浙江大学学报 (农业与生命科学版)  2017, Vol. 43 Issue (2): 183-191
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青鳉组织蛋白酶E基因全长cDNA克隆与功能预测[PDF全文]
王家庆1 , 董慧明2, 李振刚3, 李绍明1, 王若楠1, 付玉洁1    
1. 沈阳工学院生命工程学院,辽宁 抚顺 113122;
2. 辽宁省食品检验检测院,沈阳 110015;
3. 吉林大学生命科学学院, 长春 130012
摘要: 利用反转录聚合酶链式反应和cDNA末端快速扩增技术克隆青鳉(Oryzias latipes)肠道组织蛋白酶E(cathepsin E, CtpE)基因全长cDNA序列,并分析青鳉组织蛋白酶(OlCtpE)的功能。结果显示:OlCtpE基因cDNA(GenBank登录号:KP864679)全长1 301 bp,其中5'端非翻译区24 bp, 3'端非翻译区56 bp,开放阅读框1 221 bp,编码406个氨基酸;OlCtpE蛋白N端含有一个由17个氨基酸组成的信号肽,属于分泌蛋白;同源比对显示,OlCtpE蛋白由2个以活性位点“DTGT”为催化中心的同源结构域组成,对称分布的2个保守的催化中心形成“催化二联体”;三级结构分析显示,负责底物固定的“活性中心翼环”延伸到活性部位上,活性部位的发夹式裂隙可以为底物与酶的契合提供空间。
关键词: 青鳉    组织蛋白酶E    基因克隆    功能预测    
Cloning and function prediction of full-length cDNA for cathepsin E derived from medaka (Oryzias latipes)
WANG Jiaqing1 , DONG Huiming2, LI Zhengang3, LI Shaoming1, WANG Ruonan1, FU Yujie1    
1. College of Life Engineering, Shenyang Institute of Technology, Fushun 113122, Liaoning, China;
2. Liaoning Institute for Food Control, Shenyang 110015, China;
3. School of Life Science, Jilin University, Changchun 130012, China
Summary: Cathepsin is a kind of protease that mainly exists in intracellular lysosome. Under the weak acid condition, cathepsin can be activated and acts as hydrolysate protein. Based on the different mechanisms of the protein hydrolysis, cathepsin is divided into four species, including aspartic acid protease, cysteine protease, serine protease and threonine protease. Cathepsin D and cathepsin E (CtpE) both belong to aspartic proteases, while the latter is fundamental basis for life activities of mammals. Moreover, CtpE is an important enzyme in participating physiological processes of aquatic animals, such as digestion, yolk formation and immune response. However, few researches were focused on immune function of CtpE gene in fish. In this study, a full-length cDNA of CtpE was cloned from medaka (Oryzias latipes), to identify the gene and protein sequences of CtpE in medaka, and to clarify the evolutionary relationship of O. latipes CtpE (OlCtpE) with other animals, providing a theoretical foundation for further research on the physiological function of CtpE in fish. The total RNA was extracted from medaka gut tissue, using Trizol kit according to the manual steps. The quality of total RNA was extracted by agarose gel electrophoresis. Reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to clone the full-length cDNA of OlCtpE from gut tissue in medaka. According to GenBank, the CtpE gene and corresponding protein sequence of Fundulus heteroclitus, Poecilia formosa, Austrofundulus limnaeus and Larimichthys crocea were downloaded and then analyzed through the global Clustal X alignment. The conserved region of the medaka OlCtpE gene fragment was amplified using the degenerate primers PF1 and PR1. The 3' RACE specific primers PE2 and PE3 were designed according to the conserved sequence, which was amplified by degenerate primers PF1 and PR1 using DNAStar software. The 5' RACE specific primers PE4, PE5 and PE6 were designed according to the conserved sequence, which was also amplified by degenerate primers PF1 and PR1 using DNAStar software. The RT-PCR product sequence, the 3' RACE and 5' RACE product sequences were assembled by using DNAman software. Clustal X 1.81 and MEGA 4.0 softwares were used to analyze the amino acid homology. The basic physical and chemical properties of proteins were predicted by ExPASy-PROSITE and ExPASy-ProtParam. The signal peptide and the glycosylation sites were predicted by SignalP 4.1 and NetNGlyc 1.0, respectively. The tertiary structure of OlCtpE protein was predicted by homology modeling method using SWISS-MODEL software. The results showed that the full-length cDNA of the OlCtpE was 1 301 bp, containing 24 bp 5'-untranslated regions (UTR), 56 bp 3'-UTR and 1 221 bp open reading frame, presumably encoding 406 amino acids. The cloned cDNA sequence of OlCtpE gene has been submitted to the GenBank database (accession number: KP864679). The N-terminus of OlCtpE protein contained a signal peptide of 17 amino acids, and it was a secretory protein. The OlCtpE protein contained three N-linked glycosylation sites, "NPTI"(amino acids 26-29), "NFSV"(amino acids 95-98) and "NLTV"(amino acids 162-165). The sequence homology from medaka CtpE protein was 77% with those of F. heteroclitus and A. limnaeus, 74% with that of P. latipinna, and 71% with that of L. crocea. A total of 22 CtpE proteins in different fish species had the active sites of conserved aspartate protease, which were"VIFDTGSSDLWV"(amino acids 98-109) and"AIVDTGTSLIAG"(amino acids 284-295) by homology analysis. The amino acid sequence homology was 43.60% between the CtpE protein from medaka and the porcine pepsinogen, and the two tertiary structures were also very similar. The tertiary structural analysis showed that the substrate was fixed to the active site in the active center, and the hairpin of the active site could provide space for the combination of substrate and enzyme. The overall shape of CtpE protein from medaka and porcine pepsinogen showed ellipsoid, and formed a relatively independent space entity. In conclusion, OlCtpE may play a very important role in the immune processing and presentation of exogenous antigen. Cloning and function analysis of the OlCtpE provide essential evidence for further studies on immune gene function.
Key words: medaka (Oryzias latipes)    cathepsin E    gene cloning    function prediction    

