镰形扇头蜱半胱氨酸蛋白酶的鉴定及其在唾液腺细胞凋亡中的作用研究
发布时间：2018-08-11 12:25
【摘要】：半胱氨酸蛋白酶是一类古老而又保守的蛋白酶,其分布广泛,涵盖范围从病毒、细菌到哺乳动物的各个组织器官,参与胚胎发育,抗原呈递,细胞凋亡和稳态维持等多种重要的生理活动。作为专性体表寄生虫,蜱以吸血宿主血液为生,在其吸血过程中可传播细菌、真菌、病毒、支原体、原虫等多种病原体,是重要的人畜共患病传播媒介。唾液腺是宿主血液进入蜱体内最先接触的组织器官,也是蜱体内病原体传播到宿主体内的通道,因此蜱唾液腺重要分子的研究对于蜱及蜱传病控制十分重要;随着吸血过程的完成,蜱的唾液腺逐渐萎缩,这一现象是由细胞凋亡引起的,但分子机制不清。本研究以蜱的唾液腺中半胱氨酸蛋白酶分子群的鉴定为切入点,探索了半胱氨酸蛋白酶在唾液腺发育与凋亡中的作用机制。以镰形扇头蜱为研究对象,对吸血前、后唾液腺转录组进行测序及差异分析,分别测得上调基因1179个,下调基因574个,其中总转录库中预测的半胱氨酸蛋白酶表达序列标签(EST)有25个(13个上调基因EST,1个下调基因EST);经过基因克隆,共获得6个半胱氨酸蛋白酶全长序列,根据基因结构和生物信息学分析,分别命名为组织蛋白酶B(CATB)、组织蛋白酶L(CATL)、半胱天冬氨酸蛋白酶-1(CASP1)、半胱天冬氨酸蛋白酶-8(CASP8)、自噬相关蛋白酶4B(ATG4B)和自噬相关蛋白酶4D(ATG4D)。通过Real-time PCR方法检测了6种半胱氨酸蛋白酶在蜱不同发育阶段和不同吸血状态的转录水平,结果显示幼蜱和若蜱吸血后所有蛋白酶的转录量均显著性下调,而在成蜱则全部显著性上调;6种蛋白酶在成蜱唾液腺中吸血后转录量上调,尤其是CATB和CATL,上调差异非常显著,而在其他组织器官中转录水平各有不同。对5种半胱氨酸蛋白酶进行了体外重组蛋白表达和纯化(CASP8未表达),并分别制备小鼠特异抗血清。重组蛋白的酶活实验显示,各蛋白在适度浓度和PH范围内均表现一定的活性:CASP1的最适PH为5.5,可达阳性对照酶活性的60%;组织蛋白酶B和L随着浓度的增加,其活性受到抑制,其中组织蛋白酶L包含一个60个氨基酸的抑制结构域,将其切除后可明显提高组织蛋白酶L的活性,推测抑制结构域是限制组织蛋白酶L活性的重要因素,而组织蛋白酶B的高浓度抑制效应可能来自酶与底物的竞争;ATG4B和ATG4D对CASP1特异性底物具有一定活性,可达阳性对照的40%,ATG4D对组织蛋白酶特异性底物具有良好活性,高浓度下也出现抑制效应。5种重组蛋白的特异性抗血清,可分别识别成蜱体内天然蛋白酶,其中两种组织蛋白酶CATB和CATL在35~40 kDa之间均有2条带,可能为酶原和激活态,而自噬相关蛋白酶天然蛋白大小分别为~36 kDa(ATG4B)和~40 kDa(ATG4D),CASP1天然蛋白大小约为37 kDa,在70 kDa左右有类似二聚体结构,可能是其真正的活性态。利用免疫组化技术,观察5种半胱氨酸蛋白酶在唾液腺和肠腔中的定位,结果显示在唾液腺中5种蛋白酶分布广泛,而在中肠只可见ATG4D的少量分布。利用RNA干扰技术,通过电转化将合成的特异性双链RNA导入镰形扇头蜱若蜱体内,对6种半胱氨酸蛋白酶分别进行单一干扰实验,检测6种半胱氨酸蛋白酶之间是否存在相互作用关系,结果显示任一蛋白酶受到抑制均可引起其他5种蛋白酶转录水平发生变化,特别是当CATB和CATL转录受抑制可引起ATG4B和CASP1转录水平显著性上调,而CASP8和ATG4D转录受抑制时也引起CASP1转录水平显著上调;当CASP1受到抑制后,CATB的转录水平下调显著,ATG4D、ATG4B和CASP8的转录水平也受到明显抑制,但CATL的转录水平未受到影响,推测CASP1对于CASP8,CATB,ATG4B和ATG4D可能具有上游调节作用,为后续研究提供了切入点。通过解剖观察、Tunnel染色和AnnexinⅤ细胞凋亡流式检测确定了成蜱吸血过程中细胞凋亡参与唾液腺的变化过程;为了探究半胱氨酸蛋白酶在过程中的作用机制,利用显微注射技术将体外合成的干扰用CASP1双链RNA注射入镰形扇头蜱成蜱体内,24小时后接种新西兰大白兔,分别对上体后1到7天的对照组与实验组成蜱唾液腺进行分离观察,在mRNA和蛋白水平上检测7天吸血过程中半胱氨酸蛋白酶的动态变化,利用Tunnel细胞凋亡染色和AnnexinⅤ流式细胞凋亡检测技术对吸血过程中唾液腺的变化进行监测,结果表明进入快速吸血期前(上体3~4天)是唾液腺细胞诱导细胞凋亡发生的主要时相,随后随着吸血过程快速完成,唾液腺细胞逐渐坏死,CASP1作为效应性半胱天冬氨酸蛋白酶,在上体后5到7天表达量逐渐增加;CASP1的干扰可明显延长唾液腺细胞的凋亡,影响吸血过程中唾液腺细胞的坏死,并且诱导ATG4D的表达量逐渐增加,表明ATG4D与CASP1之间关系密切,暗示了自噬蛋白酶与半胱天冬氨酸蛋白酶之间可能存在互补的代偿关系,共同参与唾液腺细胞的凋亡坏死过程。综合上述结果,本研究鉴定了镰形扇头蜱6个半胱氨酸蛋白酶分子,发现它们在蜱吸血后唾液腺中上调表达,相互之间存在一定互作调节关系,其中自噬相关的半胱氨酸蛋白酶与凋亡相关的半胱氨酸蛋白酶共同参与了蜱唾液腺的凋亡过程。
[Abstract]:Cysteine proteases are ancient and conserved proteases, which are widely distributed in various tissues and organs from viruses, bacteria to mammals. They are involved in embryonic development, antigen presentation, apoptosis and homeostasis maintenance, and are important physiological activities. The salivary gland is the first contact organ for the blood of the host to enter the tick body, and it is also the channel through which the pathogen in the tick body transmits to the host body. Disease control is very important; with the completion of blood sucking, the salivary glands of ticks gradually atrophy, which is caused by apoptosis, but the molecular mechanism is unclear. Sequencing and differential analysis of salivary gland transcriptomes of Pseudostellaria falciformis before and after blood sucking showed that 1179 genes were up-regulated and 574 genes were down-regulated, of which 25 (13 up-regulated genes EST and 1 down-regulated gene EST) were predicted in the total transcription library. Six full-length sequences of cysteine proteases were obtained and named cathepsin B (CATB), cathepsin L (CATL), caspase-1 (CASP1), caspase-8 (CASP8), autophagy-associated protease 4B (ATG4B) and autophagy-related protease 4D (ATG4D) respectively according to their gene structure and bioinformatics analysis. The transcription levels of 6 cysteine