Characterization And Physiological Function Of The Chilo Suppressalis Alkaline Ceramidase And Neutral Sphingomyelinase

Alkaline ceramidases and neutral sphingomyelinases are key enzymes in the metabolism of sphingolipids.Alkaline ceramidases hydrolyze ceramides to generate sphingosine and free fatty acids.Neutral sphingomyelinases hydrolyze sphingomyelins to generate ceramides and phosphorylcholine.These sphingolipids play very important physiological roles in both prokaryote and eukaryote.Previous studies demonstrated that Drosophila alkaline ceramidase plays an important role in its development,reproduction and survival by controlling the metabolism of ceramides.Based on these achievements,we put forward the hypothesis that ceramidases and sphingomyelinases play important roles in Chilo suppressalis development and survival by regulating levels of ceramides.In this study,we focus on biochemical characterization of alkaline ceramidases and neutral sphingomyelinasesas as well as their potential physiological function in Chilo suppressalis.The main results are as follows:1.Parts of the nucleotides sequences of open reading frame(ORF)for a novel alkaline ceramidase(saCER)and neutral sphingomyelinase(snSMas)were obtained from the transcriptome sequencing data of Chilo suppressalis.Full-length cDNAs of both genes were cloned from Chilo suppressalis adults by reverse transcription-PCR(RT-PCR).The ORFs of saCER and snSMase are 807bp and 1250bp in length,encoding 269 and 409 amino acids respectively.The saCER protein exhibited a 56%identity in protein sequence to the Drosophila alkaline ceramidase,and has high similarities to the alkaline ceramidases of human,mouse and yeast as well.The snSMase protein exhibited a 70%,67%,42%and 40%identity in protein sequence to the neutral sphingomyelinases of Bombyxmori,butterfly,ant and Tribolium castaneum respectively.saCER and snSMase were also overexpressed in insect cell sf9.2.The studies on biochemical characterization of saCER are as follows:pSORTII program predicted that saCER consists of 7 putative transcription domains and is mainly located atplasma membrane and endoplasmic reticulum.We postulated that the saCER gene product may be included in the ceramidases superfamily which has multiple-transembrane domains.Overexpression of saCER in sf9 cells increases ceramidase activity in the alkaline pH range,indicating that saCER is a bona fide alkaline ceramidase.3.The studies on biochemical characterization of snSMase are as follow:pSORTII program predicted that snSMasehas only 1 putative transcription domain and is mainly located at endoplasmic reticulum,Golgi complex and mitochondria,suggesting that snSMase is a transembrane protein as well.Overexpression of snSMase increased sphingomyelinase activity at 37?,suggesting that snSMase has sphingomyelinase activity.We also found that recombinant snSMase has the highest activity at pH 7.0,suggesting that snSMase has a neutral pH optimum for its in vitro activity.4.qRT-PCR analysis showed that both of saCER and snSMase mRNA are expressed all through the development stages of the life cycle and in all of the organs of Chilo suppressalis.We also found that both of saCER and snSMase mRNA are expressed highest at pupal stage.The mRNA expression of both genes increased along with the development stages from larva to adult,suggesting that saCER and snSMase play important roles during the development of Chilo suppressalis.saCER mRNA was expressed highest in the thorax and legs,suggesting that saCER may participate in the regulation of molting hormone levels and has important physiological function in muscular system.The expression of snSMase in the ovary and thorax was significantly higher than that in other organs,suggesting that snSMase may play a role in regulating of molting hormone levels and in reproduction.In summary,saCER and snSMase,the genes that were cloned and successfully overexpressed in insect cells have the alkaline ceramidase activity and neutral sphingomyelinase activity respectively.We demonstrate that both of them play important roles in the sphingolipids metabolism,development and reproduction of Chilo suppressalis.However,the molecular mechanisms need further investigation.