Kazal型蛋白酶抑制剂结构与功能研究进展

蛋白酶抑制剂广泛存在于生物体内,在许多生命活动过程中发挥必不可少的作用,特别是对蛋白酶活性进行精确调控。其中Kazal型蛋白酶抑制剂是最重要的、研究最为广泛的酶抑制剂之一,该类抑制剂一般由一个或几个结构域组成,每一个结构域具有保守的序列和分子构象,同时发现该类抑制剂与蛋白酶作用的结合部位高度易变,它们大多数暴露于与溶剂接触的环上,其中P1部位是抑制作用的关键部位,抑制剂的专一性由P1部位氨基酸残基的性质决定,其它残基取代结合部位残基对抑制剂-酶的结合常数有显著的影响。Laskowski算法可直接从Kazal型丝氨酸蛋白酶抑制剂的序列推测其与6种丝氨酸蛋白酶之间的抑制常数(Ki)。目前在生物体内发现大量的Kazal型蛋白酶抑制剂,并证实其有重要的生物学功能。     

青蛤Kazal型丝氨酸蛋白酶抑制剂基因的克隆及其在组织间的表达分析

利用构建的SMART-cDNA文库及高通量测序方法,获得了青蛤Kazal家族丝氨酸蛋白酶抑制剂基因(Serine Protease Inhibitor,SPI)的全长序列,采用荧光定量PCR方法分析了SPI在青蛤各组织的表达情况,并在鳗弧菌胁迫下分析了SPI在青蛤血液中的时序表达关系。结果表明,青蛤SPI基因全长587 bp,CDS为67~520 bp,编码151个氨基酸,具有19个氨基酸的信号肽序列;荧光定量PCR结果显示,该基因在血液、肝脏、外套膜、鳃和闭壳肌等组织中表达差异明显,其中血液中表达水平最高,在闭壳肌中表达水平较低,其他组织表达量极少;在鳗弧菌刺激后3 h和48 h青蛤血液中SPI的表达量出现明显上调的趋势且与对照组有极显著性差异(P0.01),说明SPI基因在青蛤的免疫反应中具有重要的作用。     

重组人丝氨酸蛋白酶抑制因子Hespintor Kazal结构域的原核表达、纯化及活性鉴定

Hespintor是应用抑制消减杂交技术 (SSH) 从肝母细胞瘤细胞系HepG2中筛选得到的一未知功能蛋白,序列分析表明该蛋白属于Kazal型丝氨酸蛋白酶抑制因子 (Serine proteinase inhibitor, Serpin) 家族中的一个分泌型新成员,具有与食管癌相关基因2 (Esophageal cancer related gene 2, ECRG2) 高度同源的Serpin基本结构。为了进一步阐明Hespintor的生物学功能,必须得到纯化的Hespintor蛋白。先将Hespintor Kazal结构域编码序列亚克隆至原核表达载体pET-40b(+),转化至Rosetta (DE3) 表达宿主菌中。经 0.25 mmol/L IPTG,30 ℃诱导5 h获得了分子量约为42 kDa的Hespintor-Kazal重组融合蛋白的优化表达,Western blotting证实了重组蛋白的特异性。Hespintor-Kazal重组融合蛋白以包涵体形式在宿主菌中表达,利用金属螯合亲和层析和阴离子交换层析柱对重组蛋白进行两步纯化。初步的活性鉴定表明,纯化的Hespintor-Kazal重组融合蛋白能特异性抑制胰蛋白酶的水解活性,提示Hespintor具有作为一种新型抗肿瘤药物的潜在开发价值。     

中国林蛙皮肤丝氨酸蛋白酶抑制剂RCSPI2的基因克隆和原核表达

<正>丝氨酸蛋白酶抑制剂广泛存在于动物、植物及微生物体内[1],是一类丝氨酸蛋白酶活性调节剂,能调节生物体内许多重要的生命过程,如蛋白质折叠、血凝、补体激活、炎症反应、细胞迁移、细胞基质重建、以及肿瘤抑制等[2,3],很多已被开发为新药,在临床上有广泛应用。基于     