proteases in different stages of development and different blood-sucking states of ticks were detected by PCR. The results showed that the transcription levels of all proteases in young and nymph ticks were significantly down-regulated, while in adult ticks were significantly up-regulated. The transcription levels of 6 proteases were up-regulated in salivary glands of adult ticks, especially CAT B and C. The expression and purification of five cysteine proteases (CASP8 was not expressed) were carried out in vitro, and specific mouse antisera were prepared respectively. The activity of the recombinant proteins showed that the proteins exhibited certain activity in moderate concentration and PH range. Sex: The optimum pH of CASP1 was 5.5, reaching 60% of the activity of the positive control enzyme. Cathepsin B and L were inhibited with the increase of the concentration. Cathepsin L contained an inhibitory domain of 60 amino acids, and the activity of cathepsin L was significantly increased after excision of the domain. ATG4B and ATG4D have certain activity to the specific substrate of CASP1, up to 40% of the positive control. ATG4D has good activity to the specific substrate of cathepsin, and also has inhibitory effect at high concentration. The serum of the two cathepsin CATB and CATL had two bands between 35 kDa and 40 kDa, and the size of the autophagy-related protease was ~36 kDa (ATG4B) and ~40 kDa (ATG4D), and the size of the CASP1 protein was about 37 kDa, similar to dimerization at about 70 kDa. Immunohistochemical localization of five cysteine proteases in salivary glands and intestinal cavity showed that five proteases were widely distributed in salivary glands, while only a small amount of ATG4D was found in the midgut. In nymph ticks of Pseudostellaria falciparum, a single interference test was conducted to detect the interaction between six cysteine proteases. The results showed that inhibition of any protease could cause changes in the transcriptional levels of the other five proteases, especially AT when the transcription of CAT B and CAT L was inhibited. The transcriptional levels of G4B and CASP1 were significantly up-regulated, while those of CASP8 and ATG4D were also significantly up-regulated. When CASP1 was inhibited, the transcriptional levels of CAT B were significantly down-regulated. The transcriptional levels of ATG4D, ATG4B and CASP8 were also significantly inhibited, but the transcriptional levels of CAT L were not affected. TG4D may play an upstream regulatory role, providing a starting point for further research. Through anatomical observation, Tunnel staining and Annexin V cell apoptosis flow cytometry, we determined that apoptosis participated in the process of salivary gland changes in adult ticks during blood sucking. Disruption of biosynthesis in vitro was injected into adult ticks of Pseudostellaria falciformis with CASP1 double-stranded RNA. New Zealand rabbits were inoculated with CASP1 double-stranded RNA 24 hours later. The salivary glands of the control group and the experimental group were separated and observed 1 to 7 days later. The dynamic changes of cysteine proteinase were detected at the level of mRNA and protein during 7 days of blood sucking. Cell apoptosis staining and Annexin V flow cytometry were used to monitor the changes of salivary glands during blood sucking. The results showed that the main phase of apoptosis induced by salivary gland cells was before the fast blood sucking stage (3-4 days from the upper body). With the rapid completion of the blood sucking process, salivary