组织蛋白酶是一类主要存在于细胞内溶酶体中的蛋白酶,在弱酸性条件下被活化后可发挥水解蛋白质的功能[1]。根据其水解蛋白质的机制不同,组织蛋白酶被分为天冬氨酸蛋白酶、半胱氨酸蛋白酶、丝氨酸蛋白酶以及苏氨酸蛋白酶等。其中,天冬氨酸蛋白酶包括组织蛋白酶D和组织蛋白酶E (cathepsin E, CtpE)2种类型[2]。在哺乳动物中,CtpE参与底物水解、组织分化与再生、肿瘤入侵和转移、抗原呈递以及细胞凋亡等重要生命活动[3-4]。BENNETT等[5]早在1992年就发现CtpE具有抗原呈递功能。目前,在水产动物中发现CtpE参与消化、卵黄发生、免疫应答等生理过程[6-7]

鱼类作为一种低等脊椎动物,终生生活在水环境中。随着当前我国大部分地区水环境中各种重金属离子、赤潮和有害病菌的产生等环境恶化问题日益突出,鱼类的生存问题正面临巨大的挑战。组织蛋白酶是免疫应答过程的关键性酶类,弄清它在鱼体内的功能,有望解决目前鱼类在所面临的环境胁迫下如何能够健康生长与繁殖的难题。青鳉 (Oryzias latipes),属鳉形目,青鳉科,青鳉属。目前已被广泛作为模式鱼类应用于生命科学的诸多领域中,特别是在发育生物学、动物遗传学以及环境毒理学等方面[8]。本研究从青鳉肠道组织中克隆了CtpE基因 (OlCtpE) 的cDNA全长序列,确定了青鳉肠道中存在CtpE基因表达,并分析了青鳉与其他动物CtpE的进化关系,旨在为进一步研究鱼类组织蛋白酶的生理作用奠定理论基础。

1 材料与方法 1.1 试验材料

青鳉采集自河北省秦皇岛市海滨湿地,在实验室内暂养后进行肠道组织总RNA提取。RNA提取试剂Trizol购自美国Invitrogen公司;AMV反转录酶、Taq DNA聚合酶、末端转移酶、RNaseH、T载体、3' cDNA末端快速扩增 (rapid amplification of cDNA ends, RACE) 试剂盒等均为TaKaRa宝生物工程 (大连) 有限公司产品;凝胶回收与连接酶试剂盒均为生工生物工程 (上海) 股份有限公司产品。

