LIU Ying, XU Qiao-xian, XI Pei-yu, CHEN Hong-hao, LIU Chun-sheng *

Transcription

1 药学学报 Acta Pharmaceutica Sinica 2013, 48 (5): Cloning and characterization of a cdna coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis LIU Ying, XU Qiao-xian, XI Pei-yu, CHEN Hong-hao, LIU Chun-sheng * (School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing , China ) Abstract: The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cdna coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cdna was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro. Key words: Glycyrrhiza uralensis; HMGR; cdna; prokaryotic expression; GC-MS CLC number: R931 Document code: A Article ID: (2013) 甘草 HMGR 基因 cdna 的克隆及功能鉴定 刘 * 颖, 许巧仙, 席培宇, 陈宏昊, 刘春生 ( 北京中医药大学中药学院, 北京 ) 摘要 : 甘草为常用中药材, 具有补脾益气 清热解毒 祛痰止咳 缓急止痛和调和诸药等作用 甘草酸是甘草中的主要活性成分, 其生物合成途径受到许多酶的调控, 其中 3- 羟基 -3- 甲基戊二酰 CoA 还原酶 (3-hydroxy-3- methylglutary CoA reductase, HMGR) 催化 3- 羟基 -3- 甲基戊二酰辅酶 A (3-hydroxy-3-methylglutary CoA, HMG-CoA) 生成甲羟戊酸 (MVA), 是该途径中的第一个限速酶 本文克隆了甘草 HMGR 基因 cdna 序列, 对其进行序列分析, 并与其他物种进行序列比对 构建了原核表达载体, 诱导其外源表达及酶促反应, 并利用 TLC 及 GC-MS 对产物进行检测 结果表明本文克隆得到的 HMGR 序列能很好的完成特定的酶促反应任务 由于 HMGR 基因在甘草酸生物合成途径中的重要作用, 因此本实验的研究结果对于在分子水平上揭示高品质甘草的形成机制具有一定的意义 关键词 : 甘草 ; HMGR; cdna; 原核表达 ; GC-MS The roots of Glycyrrhiza uralensis are widely used in China for their actions of nourishing qi, alleviating Received ; Accepted Project supported by the National Natural Science Foundation ( ). *Corresponding author Tel / Fax: , max_liucs@263.net pain, tonifying spleen and stomach, eliminating phlegm and relieving coughing, heat-clearing and detoxifying [1, 2]. They are present in many Chinese herbal compound prescriptions. Besides the medicinal applications, Glycyrrhiza uralensis roots are also used as health food, industrial raw materials, and tobacco additives. The

2 774 药学学报 Acta Pharmaceutica Sinica 2013, 48 (5): reason why Glycyrrhiza uralensis has these potent actions and wide applications is its content of various natural active components, including flavonoids and triterpenoids. Among them, glycyrrhizic acid is considered to be the main effective constituent in Glycyrrhiza uralensis and treated as an indicator component to characterize the quality of this herb. Many studies in vivo and in vitro have shown that glycyrrhizic acid exhibits various biological activities, such as anti-inflammatory, anticancer and strengthening immunity, etc [3 5]. Recent irresponsible excessive excavation of wild Glycyrrhiza uralensis plants has resulted in the decrease and extinction of wild Glycyrrhiza uralensis resources. In 2000, Chinese government imposed restrictions on the collection of wild Glycyrrhiza uralensis plants. As a result, cultivar became the main source of this herb. However, the degradation of quality and low content of glycyrrhizic acid are widely present in the cultivar of Glycyrrhiza uralensis. So improving the quality of cultivar of Glycyrrhiza uralensis has became the key problem of their sustainable development. Up to now the biosynthetic pathway for forming glycyrrhizic acid has been clear. 3-Hydroxy-3-methylglutary CoA reductase (HMGR) is considered to play an important role in this pathway [6 8]. HMGR can catalyze HMG-CoA and NADPH to form MVA, which is the first key reaction in glycyrrizic acid biosynthesis. So HMGR is the rate-limiting enzyme in the upstream biosynthesis pathway of glycyrrhizic acid. This work describes the cloning and biochemical characterization of a HMGR cdna from Glycyrrhiza uralensis. The finding and investigation of HMGR in Glycyrrhiza uralensis will provide an efficient route for improving the quality of cultivar of Glycyrrhiza uralensis and revealing the molecular mechanism of the biosynthetic pathway of glycyrrhizic acid. Materials and methods Plant material and RNA extraction The roots of Glycyrrhiza uralensis were collected from the herb garden in Beijing University of Chinese Medicine, Beijing, China. Samples were immersed in liquid nitrogen during the field collection and identified by Prof. LIU Chun-sheng. Voucher specimens were deposited in the herbarium at the School of Chinese Pharmacy, Beijing University of Chinese Medicine. Total RNA of Glycyrrhiza uralensis was extracted from approximately 1 g fresh tissue using the Trizol reagent (Beijing MeiLaiBo Medical Technology Co., LTD), following