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.
| Original language | English |
|---|---|
| Pages (from-to) | 229-236 |
| Number of pages | 8 |
| Journal | Journal of biochemistry |
| Volume | 132 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Aug 2002 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
Cite this
Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster. / Yoshimura, Yukihiro; Okino, Nozomu; Tani, Motohiro; Ito, Makoto.
In: Journal of biochemistry, Vol. 132, No. 2, 08.2002, p. 229-236.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster
AU - Yoshimura, Yukihiro
AU - Okino, Nozomu
AU - Tani, Motohiro
AU - Ito, Makoto
PY - 2002/8
Y1 - 2002/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0036684043&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036684043&partnerID=8YFLogxK
U2 - 10.1093/oxfordjournals.jbchem.a003215
DO - 10.1093/oxfordjournals.jbchem.a003215
M3 - Article
C2 - 12153720
AN - SCOPUS:0036684043
VL - 132
SP - 229
EP - 236
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 2
ER -