Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

Yukihiro Yoshimura, Nozomu Okino, Motohiro Tani, Makoto Ito

Research output: Contribution to journalArticle

47 Citations (Scopus)

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 languageEnglish
Pages (from-to)229-236
Number of pages8
JournalJournal of biochemistry
Volume132
Issue number2
DOIs
Publication statusPublished - Aug 2002

Fingerprint

Neutral Ceramidase
Ceramidases
Cloning
Molecular Cloning
Drosophila melanogaster
Drosophila
Enzymes
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Galactosylceramides
Imaginal Discs
Glycosylation
Sphingomyelins
Nucleic Acid Databases
Protein Sorting Signals
RNA Interference
Gene Library
Open Reading Frames
Polysaccharides
Nucleotides
Genes

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 journalArticle

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