1. 中南林业科技大学 林业生物技术湖南省重点实验室，湖南 长沙 410004;
2. 中南林业科技大学 森林资源生物技术湖南省国际科技创新合作基地，湖南 长沙 410004
收稿日期：2021-04-07；接收日期：2021-06-03；网络出版时间：2021-06-09
基金项目：国家自然科学基金(Nos. 31900027，31772374，32071673)，中国博士后科学基金(No. 2020M682601)，湖南省自然科学基金(No. 2020JJ5972)，湖南省科技创新计划(No. 2020RC2059)，湖南省教育厅科学研究项目(No. 18B167)，中南林业科技大学研究生科技创新基金(No.CX20202014) 资助
摘要：灵芝是名贵药用真菌，三萜是灵芝的关键药效成分。前期研究发现，磷脂酶D (Phospholipase D，PLD) 产生的磷脂酸(Phosphatidic acid，PA) 可调控三萜合成，为进一步阐明PA调控灵芝三萜合成的分子机制，研究采用PA-beads富集结合LC-MS/MS技术，鉴定灵芝细胞中PA互作蛋白，结果共鉴定到了19个PA互作蛋白，主要包括细胞色素P450单加氧酶(GL22084)、特异性蛋白激酶MAPK (GL23765)、过氧化氢酶和细胞表面疏水性蛋白等。通过基因克隆、原核表达载体构建、蛋白诱导表达和分离纯化，获得了融合GST标签的GL22084和GL23765蛋白，采用GST-pull down实验，验证了灵芝GL22084和GL23765蛋白与PA互作。研究结果揭示了灵芝细胞中PA互作蛋白，为后续解析PLD介导的PA信号分子调控灵芝三萜合成的分子机理奠定了基础；同时，鉴定到的PA互作蛋白也为其他物种的PLD/PA信号通路相关研究提供借鉴。
关键词：磷脂酸互作蛋白灵芝三萜合成蛋白鉴定谷胱甘肽-S-转移酶蛋白下拉实验
Identification of phosphatidic acid interacting proteins in Ganoderma lingzhi
Yongnan Liu1,2, Yuanyuan Yin1,2, Hongwei Hao1,2, Rui Wang1,2, Zhe He1,2, Renyuan Tian1,2, Gaoqiang Liu1,2
1. Hunan Provincial Key Laboratory of Forestry Biotechnology, Central South University of Forestry & Technology, Changsha 410004, Hunan, China;
2. International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province, Central South University of Forestry & Technology, Changsha 410004, Hunan, China
Received: April 7, 2021; Accepted: June 3, 2021; Published: June 9, 2021
Supported by: National Natural Science Foundation of China (Nos. 31900027, 31772374, 32071673), China Postdoctoral Science Foundation (No. 2020M682601), Natural Science Foundation of Hunan Province, China (No. 2020JJ5972), Science and Technology Innovation Program of Hunan Province, China (No. 2020RC2059), Scientific Research Fund of Hunan Provincial Education Department, China (No. 18B167), Scientific Innovation Fund for Post-graduates of Central South University of Forestry and Technology, China (No. CX20202014)
Corresponding author: Gaoqiang Liu. Tel/Fax: +86-731-85623486; E-mail: gaoliuedu@csuft.edu.cn.
Abstract: Ganoderma lingzhi is widely recognized as a medicinal basidiomycetes. Triterpene acids (TAs) are the key bioactive medicinal components of G. lingzhi. Our previous studies have shown that phospholipid acid (PA) produced by phospholipase D (PLD) plays a regulatory role in TA synthesis. In order to further elucidate the molecular mechanism how PA regulates TA synthesis in G. lingzhi, PA beads enrichment combined with LC-MS/MS technology was used to identify PA interacting proteins in G. lingzhi. A total of 19 PA interacting proteins were identified, including cytochrome P450 monooxygenase (GL22084), specific protein kinase MAPK (GL23765), catalase and cell surface hydrophobicity-associated protein. GST tagged GL22084 and GL23765 proteins were obtained through gene cloning, heterologous expression, and purification. The interactions between GL22084/GL23765 and PA were verified by GST pull down assay. The identification of PA interacting proteins provides a basis for further understanding the molecular mechanism how PLD-mediated PA signaling molecules regulates the TA synthesis in G. lingzhi. Moreover, the PA interacting proteins identified in this study can also provide clues for the research of PLD/PA signaling pathway in other species.
