        to ceramide hydrolysis in hepatocytes.
      action: ACCEPT
      reason: >-
        Functional demonstration of ceramidase activity in cellular context.
      supported_by:
        - reference_id: PMID:15946935
          supporting_text: "Overexpression of neutral CDase (NCDase) inhibited the
            TNF-alpha-induced increase of C16-ceramide"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IDA
    original_reference_id: PMID:17475390
    review:
      summary: >-
        IDA annotation based on purification and direct enzymatic
        characterization of human intestinal neutral ceramidase.
      action: ACCEPT
      reason: >-
        Direct biochemical characterization of the purified enzyme with
        defined kinetic parameters. Core molecular function.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "The enzyme has neutral pH optimum and catalyses both hydrolysis
            and formation of ceramide"
  - term:
      id: GO:0046513
      label: ceramide biosynthetic process
    evidence_type: IDA
    original_reference_id: PMID:17475390
    review:
      summary: >-
        IDA annotation based on demonstrating the reverse (synthetic) activity
        of purified human intestinal neutral ceramidase.
      action: ACCEPT
      reason: >-
        The enzyme can catalyze ceramide synthesis in addition to hydrolysis.
        This reverse activity has been biochemically characterized.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: "The enzyme has neutral pH optimum and catalyses both hydrolysis
            and formation of ceramide"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IMP
    original_reference_id: PMID:10781606
    review:
      summary: >-
        IMP annotation based on demonstrating ceramide hydrolysis by
        overexpressed ASAH2.
      action: ACCEPT
      reason: >-
        Functional demonstration of ceramide catabolism. Core biological
        process function.
      supported_by:
        - reference_id: PMID:10781606
          supporting_text: "the enzyme catalyzes the hydrolysis of ceramide in the
            neutral alkaline range"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IMP
    original_reference_id: PMID:15946935
    review:
      summary: >-
        IMP annotation based on showing reduced ceramide levels upon ASAH2
        overexpression in hepatocytes.
      action: ACCEPT
      reason: >-
        Demonstrates functional ceramide catabolism in cellular context.
      supported_by:
        - reference_id: PMID:15946935
          supporting_text: "Overexpression of neutral CDase (NCDase) inhibited the
            TNF-alpha-induced increase of C16-ceramide"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IDA
    original_reference_id: PMID:16229686
    review:
      summary: >-
        IDA annotation based on direct biochemical characterization of
        ceramide hydrolysis by purified/recombinant enzyme.
      action: ACCEPT
      reason: >-
        Direct demonstration of ceramide catabolism with defined kinetics.
      supported_by:
        - reference_id: PMID:16229686
          supporting_text: "the enzyme exhibited classical Michaelis-Menten kinetics,
            with an optimum activity at pH 7.5"
  - term:
      id: GO:0046514
      label: ceramide catabolic process
    evidence_type: IDA
    original_reference_id: PMID:17475390
    review:
      summary: >-
        IDA annotation based on characterization of purified human intestinal
        neutral ceramidase.
      action: ACCEPT
      reason: >-
        Direct biochemical demonstration of ceramide catabolism.
      supported_by:
        - reference_id: PMID:17475390
          supporting_text: Mar 19. Purification and characterization of human 
            intestinal neutral ceramidase.
  - term:
      id: GO:2001234
      label: negative regulation of apoptotic signaling pathway
    evidence_type: IMP
    original_reference_id: PMID:15946935
    review:
      summary: >-
        IMP annotation based on showing that ASAH2 overexpression protects
        hepatocytes from TNF-alpha-induced apoptosis.
      action: KEEP_AS_NON_CORE
      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.
      supported_by:
        - reference_id: PMID:15946935
          supporting_text: "NCDase prevented apoptosis both by reducing C16-ceramide
            and by activation of AKT through S1P formation"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IMP
    original_reference_id: PMID:11278489
    review:
      summary: >-
        IMP annotation based on biochemical characterization of the reverse
        (ceramide synthase) activity, which demonstrates the bidirectional
        nature of the ceramidase/synthase activity.
      action: ACCEPT
      reason: >-
        The study characterizes the reverse reaction but this demonstrates
        the bidirectional catalytic capability of the enzyme at the same
        active site.
      supported_by:
        - reference_id: PMID:11278489
          supporting_text: "the same enzyme is able to catalyze the reverse reaction
            of ceramide synthesis"
  - term:
      id: GO:0046513
      label: ceramide biosynthetic process
    evidence_type: IMP
    original_reference_id: PMID:11278489
    review:
      summary: >-
        IMP annotation based on demonstrating that ASAH2 can synthesize
        ceramide from sphingosine and fatty acids (reverse activity).
      action: ACCEPT
      reason: >-
        The reverse ceramide synthase activity of ASAH2 is well-characterized.
