Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney.
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.
Mitsutake
S
S
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Tani
M
M
Okino
N
N
Mori
K
K
Ichinose
S
S
Omori
A
A
Iida
H
H
Nakamura
T
T
Ito
M
M
D000581:Amidohydrolases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055572:Ceramidases; D003001:Cloning, Molecular; D004789:Enzyme Activation; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D051381:Rats; D016415:Sequence Alignment; D017421:Sequence Analysis
D000581:Amidohydrolases; C526747:Asah2 protein, mouse; C526748:Asah2 protein, rat; D055572:Ceramidases; D055576:Neutral Ceramidase