Keywords: phospholipid acid interacting proteinGanoderma lingzhitriterpenoid biosynthesisprotein identificationGST-pull down
中国灵芝Ganoderma lingzhi S.H. Wu, Y. Cao & Y.C. Dai，原学名Ganoderma lucidum (Curtis) P. Karst.[1]，为名贵药用担子菌，具有抑制肿瘤生长[2]、保护肝脏[3]和抗人类免疫缺陷病毒[4]等药理功能。近年来，国内外****对灵芝的化学成分、药理作用及临床效果等进行了研究，目前已从灵芝中分离得到160多种化合物，其中三萜类化合物(特别是灵芝酸Ganoderic acid，GA) 具有显著的药理作用，是灵芝的关键药理活性物质，也是决定灵芝药效高低的重要指标[5-6]。
目前，通过控制发酵策略[7-8]、添加化学诱导剂[9-10]和基因工程[11-12]等方法，提高灵芝中GA产量的相关研究已取得了一定进展。灵芝三萜合成的分子调控机制领域也取得了一些成果，初步阐明了活性氧[13]、钙离子[14]、cAMP[15]和磷脂信号[16-17]在三萜生物合成中的作用。然而，这些已知信号分子的下游通路(位点/靶点) 对于三萜生物合成的调控机制仍知之甚少。
磷脂酸(Phosphatidic acid，PA) 是生物细胞内天然存在的最简单的甘油磷脂，也是细胞膜的一个次要组成部分，由于其具有多种生物功能而受到越来越多的关注。例如，作为合成所有膜甘油磷脂的关键中间代谢物，PA有助于细胞膜的形成，从而在活细胞中发挥重要的膜结构基础作用[18]。此外，PA还被认为是一类脂质信号分子，通过与蛋白质结合，包括转录因子、蛋白激酶、脂类激酶、蛋白磷酸酶等，参与各种植物过程[19]。例如，磷脂酶D (Phospholipase D，PLD) 介导产生的PA可与组成型三重反应蛋白(Constitutive triple response，CTR) 相互作用，参与缺氧胁迫条件下的植物乙烯信号转导[20-21]。PA与植物两个生物钟(昼夜节律) 相关蛋白(Late elongated hypocotyl，LHY；circadian clock associated，CCA)相互作用并调节其功能，以调节植物昼夜节律生物钟[22]。然而，在微生物中，对PA的生物调节功能的研究却很少，PA互作蛋白的研究也完全不清楚。
我们先前的研究发现PLD介导产生的PA信号在灵芝三萜生物合成中发挥关键调控作用[17]，为进一步阐明PA调控灵芝三萜合成的分子机制，本研究采用PA-beads富集灵芝细胞PA互作蛋白，结合LC-MS/MS技术，鉴定灵芝细胞中PA互作蛋白，进一步利用GST-pull down进行免疫学验证，为后续深入研究PA调控灵芝三萜生物合成的分子机制奠定基础，也为其他物种的PA信号通路相关研究提供借鉴。
1 材料与方法1.1 材料与试剂1.1.1 菌株与培养基供试灵芝菌株为SCIM 1005 (Strain Collection of Industrial Microorganisms)；
培养基(g/L)：葡萄糖(44.0)、玉米粉(0.5)、蛋白胨(6.5)、磷酸二氢钾(0.75)，MgSO4·7H2O (0.45) 和维生素B1 (0.01)。
1.1.2 主要试剂蛋白提取液：10 mmol/L HEPES-KOH (pH 7.9)，1.5 mmol/L MgCl2，10 mmol/L KCl。
PA-beads：购自Echelon Biosciences公司，P-B0PA。
洗涤缓冲液：10 mmol/L HEPES-KOH (pH 7.4)，150 mmol/L NaCl，0.25% Igepal。
2× Laemmli sample buffer：4% SDS、20%甘油、10% 2-巯基乙醇、0.004%溴酚蓝和0.125 mol/L Tris-HCl (pH 6.8) (Sigma，S3401)。
1.2 方法1.2.1 灵芝培养条件灵芝菌丝在液体培养基中，27 ℃、160 r/min黑暗培养7 d，过滤收集菌丝体(设置3个重复)，预冷PBS清洗2遍。
1.2.2 蛋白提取菌丝体样品进行液氮研磨，使用蛋白提取液提取灵芝总蛋白，总蛋白液用Bradford法进行定量后，进行PA互作蛋白富集。
1.2.3 PA互作蛋白富集取10 μg蛋白用Washing buffer稀释，加入100 μL PA-beads试剂，蛋白与PA-beads置于4 ℃摇床温和孵育3 h。离心去上清，加入10倍体积洗涤缓冲液清洗PA-beads三遍。加入等体积的2× Laemmli sample buffer，95 ℃反应10 min洗脱PA互作蛋白，离心取上清蛋白液用于后续分析。
1.2.4 PA互作蛋白鉴定上述蛋白样品进行SDS-PAGE分离，将SDS-PAGE中的蛋白条带进行切割，切割条带用蛋白胶回收试剂盒进行蛋白回收纯化，蛋白经过酶解后，进行Q Exactive-HF质谱仪上机检测，ProteomeDiscover 2.4软件进行检索，鉴定结果比对数据库为已发表的灵芝基因组翻译而来的蛋白序列[23]，实验简要流程如下所示：
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表 1 克隆GL22084和GL23765基因的引物信息Table 1 Primers for cloning GL22084 and GL23765 genes
