Cloning, Expression And Characterization Of Sphingomyelin Synthase Family Gene (TcSMS1,CsSMSr) From Two Insects

Sphingomyelin synthase (SMS, EC2.7.8.27) that transfers the phosphocholine moiety from phosphatidylcholine (PC) to ceramide resulting in sphingomyelin (SM) and diacylglycerol (DAG) is a vital enzyme in sphingolipid metabolism.It plays an important role in cell growth and survival by regulating the level of pro-apoptotic factor ceramide and mitogenic factor DAG. Here we report the cloning, identification of a SMS1from the red flour beetle Tribolium castaneum and a SMSr gene from Chilo suppressalis (Walker), and biochemical characterization of Tribolium SMS1. The primary results are as follows:1. The full-lenth cDNA encoding Tribolium SMS1gene was obtained by reverse transcription-PCR, and the coding region of SMS1cDNA was1308bp, encoding a436amino-acid protein. It was found that the Tribolium SMS1gene product contains four highly conserved sequences motifs, designated D1-D4shared among these SMS1. Among them, Motifs D3and D4include the His314, His357, Asp361which form a catalytic triad. Because the SMART program predicted that the Tribolium SMS1consists of6putative transmembrane domains just like the other SMS genes, but the SAM domain was not found, it was named as TcSMSl.2. Overexpression of TcSMS1increased SM synthase activity in High Five cells, suggesting that that it is a bona fide SMS. TcSMS1has an optimal temperature of30℃and a broad pH optimum of3-7for its in vitro activity. Zn2+and Ni2+abolished TcSMSl enzymatic activity whereas EDTA, EGTA, Fe2+enhanced its activity. TcSMS1could use non-choline phospholipids in paticular phosphatidylethanolamine (PE) as substrates. SM synthase activities were inhibited dose-dependently by the phosphatidylcholine (PC)-phospholipase C inhibitor, D609(50%at6.25mg/ml D609).3. qRT-PCR analysis showed TcSMS1mRNA levels are the highest in the larval stage among the stages of the life cycle, suggesting that TcSMSl may have a physiological role in Tribolium castaneum development. TcSMS1mRNA levels are the highest in the head among the tissues examined, including the head, thorax, midgut, and reproductive organs, indicating that this enzyme may have a prominent role in the central nerve system.4. The full-lenth cDNA encoding SMSr gene (CsSMSr) was obtained by using RT-PCR and rapid amplification of cDNA ends(RACE) stategies, and the coding region of CsSMSr cDNA was1587bp, encoding a528amino-acid protein. CsSMSr contains a N-terminal sterile alpha motif (SAM) domain and six transmembranes, it also share four highly conserved sequences motifs, designated D1-D4. Among them, Motifs D3and D4include the His302, His345, Asp349which form a catalytic triad. CsSMSr gene was successfully eukaryotically expressed in Tn cell, the recombinant protein molecular weight was60kDa.5. RT-PCR was carried out to analyze the gene temporal and spatial expression patterns, the results showed that the expression level of CsSMSr was much higher in the adult stage than in pre-adult stages, and highly expressed at highest levels in reproductive organs of male larvae female adults, furthermore the expression level of CsSMSr was much higher in testis, but not significant different from midgut and Malpighian tubules in male adults, suggesting that CsSMSr gene may play a key role in organ maturity and reproductive function. All these results paved the foundament for the further research on the physiological and population ecological functions of CsSMSr gene.