gland cells gradually necrosis, and CASP1 acted as an effect. The expression of caspase increased gradually from 5 to 7 days after supernatant. The interference of CASP1 could significantly prolong the apoptosis of salivary gland cells, affect the necrosis of salivary gland cells during blood sucking, and induce the expression of ATG4D to increase gradually, indicating that there was a close relationship between ATG4D and CASP1, suggesting that autophagy protease and caspase were closely related. There may be complementary compensatory relationship between proteases involved in the process of apoptosis and necrosis of salivary gland cells. Based on the above results, six cysteine proteinase molecules from ticks were identified. They were up-regulated in the salivary gland of ticks after blood sucking, and there was a certain interaction between them, among which autophagy was related. Cysteine protease and apoptosis-related cysteine protease participate in the apoptosis process of tick salivary gland.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.7
，
本文编号：2176986
[Abstract]:Cysteine proteases are ancient and conserved proteases, which are widely distributed in various tissues and organs from viruses, bacteria to mammals. They are involved in embryonic development, antigen presentation, apoptosis and homeostasis maintenance, and are important physiological activities. The salivary gland is the first contact organ for the blood of the host to enter the tick body, and it is also the channel through which the pathogen in the tick body transmits to the host body. Disease control is very important; with the completion of blood sucking, the salivary glands of ticks gradually atrophy, which is caused by apoptosis, but the molecular mechanism is unclear. Sequencing and differential analysis of salivary gland transcriptomes of Pseudostellaria falciformis before and after blood sucking showed that 1179 genes were up-regulated and 574 genes were down-regulated, of which 25 (13 up-regulated genes EST and 1 down-regulated gene EST) were predicted in the total transcription library. Six full-length sequences of cysteine proteases were obtained and named cathepsin B (CATB), cathepsin L (CATL), caspase-1 (CASP1), caspase-8 (CASP8), autophagy-associated protease 4B (ATG4B) and autophagy-related protease 4D (ATG4D) respectively according to their gene structure and bioinformatics analysis. The transcription levels of 6 cysteine proteases in different stages of development and different blood-sucking states of ticks were detected by PCR. The results showed that the transcription levels of all proteases in young and nymph ticks were significantly down-regulated, while in adult ticks were significantly up-regulated. The transcription levels of 6 proteases were up-regulated in salivary glands of adult ticks, especially CAT B and C. The expression and purification of five cysteine proteases (CASP8 was not expressed) were carried out in vitro, and specific mouse antisera were prepared respectively. The activity of the recombinant proteins showed that the proteins exhibited certain activity in moderate concentration and PH range. Sex: The optimum pH of CASP1 was 5.5, reaching 60% of the activity of the positive control enzyme. Cathepsin B and L were inhibited with the increase of the concentration. Cathepsin L contained an inhibitory domain of 60 amino acids, and the activity of cathepsin L was significantly increased after excision of the domain. ATG4B and ATG4D have certain activity to the specific substrate of CASP1, up to 40% of the positive