Sal synthase cDNA was synthesized by Sangon Biotech, China. The pcDNA3.1(+), pEGFP N2 vectors were chosen to construct the Sal synthase recombinant plasmids, and empty vectors were used as the control group, respectively. Primers 1 [5’-CCGCTCGAG (Xho1) ATGCAAATCTTCGTG (Forward) and 5’-CGCGGATCC (BamH1) ATAGTCAACCCT-3’ (Reverse)] were used for the plasmid construction of pcDNA3.1(+)-Sal synthase. Primer 2 [5’-CCGCTCGAG (Xho1) ATGCAAATCTTCGTG-3’ (Forward) and 5’-CGCGGATCC-(BamH1) ATAGTCAACCCT-3’ (Reverse)] was used for pEGFP N2-Sal synthase construction.

The rat pheochromocytoma cell line (PC12) was obtained from cell center of the Chinese Academy of Sciences (Shanghai, China). PC12 cells were cultured in 1640 medium (Gibco, USA) with 5% fetal bovine serum (Wisent, Canada) and 10% horse serum (Gibco, USA), and were incubated with 5% CO${}_2$ and maximal humidity at 37 ${}^{\circ }$C. PC12 cells were grown to about 80% confluent for 48 h transfection, which was performed using Lipofectamine 3000 according to the manufacture’s direction (Invitrogen, USA).

The transfected cells were ultra-sonicated for 6 min in 50 mM Tris-HCl (pH 7.4). After quantification, proteins were removed using 1 M perchloric acid (PCA) solution (1 M HClO${}_4$, 10 mM Na${}_2$S${}_2$O${}_5$, 10 mM EDTA) [13]. The supernatants were collected and filtered through Millipore 0.22 $\mu$m filters. Afterwards, quantitative analysis of Sal and NM-Sal was completed on a Discovery®HS F5-5 column by HPLC-MS/MS. The mobile phase was delivered at flow rate of 0.2 mL/min, which consisted of methanol-water (25/75, v/v) with 10 mM ammonium formate (pH 3.5). The injection volume of each sample was 5 $\mu$L. Isoproterenol (ISOP) was used as an internal standard. The quantitative analysis of Sal and NM-Sal was detected according to methods described previously [14].

The transfected cells were lysed in RIPA buffer, and then centrifuged at 4 ${}^{\circ }$C at 12,000 g for 10 min and the supernatants were used as the crude enzyme of Sal synthase. The crude enzyme was incubated with 1 mM DA and 1 mM AcH in Tris-HCl (50 mM, pH 7.4) for 40 min at 37 ${}^{\circ }$C. PCA was added to stop the reaction. After centrifugation, the supernatants were filtered with 0.22 $\mu$m filters, and the content of Sal was measured by HPLC-MS/MS. Sal synthase’s catalytic activity was estimated from the increase of Sal (per mg protein per minute, ng$\cdot$mg${}^{-1}$$\cdot$min${}^{-1}$). The Sal produced by the Pictet-Spengler reaction was deducted from the enzyme-activity calculation.

After a 48 h transfection, Sal synthase overexpression cells and control group cells were collected for the detection of mitochondrial membrane potential (MMP) and apoptosis. The fluorescent probe JC-1 (Beyotime, China) and an Annexin V-FITC/PI Apoptosis Detection Kit (DOJINDO, Japan) were utilized as described previously [15], and the final detection was determined with flow cytometry (Becton-Dickinson, USA).

Protein samples obtained from cell lysates were mixed with 5$\times$ protein loading buffer and boiled at 100 ${}^{\circ }$C for 5 min. The samples were separated on 12% SDS-PAGE, and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Germany). The membranes were blocked by 5% non-fat milk in TBS-Tween buffer. The primary antibodies of Bax, Bcl-2 and GAPDH were acquired from Beyotime of China, while $\beta$-actin was from Sigma. The Sal synthase antibody was prepared by Huada Beijing Protein Innovation of China. Primary antibodies were diluted in TBST or primary antibody dilution buffer at 4 ${}^{\circ }$C overnight and the HRP-conjugated secondary antibody at RT for 2 h. The bands were visualized using ECL (enhanced chemiluminescence) reagents (Millipore, Germany) and an autoradiographic film (Tanon, China). The quantitative analysis was performed by Image J, and signals were normalized for $\beta$-actin or GAPDH as loading controls.

Data were presented as mean $\pm$ standard deviation (SD) values from at least three independent experiments. Statistical significance was evaluated using unpaired t-test with Welch’s correction. p-value 0.05 (*) or 0.01 (**) were considered as statistically significant.

PC12 is a rat adrenal pheochromaocytoma-derived cell line that produces and releases dopamine [16], and has been commonly used for studies of cell toxicology, neuronal development, and neurodegenerative diseases. In our research, it was used to investigate the function of Sal synthase. The amino acid sequence of Sal synthase was reverse transcripted to cDNA to construct the recombinant plasmid [pcDNA3.1(+)-Sal synthase], which then transfects PC12 cells for Sal synthase overexpression (Fig. 1A). After a 48 h transfection, the Sal synthase protein level was determined. The results indicated that Sal synthase was successfully overexpressed in PC12 cells at a high level compared with the control which used empty vectors (Fig. 1B). Consistent with our previous study, endogenous Sal synthase in control PC12 cells was difficult to detect with western blot [13]. In addition, the soluble whole proteins of PC12 cells were extracted and evaluated the catalytic activity of Sal synthase by HPLC-MS/MS. Compared with control, Sal synthase’s activity increased by approximately 20% in overexpressed PC12 cells (p = 0.0060, Fig. 1C). This result confirmed that the overexpressed Sal synthase maintains catalytic activity.