        This is a CoA-independent ceramide synthase activity distinct from
        classical ceramide synthases.
      supported_by:
        - reference_id: PMID:11278489
          supporting_text: "A CoA-independent and fumonisin B1-insensitive ceramide
            synthase"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IDA
    original_reference_id: PMID:16229686
    review:
      summary: >-
        IDA annotation based on detailed biochemical characterization including
        identification of the novel amidase motif and catalytic residues.
      action: ACCEPT
      reason: >-
        Definitive enzymatic characterization with identification of catalytic
        mechanism and key residues.
      supported_by:
        - reference_id: PMID:16229686
          supporting_text: "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"
  - term:
      id: GO:0005509
      label: calcium ion binding
    evidence_type: IDA
    original_reference_id: PMID:26190575
    review:
      summary: >-
        IDA annotation based on crystal structure showing Ca2+ bound to ASAH2
        at defined coordination sites.
      action: ACCEPT
      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).
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "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"
  - term:
      id: GO:0006670
      label: sphingosine metabolic process
    evidence_type: IDA
    original_reference_id: PMID:26190575
    review:
      summary: >-
        IDA annotation based on structural characterization showing how the
        enzyme binds and hydrolyzes ceramide to produce sphingosine.
      action: ACCEPT
      reason: >-
        The structural study provides mechanistic insight into sphingosine
        production from ceramide.
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "Neutral ceramidase (nCDase) catalyzes conversion of the
            apoptosis-associated lipid ceramide to sphingosine, the precursor for
            the proliferative factor sphingosine-1-phosphate"
  - term:
      id: GO:0006672
      label: ceramide metabolic process
    evidence_type: IDA
    original_reference_id: PMID:26190575
    review:
      summary: >-
        IDA annotation based on structural characterization of ceramide
        binding and hydrolysis mechanism.
      action: ACCEPT
      reason: >-
        The structural study reveals how ASAH2 recognizes and metabolizes
        ceramide through its hydrophobic active site pocket.
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "generates ceramide specificity by sterically excluding
            sphingolipids with bulky headgroups and specifically recognizing the small
            hydroxyl head group of ceramide"
  - term:
      id: GO:0008270
      label: zinc ion binding
    evidence_type: IDA
    original_reference_id: PMID:26190575
    review:
      summary: >-
        IDA annotation based on crystal structure showing Zn2+ coordination
        at the active site, essential for catalysis.
      action: ACCEPT
      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.
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "nCDase uses a new catalytic strategy for Zn(2+)-dependent
            amidases"
  - term:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    evidence_type: IDA
    original_reference_id: PMID:26190575
    review:
      summary: >-
        IDA annotation based on definitive structural and biochemical
        characterization of the ceramidase activity mechanism.
      action: ACCEPT
      reason: >-
        The crystal structure provides molecular-level understanding of
        the ceramidase catalytic mechanism.
      supported_by:
        - reference_id: PMID:26190575
          supporting_text: "Structural Basis for Ceramide Recognition and Hydrolysis
            by Human Neutral Ceramidase"
references:
  - id: GO_REF:0000024
    title: Manual transfer of experimentally-verified manual GO annotation data 
      to orthologs by curator judgment of sequence similarity
    findings: []
  - id: GO_REF:0000033
    title: Annotation inferences using phylogenetic trees
    findings: []
  - id: GO_REF:0000043
    title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword 
      mapping
    findings: []
  - id: GO_REF:0000044
    title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular 
      Location vocabulary mapping, accompanied by conservative changes to GO 
      terms applied by UniProt
    findings: []
  - id: GO_REF:0000117
    title: Electronic Gene Ontology annotations created by ARBA machine learning
      models
    findings: []
  - id: GO_REF:0000120
    title: Combined Automated Annotation using Multiple IEA Methods
    findings: []
  - id: PMID:10781606
    title: Molecular cloning and characterization of a human mitochondrial 
      ceramidase.
    findings:
      - statement: Cloning and initial characterization of human ASAH2
      - statement: Demonstrated ceramidase activity at neutral-alkaline pH
      - statement: Reported mitochondrial localization (later disputed)
      - statement: Showed ubiquitous expression with higher levels in kidney, 
          skeletal muscle, heart
  - id: PMID:11278489
    title: Biochemical characterization of the reverse activity of rat brain 
      ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide 
      synthase.
    findings:
      - statement: ASAH2 catalyzes reverse reaction synthesizing ceramide from 
          sphingosine and fatty acids
      - statement: CoA-independent ceramide synthase activity
      - statement: Not inhibited by fumonisin B1
  - id: PMID:15946935
    title: Roles for C16-ceramide and sphingosine 1-phosphate in regulating 
      hepatocyte apoptosis in response to tumor necrosis factor-alpha.
