Study on the Properties of Recombinant Prolyl Endopeptidase from Abalone (Haliotis discus hannai) Muscle
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摘要: 为研究皱纹盘鲍（Haliotis discus hannai）中脯氨酰内肽酶（Prolyl endopeptidase，Hdh-PEP)的酶学特性与结构特性，利用基因工程技术重组并在大肠杆菌中高效表达了皱纹盘鲍PEP。原核表达的Hdh-PEP分子量为85 kDa，在pH2～6、温度20～60 ℃条件下，Hdh-PEP的表面疏水性明显升高。氨基酸序列同源性分析结果表明，Hdh-PEP催化结构域中有三个高度保守的氨基酸序列：Seq 1：K-D-G-T-K/R-I-P、Seq 2：Y-G-Y-G-G-F和Seq 3：I-R-G-G-E-Y/F。酶动力学研究表明，Hdh-PEP的米氏常数Km为5.32 μmol/L，催化常数kcat值为15.7 s−1。PEP的特异性抑制剂SUAM-14746和ZPP对Hdh-PEP酶活力具有强抑制作用，丝氨酸蛋白酶抑制（PMSF）对Hdh-PEP酶活力也有较大程度的抑制作用。本实验制备了高特异性抗Hdh-PEP多克隆抗体，可检测鲍鱼肌肉中天然PEP的存在情况。Hdh-PEP的体外高效表达和特异性多克隆抗体制备为后续深入研究Hdh-PEP的性质提供了重要参考。Abstract: In order to study the enzymatic characteristics and structure of prolyl endopeptidase from Haliotis discus hannai (Hdh-PEP), recombinant PEP was cloned and highly expressed in E.coli. Hdh-PEP with molecular weight of 85 kDa was successfully purified and its surface hydrophobicity was greatly affected by pH at low value (pH2～6) and temperature (20～60 ℃). Amino acid sequence homology analysis showed that there were three highly conserved sequences in the catalytic domain of Hdh-PEP: Seq 1: K-D-G-T-K/R-I-P, Seq 2: Y-G-Y-G-G-F and Seq 3: I-R-G-G-E-Y/F. Kinetic study revealed that the Km and kcat of Hdh-PEP were 5.32 μmol/L and 15.7 s−1, respectively. Specific inhibitors SUAM-14746 and ZPP of PEP had strong inhibition on Hdh-PEP activity, and serine protease inhibitor (PMSF) also exhibited inhibition. A high specific polyclonal antibody toward Hdh-PEP was prepared which could be applied for the detection of native PEP in abalone muscle. The successful in vitro expression of Hdh-PEP and preparation of a specific polyclonal antibody against Hdh-PEP provided an important reference for subsequent investigation on Hdh-PEP.
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图 1 Hdh-PEP在鲍鱼不同组织中的相对酶活
Figure 1. Relative enzymatic activity of Hdh-PEP in different tissues
图 2 纯化Hdh-PEP的SDS-PAGE分析
注：M，标准蛋白；1，表达菌株的全菌液；2，表达菌株的上清液；3，纯化后的Hdh-PEP。
Figure 2. SDS-PAGE analysis of purified Hdh-PEP
图 3 PEP的氨基酸序列和二级结构比对
注：1，Hdh-PEP；2，Gm-PEP；3，Pc-PEP；4，Hm-PEP；5，Mx-PEP；蓝色实线，催化结构域；蓝色虚线，β-螺旋桨结构域；红色，保守的氨基酸序列。
Figure 3. Amino acid sequence and secondary structure comparison of PEPs
图 4 Hdh-PEP的Lineweaver-Burk双倒数图
Figure 4. Lineweaver-Burk double reciprocal of Hdh-PEP
图 5 pH和温度对Hdh-PEP表面疏水性的影响
注：A、C，pH和温度对荧光强度的影响；B、D，pH和温度对疏水指数的影响。
Figure 5. Effect of pH and temperature on the surface hydrophobicity of Hdh-PEP
图 6 Hdh-PEP多克隆抗体的SDS-PAGE分析和Hdh-PEP的Western blotting分析
注：A，Hdh-PEP多克隆抗体的SDS-PAGE；B，Hdh-PEP的Western blotting分析。泳道M，标准蛋白；1，Hdh-PEP多克隆抗体；2，鲍鱼肌肉蛋白；3，天然Hdh-PEP的Western blotting分析。
Figure 6. SDS-PAGE analysis of Hdh-PEP polyclonal antibody and western blotting analysis of Hdh-PEP
表 1 蛋白酶抑制剂对Hdh-PEP的作用
Table 1. Effect of proteinase inhibitors on the activity of Hdh-PEP
抑制剂 终浓度（μmol/L） 相对酶活（%） 对照组 0 100.0 SUAM-14746 5 5.7 ZPP 5 6.9 PMSF 5 58.5 EDTA 2 86.9 EGTA 5 98.2 E-64 5 89.5 Benzamidine 1 96.7 Leupeptin 5 94.1 
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[1] 农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会编制. 中国渔业统计年鉴-2019[M]. 北京: 中国农业出版社, 2019. [2] 朱蓓薇. 海珍品加工理论与技术的研究[M]. 北京: 科学出版社, 2010: 152-158. [3] Yadav P, Goyal V D, Gaur N K, et al. Carboxypeptidase in prolyl oligopeptidase family: Unique enzyme activation and substrate-screening mechanisms[J]. The Journal of Biological Chemistry,2019,294(1):89−100. doi: 10.1074/jbc.RA118.004254 [4] Babkova K, Korabecny J, Soukup O, et al. Prolyl oligopeptidase and its role in the organism: Attention to the most promising and clinically relevant inhibitors[J]. Future Medicinal Chemistry,2017,9(10):1015−1038. doi: 10.4155/fmc-2017-0030 [5] Männistö P T, García-Horsman J A. Mechanism of action of prolyl oligopeptidase (PREP) in degenerative brain diseases: Has peptidase activity only a modulatory role on the interactions of PREP with proteins?