2 National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
3 School of Pharmaceutical Science, Capital Medical University, Beijing 100069, China;
4 School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China;
5 Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing 100069, China
Tripterygium wilfordii (Hook. f.) is a perennial vine plant belonging to the family Celastraceae, which has been widely used to treat rheumatoid arthritis and other inflammatory diseases [1-2]. The medicinal properties of T. wilfordii are attributed to terpenes which mainly include di- and triterpenes. Triptolide is a diterpene triepoxide isolated from T. wilfordii that exhibits strong antitumor activity. The potential mechanism involves the inhibition of the general RNAPII-mediated transcription . A representative T. wilfordii triterpene is the structurally unique celastrol, which hold promise as a powerful anti-obesity agent, a potent anti-inflammatory drug, and a breast cancer treatment [4-6].
Terpenes are derived from the universal C5 precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) , which are provided by the mevalonate (MVA) pathway and the 2-methyl-Derythritol-4-phosphate (MEP) pathway . The enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is responsible for the first committed step in the MVA pathway, catalyzing the aldol-type condensation of acetoacetyl-CoA and acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) . HMGS plays a critical role in the biosynthesis of terpenes in medicinal plants and provides the possibility of regulating the production of target terpene metabolites. The HMGS of Ginkgo biloba has been isolated and characterized via functional complementation of GbHMGS1 in Saccharomyces cerevisiae . An Escherichia coli strain that was transformed with a codon-optimized HMGS gene exhibited significantly improved bisabolene production over the control bacteria . LIU et al. cloned the full-length cDNA of TwHMGS gene and proved that the enzyme participated in yeast terpene biosynthesis in a functional complement assay .
However, the unambiguous correlation between TwHMGS expression and terpene biosynthesis in T. wilfordii has not yet been studied in detail. Overexpression and RNA interference (RNAi) genes encoding the presumptive enzymes directly regulate the transcription levels and change the flux towards the desired end-products. Overexpressing the rate-limiting enzyme HMGR in tobacco (Nicotiana benthamiana) led to a 20- to 40-fold increase of sesquiterpenes and a 6-fold increase of triterpenes. The production of α-Bisabolol and β-amyrin finally reached 28.8 and 9.8 mg·g–1 dry weight, respectively . Manipulation of Brassica juncea HMGS1 in transgenic tomato not only up-regulated several genes related to terpene biosynthesis, but also increased the accumulation of MVA- derived squalene and phytosterols, as well as MEP-derived α-tocopherol and carotenoids . It stands to reason that the overexpression of HMGS endowed the transgenic tomato plants with a higher terpene accumulation capacity through both MVA and MEP pathways. RNAi provides an effective post-transcriptional silencing method to inhibit the expression of endogenous genes. In this approach, the RNA-induced silencing complex, an siRNA-containing effector complex, recognizes and cleaves the complementary target RNAs .
In this study, we analyzed the phylogenetic position of TwHMGS. Furthermore, TwHMGS was overexpressed and silenced using RNAi in T. wilfordii suspension cells. The expression of relevant genes in the terpene biosynthetic pathway and accumulation of triptolide and celastrol were analyzed in the TwHMGS overexpression and RNAi groups. The increase and decrease of triptolide content was consistent with the up- and downregulation of TwHMGS, suggesting that the encoded enzyme plays an important role in the biosynthesis of this important bioactive molecule. On the basis of molecular docking, we obtained the binding state of the TwHMGS complex with acetyl-CoA and analyzed key residues around the substrate which were suggested to participate in the acetylation/deacetylation, condensation/cleavage and hydrolysis/dehydration processes of the catalytic reaction . Thess findings add to the fundamental knowledge of HMGS and the production of terpenes with pharmacological properties.Materials and Methods Suspension cells
The T. wilfordii suspension cells used in this study were cultured as previously reported .Bioinformatics analysis
The phylogenetic analysis of TwHMGS and other HMGSs from diverse species was conducted using MEGA 5.1 software (Arizona State University, USA). The multiple alignment analysis of TwHMGS and nine other HMGSs was especially performed using ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/) and ESPript 3 (http://espript.ibcp.fr/ESPript/ESPript/). The nine HMGSs from different species were obtained in PDB (http://www.rcsb.org/).Construction of the entry- and expression vectors
The entry vectors were constructed using pENTRTM/D- TOPO® Cloning Kit (Invitrogen, USA). The overexpression fragment corresponded to the open reading frame (ORF) of TwHMGS, while the RNAi fragment contained a specific 515 bp fragment from 880-1394 bp of the TwHMGS ORF. All the primers were designed using Primer Premier 5.0 software (PREMIER Biosoft, Canada). Both the overexpression fragment and RNAi fragment were amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs, USA), with the TwHMGS plasmid being as the template. Subsequently, the purified target fragments were ligated into the pENTRTM/D-TOPO entry vector according to the manufacturer's instructions (Invitrogen, USA). The entry vectors pENTR-TwHMGSOE and pENTR-TwHMGSRi were confirmed by sequencing.
