Characterization And Function Analysis Of Enzymes Related With Insect Sphingolipid Metabolism

Neutral ceramidase and neutral sphingomyelinase are key enzymes in sphingolipid metabolism. Neutral sphingomyelinase hydrolyzes sphingomyelin to generate ceramide. Neutral ceramidase hydrolyzes ceramide, forming sphingosine. We focused on the sphingolipid metabolism of model insect Tribolium castaneum and rice pest the small brown planthopper Laodelphax striatellus. In these researches, we studied these two enzymes of Tribolium castaneum and Laodelphax striatellus. The main results we got were:1. We identified the neutral ceramidase of Tribolium castaneum (Tncer) from three candidate homologue genes. The amino acid sequence of Tncer has49.1%,38.1%,36.8%,33.4%similarities to the neutral ceramidase of Drosophila melanogaster, rice, human and Pseudomonas aeruginosa. Tncer showed unique biochemical properties. The optimum pH for Tncer activity is about pH5. From pH5to pH12, the ceramidase activity did not change too much. Tncer could hydrolyze various ceramidase from C6-ceramide to C24:1-ceramide. However the hydrolyzed ability to C6-ceramide is lower. The best temperature for its in vitro activity is about37℃. Tncer is a cation independent enzyme, Fe2+can inhibit its activity. The mRNA level of Tncer is highest in spermary and ovary of Tribolium castaneum. And the mRNA level of Tncer is increased with the maturation of ovary. These suggest that Tncer may paly roles in the oogenesis. Tncer is mainly located in the plasma membrane in High Five cells.2. With the sequence assembly, we got the neutral ceramidase homologue (LsnCer) from the transcriptome database of Laodelphax striatellus. The sequence alignment showed that the sequence similarities between LsnCer and neutral ceramidase of Tribolium castaneum, Drosophila melanogaster, human, Pseudomonas aeruginosa and rice is50.62%,47.76%,37.11%,36.14%and36.84%respectively. The optimum pH for LsnCer is pH8. Little activity was detected in the strong acid and alkaline pH environment. It prefers medium-chain to long-chain ceramide as substrates. Short chain-ceramides such as C2-ceramide and super long chain-ceramide are resistant to LsnCer. The activity of LsnCer could be inhibited significantly by Zn2+The best temperature for its in vitro activity is also37℃. The mRNA level of LsnCer is highest in spermary and ovary of Laodelphax striatellus. It’s a transmembrane protein and located in the plasma membrane in the High Five cells.3. Rice stripe virus (RSV) can stimulate the expression of LsnCer. The ceramidase activity of LsnCer around pH8is also higher in RSV-infected Laodelphax striatellus. mRNA level of LsnCer as well as the neutral ceramidase activity were stimulated significantly after treated with insecticide4hours later.4. We cloned neutral ceramidase1homologue (LsnSMase) from Laodelphax striatellus.323-342AA and355-374AA are two transmembrane domains. With the bioinformatic analysis and reported results, we presumed LsnSMase is the main sphingomyelinase in Laodelphax striatellus. The activity of LsnSMase is dependent on Mg2+, DTT can also increased the activity of LsnSMase. EDTA inhibited LsnSMase activity significantly. However, EGTA is better stimulator of LsnSMase than Mg2+. The co-operation of Mg2+and EGTA or Mg2+and EDTA or Mg2+and DTT could increase the LsnSMase activity sharply. mRNA level of LsnSMase is highest in ovary of Laodelphax striatellus.In conclusion, we first identified biochemical properties of neutral ceramidase and neutral sphingomyelinase of agriculture pest. The function of these enzymes are also uncovered in our research.