RGD Reference Report - Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney. - Rat Genome Database

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Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney.

Authors: Mitsutake, S  Tani, M  Okino, N  Mori, K  Ichinose, S  Omori, A  Iida, H  Nakamura, T  Ito, M 
Citation: Mitsutake S, etal., J Biol Chem 2001 Jul 13;276(28):26249-59.
RGD ID: 68675
Pubmed: PMID:11328816   (View Abstract at PubMed)
DOI: DOI:10.1074/jbc.M102233200   (Journal Full-text)

Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229--11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.

Gene Ontology Annotations    Click to see Annotation Detail View

Cellular Component

Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Asah2Ratmembrane raft located_inIDA PMID:11328816UniProt 
Asah2Ratplasma membrane located_inIDA PMID:11328816UniProt 

Molecular Function

Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Asah2RatN-acylsphingosine amidohydrolase activity enablesIDA PMID:11328816UniProt 
Asah2Ratobsolete dihydroceramidase activity enablesIDA PMID:11328816UniProt 
Asah2Ratobsolete phytoceramidase activity enablesIDA PMID:11328816UniProt 

Objects Annotated

Genes (Rattus norvegicus)
Asah2  (N-acylsphingosine amidohydrolase 2)

Objects referenced in this article
Gene Asah2 N-acylsphingosine amidohydrolase 2 Mus musculus

Additional Information