The expression vectors were constructed using Gateway LR ClonaseTM Ⅱ Enzyme Mix (Invitrogen, USA). The reaction mixture was composed of pENTR-TwHMGSOE (100 ng), pH7WG2D (150 ng), 1 μL of LR Clonase, and 0.6 μL of TE buffer (pH 8.0). The reaction was carried out at 25 ℃ for 2 h, after which 0.5 μL of Proteinase K Solution was added and incubated at 37 ℃ for 10 min. pENTR-TwHMGSRi and pK7GWIWG2D were used to construct the RNAi expression vector. After screening and sequencing, the recombinant expression vectors pH7WG2D-TwHMGSOE and pK7GWIWG 2D-TwHMGSRi were enriched and extracted using EZNA® Plasmid Maxi Kit (Omega Bio-tek Inc., USA).Genetic transformation of T. wilfordii suspension cells using a biolistic-gun
The T. wilfordii suspension cells were cultured in MS solid medium. We used a PDS, 100/He gene gun (Bio-Rad, USA) to deliver the expression vectors pH7WG2D- TwHMGSOE and pK7GWIWG2D-TwHMGSRi mixed with 1 μm gold micro-particles into the T. wilfordii suspension cells. The empty vectors pH7WG2D and pK7GWIWG2D were also performed as negative controls. The detailed protocol has been published previously . Each vector transformation was performed in five biological replicates.Verification of successful transformation with the expression vectors
After genetic transformation, the suspension cells were cultured for 5-7 days to allow sufficient gene expression and metabolite accumulation. To verify the successful transformation, total RNA of suspension cells was isolated from the suspension cells using the Total RNA Extraction Kit (Promega, Shanghai, China), after which the RNAs were reverse-transcribed to first-strand cDNAs as templates for PCR using the FastQuant RT kit (with gDNase, Tiangen Biotech, Beijing, China). The pH7WG2D vector encodes hygromycin resistance (Hyg) and spectinomycin resistance, while the pK7G WIWG2D vector contains a kanamycin resistance (Kan) cassette. Specific primers were designed to amplify the Hyg fragment and the Kan fragment with the cDNA as templates. The PCR products were detected by gel electrophoresis to verify the successful transformation of the suspension cells with the expression vectors.Transcriptional expression analysis of TwHMGS and other selected genes
Transcript levels of TwHMGS and a number of other relevant genes were analyzed using real-time quantitative polymerase chain reactions (RT-qPCR). The LightCycler 480 Ⅱ (Roche, Switzerland) and KAPA SYBR® FAST qPCR Master Mix Kit (KAPA Biosystems, USA) were used to carry out the expression analysis. The house-keeping gene β-actin was used as internal control in each reaction. The RT-qPCR primers for TwHMGS, TwHMGR, TwDXS, TwDXR, TwMCT, TwGGPS, and TwTPS7v2 are listed in Supplementary Table. The RT-qPCR reaction mixture contained 10 μL of 2 × qPCR Master Mix, 1 μL of cDNA (10 ng), and 0.4 μL of primers (10 μmol·L-1) and 8.2 μL of ddH2O. The RT-qPCR conditions encompassed enzyme activation for 3 min at 95 ℃, followed by denaturation for 3 s at 95 ℃, annealing, extension and data acquisition for 30 s at 60 ℃. These conditions were repeated for 40 cycles. Each sample was run in triplicate to reduce data errors, and the relative expression levels were calculated using the 2–△△Ct method.Extraction and determination of biologically active terpenes in T. wilfordii