| Primer name | Primer sequence (5'–3')* |
| GL22084-F | AATCGGATCTGGTTCCGCGTggatccATGTCCTCTGCTCGTGTTTGG |
| GL22084-R | GGCCGCTCGAGTCGACCCGGgaattcCTAATTGGTATCGTATCGAAGA |
| GL23765-F | AATCGGATCTGGTTCCGCGTggatccATGGCGACCGTGACCCCCG |
| GL23765-R | GGCCGCTCGAGTCGACCCGGgaattcCTAGAACGTCGACGCCCACGCC |
| *The large and small letters in italics of each primer are homologous DNA sequences and restriction sites, respectively. | |
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1.2.6 GL22084和GL23765蛋白诱导表达与分离纯化将测序正确的BL21菌株接种于液体LB培养基中，摇床培养(37 ℃、200 r/min) 至OD600为0.55–0.6之间，取1 mL菌液作为对照，在剩余的菌液中加入1 mmol/L的异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactoside，IPTG) 于20 ℃摇床200 r/min培养12 h诱导表达。采用GST融合蛋白纯化磁珠(苏州英芮诚生化科技) 进行蛋白纯化，纯化步骤按照说明书进行，纯化获得的蛋白进行SDS-PAGE检测。
1.2.7 GST-pull down验证将纯化获得的蛋白液与PA-beads孵育洗脱(方法同1.2.3)，获得的蛋白液进行Western blotting分析(未与PA-Beads孵育的蛋白样品为input阳性对照，GST标签蛋白为阴性对照)：采用SDS-PAGE分离蛋白样品，利用电转膜仪(恒流250 mA，2 h) 将PAGE胶中的蛋白转移到聚偏二氧乙烯(Polyvinylidene fluoride，PVDF)膜上；使用5%脱脂牛奶室温封闭PVDF膜1 h后，加入GST一抗(Engibody)，室温条件下摇床摇动1 h，然后4 ℃过夜；采用PBST洗膜3次(10 min/次)，加入HRP二抗(Engibody)，孵育1 h；之后PBST缓冲液洗膜2次，PBS缓冲液洗膜一次(10 min/次)；最后将PVDF膜置于成像系统进行显色分析。
2 结果与分析2.1 质谱鉴定PA互作蛋白经质谱鉴定并与灵芝基因组数据库比对，共获得19个PA互作蛋白，进一步利用NCBI BLAST分析PA互作蛋白的功能，所得结果如表 2所示。检测出的灵芝蛋白主要有细胞色素P450单加氧酶、特异性蛋白激酶(丝裂原激活蛋白激酶)、过氧化氢酶、细胞表面疏水性蛋白、磷脂酸胞苷酰转移酶、转录延伸因子、rRNA加工蛋白和核糖体蛋白等(鉴定到的蛋白氨基酸序列见《生物工程学报》网络版附件2)。
表 2 质谱鉴定到的灵芝PA互作蛋白Table 2 Identification of PA interacting proteins by mass spectrometry
| Protein accession | Description | Amino acid residues | Protein molecular weight (kDa) |
| GL29943-R1_1 | Translation elongation factor | 1 101 | 119.4 |
| GL26723-R1_1 | Pre-rRNA-processing protein | 307 | 34 |
| GL30114-R1_1 | Phosphopyruvate hydratase | 442 | 46.9 |
| GL25415-R1_1 | Phosphatidate cytidylyltransferase | 471 | 51.4 |
| GL22084-R1_1 | Cytochrome P450 monooxygenase | 341 | 37.7 |
| GL20732-R1_1 | Glyoxaloxidase | 230 | 24.9 |
| GL23765-R1_1 | Specificity protein kinase, mitogen-activated protein kinase | 462 | 50.7 |
| GL25601-R1_1 | β-D-glucanases, glycoside hydrolase family 16 protein (GH16) | 387 | 40.6 |
| GL17383-R1_1 | Cell surface hydrophobicity-associated protein | 273 | 30.5 |
| GL22189-R1_1 | Catalase | 532 | 59.4 |
| GL22047-R1_1 | Carbohydrate-binding protein, ricin family protein | 139 | 15.8 |
| GL30174-R1_1 | Aldehyde dehydrogenase | 526 | 57.7 |
| GL31438-R1_1 | 40S ribosomal protein S7 | 196 | 22.3 |