[J]. Frontiers in Aging Neuroscience,2017,9:27. [6] García-Horsman J A, Männistö P T, Venäläinen J I. On the role of prolyl oligopeptidase in health and disease[J]. Neuropeptides,2007,41(1):1−24. doi: 10.1016/j.npep.2006.10.004 [7] Myöhänen T T, Pyykkö E, Männistö P T, et al. Distribution of prolyl oligopeptidase in human peripheral tissues and in ovarian and colorectal tumors[J]. Journal of Histochemistry & Cytochemistry,2012,60(9):706−715. [8] Venditti M, Aniello F, Santillo A, et al. Study on PREP localization in mouse seminal vesicles and its possible involvement during regulated exocytosis[J]. Zygote (Cambridge, England),2019,27(3):160−165. doi: 10.1017/S0967199419000194 [9] Campbell D J. Neprilysin inhibitors and bradykinin[J]. Frontiers in Medicine,2018,5:257. doi: 10.3389/fmed.2018.00257 [10] Prades R, Munarriz-Cuezva E, Urigüen L, et al. IPR-166: A potent and selective prolyl oligopeptidase inhibitor ameliorates cognitive symptoms associated to schizophrenia after oral administration[J]. European Neuropsychopharmacology,2019,29:S244. [11] Colgrave M L, Byrne K, Howitt C A. Liquid chromatography-mass spectrometry analysis reveals hydrolyzed gluten in beers crafted to remove gluten[J]. Journal of Agricultural and Food Chemistry,2017,65(44):9715−9725. doi: 10.1021/acs.jafc.7b03742 [12] Luoto S, Jiang Z Q, Brinck O, et al. Malt hydrolysates for gluten-free applications: Autolytic and proline endopeptidase assisted removal of prolamins from wheat, barley and rye[J]. Journal of Cereal Science,2012,56(2):504−509. doi: 10.1016/j.jcs.2012.06.004 [13] Walter T, Wieser H, Koehler P. Production of gluten-free wheat starch by peptidase treatment[J]. Journal of Cereal Science,2014,60(1):202−209. doi: 10.1016/j.jcs.2014.02.012 [14] 翁凌, 林韵, 张玲, 等. 草鱼肌肉脯氨酸内肽酶的研究[J]. 中国食品学报,2015,15(9):23−31. [15] 王梦想. 鲤鱼肌肉脯氨酸内肽酶的分离纯化及性质分析[D]. 厦门: 集美大学, 2015. [16] 肖琳琳. 罗非鱼和鲈鱼骨骼肌脯氨酸内肽酶的研究[D]. 厦门: 集美大学, 2017. [17] 唐俊, 颜龙杰, 翁凌, 等. 蓝圆鲹骨骼肌脯氨酸内肽酶的分离纯化及其对胶原肽的作用[J]. 现代食品科技,2016,32(6):122−129. [18] 颜龙杰. 刺参(Stichopus japonicas)胶原蛋白及自溶相关蛋白酶的研究[D]. 厦门: 集美大学, 2019. [19] 郑杰, 宋志远,于笛, 等. 水产动物自溶研究进展[J]. 食品工业科技,2015,36(13):397−400. [20] 谢雪琼. 鲢鱼肌肉脯氨酰内肽酶及抑制肽的研究[D]. 厦门: 集美大学, 2019. [21] 肖琳琳, 翁凌, 钟婵, 等. 罗非鱼脯氨酸内肽酶的分离及抑制剂对其的作用机制[J]. 集美大学学报(自然科学版),2017,22(1):10−20. [22] Li W Y, Li Y, Chen Y L, et al. Characterization and crystal structure of prolyl endopeptidase from abalone (Haliotis discus hannai)[J]. Food Chemistry,2020,333:127452. doi: 10.1016/j.foodchem.2020.127452 [23] 李越. 皱纹盘鲍脯氨酰内肽酶的分子克隆、体外表达及相关性质分析[D]. 厦门: 集美大学, 2018. [24] 杨阳. 拟穴青蟹(Scylla paramamosain)肌浆蛋白过敏原的致敏性研究[D]. 厦门: 集美大学, 2018. [25] Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server[J]. Nucleic Acids Research,2014,42(W1):W320−W324. doi: 10.1093/nar/gku316 [26] Kato A, Nakai S. Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins[J]. Biochimica et Biophysica Acta (BBA) - Protein Structure,1980,624(1):13−20. doi: 10.1016/0005-2795(80)90220-2 [27] 邹睿. 贝类副肌球蛋白的分离纯化、性质研究与分子克隆[D]. 厦门: 集美大学, 2019. [28] Ohta N, Takahashi T, Mori T K, et al. Hormonal modulation of prolyl endopeptidase and dipeptidyl peptidase IV activities in the mouse uterus and ovary[J]. Acta Endocrinologica,1992,127(3):262−266. doi: 10.1530/acta.0.1270262 [29] Yoshida K, Inaba K, Ohtake H, et al. Purification and characterization of prolyl endopeptidase from the Pacific herring, Clupea pallasi, and its role in the activation of sperm motility[J]. Development, Growth & Differentiation,1999,41(2):217−225. [30] Wang M X, Zhong C, Cai Q F, et al. Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)[J]. Process Biochemistry,2012,47(12):2211−2218. doi: 10.1016/j.procbio.2012.08.016 -
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