Identification and biochemical characterization of Laodelphax striatellus neutral ceramidase

Abstract

Ceramidases are a group of enzymes that catalyse hydrolysis of ceramides to generate fatty acid and sphingosine. In this study, we report the cloning and characterization of the rice small brown planthopper Laodelphax striatellus neutral ceramidase (nCDase), LsnCer. LsnCer was identified by sequencing the transcriptome of L. striatellus and is a protein of 717 amino acids with a predicted molecular weight of 79.3 kDa. Similarly to other known nCDases, the optimum pH for LsnCer is 8.0 and the optimum temperature is 37 °C for its in vitro activity. LsnCer activity is inhibited by Zn(2+) significantly and Fe(2+) slightly. LsnCer has broad substrate specificity with a preference for ceramides with a medium acyl-chain or a monounsaturated long acyl-chain. Infection with rice strip virus (RSV) or treatment with insecticides significantly increased LsnCer mRNA expression and its enzymatic activity in L. striatellus. These results suggest that LsnCer is a bona fide nCDase that may have a role in adaption of L. striatellus to environmental stresses.

Keywords: LsnCer; activity; insecticide; mRNA; nCDase; rice strip virus.

Figure 1. Sequence analysis of LsnCer

(A) The LsnCer coding and protein sequences. The deduced amino acid sequence of LsnCer is shown in one letter symbols below the nucleotide sequence. (B) Phylogenetic analysis of LsnCer. Classification of fungi, plant, insect, and mammalian nCDases was based on amino acid sequence similarities. Neighbor-joining tree was generated using MAGE 4 software. The numerals represent the confidence level from 1000 replicate bootstrap samplings.

Figure 1. Sequence analysis of LsnCer

(A) The LsnCer coding and protein sequences. The deduced amino acid sequence of LsnCer is shown in one letter symbols below the nucleotide sequence. (B) Phylogenetic analysis of LsnCer. Classification of fungi, plant, insect, and mammalian nCDases was based on amino acid sequence similarities. Neighbor-joining tree was generated using MAGE 4 software. The numerals represent the confidence level from 1000 replicate bootstrap samplings.

Figure 2. LsnCer was localized to the plasma membrane

(A) Membranes and medium were prepared from pFastBac-HTB/LsnCer (LsnCer) and pFastBac-HTB (vector) cells and subjected to Western blot analysis with the anti-His×6 antibody (1:1,000). (B) LsnCer-eGFP fusion protein was observed in green under confocal fluorescence microscope. (C) Plasma membrane was stained by 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18(3)). (D) Merged. Extensive co-localization of LsnCer-eGFP with plasma membrane marker was observed in yellow.

Figure 3. Tissue specific expression of LsnCer

(A) mRNA levels of female adult L. striatellus nCDase of different organs. (B) mRNA levels of male adult L. striatellus nCDase of different organs. Transcript abundance was calculated based on the difference in threshold cycle (Ct) values between LsnCer and actin transcripts based on the normalized relative quantification 2^−ΔΔCt method. Data represent the mean value ± SE of three independent experiments performed in duplicate.

Figure 4. Biochemical properties of LsnCer

(A) The total membrane fractions from cells transfected with pFast-HTB/LsnCer (LsnCer) or cells transfected with pFast-HTB (vector) were assayed for ceramidase activity at pH 7.4. (B) LsnCer activity in the membranes of pFast-HTB/LsnCer and pFast-HTB cells were assayed at different pH values. pH was adjusted by adding the following buffer: NaAc (pH 5–6), Tris-HCl (pH 7–8), Glycine (pH 9–10). Ceramidase activity of the recombinant LsnCer at each pH value was computed by subtracting ceramidase activity in pFast-HTB membranes from that in pFast-HTB/LsnCer membranes. The LsnCer ceramidase activity at pH 8 was highest and set as 100%, and ceramidase activity at other pH values was expressed as % of the maximal activity. (C) Temperature dependence of LsnCer. The membranes were assayed for ceramidase activity at indicated temperature. Ceramidase activity of the recombinant LsnCer at each temperature was computed as mentioned. The LsnCer ceramidase activity at 37 °C was highest and set as 100%, and ceramidase activity at other temperature values was expressed as % of the maximal activity. (D) Substrate specificity of LsnCer. The membranes of pFastBac HTB/LsnCer and pFastBac HTB were assayed for ceramidase activity using indicated ceramide as substrate. Ceramidase activity was computed as mentioned. The LsnCer ceramidase activity was highest with substrate of C₁₂ ceramide and set as 100%, and ceramidase activity on other substrates was expressed as % of the maximal activity. (E) LsnCer activity was inhibited by different cations. The membranes of pFastBac HTB/LsnCer and pFastBac HTB were assayed for ceramidase activity in the presence of indicated cations. The LsnCer ceramidase activity with no cation added was highest and set as 100%, and ceramidase activity in the presence of different cations was expressed as % of the maximal activity. All data represent the mean value of three independent experiments performed in duplicate.

Figure 5. Higher LsnCer mRNA level and nCDase activity were found in rice stripe virus infected L. striatellus

(A) mRNA level of LsnCer was higher in different life stage and forms of rice stripe virus infected L. striatellus. Transcript abundance was calculated based on the difference in threshold cycle (Ct) values between LsnCer and actin transcripts based on the normalized relative quantification 2^−ΔΔCt method. Data represent the mean value ± SE of three independent experiments performed in duplicate. (B) NCDase activity of rice stripe virus infected L. striatellus nymphs was higher than that of rice stripe virus free one. The ceramidase was assay as mentioned at pH 8.

Figure 6. Insecticide stimulated the LsnCer expression

(A) 4-instar nymphs were treated with fipronil, imidacloprid and chlorpyrifos with LD50. Nymphs treated with acetone were used as control. The relative mRNA level of LsnCer was analyzed by qRT-PCR at indicated time points. Transcript abundance was calculated based on the difference in threshold cycle (Ct) values between LsnCer and actin transcripts based on the normalized relative quantification 2^−ΔΔCt method. The mRNA level of acetone treated nymphs was set as 1 at each time point, the relative mRNA level of each insecticide treated insects was calculated by comparing with mRNA level of acetone treated ones. (B) The nCDase activity of imidacloprid treated 4-instar nymphs analyzed at indicated time points. Ceramidase activity was assayed at pH 8. The nCDase activity in acetone-treated nymphs was set as 100% at each time point. Ceramidase activity of imidacloprid treated nymphs was expressed as % of the activity of the acetone-treated insects. Data represent the mean value ± SE of three independent experiments performed in duplicate.