| GL25761-R1_1 | 40S ribosomal protein S5 | 215 | 23.8 |
| GL19949-R1_1 | 40S ribosomal protein S2 | 305 | 33 |
| GL17407-R1_1 | Hypothetical protein | 517 | 56.1 |
| GL17187-R1_1 | Hypothetical protein | 800 | 87.5 |
| GL15423-R1_1 | Hypothetical protein | 381 | 41.9 |
| GL16183-R1_1 | Hypothetical protein | 372 | 40.4 |
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2.2 GST-pull down验证为了验证质谱鉴定到的灵芝PA互作蛋白，我们选择细胞色素P450单加氧酶(GL22084) 和丝裂原活化蛋白激酶MAPK (GL23765) 进行免疫学GST-pull down验证。通过诱导表达和SDS-PAGE分析(图 1A)，GL23765和GL22084蛋白成功诱导表达；利用GST融合蛋白纯化磁珠纯化和SDS-PAGE分析，获得了纯化的GL23765和GL22084蛋白(图 1B)；进一步采用GST-pull down实验分析，结果显示GL23765和GL22084蛋白与PA互作(图 1C)。以上结果与质谱鉴定结果一致。
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| 图 1 GST-pull down验证两个蛋白GL23765和GL22084与PA互作 Fig. 1 Verification of interactions between two proteins GL23765 and GL22084 to PA by GST-pull down. (A) GL23765 (lanes a2–a4) and GL22084 (lanes b2–b4) were successfully induced to express. Lanes a1 and b1 were pre-induced proteins. (B) After purification, GL23765 (lanes a1–a4) and GL22084 (lanes b1–b4) proteins were obtained. (C) The purified GL23765 and GL22084 proteins were incubated with PA beads, eluted and the obtained proteins was analyzed by Western blotting with GST antibody. GL23765 and GL22084 proteins not incubated with PA beads were set as parallel control (input), and GST protein was used as a negative control. |
| 图选项 |
3 讨论灵芝三萜的合成从乙酰辅酶A形成甲羟戊酸开始，经过甲羟戊酸途径中的酶催化反应，甲羟戊酸被催化合成羊角甾醇(合成途径明确)，羊角甾醇进一步反应形成三萜的催化过程还不清楚，但很可能包括细胞色素P450超家族的一系列氧化、还原和酰化反应等；灵芝中有78个细胞色素P450基因与羊角甾醇合成酶基因共表达，其中16个最有可能直接参与灵芝三萜合成[23]。最近的研究发现，cyp5150l8、cyp512a2、cyp512v2和cyp512a13与灵芝三萜单体合成直接相关[12, 24]。本研究鉴定到一个PA互作的细胞色素P450蛋白(GL22084)，为后续深入解析PA直接调控三萜生物合成相关酶(CYP450) 的机制研究提供了借鉴。
MAPK磷酸化级联反应链是真核生物信号传递网络中的重要途径之一，调节着细胞的生长、分化、对环境的应激适应、炎症反应和基因表达等多种重要的细胞生理/病理过程[25]。在植物盐胁迫下，PLD产生的PA与植物MAP6结合，并促进其活性，调控细胞骨架微管的聚合和成束，参与耐盐响应[26-27]。相一致的是，本研究在灵芝中也鉴定到一个PA互作的MAPK蛋白(GL22084)，但GL22084在灵芝中的具体功能，尤其是在三萜合成中的调控作用，还有待后续研究。
鉴于脂质信号分子的重要性，采用不同的方法鉴定脂质分子互作蛋白研究已有报道，主要利用磁珠富集或脂质体富集结合质谱鉴定的胞外鉴定方法。例如，Jungmichel等利用磁珠法开展了动物肌醇磷脂互作蛋白的鉴定[28]，并采用GST-pull down实验进行了验证，该研究方法与本研究方案一致。Kim等利用主要含有PA的脂质体开展了植物PA互作的转录因子蛋白鉴定，并进一步利用鉴定蛋白的特异性抗体进行免疫共沉淀，通过脂质提取和脂类分子质谱鉴定，证明了鉴定蛋白与PA在胞内互作[22]。然而，胞内鉴定脂质分子互作蛋白依赖特异性蛋白抗体等因素，实验成功率较低，实验难度大，相关报道相对较少。此外，最近的研究还开发了基于荧光信号进行活细胞观测PA分子和监测PLD酶活的方法，为活细胞研究PLD介导的PA信号分子的生理功能奠定了基础[29-30]。
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