the manufacturer s instructions. Final RNA concentrations were determined by spectrophotometry and their integrity was examined by electrophoresis in 1% (w/v) agarose gel. Molecular cloning of the full-length Glycyrrhiza uralensis HMGR cdna Single-stranded cdna was synthesized from 12 µl of total RNA using the primers oligo (dt) and reverse transcriptase M-MLV (TaKaRa) in 20 µl reactions, following the manufacturer s instructions. After aligning HMGR sequences of Leguminous plant recorded in GenBank (accession number: XM and GI ), the first amplification step was performed using primers HMGR-F1 and HMGR-R1 (Table 1) designed using conserved regions. The cycling parameters were as follows: 94 for 5 min; 35 cycles of 94 for 30 s, annealing at 5 for 3 s, extension at 72 for 2 min; a final extension at 72 for 7 min. The partial cdna sequences were determined. To obtain the 3'-end cdna sequence, RACE- PCR was carried out. The total RNA of Glycyrrhiza uralensis was used as template, HMGR-3CDS was used as primer (Table 1), then the first stranded cdna was synthesized following the manufacturer s instructions of SMART TM RACE cdna Amplification Kit and used as template of the following two round PCR. In the first round, HMGR-3GSP1 and HMGR-3R1 were used as primers (Table 1), and the cycling parameters were as follows: 94 for 5 min; 35 cycles of 94 for 30 s, annealing at 63 for 30 s, extension at 72 for 2 min; a final extension at 72 for 7 min. In the second round, HMGR-3GSP2 and HMGR-3R2 were used as primers (Table 1), and the cycling parameters were as follows: 94 for 5 min; 35 cycles of 94 for 30 s, annealing at 62 for 30 s, extension at 72 for 2 min; a final extension at 72 for 7 min. To obtain the 5'-end cdna sequence, RACE- PCR was carried out. The total RNA of Glycyrrhiza uralensis was used as template, HMGR-5CDS and HMGR-BD were used as primers (Table 1), then the first stranded cdna was synthesized following the manufacturer s instructions of SMART TM RACE cdna Amplification Kit. In the first round, HMGR- 5GSP1 and HMGR-5UPM mixture (HMGR-5UPM-L: HMGR-5UPM-S = 1 5, Table 1) were used as primers, and the cycling parameters were as follows: 94 for 5 min; 35 cycles of 94 for 30 s, annealing at 60

3 LIU Ying, et al: Cloning and characterization of a cdna coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis 775 Table 1 Primer sequences used in the cloning of Glycyrrhiza uralensis HMGR cdna Primer Sequence (5' 3') HMGR-F1 TTCTTCTCCGTCGCGTACTTTCT HMGR-R1 TTTTGATACCATGTTCATCCCCAT HMGR-3CDS GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)18 HMGR-3GSP1 TTTCGGGCTCAATTCCGTCGTAC HMGR-3R1 GCTGTCAACGATACGCTACGTAACG HMGR-3GSP2 CAGATTCGCCAGGTTGCAGAGTA HMGR-3R2 CGCTACGTAACGGCATGACAGTG HMGR-5BD AAGCAGTGGTATCAACGCAGAGTACGCGGG HMGR-5CDS (T)18VN, (N=A, C, G or T; V=A, G or C) HMGR-5GSP1 GCATTGAACCCACCAAGAGCACC HMGR-5UMP-L CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT HMGR-5UMP-S CTAATACGACTCACTATAGGGC HMGR-5NGSP1 AAGAAGCCGAGGAGGTAGACG HMGR-5NUP AAGCAGTGGTATCAACGCAGAGT HMGR-GHF GGGTCGGAAAATGGACGT HMGR-GHR GGAGGCTTTCGTTATTGGTC HMGR-F CGGGGTACCATGGACGTTCGCCGGAG (Hind III) HMGR-R CCCAAGCTTTGGAGGCTTTCGTTATTGGT (Kpn I) for 30 s, extension at 72 for 2 min; a final extension at 72 for 7 min. In the second round, HMGR- 5NGSP1 and HMGR-5NUP were used as primers (Table 1), and the cycling parameters were same as the first round. All of the obtained fragments mentioned above were sequenced and spliced together, then the fulllength HMGR cdna was determined and used to design specific primers for full-length cdna isolation. HMGR-GHF and HMGR-GHR were used as primers (Table 1), and the cycling parameters were as follows: 94 for 5 min; 35 cycles of 94 for 30 s, annealing at 55 for 30 s, extension at 72 for 2 min; a final extension at 72 for 7 min. The full-length HMGR cdna was obtained and sequenced. Glycyrrhiza uralensis HMGR full-length deduced amino acid sequence was aligned with the publicly available HMGR groups using ClustalX and MEGA version 5.0. A molecular phylogenetic tree was constructed based on aligning publicly available HMGR sequences. Heterologous expression of Glycyrrhiza uralensis HMGR in E. coli Glycyrrhiza uralensis HMGR ORF was amplified using HMGR-F and HMGR-R primers (Table 1), which contained restriction enzyme cutting sites (Hind