control. ATG4D has good activity to the specific substrate of cathepsin, and also has inhibitory effect at high concentration. The serum of the two cathepsin CATB and CATL had two bands between 35 kDa and 40 kDa, and the size of the autophagy-related protease was ~36 kDa (ATG4B) and ~40 kDa (ATG4D), and the size of the CASP1 protein was about 37 kDa, similar to dimerization at about 70 kDa. Immunohistochemical localization of five cysteine proteases in salivary glands and intestinal cavity showed that five proteases were widely distributed in salivary glands, while only a small amount of ATG4D was found in the midgut. In nymph ticks of Pseudostellaria falciparum, a single interference test was conducted to detect the interaction between six cysteine proteases. The results showed that inhibition of any protease could cause changes in the transcriptional levels of the other five proteases, especially AT when the transcription of CAT B and CAT L was inhibited. The transcriptional levels of G4B and CASP1 were significantly up-regulated, while those of CASP8 and ATG4D were also significantly up-regulated. When CASP1 was inhibited, the transcriptional levels of CAT B were significantly down-regulated. The transcriptional levels of ATG4D, ATG4B and CASP8 were also significantly inhibited, but the transcriptional levels of CAT L were not affected. TG4D may play an upstream regulatory role, providing a starting point for further research. Through anatomical observation, Tunnel staining and Annexin V cell apoptosis flow cytometry, we determined that apoptosis participated in the process of salivary gland changes in adult ticks during blood sucking. Disruption of biosynthesis in vitro was injected into adult ticks of Pseudostellaria falciformis with CASP1 double-stranded RNA. New Zealand rabbits were inoculated with CASP1 double-stranded RNA 24 hours later. The salivary glands of the control group and the experimental group were separated and observed 1 to 7 days later. The dynamic changes of cysteine proteinase were detected at the level of mRNA and protein during 7 days of blood sucking. Cell apoptosis staining and Annexin V flow cytometry were used to monitor the changes of salivary glands during blood sucking. The results showed that the main phase of apoptosis induced by salivary gland cells was before the fast blood sucking stage (3-4 days from the upper body). With the rapid completion of the blood sucking process, salivary gland cells gradually necrosis, and CASP1 acted as an effect. The expression of caspase increased gradually from 5 to 7 days after supernatant. The interference of CASP1 could significantly prolong the apoptosis of salivary gland cells, affect the necrosis of salivary gland cells during blood sucking, and induce the expression of ATG4D to increase gradually, indicating that there was a close relationship between ATG4D and CASP1, suggesting that autophagy protease and caspase were closely related. There may be complementary compensatory relationship between proteases involved in the process of apoptosis and necrosis of salivary gland cells. Based on the above results, six cysteine proteinase molecules from ticks were identified. They were up-regulated in the salivary gland of ticks after blood sucking, and there was a certain interaction between them, among which autophagy was related. Cysteine protease and apoptosis-related cysteine protease participate in the apoptosis process of tick salivary gland.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.7
，
本文编号：2176986
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