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

Previously, we reported two types of neutral ceramidase in mice, one solubi lized by freeze-thawing and one not. The former was purified as a 94-kDa pr otein 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 di stribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, wh ich was completely insoluble by freeze-thawing. The open reading frame of t he enzyme encoded a polypeptide of 761 amino acids having nine putative N-g lycosylation 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 pr oteins were converted to a 87-kDa protein concomitantly with loss of activi ty when expressed in the presence of tunicamycin, indicating that the N-gly cosylation process is indispensable for the expression of the enzyme activi ty. Immunohistochemical analysis clearly showed that the ceramidase was mai nly localized at the apical membrane of proximal tubules, distal tubules, a nd collecting ducts in rat kidney, while in liver the enzyme was distribute d with endosome-like organelles in hepatocytes. Interestingly, the kidney c eramidase was found to be enriched in the raft microdomains with cholestero l and GM1 ganglioside.