1.2 引物设计

根据GenBank数据库中已知的底鳉 (Fundulus heteroclitus)、帆鳍鳉 (Poeciliaformosa)、澳鳉 (Austrofundulus limnaeus) 及大黄鱼 (Larimichthys crocea) 等鱼类的CtpE基因序列和对应的蛋白质序列,通过Clustal X全局比对,在CtpE基因的保守区域设计简并引物PF1和PR1,用于扩增青鳉OlCtpE基因片段;引物PE2和PE3是根据简并引物PF1和PR1扩增出的保守序列,使用DNAStar软件设计的用于3' RACE的特异引物;引物PE4、PE5和PE6是根据简并引物PF1和PR1扩增出的保守序列而设计的用于5' RACE的特异引物;衔接头引物AP及OligodT-AP均来自试剂盒。各引物序列见表 1,委托TaKaRa生物公司合成。

表1 扩增试验所用引物及其序列 Table 1 Primers and their sequences used in this study
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1.3 组织总RNA抽提

取青鳉肠道组织研磨破碎,按Trizol试剂盒说明书步骤提取总RNA。用琼脂糖凝胶电泳检验所提取的总RNA质量。

1.4 OlCtpE基因保守编码区扩增

取成功提取的总RNA 5 μg, 使用引物OligodTAP,按照AMV反转录酶操作手册进行反转录聚合酶链式反应 (reverse transcription-polymerase chain reaction, RT-PCR),再用引物PF1和PR1,以RT-PCR产物为模板扩增OlCtpE基因约600 bp的保守区。PCR扩增体系:总体积25 μL, 包含2.5 μL 10×PCR反应缓冲液,2 μmol/L MgCl2, 200 μmol/L dNTP,引物各0.1 μmol/L, 0.125 U Taq DNA聚合酶。PCR扩增条件:94 ℃预变性5 min; 94 ℃变性1 min, 59 ℃复性1 min, 72 ℃延伸1.5 min,31个循环;最后,72 ℃ 10 min;于4 ℃下贮存,待用。PCR扩增产物以1%琼脂糖凝胶电泳分离,经切胶回收后,再以T-A克隆法将其克隆至T载体并测序。根据上述测序序列再次设计特异性的3' RACE引物和5' RACE引物,用于扩增OlCtpE基因的3'和5'端序列。

1.5 3' RACE技术扩增3'端非翻译区

取上述AMV反转录酶的反转录产物, 用引物AP和3' RACE特异引物PE2进行扩增,反应体系按操作指南进行。扩增条件:94 ℃ 4 min;94 ℃ 1 min,59 ℃ 1 min,72 ℃ 1 min,32个循环;最后,72 ℃延伸10 min;于4 ℃下贮存,待用。为确保扩增的特异性, 将上述PCR产物稀释10倍后,再取2 μL作为模板, 用引物AP和引物PE3再次进行扩增,扩增产物用1%琼脂糖凝胶电泳分离回收,并送至生工生物工程 (上海) 股份有限公司测序。

1.6 5' RACE技术扩增5'端非翻译区

5' RACE技术扩增原理参照文献[9],以PE4为引物,反转录过程同3' RACE,通过RNaseH分解mRNA,回收cDNA后再用末端转移酶在cDNA 3'端添加多聚腺苷化位点[poly (A)],再回收具有poly (A) 尾的cDNA;以此为模板,用OligodT-AP及PE5为引物进行扩增。将上述扩增产物稀释10倍,取2 μL为模板,用PE6及AP引物再次进行扩增,扩增产物用1%琼脂糖凝胶电泳分离回收,并送至生工生物工程 (上海) 股份有限公司测序。

1.7 核酸序列拼接与蛋白质序列分析

用DNAman软件拼接上述RT-PCR产物、3' RACE产物和5' RACE产物序列。用Clustal X 1.81和MEGA 4.0软件进行氨基酸同源性比对。用在线工具ExPASy-PROSITE和ExPASy-ProtParam (http://www.expasy.org) 预测蛋白质的基本理化性质。用SignalP 4.1预测信号肽,用NetNGlyc 1.0预测糖基化位点。利用SWISSMODEL软件 (http://swissmodel.expasy.org) 选择同源建模法构建OlCtpE蛋白的三级结构。

2 结果与分析 2.1 RT-PCR克隆OlCtpE基因保守cDNA序列的结果

根据已知几种鱼类CtpE的保守序列设计的简并引物,通过RT-PCR扩增OlCtpE基因得到约近600 bp的电泳条带 (图 1);将扩增产物连入T载体转化大肠埃希菌,酶切鉴定并测序得到584 bp的cDNA保守片段。经NCBI核酸同源性比对,证实该扩增产物即为青鳉肠道组织OlCtpE基因的部分cDNA序列。