III and Kpn I) that marked by underline. The PCR procedure was as follows: 94 for 5 min; 35 cycles of 94 for 30 s, annealing at 55 for 30 s, extension at 72 for 2 min; a final extension at 72 for 7 min. The amplified fragments were subcloned into pmd19-t, and the recombinant plasmid HMGR-T was obtained, which was transformed into E.coli DH5α competent cells, and DNA sequencing indicated that it was correct. Then HMGR-T was digested with Hind III and Kpn I (3 h at 37 ). After digestion, the fragments were ligated into the cloning site of Hind III- Kpn I digested pet-32a plasmid for expression of recombinant fusion protein. The resulting recombinant pet-hmgr plasmid was transferred into E.coli BL21 competent cells, then purified and sequenced to check for correct insertion. Individual correct colonies were transferred to 8 ml overnight cultures at 37 and 150 r min 1 in Luria-Bertani medium supplemented with ampicillin (50 mg ml 1 ). Culture conditions and cell lysis method were as previously described in the literature [9]. Both supernatant and pellet were examined by SDS-PAGE (10% resolving gel, 5% stacking gel) using Coomassie brilliant blue staining. Negative controls used possessed a void vector. The fusion proteins were purified as follows: the host bacteria were collected by centrifugation and resuspended with PBS after inducing, which was frozenthawed three times using liquid nitrogen. Then the target proteins were released from the cells by sonication (6 times, 10 s each time, interval was 10 s, voltage was V). Centrifugation was carried out for 20 min at 4, r min 1. Supernatant was purified by flowing through the Ni 2+ -NTA column (using wash buffer to elute the impurity proteins, using

4 776 药学学报 Acta Pharmaceutica Sinica 2013, 48 (5): elution buffer to elute the target protein with 6-His). Enzyme assay and product analysis of Glycyrrhiza uralensis HMGR For enzymatic assay of HMGR, 1 ml of a reaction mixture containing 25 µl K 2 HPO 4 (1 mol L 1, ph = 7.2), 50 µl KCl (1 mol L 1 ), 2 µl EDTA (0.5 mol L 1 ), 5 µl DTT (1 mol L 1 ), 713 µl ddh 2 O, 30 µl NADPH (10 mmol L 1 ), 112 µl HMG-CoA (2.67 mmol L 1 ), and 63 µl HMGR protein was used. Reactions were initiated by crude extract addition, incubated at 30 for 30 min, and terminated by adding 100 µl 6 mol L 1 HCl. Negative control was performed under the same conditions but using the extract form E. coli carrying an empty vector. After standing at 25 for 1 h, two times volume of ethyl acetate was used to extract. Qualitative investigation of HMGR reaction products by TLC analysis was performed using benzene/acetone (2 1) as the developer and vanillic aldehyde as the colour-developing agent. Quantitative analysis of HMGR reaction products by GC-MS analysis was performed using Trace GC and Trace DSQ. The chromatographic column was DB5-MS column (Agilent). Temperature procedure was as follows: standing at 75 for 30 s, warming up to 150 at 25 C min 1, warming up to 200 at 15 min 1, then warming up to 275 at 30 min 1, standing for 5 min. Inlet temperature was 290, carrier gas was high purity helium, flow rate of carrier gas was 1 ml min 1, sampling injection mode was splitless sampling and the sample size was 1 µl. Mass spectrum conditions were as follows: GC-MS interface temperature was 290, ion source temperature was 200, ionization way was EI, and ionization voltage was 70 ev. The structure of production was searched using Agilent Technologies Corp. HP6890G/5973 MS, wiley7n.l. Results 1 Molecular cloning of the full-length Glycyrrhiza uralensis HMGR cdna Using a pair of primers, HMGR-F1 and HMGR- R1 (Table 1), a 915 bp fragment was amplified. After Blast analysis in NCBI, this fragment was determined to be the central fragment of Glycyrrhiza uralensis HMGR gene. To obtain the 3'-end cdna sequence, RACE-PCR was carried out and a 825 bp fragment was isolated. In the 3'-end of the fragment, a poly-a sequence was present. After Blast analysis, the fragment was determined to be the 3'-end of Glycyrrhiza uralensis HMGR gene. Then RACE-PCR was used for the 5'-end of the gene, and a 284 bp fragment was obtained and determined to be the 5'-end of Glycyrrhiza uralensis HMGR gene. All of the obtained fragments mentioned above were sequenced and spliced together, then the full-length HMGR cdna was determined