Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 26249-26259 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 276 |
| Issue number | 28 |
| DOIs | |
| Publication status | Published - Jul 13 2001 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney. / Mitsutake, Susumu; Tani, Motohiro; Okino, Nozomu; Mori, Kaoru; Ichinose, Sachiyo; Omori, Akira; Iida, Hiroshi; Nakamura, Takashi; Ito, Makoto.
In: Journal of Biological Chemistry, Vol. 276, No. 28, 13.07.2001, p. 26249-26259.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney
AU - Mitsutake, Susumu
AU - Tani, Motohiro
AU - Okino, Nozomu
AU - Mori, Kaoru
AU - Ichinose, Sachiyo
AU - Omori, Akira
AU - Iida, Hiroshi
AU - Nakamura, Takashi
AU - Ito, Makoto
PY - 2001/7/13
Y1 - 2001/7/13
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0035854705&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035854705&partnerID=8YFLogxK
U2 - 10.1074/jbc.M102233200
DO - 10.1074/jbc.M102233200
M3 - Article
C2 - 11328816
AN - SCOPUS:0035854705
VL - 276
SP - 26249
EP - 26259
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 28
ER -