泳道1:引物PF1与PR1的扩增产物;泳道M:DNA分子质量标志物DL2000。 Lane 1: Product amplified by primers PF1 and PR1; Lane M: DL2000 marker. 图1 RT-PCR扩增OlCtpE基因的琼脂糖凝胶电泳结果 Fig. 1 Agarose gel electrophoresis result of RT-PCR product for OlCtpE gene
2.2 3' RACE与5' RACE克隆OlCtpE基因全长cDNA序列的结果

利用引物PE3和AP,经3' RACE技术扩增到约450 bp的电泳条带 (图 2),经转化T质粒载体并双酶切测序后获得440 bp的cDNA片段;利用引物PE6及AP,经5' RACE技术扩增,获得约400 bp的特异性条带 (图 2),经转化T质粒载体并双酶切测序后获得401 bp的cDNA片段。

泳道1:3 ' RACE技术扩增产物;泳道2:空白对照;泳道3:5 ' RACE技术扩增产物;泳道M:DNA分子质量标志物DL2000。 Lane 1: 3'RACE product; Lane 2: Blank control; Lane 3: 5 'RACE product; Lane M: DL2000 marker. 图2 RACE技术扩增OlCtpE基因的琼脂糖凝胶电泳结果 Fig. 2 Agarose gel electrophoresis result of RACE product for OlCtpE gene
2.3 序列拼接与蛋白质功能位点分析

将上述扩增获得的保守序列片段、3' RACE及5' RACE片段进行拼接后,获得青鳉OlCtpE基因的全长cDNA编码序列 (图 3)。该基因cDNA全长1 301 bp,其中,5'非翻译区(untranslated regions,UTR)长24 bp,3' UTR长56 bp,开放阅读框长1 221 bp,编码406个氨基酸。预测的蛋白质分子质量为45.556 kDa,理论等电点为6.83;分子式为C2066H3168N548O587S15;SignalP 4.1软件预测该蛋白质含有一个由17个氨基酸组成的信号肽 (图 3),证实Ol-CtpE为分泌蛋白。NetNGlyc 1.0服务器预测OlCtpE蛋白质含有3个N-连接糖基化位点,分别为“NPTI” (氨基酸26~29)、“NFSV”(氨基酸95~98) 和“NLTV” (氨基酸162~165)。所克隆的OlCtpE基因的cDNA序列已提交至GenBank数据库,登录号为KP864679。

方框表示起始密码子;下划线处表示信号肽序列;加粗字表示3 '端Poly(A)信号(AATAAA);阴影部分氨基酸表示天冬氨酸蛋白酶家族ASP保守结构域;*表示终止翻译。 The start codons are boxed; the putative signal peptide is underlined; the 3 ' poly (A) signal (AATAAA) is highlighted in boldface; the shaded amino acids show the major aspartic proteinase conserved domain; single asterisk (*) shows termination of translation. 图3 青鳉OlCtpE基因cDNA及其推导的氨基酸序列 Fig. 3 cDNA and deduced amino acid sequence of the medaka OlCtpE gene
2.4 蛋白质序列同源比对

经BLASTP同源性比对,青鳉OlCtpE蛋白序列与底鳉和澳鳉的CtpE蛋白同源性为77%, 与茉莉花鳉 (P. latipinna) 的同源性为74%,与大黄鱼的同源性为71%。本研究对22种鱼类的CtpE蛋白 (表 2) 同源性进行分析显示:它们均存在保守的天冬氨酸蛋白酶活性位点,分别为“VIFDTGSSDLWV” (氨基酸98~109) 和“AIVDTGTSLIAG”(氨基酸284~295) (图 4中矩形网格所示);2个保守的半胱氨酸C321与C358被36个氨基酸所分隔,形成一个保守的二硫键“C-x*-C” (x为任意氨基酸)(图 4)。

表2 不同鱼类CtpE蛋白序列信息 Table 2 Sequence information of CtpE protein in different fish species
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图中物种拉丁名及序列登录号见表 2 The latin name and accession number of species are shown in Table 2. 图4 鱼类CtpE蛋白同源性比对结果 Fig. 4 Homology alignment of CtpE proteins in fish
2.5 蛋白质三级结构的同源建模