The remaining suspension cells were ground into powder in liquid nitrogen and freeze-dried for 36 h. Approximately 20 mg dry powder were weighed and soaked in 1 mL of 80% (V/V) methanol overnight at 4 ℃, followed ultrasonication at 60 kHz and 25 ℃ for 30 min. Subsequently, the supernatant obtained by centrifugation at 12 000 g for 10 min was filtered through a 0.22 μm PTFE microporous membrane. The contents of triptolide and celastrol were determined using an ACQUITY UPLCTM I-Class System (Waters, Milford, MA, USA) that included a PDA detector. An ACQUITY UPLC HSS T3 chromatographic column (1.8 μm, 2.1 mm ×100 mm, Waters, USA) was used to separate ingredients, keeping the column temperature at 40 ℃. The mobile phase was a mixture containing 0. 1% (V/V) formic acid in acetonitrile (mobile phase A) and 0.1% (V/V) formic acid in water (mobile phase B). The elution programme comprised 30% (A) at the beginning, 30%-35% (A) at 0-5 min, 35% (A) at 5-8 min, 35%-70% (A) at 8-15 min, 70%-90% (A) at 15-21 min, 90% (A) until 24 min, and the flow rate was set at 0.4 mL·min–1. The standard triptolide (> 98%, DESITE, China) and celastrol (> 98%, DESITE, China) were used to generate standard curves to quantify their contents in each suspension cells. The standard curve was plotted by corrected peak area (Y) for every standard against its concentration (X/mg·L–1), generating relative equations to quantify the triptolide and celastrol.Molecular docking of TwHMGS-ligand complexes
The HMGS from Brassica juncea is the only plant-derived HMGS with an available crystal structure. Thus, the model structure of TwHMGS was constructed using the SWISS Model (https://swissmodel.expasy.org/) with BjHMGS (PDB: 2FA3) as the template. The value of GMQE and identity calculated by SWISS Model proved the reliability of modeling. The 2D structure of the substrate acetyl-CoA was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/acetyl-CoA) and refined using the elbow program in the PHENIX suite. Then, the acetyl-CoA was prepared and docked to the receptor grid of the prepared TwHMGS as described previously . The analysis of the docking result was performed in PyMOL (DeLano Scientific LLC, USA).Results Sequence analysis of TwHMGS
TwHMGS catalyzes the first committed step of the MVA pathway for terpene biosynthesis in T. wilfordii. The MVA pathway genes which were derived from archaeal evolved through lateral transfer from bacteria and other domain of life . The evolutionary position of TwHMGS was exhibited in a phylogenetic tree of the HMGSs from various species (Fig. 1). The TwHMGS has arisen from a common ancestral gene that evolved into the five groups of enzymes from bacteria, animals, fungi, angiosperms and gymnosperms, which suggests an evolutionary relationship from lower organisms to higher organisms. In the angiosperm group, TwHMGS was 81.74% identical to Camptotheca acuminata HMGS .
To date, the crystal structures of nine HMGSs have been reported, including those from Homo sapiens, bacteria and plant [22-24]. To explore the probable relationship of function and secondary structure, we aligned the sequence of TwHMGS with the nine known HMGSs. The details of secondary structures elements were shown in Fig. 2. Multiple sequence alignment among the HMGS from Brassica juncea showed 81% identity, from bacteria for Myxococcus xanthus (strain DK 1622), Enterococcus faecalis, Moorea producens 3L, showed 28.36%, 24.25% and 19.20% identity, respectively, from Homo sapiens exhibited 46.65% identity.
The TwHMGS overexpression vector harbored the ORF fragment of TwHMGS with 1398 bp, while the RNAi expression vector contained partial fragment of TwHMGS with 515 bp. Electrophoresis of the target bands was shown in Fig. 3B. Specific primers were designed to amplify the cDNA of each sample. The pH7WG2D and pH7WG2D-TwHMGSOE were identified via the Hyg fragment of 1787 bp, while pK7GWIWG2D and pK7GWIWG2D-TwHMGSRi were verified by the Kan fragment of 1397 bp (Fig. 3). The electrophoresis bands were shown in Fig. 3C. These results indicated that the suspension cells were successfully transformed with the pH7WG2D-TwHMGSOE, pK7GWIWG2D- TwHMGSRi, pH7WG2D and pK7GWIWG2D vectors, respectively.