家蚕丝氨酸蛋白酶抑制剂20的表达特征分析

【目的】原核表达家蚕Bombyx mori丝氨酸蛋白酶抑制剂20(Serine proteinase inhibitor 20,Bm Serpin-20),分析Bm Serpin-20基因和蛋白组织表达分布特点,以及不同外源病原物刺激家蚕后Bm Serpin-20基因的表达模式。【方法】利用p ET-28a表达载体在大肠杆菌Transetta(DE3)中融合表达Bm Serpin-20蛋白,利用纯化的重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,利用实时定量PCR技术及Western blot方法分别分析Bm Serpin-20基因和蛋白的组织表达水平,以及病原物免疫刺激后的表达模式。【结果】在大肠杆菌中正确表达和纯化了融合Bm Serpin-20蛋白,并制备了多克隆抗体;Bm Serpin-20基因和蛋白在脂肪体、中肠、血细胞、马氏管、表皮、睾丸、卵巢中均有表达,且在表皮中表达量最高;家蚕5龄幼虫经核型多角体病毒(Nuclear polyhedrosis viruses)、藤黄微球菌Micrococcus luteus、大肠杆菌Escherichia coli、白僵菌Beauveria bassiana处理后,Bm Serpin-20基因表达量先下调而后上调,但不同病原物在不同时间的影响不尽相同。【结论】研究了Bm Serpin-20基因和蛋白的表达模式,为下一步研究其在家蚕免疫系统中的作用奠定了基础。     

褐飞虱丝氨酸蛋白酶抑制剂基因Nlserpin4的克隆及表达模式分析

【目的】克隆和分析褐飞虱Nilaparvata lugens丝氨酸蛋白酶抑制剂基因Nlserpin4,并探明其时空表达谱和病原真菌诱导表达模式。【方法】基于褐飞虱转录组和全基因组序列数据,利用PCR技术克隆得到褐飞虱Nlserpin4基因的全长cDNA序列;利用生物信息学手段分析其核苷酸和蛋白质序列特征;通过qRT-PCR技术检测其在褐飞虱不同发育时期(卵、1-5龄若虫和初羽化雌雄成虫)和5龄若虫不同组织(脂肪体、肠道、血淋巴和剩余虫体)中的时空表达谱,以及金龟子绿僵菌Metarhizium anisopliae注射感染褐飞虱5龄若虫不同时间后的诱导表达模式。【结果】克隆获得褐飞虱Nlserpin4基因全长cDNA序列(GenBank登录号:MN822802),其开放阅读框长1 227 bp,编码408个氨基酸,蛋白的相对分子质量和等电点分别为45.91 kD和6.23。氨基酸序列分析表明,Nlserpin4蛋白无糖基化位点,N端包含一段由23个氨基酸残基组成的信号肽,C端具有serpin蛋白家族典型的RCL区,且含有能被靶标蛋白酶识别的活性裂解位点。系统发育分析表明,Nlserpin...     

家蝇3种丝氨酸蛋白酶抑制剂Serpin基因克隆、序列分析及表达模式

对家蝇3种丝氨酸蛋白酶抑制剂Serpin基因SP2、SP13和SP16进行克隆、序列分析和时空表达模式检测。运用瑞士生物信息学研究所的蛋白分析专家系统（ExPASy）和美国国家生物技术信息中心（NCBI）等有关的生物信息学分析工具,预测和分析3条Serpin基因编码蛋白质的结构与生物学功能。以RPS18（核糖体S18蛋白）为内参基因,采用实时荧光定量PCR（Real-Time PCR）技术检测3条 Serpin 基因在家蝇不同发育时期,以及家蝇3龄幼虫中脂肪体、唾液腺、中肠、马氏管和体壁的表达特征。SP2、SP13、SP16基因序列的开放阅读框ORF全长分别为1191 bp、1137 bp和1212 bp,分别编码含396、378和403个氨基酸残基的蛋白质,其理论分子量分别为45315.3 Da、43701.5 Da和45011.5 Da,等电点分别为9.01、5.98和6.04,蛋白质的N端都含有一段信号肽,并具有Serpin基因的保守结构域,属于Serpin家族。SP2和SP16在蛋白质的C端含反应中心环（Reactive center loop,RCL),而SP13 C端不含RCL。时空表达模式分析结果显示,SP2基因在2龄幼虫中的表达量最高,而雄蝇的表达量与卵期相比有所下调;SP13基因在蛹中的表达量最高,1龄幼虫和雌蝇中的表达量与卵期相近;SP16基因在2龄幼虫和3龄幼虫中的表达量最高,雄蝇与雌蝇的表达量与卵期相比有所下调。在家蝇3龄幼虫的各主要组织中,以体壁为参照,SP2基因在唾液腺表达水平最高,其次是脂肪体,中肠和马氏管均低于体壁;SP13基因在马氏管和脂肪体中的表达水平最高,中肠和唾液腺的表达量低于体壁;SP16基因在唾液腺、中肠、脂肪体和马氏管的表达量均低于体壁。推测Serpin基因在家蝇个体发育和免疫调节中起不同作用。     