利用SWISS-MODEL分子模建服务系统,通过自动搜索SWISS蛋白分子模型PDB数据库,选择同源建模方法。结果(图 5)显示:青鳉OlCtpE蛋白与猪胃蛋白酶原(pepsinogen)(PDB号:2psg.1.A)的氨基酸序列同源性为43.60%, 两者的三级结构极为相似;青鳉OlCtpE主要以无规则卷曲(47.6%)为主,α-螺旋(31.5%)和β-延伸链(20.9%)散布于整个蛋白质中;在建模区域内,2种蛋白均包括8个明显的α-螺旋与19个β-片层,除中心区域的一个由α-螺旋构成的“活性中心翼环”重叠度略低外,2种蛋白的其他部位均具有高度的重叠度; 青鳉OlCtpE蛋白与猪胃蛋白酶原整体均呈椭球形,并形成相对独立的空间实体。

图5 青鳉OlCtpE蛋白与猪胃蛋白酶原的三级结构比较 Fig. 5 Comparison of tertiary structure of medaka OlCtpE and porcine pepsinogen protein
3 讨论

本研究通过反转录PCR及RACE技术克隆到青鳉天冬氨酸蛋白酶OlCtpE基因全长cDNA序列。该基因全长cDNA为1 301 bp,编码一个由406个氨基酸组成的多肽。本研究获得的OlCtpE属于细胞内溶酶体中的蛋白酶。在通常情况下,糖基化的蛋白酶可以避免其自身在酸性环境下被水解而失活。本研究发现,OlCtpE蛋白含有3个可信度高的N-连接糖基化位点。这与CAPASSO等[10]的研究结果一致。更为重要的是,OlCtpE可以利用这些糖基化位点经6-磷酸甘露糖受体途径转运至溶酶体腔内。此外,OlCtpE的N端具有一个由17个氨基酸残基构成的信号肽,可以为蛋白酶分泌提供信号。天冬氨酸蛋白酶的信号肽可以引导新生成的蛋白酶前体酶原转运至内质网上,信号肽断裂并释放出成熟肽,通过折叠卷曲形成二硫键[11]。对青鳉OlCtpE基因编码蛋白质序列进行同源性分析表明,青鳉OlCtpE含有2个保守的半胱氨酸C321与C358,两者通过二硫键形成一个保守的“C-x*-C”结构,对维持OlCtpE蛋白的空间结构发挥关键作用。

本研究结果显示,与其他类型的天冬氨酸蛋白酶相似,青鳉OlCtpE蛋白也由2个同源结构域组成,每个结构域的活性位点均为一段十分保守的“DTGT” 4个氨基酸残基[12]。保守的“DTGT”能够决定酶活性及酶的激活[13]。刘剑[14]通过定点突变技术将基因激活位点“DTGT”中的天冬氨酸残基 (D) 用丙氨酸 (A) 替代而获得变异体,并将大鼠的野生型与变异型的组织蛋白酶E基因异源表达于人胚胎肾细胞中,结果显示,突变体酶原在酸性环境下不能转变为酶,也无酶活性。这表明组织蛋白酶E的激活位点“DTGT”中的天冬氨酸残基 (D) 是酶的催化性残基,对酶的催化活性起决定性作用。

本研究以NCBI的PDB数据库中的猪胃蛋白酶原 (2psg.1.A) 为模板,通过同源建模得到青鳉OlCtpE的三级结构。结果显示,所获得的青鳉OlCtpE蛋白酶中2个保守的催化中心是对称分布的;推测每个催化中心的2个天冬氨酸残基可能发挥“催化二联体”的作用。活性中心翼环延伸到活性部位上,可能对活性部位的底物固定发挥一定的作用,活性部位形成的发夹式裂隙 (类似于催化口袋) 为底物结合提供空间[15]。青鳉OlCtpE蛋白与猪胃蛋白酶原在中心区域的一个“活性中心翼环”的重叠度较低,暗示两者的催化底物是不同的;除此之外,其他部位则具有高度的重叠度,这与两者同属于具有椭球形实体三维结构特点的胃蛋白酶家族成员这一事实相符。

由于动物的组织蛋白酶E在自然状态下的生理性底物还不清楚,其生理功能还需进一步阐明;因此,对组织蛋白酶E的催化活性和蛋白质三维结构开展研究具有重要意义。本文在青鳉消化道中克隆了OlCtpE基因,并分析了OlCtpE蛋白酶的功能,对于揭示鱼类组织蛋白酶E基因与蛋白质的进化历程,明晰这些基因与蛋白质的功能,透视水生低等脊椎动物的组织蛋白酶E参与抗原呈递等免疫作用机制都具有重要的参考价值。

