Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster |
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Authors: | Yoshimura Yukihiro Okino Nozomu Tani Motohiro Ito Makoto |
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Affiliation: | Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. |
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Abstract: | We report here the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue (AE003774) was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product. |
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