褐飞虱丝氨酸蛋白酶抑制剂基因Nlserpin4的克隆及表达模式分析

摘要: 【目的】克隆和分析褐飞虱Nilaparvata lugens丝氨酸蛋白酶抑制剂基因Nlserpin4,并探明其时空表达谱和病原真菌诱导表达模式。【方法】基于褐飞虱转录组和全基因组序列数据,利用PCR技术克隆得到褐飞虱Nlserpin4基因的全长cDNA序列;利用生物信息学手段分析其核苷酸和蛋白质序列特征;通过qRT-PCR技术检测其在褐飞虱不同发育时期(卵、1-5龄若虫和初羽化雌雄成虫)和5龄若虫不同组织(脂肪体、肠道、血淋巴和剩余虫体)中的时空表达谱,以及金龟子绿僵菌Metarhizium anisopliae注射感染褐飞虱5龄若虫不同时间后的诱导表达模式。【结果】克隆获得褐飞虱Nlserpin4基因全长cDNA序列(GenBank登录号: MN822802),其开放阅读框长1 227 bp,编码408个氨基酸,蛋白的相对分子质量和等电点分别为45.91 kD和6.23。氨基酸序列分析表明,Nlserpin4蛋白无糖基化位点,N端包含一段由23个氨基酸残基组成的信号肽,C端具有serpin蛋白家族典型的RCL区,且含有能被靶标蛋白酶识别的活性裂解位点。系统发育分析表明,Nlserpin4与半翅目其他昆虫的serpin亲缘关系较近,其中与蔗黄伪毛蚜Sipha flava serpin4的亲缘关系最近。qRT-PCR分析表明,Nlserpin4基因表达具有明显的时空特异性,其在成虫中的表达量显著高于在其他龄期的,且在雄成虫中表达量最高;Nlserpin4基因在褐飞虱5龄若虫脂肪体、肠道、血淋巴和剩余虫体中均有表达,且在剩余虫体组织中表达量最高;病原真菌金龟子绿僵菌诱导48 h内Nlserpin4表达量均显著下调,但随着诱导时间的增加,Nlserpin4表达量呈回升趋势。【结论】褐飞虱Nlserpin4基因在褐飞虱不同发育阶段、不同组织以及病原真菌金龟子绿僵菌诱导不同时间下差异表达。研究结果为深入研究Nlserpin4在褐飞虱生长发育和免疫调节中的功能奠定了基础。     

家蚕丝氨酸蛋白酶抑制剂4(serpin-4)的基因克隆、原核表达和多克隆抗体制备

丝氨酸蛋白酶抑制剂4(serpin-4)为丝氨酸蛋白酶抑制剂家族中的一员.本研究旨在研制高效价的家蚕Bombyx mori serpin-4多克隆抗体,为深入研究serpin-4基因的生理功能打下物质基础.首先于家蚕脂肪体中克隆了serpin-4基因,利用基因重组技术构建了pET28a-serpin-4原核表达载体,经...     

Truncated reactive center loop decrease the inhibitory activity of Antheraea pernyi serine protease inhibitor 6

Here, we assessed the effect of a systematic change in reactive center loop (RCL) length, N-terminal to the reactive center, on the inhibitory activity of the recombinant Apserpin-6. The domain prediction results indicated that the RCL is located between the amino acid numbered 359–379 at the C-terminal of Apserpin-6. The N-terminal variable region for amino acid positions P7–P1 of the RCL of Apserpin-6 was truncated or extended by residue deletion or insertion using site-directed mutagenesis. The recombinant Apserpin-6 with one or two residues insertion in RCL had no effect on prophenoloxidase (proPO) activity, whereas deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6. The results of this study will facilitate the understanding of inhibition mechanism of RCL on proPO activity.     