Fig. 1.

Overexpression and the activity detection of Sal synthase in PC12 cells. (A) Recombinant plasmid was constructed based on the amino acid sequence of Sal synthase and transfected in PC12 cells for 48 h. (B) Quantification of overexpressed Sal synthase by western blot. (C) The activity of Sal synthase was increased in overexpressed PC12 cells. Error bar indicates means $\pm$ SD (n = 3), **p 0.01.

In order to detect the subcellular distribution of Sal synthase, co-expression plasmids containing both Sal synthase and Enhanced Green Fluorescent Protein (EGFP) were used to determine its subcellular localization. The EGFP representing the fusion protein of Sal synthase and Hoechst 33258 was used for nuclear staining. As shown in Fig. 2, Sal synthase-EGFP fusion protein was found within the cytoplasm of PC12 cells, suggesting it is one type of cytoplasmic protein and has enzymatic functions.

Fig. 2.

Subcellular localization of Sal synthase in PC12 cells. After transfection for 48 h, a laser scanning confocal microscope was used to observe the subcellular distribution of Sal synthase. Green fluorescence indicated Sal synthase and the blue fluorescence represented cell nucleus.

To investigate the biocatalytic function of Sal synthase, the level of Sal was detected by HPLC-MS/MS using ISOP as an internal standard. In our previous study, (R)-Sal exhibits an approximately 1.49-fold increase compared to the production of (S)-Sal in enzymatic reaction [12]. This indicated that the (R)-Sal in excess was biosynthesized by Sal synthase and two pathways (the other one being the Pictet-Spengler reaction) of Sal production may co-existed. As it is difficult to completely prevent the Pictet-Spengler reaction inside PC12 cells, we measured the total Sal instead of (R)-Sal in this study. Sal may be further metabolized by N-methyltransferase to NM-Sal, so we also determined the content of NM-Sal in PC12 cells.

Typical MRM ion chromatograms of ISOP, Sal and NM-Sal are shown in Fig. 3A. The retention time of Sal (m/z 180.1/117.1) was 6.22 min and NM-Sal (m/z 194/145) was 7.52 min. The levels of Sal and NM-Sal are presented as pg per mg protein (pg/mg protein). Results indicate that both Sal and NM-Sal were increased in Sal synthase overexpressed PC12 cells (Sal, p = 0.0387; NM-Sal, p = 0.0340) (Fig. 3B). This result indicated that Sal synthase could induce the production of Sal, and the accumulation of Sal could be further metabolized to NM-Sal.

Fig. 3.

Sal synthase improved the production of Sal and NM-Sal. (A) Typical MRM ion chromatograms of ISOP, Sal and NM-Sal detected by HPLC-MS/MS. (B) Quantification of Sal and NM-Sal in PC12 cells. Error bars indicate means $\pm$ SD (n = 3), *p 0.05.

Previous studies suggest that the excessive formation of Sal and NM-Sal can induce oxidative stress and neurotoxicity in numerous cells [17, 18], and Sal can also cause apoptosis in neural stem cells [19]. In order to evaluate the potential mechanism of Sal synthase, mitochondria damage and apoptosis were evaluated in Sal synthase overexpressed PC12 cells. For mitochondria damage detection, we estimated the MMP by loading the JC-1 probe. In normal mitochondria, JC-1 assembled in the matrix and the polymer emitted red fluorescence. However, when mitochondria were damaged and MMP decreased, the JC-1 monomer dissociated in the cytoplasm and displayed green fluorescence. Therefore, the ratio of green and red fluorescence intensity reflected the change of MMP. As shown in Fig. 4A,B, Sal synthase significantly increased the ratio of green/red fluorescence (p = 0.0140), which indicated mitochondrial damage.

We next investigated the apoptotic rate using an Annexin V-FITC/PI Apoptosis Detection Kit (Fig. 4C). The apoptosis rate was significantly increased (twelve-fold) compared with the negative control (p = 0.0081), indicating the substantial neurotoxicity of Sal synthase. Additionally, the expression of the anti-apoptotic protein Bcl-2 and the correlated pro-apoptotic protein Bax were detected. The results illustrate a remarkable decrease of Bcl-2 in overexpressed group (Bcl-2, p = 0.0132), along with no significant change in Bax (Fig. 4D). The significant decrease in the Bcl-2/Bax ratio caused by Sal synthase was in accordance with the Sal exposure [19], which demonstrated that the cytotoxicity of Sal synthase may be caused by the toxicity of Sal.

Fig. 4.

The effect of Sal synthase on MMP levels and apoptosis. (A,B) MMP levels were estimated by flow cytometry using JC-1 probe, and the ratio of green and red fluorescence intensity reflected the change of MMP. (C) After transfection, PC12 cells were dually stained with Annexin V/PI (Propidium iodide, PI) to quantify the apoptotic cells. (D) Detection and quantification of Bcl-2 (26 kD, the upper band; the band below is non-specific band as using the polyclonal antibody) and Bax by western blot. Error bars indicate means $\pm$ SD (n = 3), *p 0.05, **p 0.01.