The relative transcription level of TwHMGS was determined by RT-qPCR. Compared to the suspension cells transformed with the empty vector, the overexpression group exhibited a 2-fold increase (Fig. 4A). Conversely, the expression of TwHMGS in the RNAi group was reduced to approximately 70% of the control (Fig. 4B).
Apart from the expression analysis of TwHMGS, we focused on the expression of genes in the MEP pathway and further downstream in the biosynthesis of terpene (Fig. 4). HMG-CoA reductase (HMGR) and HMGS are adjacent enzymes in the cytosolic MVA pathway. The HMGR converts HMG-CoA, the product of HMGS, to mevalonate. In the TwHMGS overexpression group, the TwHMGR mRNA was induced, reaching an approximately 1.9-fold high value that that of the control. In the RNAi group, the TwHMGR mRNA level decreased to about half that of the control (Fig. 4). The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) are the first two rate-limiting enzymes in the plastidic MEP pathway. Additionally, 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT or IspD) is another key enzyme in the MEP pathway. The expression of TwDXS, TwDXR and TwMCT decreased in all RNAi suspension cells, whereas the levels of TwDXS and TwMCT in the overexpression group remained unaffected. Among these, the mRNA of TwDXR had the largest increment during TwHMGS overexpression (Fig. 4A). TwTPS7v2 is the class I diterpene cyclase in the downstream pathway of terpene synthesis . Approximately 1.8-fold increasing was observed in the expression of TwTPS7v2 when TwHMGS was overexpressed (Fig. 4). The expression of TwGGPS did not change significantly during TwHMGS overexpression and RNAi.Accumulation of triptolide and celastrol in the suspension cells
Triptolide and celastrol are pharmacologically active diterpene and triterpene natural products of T. wilfordii, respectively. We detected the content of triptolide and celastrol in the overexpression groups, RNAi groups and the control groups. The methanol extracts from independent samples were analyzed by UPLC. The chromatogram of triptolide and celastrol was shown in Figs. 5A and 5B, where peak 1 detected at 425 nm corresponded to triptolide and peak 2 detected at 219.5 nm to celastrol. The standard curve equations for quantifying triptolide and celastrol were Y = 10 849X + 6075.8 (R2 = 0.9964) and Y = 2678.4X – 261.29 (R2 = 0.9998), respectively. Overexpression of TwHMGS promoted the synthesis of triptolide by 2-fold and had no significant effect on the celastrol content compared to the control groups (Fig. 5C). In the RNAi and pK7GWIWG2D groups, the contents of triptolide were 5.51 ± 1.87 and 14.80 ± 2.17 μg·g–1, respectively, while those of celastrol were 41.50 ± 5.76 and 50.56 ± 10.95 μg·g–1. The limited culture time of the suspension cells that were transformed with expression vectors may be a bit short for the accumulation of celastrol. These results supported the vital function of TwHMGS in the biosynthesis of terpenes in T. wilfordii suspension cells.
The crystal structures of BjHMGS in the apo-form and BjHMGS in complex with acetyl-CoA exhibited an open- closed change of conformation . Therefore, the structure of BjHMGS in complex with acetyl-CoA structure was used as template to construct the three-dimensional homology model of TwHMGS. The values of GMQE and identity were 0.92% and 83.93%, respectively, which support the reliability of modeling. The overall structure of TwHMGS was composed of helices, sheets and loops, and as similar to the reported structures BjHMGS and bacterial HMGS with the conserved αβαβα catalytic fold (Fig. 6A) [26-29]. Depending on the ligand docking result, the substrate acetyl-CoA and some residues, including the Arg296, Lys256, and His247, potentially existed mutual polar contacts excluding solvent. The His247 was reported to be a highly conserved site (Fig. 2), serving as a hydrogen donor in the deacetylation process of BjHMGS . In TwHMGS, the distance between His-247 and the acetyl oxygen atom was 2.9 Å, which may share the same mechanism reported for BjHMGS. The phosphate groups of acetyl- CoA were bound to basic residues, such as K256, K34 and R296 through electrostatic interactions (Fig. 6B). In addition, the CoA was anchored by an H-bond interaction between the adenine moiety and the hydroxyl side chain of Ser31 (Fig. 6B).