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3h post-challenge. After a decrease at 6h, the expression level increased again and reached 207.8-fold at 12h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was 173 nmol L(-1). These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response.     

叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析

为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征,为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列,使用生物信息学软件进行序列分析以及系统进化分析,采用实时荧光定量PCR (Real time quantitativate PCR,RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明,EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp,分别编码125个氨基酸和306个氨基酸,预测均为亲水蛋白质,理论分子量分别为13.48 kDa和33.82 kDa,等电点分别为6.68和5.80,分别有30个和23个氨基酸残基的信号肽序列,EfSPI20有跨膜结构域,EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高,氨基酸序列一致性达58%;EfSP1与稻绿蝽Nezara viridula SP同源性最高,氨基酸序列一致性达66%;系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达,推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关;EfSP1基因在卵期、卵巢和肠道中高表达,推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。     

柞蚕Septin基因的克隆及表达分析

Septin蛋白参与了细胞分裂、细胞内物质转运、细胞周期调控及细胞凋亡等生理反应,并且发现与肿瘤发生、神经功能障碍和病原物侵染的过程直接相关。研究克隆了柞蚕septin基因全长,序列分析表明septin基因全长1904 bp,开放阅读框长度为1137 bp,编码378个氨基酸。多重序列比对分析表明柞蚕septin与家蚕及黑脉金斑蝶septin相似性最高,聚为一类。实时定量PCR结果表明柞蚕septin基因在各个组织中都有表达,但在血细胞中表达量最高,其次是表皮,而在丝腺中表达量最低。通过构建原核表达载体,柞蚕septin蛋白在大肠杆菌中成功诱导表达。     

Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6

Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K_i around 1 nM, KLK4 with K_i = 27.3 nM, KLK6 with K_i = 140 nM, caspase-14 with a K_i approximating 1 μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members.     

Molecular cloning,expression, and anti-tumor activity of a novel serine protease from Arenicola cristata

Arenicola cristata, a marine annelid, is a wellknown and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease ofA. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA （cDNA） of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the pro posed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was 26 kDa after overexpression in E. coli. The recombinant pro tein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma （ESCC） in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activa tion of caspase9 as well as downregulation of Mcl1 and Bcl2. Taken together, our findings indicated that the recom binant serine protease ofA. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.     

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.     

菜粉蝶丝氨酸蛋白酶基因Pr-SP1的克隆及其表达谱分析

昆虫主要依靠先天免疫反应来抵御外源异物的入侵, 而与血淋巴黑化及抗菌肽合成等过程密切相关的丝氨酸蛋白酶激活级联反应在其中起着重要作用。为阐明丝氨酸蛋白酶在菜粉蝶Pieris rapae免疫中的作用, 本文通过简并引物RT-PCR克隆获得了菜粉蝶丝氨酸蛋白酶家族基因Pr-SP1的cDNA片段, 并利用RACE法扩增获得其全序列。该cDNA序列长1 489 bp, 其中开放阅读框长1 059 bp, 共编码353个氨基酸残基。Pr-SP1含一长度为20个氨基酸残基的信号肽序列, 其蛋白理论分子量为36.85 kDa, 理论等电点为6.41。多序列比对结果表明, Pr-SP1与其他昆虫的同源蛋白基因序列上存在较高一致性, 在N端有一个发夹结构域, 而C端是一个具有催化活性的结构域。实时荧光定量RT-PCR及免疫印迹结果表明, 蛹期Pr-SP1主要在颗粒血细胞内进行转录, 其蛋白产物主要定位在血浆; Pr-SP1在不同虫态及虫龄都有转录, 其蛋白产物在不同虫态及虫龄都有表达, 其中5龄幼虫最高, 卵期最低; Pr-SP1的转录水平及其蛋白产物的表达水平均会被大肠杆菌Escherichia coli、 藤黄微球菌Micrococcus luteus和巴斯德毕赤酵母Pichia pastoris诱导。根据这些结果分析认为, Pr-SP1属于Spätzle蛋白酶前体激活酶, 并参与菜粉蝶的先天免疫反应。