参考文献
[1] KADOWAKI T, KIDO M A, HATAKEYAMA J, et al. Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet. Biochemical and Biophysical Research Communications, 2014,446 (1):212–217. DOI: 10.1016/j.bbrc.2014.02.089.
[2] NI J J, WU Z, PETERTS C, et al. The critical role of proteolytic relay through cathepsins B and E in the phenotypic change of microglia/ macrophage. The Journal of Neuroscience, 2015,35 (36):12488–12501. DOI: 10.1523/JNEUROSCI.1599-15.2015.
[3] KAWAKUBO T, YASUKOCHI A, TOYAMA T, et al. Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer. Carcinogenesis, 2013,35 (3):714–726.
[4] ZHANG X C, SHAN P Y, HOMER R, et al. Cathepsin E promotes pulmonary emphysema via mitochondrial fission. The American Journal of Clinical Pathology, 2014,184 (10):2730–2741. DOI: 10.1016/j.ajpath.2014.06.017.
[5] BENNETT K, LEVINE T, ELLIS J S, et al. Antigen processing for presentation by class Ⅱ major histocompatibility complex requires cleavage by cathepsin E. European Journal of Immunology, 1992,22 :1519–1524. DOI: 10.1002/(ISSN)1521-4141.
[6] XUE Y, ZHAO J L, DENG Y F, et al. Cloning and spatiotemporal expression of pepsinogen and gastric proton pump genes from mandarin fish (Siniperca chuatsi) during early ontogeny. Fish Physiology and Biochemistry, 2013,39 (4):881–893. DOI: 10.1007/s10695-012-9748-4.
[7] JIAO Y, LI C H, LU X J, et al. Characterization and expression of sweetfish (Plecoglossus altivelis) cathepsin D. Zoological Research, 2014,35 (4):294–299.
[8] 李林春, 王家庆, 何振峰, 等.青鳉鱼DBI基因全长cDNA的克隆与蛋白结构分析. 基因组学与应用生物学,2010,29 (5):885–889.
LI L C, WANG J Q, HE Z F, et al. The full-length cDNA cloning of DBI gene and its protein structure analysis derived from medaka (Oryzias latipes). Genomics and Applied Biology, 2010,29 (5):885–889. (in Chinese with English abstract)
[9] 王家庆, 边佳, 李代宗, 等.虹鳟Ndufb2基因全长cDNA序列的克隆与分析. 浙江大学学报 (农业与生命科学版),2013,39 (6):600–606.
WANG J Q, BIAN J, LI D Z, et al. Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss. Journal of Zhejiang University (Agriculture and Life Sciences), 2013,39 (6):600–606. (in Chinese with English abstract)
[10] CAPASSO C, RIGGIO M, SCUDIERO R, et al. Molecular cloning and sequence determination of a novel aspartic proteinase from Antarctic fish. Biochimica et Biophysica Acta: Protein Structure and Molecular Enzymology, 1998,1387 (1/2):457–461.
[11] LI H, LI Y D, CUI L, et al. Monitoring pancreatic carcinogenesis by the molecular imaging of cathepsin E in vivo using confocal laser endomicroscopy. PLoS One, 2014,9 (9):e106566. DOI: 10.1371/journal.pone.0106566.
[12] ZELEZNIK T Z, PUIZDAR V, DOLENC I, et al. Expression, purification and auto-activation of cathepsin E from insect cells. Protein and Peptide Letters, 2015,22 (6):525–531. DOI: 10.2174/0929866522666150506094458.
[13] ŽELEZNIK T Z, KADIN A, TURK V, et al. Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells. Biochemical and Biophysical Research Communications, 2015,465 (2):213–217. DOI: 10.1016/j.bbrc.2015.07.155.
[14] 刘剑.一种天门冬氨酸蛋白酶活性部位基序中酸性氨基酸的作用. 中国组织工程研究与临床康复,2009,13 (28):5558–5561.
LIU J. An acidic amino acid in aspartic proteinase active site motif. Journal of Clinical Rehabilitative Tissue Engineering Research, 2009,13 (28):5558–5561. (in Chinese with English abstract) DOI: 10.3969/j.issn.1673-8225.2009.28.033.
[15] CHOI K M, SHIM S H, AN C M, et al. Cloning, characterisation, and expression analysis of the cathepsin D gene from rock bream (Oplegnathus fasciatus). Fish & Shellfish Immunology, 2014,40 (1):253–258.