    findings:
      - statement: ASAH2 overexpression protects hepatocytes from 
          TNF-alpha-induced apoptosis
      - statement: Protection via ceramide reduction and S1P/AKT activation
      - statement: Demonstrates ceramide/S1P rheostat in apoptosis regulation
  - id: PMID:16229686
    title: Identification of a novel amidase motif in neutral ceramidase.
    findings:
      - statement: Identified novel amidase motif with critical Ser354 residue
      - statement: Characterized kinetic parameters at pH 7.5
      - statement: Identified conserved catalytic residues through mutagenesis
  - id: PMID:17475390
    title: Purification and characterization of human intestinal neutral 
      ceramidase.
    findings:
      - statement: Purified ASAH2 from human ileostomy content
      - statement: Confirmed both hydrolytic and synthetic activities
      - statement: Demonstrated major role in ceramide metabolism in human gut
      - statement: Inhibited by Cu2+, Zn2+, and cholesterol
  - id: PMID:19345744
    title: 'Downregulation of neutral ceramidase by gemcitabine: Implications for
      cell cycle regulation.'
    findings:
      - statement: ASAH2 knockdown causes cell cycle arrest at G0/G1
      - statement: ASAH2 loss leads to ceramide accumulation
      - statement: Demonstrates link between ceramide levels and cell cycle
  - id: PMID:24798654
    title: Low-dose cytokine-induced neutral ceramidase secretion from INS-1 
      cells via exosomes and its anti-apoptotic effect.
    findings:
      - statement: ASAH2 secreted via exosomes upon low-dose cytokine treatment
      - statement: Exosomal ASAH2 has anti-apoptotic effect via S1P-S1P receptor
          2 signaling
  - id: PMID:26190575
    title: Structural Basis for Ceramide Recognition and Hydrolysis by Human 
      Neutral Ceramidase.
    findings:
      - statement: 2.6 Angstrom crystal structure of human ASAH2
      - statement: Revealed 20 Angstrom deep hydrophobic active site pocket
      - statement: Zn2+-dependent amidase mechanism
      - statement: Explained ceramide specificity via headgroup recognition
      - statement: Identified Ca2+ binding sites
  - id: PMID:30154232
    title: Functions of neutral ceramidase in the Golgi apparatus.
    findings:
      - statement: ASAH2 localized to both plasma membrane and Golgi
      - statement: Golgi ASAH2 metabolizes ceramide and protects from 
          C6-ceramide-induced cell death
      - statement: Compartmentalized sphingolipid metabolism
  - id: file:human/ASAH2/ASAH2-deep-research-falcon.md
    title: Deep research report on ASAH2
    findings: []
core_functions:
  - description: N-acylsphingosine amidohydrolase (ceramidase) activity - 
      hydrolyzes ceramide to sphingosine and fatty acid
    molecular_function:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    directly_involved_in:
      - id: GO:0046514
        label: ceramide catabolic process
      - id: GO:0046512
        label: sphingosine biosynthetic process
    locations:
      - id: GO:0005886
        label: plasma membrane
      - id: GO:0005794
        label: Golgi apparatus
  - description: Lipid digestion - physiological role in intestinal sphingolipid
      digestion at brush border
    molecular_function:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    directly_involved_in:
      - id: GO:0044241
        label: lipid digestion
    locations:
      - id: GO:0005576
        label: extracellular region
  - description: Reverse ceramide synthase activity - can synthesize ceramide 
      from sphingosine and fatty acids
    molecular_function:
      id: GO:0017040
      label: N-acylsphingosine amidohydrolase activity
    directly_involved_in:
      - id: GO:0046513
        label: ceramide biosynthetic process
proposed_new_terms: []
suggested_questions:
  - question: What is the relative contribution of ASAH2 vs. ASAH1 (acid 
      ceramidase) to cellular ceramide homeostasis in different tissues?
  - question: Does ASAH2 have physiologically relevant mitochondrial 
      localization in humans, or is this specific to rodent orthologs?
  - question: What are the specific ceramide species (chain lengths) preferred 
      by human ASAH2 in intestinal vs. other tissue contexts?
suggested_experiments:
  - description: Endogenous localization studies of ASAH2 in human intestinal 
      tissue using validated antibodies without overexpression
  - description: Lipidomics analysis comparing ceramide/sphingosine profiles in 
      ASAH2 knockout vs. wild-type human intestinal organoids
  - description: In vivo studies of dietary sphingolipid absorption in 
      tissue-specific ASAH2 knockout models