Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster.
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.
Yoshimura
Yukihiro
Y
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Okino
Nozomu
N
Tani
Motohiro
M
Ito
Makoto
M
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A / Q000378:metabolism; D002478:Cells, Cultured; D055572:Ceramidases; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D016366:Open Reading Frames; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D034622:RNA Interference; D016415:Sequence Alignment; D013379:Substrate Specificity
D029721:Drosophila Proteins; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D000581:Amidohydrolases; C526744:ASAH2 protein, human; C472265:CDase protein, Drosophila; D055572:Ceramidases; D055576:Neutral Ceramidase