HMGS is the rate-limiting enzyme in the biosynthesis of isoprenoids, the C5 precursor of all terpenes. The crystal structures of HMGS from Staphylococcus aureus  and from Brassica juncea , in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis. The reaction catalyzed by HMGS consists of three steps namely acetylation, condensation and dehydration. We noticed that key residue R296 was not conserved among the seven bacteria including Myxococcus xanthus and Enterococcus faecalis (Fig. 2). Furthermore, the residue K256 of TwHMGS was not completely conserved in other HMGS, especially that from Moorea producens 3 L . This phenomenon suggested that the residues may be responsible for some of the structural differences between bacterial and plant enzymes. Overexpression of Brassica juncea HMGS has been studied in Arabidopsis. The stigmasterol and sitosterol contents were significantly increased in leaves and seedlings of the overexpression lines . A similar result was shown in Fig. 5. However, neither the overexpression group nor the RNAi group showed significant changes in the accumulation of celastrol, the main bioactive triterpene of T. wilfordii. Generally speaking, triterpene biosynthesis relies on the cytosolic MVA pathway, while diterpene biosynthesis mainly uses the plastidial MEP pathway. However, metabolic cross-talk has been reported to occur between the MVA and MEP pathways, suggesting that isoprenoids flow between them [32-34]. This may explain the significant changes in the content of triptolide. In addition, the ratio of DMAPP/IPP influences the isoprene flux, and the differential expression of TwHMGS may interfere with the dynamic equilibrium between IPP and DMAPP , which we assumed to affect triptolide and celasol biosyntehsis. ZHANG et al. and SU et al. measured the terpenes of T. wilfordii suspension cells and found that the content of celastrol was higher than that of triptolide [25, 36-37]. The differential expression of single gene may prefer metabolite flux to compounds with low content. It was reported that the overexpression and RNAi of TwDXR and TwIDI affected the feedback regulation of other genes in the terpene synthesis pathway [36, 38]. The TwHMGS in our research just exhibited a synergistic regulation (Fig. 7).
The His188 and Ser359 of BjHMGS play a particularly important role in the catalytic activity of this enzyme . The H188N mutation abrogated the substrate inhibition by acetoacetyl-CoA, while the S359A mutation highly increased BjHMGS activity. We noticed that the Ser359 in TwHMGS was close to the substrate acetyl-CoA (Fig. 6B). Overexpression of the wild type BjHMGS, the H188N mutant, S359A mutant and H188N/S359A double mutant in Arabidopsis improved both sterol production and stress tolerance . Furthermore, the introduction of BjHMGS: S359A into transgenic tomato obviously enhanced the α-tocopherol, carotenoid, squalene and phytosterol contents in the fruits .Conclusion
TwHMGS enzyme catalyzes the condensation of acetoacetyl-CoA to form HMG-CoA in the MVA pathway of terpene synthesis. RNAi and overexpression vectors targeting TwHMGS gene were respectively introduced into T. wilfordii suspension cells via biolistic-gun transformation and verified by PCR. TwHMGS overexpression in T. wilfordii not only elevated the expression of adjacent TwHMGR, but also TwDXR in the MEP pathway, and further downstream, the terpene cyclase gene TwTPS7v2 related to diterpene biosynthesis was also upregulated. Meanwhile, a synergistic downregulation of TwDXS, TwDXR, TwMCT and TwHMGR occurred in the TwHMGS RNAi group (Fig. 7). Subsequently, changes in the diterpene end-product triptolide were investigated, providing an important reference for regulating the key genes in the biosynthesis pathway to generate pharmacologically active ingredients. The potential mechanism of HMGS catalytic activity was analyzed on the basis of molecular docking, exploring the binding state of TwHMGS in complex with acetyl-CoA.Acknowledgments
The authors acknowledge XIANG Yu-Hong in Capital Normal University for supporting SYBYL-based computing services.
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