Conserved Amino Acid Residues in the COOH-terminal Tail Are Indispensable for the Correct Folding and Localization and Enzyme Activity of Neutral Ceramidase

Abstract

Several lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling. Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH 2 -terminal anchoring region (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523-10530). We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase. Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells. Point mutation analysis revealed that Ile 758 , the 4 th amino acid residue from the COOH terminus, and Phe 756 are essential for the enzyme to function. The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus. Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome. It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution.

Role of Ceramidases in Sphingolipid Metabolism and Human Diseases

“…Human ASAH2 was first identified in the small intestine ( Figure 2A, lower panel) [33,60,61]. In human embryonic kidney cells, which exogenously overexpress ASAH2, the protein was found to localize to mitochondria [31].…”

Mechanistic Insights into the Hydrolysis and Synthesis of Ceramide by Neutral Ceramidase

“…Both the V641D mutant and the deletion mutant that lacks four residues at the C-terminal end of PaCD were 10 times as sensitive to trypsin compared with the wild-type enzyme (23). Although the position of the C ␣ (Val-641) is far from the Mg 2ϩ /Ca 2ϩ and Zn 2ϩ binding sites, with distances of 19 and 42 Å, respectively, the point mutation V641D may affect the structure of S5, destabilizing not only the ␤-sandwich fold but also the Mg 2ϩ /Ca 2ϩ binding site.…”

“…The cellular location of the enzyme may influence the function of the protein. In contrast to the N-terminal region, the C-terminal tail of nCDase is strictly conserved through bacteria to humans and is indispensable for the correct folding, localization, and enzyme activity; however, the molecular basis is poorly understood (23).…”

Ceramidase Enhances Phospholipase C-induced Hemolysis by Pseudomonas aeruginosa

“…Anti-Pseudomonas CDase antibody was raised in a rabbit using recombinant Pseudomonas CDase as the antigen (23). SMase from Bacillus cereus was obtained from Funakoshi (Tokyo, Japan).…”