Characterization Of The Drosophila Alkaline Ceramidase (DaCER) And Its Role In The Fly Development, Survival And Reproduction

Ceramidases catalyze the hydrolysis of ceramides to generate sphingosine (SPH)and fatty acids. The metabolism of these sphingolipids is implicated in biologicalresponses in various organisms ranging from yeast to mammals. In this study, wedemonstrate that the protein product (DaCER) of the Drosophila BWA gene hasalkaline ceramidase activity and that it plays an important role in the metabolism ofsphingolipids, development, survival and reproduction in Drosophila melangaster.The primary results are as follows:1. A Blast search of Drosophila melanogaster genomic database using humanalkaline ceramidases as queries revealed a putative protein encoded by the brainwashing gene (BWA) that exhibited a 35%, 46% and 26% identity in protein sequenceto the human alkaline ceramidases ACER1, ACER2 and ACER3, respectively. A full-lengthcDNA of BWA was cloned from Drosophila adults by reverse transcription - PCR(RT-PCR). It was also found that the BWA gene product contains multipleconserved domains shared among these alkaline ceramidases. Because the pSORTIIprogram predicted that the BWA consists of 5 putative transmembrane domains, thisgene may be a member of the multiple-transmembrane domain (MTD) ceramidasesuperfamily.2. Overexpression of BWA in Tn cells increased ceramidase activity onC24:1-ceramide at pH 8.0, suggesting that BWA has alkaline ceramidase activity. Thuswe renamed it the Drosophila alkaline ceramidase, DaCER2. The optimal temperaturefor DaCER activity is 35℃. We also found that DaCER has a much broader pHoptimum, with highest activity at pH8.0.3. qRT-PCR analysis showed that DaCER mRNA is expressed highest in theovary, moderately in the brain and midgut, and slightly in other organs, and thatDaCER expression is up-regulated at pupal stage during the development ofDrosophila. The plasmid pEGFP-Cl-DaCER was constructed and transfected intoHeLa cells. Fluorescence microscopy analysis showed that DaCER is localized tothe plasma membrane and Golgi complex. 4. DaCER inactivation leads to a delayed pre-adult development but anincreased lifespan and fecundity in Drosophila. These results suggest that DaCER hasan important role in the Drosophila development, longevity, and fecundity.5. An inactivation of DaCER by insertional mutagenesis caused a decrease inthe levels of sphingosine and dihydrosphingosine, especially the C₁₆ sphingosine. Thiswas consistent with in vitro assay for detemining the effect of MAPP on sphingosineslevels. The level of C₁₆ sphingosine decreased significantly after 72h treatment ofMAPP. These results suggest that DaCER inactivation alters the metabolism ofsphingolipids.6. To investigate whether DaCER down-regulation would have positive effectin vitro. Expression of DaCER in S2 cells was down-regulated by RNAi. qRT-PCRanalysis demonstrated that transfection of S2 cells with a dsRNA specifically againstDaCER caused a significant decrease in the level of DaCER mRNA and a littleincrease in the level of neutral ceramidase. However, FACS analysis revealed thatDaCER knockdown had no effect on the percentage of cells in the S-phase of the cellcycle.In summary, Drosophila alkaline ceramidase (DaCER) plays an important role inthe fly sphingolipids metabolism, development, survival and reproduction.