Subcellular localization of human neutral ceramidase expressed in HEK293 cells.
We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.
Hwang
Young-ha
YH
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
Motohiro
M
Nakagawa
Tetsuto
T
Okino
Nozomu
N
Ito
Makoto
M
D000581:Amidohydrolases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D055572:Ceramidases; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D016415:Sequence Alignment
D000581:Amidohydrolases; C526744:ASAH2 protein, human; C526747:Asah2 protein, mouse; C526748:Asah2 protein, rat; D055572:Ceramidases; D055576:Neutral Ceramidase