and used to design specific primers, HMGR-GHF and HMGR-GHR (Table 1), for full-length cdna isolation. As a result, the bp full-length cdna sequence (GQ845405) encoding a 573-residue protein was obtained. Using ORF finder to search for the ORF of the gene, the results showed that a bp ORF was present between 39 bp to bp, including a 38 bp 5'-UTR and a 82 bp 3'-UTR. 2 Comparison of the deduced amino acid sequences of HMGR from Glycyrrhiza uralensis and other species Using DNAman software to analyse the amino acid sequence of Glycyrrhiza uralensis HMGR and other publicly available HMGR sequences, such as Pisum sativum, Medicago sativa, Nicotiana tabacum, Eucommia ulmoides, Camptotheca acuminat, Gentiana scabra, Catharanthus roseus, Ginkgo biloba, Arabidopsis thaliana and Artemisia annua, reveals that the most closely related sequence to Glycyrrhiza uralensis HMGR is a sequence from Pisum sativum, with which it shares 84% similarity. The next most closely related sequence to Glycyrrhiza uralensis HMGR is a Medicago sativa HMGR (82.8% similarity). And the similarity with other species are separately 75.1%, 74%, 72.7%, 71.8%, 70.4%, 69.1%, 66.1% and 65.8% from high to low. At the same time, the typical polypeptide sites, which are present in all HMGR sequences to be necessary for the activity of HMGR family, two HMG-CoA binding domains (EMP (V/I) GY (V/I) QIP and TTEGCLVA) and two NADPH binding domains (DAMGMNM and GTVGGGT), also present in the amino acid sequence of Glycyrrhiza uralensis HMGR (Figure 1). These results reveal that functional domains are stable in the process of molecular evolution of HMGR. In Figure 1 the red boxes indicate the functional domains in the HMGR family. The black parts indicate that the amino acid sequences are the same, the pink parts indicate that the similarity of amino acid sequences are more than 75%, the blue parts are about 50% 75%, the yellow parts are about 30% 50%, while the white parts are less than 30%. The molecular phylogenetic tree (Figure 2),

5 LIU Ying, et al: Cloning and characterization of a cdna coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis constructed by the neighbor-joining method in MEGA version 5.0, based on aligning publicly available HMGR sequences, reveals that the most closely related sequence to Glycyrrhiza uralensis HMGR is a sequence from P. sativum, which also belongs to Leguminosae. 777 Furthermore, all the HMGR sequences from plants, fungus, insects and animals separately gather to the same branch, which is consistent with the results of classical taxonomy. The phylogenetic tree shows that all the HMGR proteins come from the same ancestor and

6 778 药学学报 Acta Pharmaceutica Sinica 2013, 48 (5): Figure 1 Comparison of the deduced amino acid sequences of HMGR from Glycyrrhiza uralensis and other species. 1: Glycyrrhiza uralensis; 2: Pisum sativum; 3: Medicago sativa; 4: Artemisia annua; 5: Arabidopsis thaliana; 6: Ginkgo biloba; 7: Camptotheca acuminate; 8: Nicotiana tabacum; 9: Catharanthus roseus; 10: Eucommia ulmoides; 11: Gentiana scabra Figure 2 Phylogenetic tree showing the relationship between HMGR from Glycyrrhiza uralensis and some other species retrieved from the GenBank process the similar function. 3 Heterologous expression of Glycyrrhiza uralensis HMGR in E. coli We expressed the isolated cdna in E. coli as fusion proteins. SDS-PAGE analysis demonstrated the presence of a recombinant protein of apparent molecular mass 60 kd (consistent with the predicted size of the transgene translation product). This protein was absent from the equivalent fractions of cultures of bacteria carrying a void pet32a plasmid. 4 Enzyme assay and product analysis of Glycyrrhiza uralensis HMGR To examine the activity of the recombinant HMGR, the crude extract from induced E. coli with recombinant pet-hmgr plasmid was used for the MVA synthesis reaction. The reaction carried out with the extract from E. coli with a void vector was treated as a negative control. Because MVA was very easy to be lactonizated to form MVL, so in this study we select MVL as the testing index. TLC analysis demonstrated that a new product was produced in the synthesis reaction, while it was not present in the negative control carrying an empty vector. A standard sample of MVL and the peak present in the E. coli transformed with pet-hmgr but absent when transformed with empty pet32a were analyzed by GC-MS. In total ion chromatorgraphy of

7 LIU Ying, et al: Cloning and characterization of a cdna coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis 779 GC-MS, the characteristic peak of reference substance MVL (retention time 6.09 min) was only present in the E. coli transformed with pet-hmgr but absent in the negative control. Meanwhile in MS spectrogram, the results reveal that the characteristic peaks of the novel product were the same as that of the authentic MVL. Discussion A cdna coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis has been obtained and characterized. RACE-PCR was used to obtain the 5'-end and 3'-end of the gene. As a result, the bp full-length cdna sequence encoding a 573-residue protein was obtained. A bp ORF was present between 39 bp to bp, including a 38 bp 5'-UTR and a 82 bp 3'-UTR. We compared the amino acid sequences of HMGR from Glycyrrhiza uralensis and other species, such as Pisum sativum, Medicago sativa, Artemisia annua, etc. The results showed that the functional domains in different HMGR amino acid sequences were close to each other. The typical polypeptide sites, which are present in all HMGR sequences to be necessary for the activity of HMGR family also present in the amino acid sequence of Glycyrrhiza uralensis HMGR. Up to now HMGR gene has been cloned in Salvia miltiorrhiza [7], Ginkgo biloba [10], Ganoderma lucidum [8] and many other species. All these HMGR genes showed the very important function in the biosynthetic pathway of terpenoid. However, HMGR gene in plant belongs to a gene family containing at least two members with different expression patterns. In these studies, we are still not clear the HMGR gene we obtained belong to which one. And this needs a further deep research. Both TLC analysis and GC/MS demonstrated that a new product was produced in the synthesis reaction, while it was not present in the negative control carrying an empty vector. So we could come to a conclusion that the expression product of the gene isolated in this study could catalyze HMG-CoA and NADPH to form MVA, which is the first key reaction in glycyrrizic acid biosynthesis. This study can be used as an important basis for further work on improving the efficacy of Glycyrrhiza uralensis by means of a higher glycyrrhizic acid content, and exploring the biosynthesis of glycyrrhizic acid in vitro. Acknowledgments: We would like to thank Prof. LIU Yong (Beijing University of Chinese Medicine, Beijing, China) for his critical review of the manuscript. References [1] Liu Y, Liu DJ, Liu CS, et al. Mechanism of genuineness of liquorice Glycyrrhiza uralensis based on CNVs of HMGR, SQS1 and β-as gene [J]. Acta Pharm Sin ( 药学学报 ), 2012, 47: [2] State Pharmacopoeia Committee. Pharmacopoeia of People s Republic of China ( 中华人民共和国药典 ) [M]. Part 1. Beijing: China Medical Sciences Press, 2010: [3] Cherng JM, Lin HJ, Hsu YH, et al. A quantitative bioassay for HIV-1 gene expression based on UV activation effect of glycyrrhizic acid [J]. Antivir Res, 2004, 62: [4] Hoever G, Bzltina L, Michaelis M. Antiviral activity of glycyrrhizic acid derivatives against SARS-coronavirus [J]. Med Chem, 2005, 48: [5] Liu DJ. Researches on the Correlation Between CNVs of HMGR, SQS1, β-as Gene and Origin and Morphology of Glycyrrhiza uralensis ( 甘草 HMGR, SQS1, β-as 合酶基因 CNVs 与产地 形态的相关性研究 ) [D]. Beijing: Beijing University of Chinese Medicine, 2011: [6] Ge X, Wu J. Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag + and yeast elicitor [J]. Plant Sci, 2005, 168: [7] Dai Z, Cui GH, Huang LQ. Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation [J]. J Plant Physiol, 2011, 168: [8] Shang CH, Zhu F, Li N, et al. Cloning and characterization of a gene encoding HMG-CoA reductase from Ganoderma lucidum and its functional identification in yeast [J]. Biotechnol Biochem, 2008, 72: J Biosci [9] Comino C, Lanteri S, Portis E, et al. Isolation and functional characterization of a cdna coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L. [J]. BMC Plant Biol, 2007, 7: [10] Shen G, Pang Y, Tang K, et al. Cloning and characterization of a root specific expressing gene encoding 3-hydroxy-3- methylglutaryl coenzyme A reductase from Ginkgo biloba [J]. Mol Biol Rep, 2006, 33: