重组七鳃鳗富含半胱氨酸RGD蛋白rLj-RGD1对人肝癌HepG2细胞的抑制作用

rLj-RGD1为源于日本七鳃鳗口腔腺的基因重组蛋白,其富含半胱氨酸并具有一个RGD(Arg-Gly-Asp)模体.前期工作表明,rLj-RGD1具有抑制血小板聚集及血管新生的RGD毒素蛋白典型功能.为了研究rLj RGD1是否具有RGD毒素蛋白的另一典型抗肿瘤功能,以人肝癌HepG2细胞为模型,对rLj RGD1进行了活性研究.MTT法结果显示,rLj-RGD1呈剂量依赖方式抑制HepG2细胞的增殖,其半抑制浓度(IC50)为36 μmol/L;细胞迁移与浸润实验结果显示,rLj-RGD1能以剂量依赖方式抑制HepG2细胞碱性成纤维生长因子（bFGF）诱导的迁移与浸润.Hoechst染色和DNA Ladder等细胞凋亡实验表明,rLj-RGD1能够以剂量依赖方式诱导HepG2细胞发生凋亡.细胞黏附实验表明,rLj-RGD1以剂量依赖方式抑制HepG2细胞与玻连蛋白(vitronectin, VN)的黏附.上述结果表明,rLj-RGD1具有抑制人肝癌HepG2细胞增殖、迁移和浸润的功能,并可诱导其发生失巢凋亡. 本研究结果提示,rLj-RGD1具有典型的RGD毒素蛋白抗肿瘤功能,其未来具有成为抗肿瘤药物的潜力.     

骨桥蛋白RGD黏附序列多拷贝短肽的表达、纯化及生物学活性测定

目的:用大肠杆菌表达骨桥蛋白RGD黏附序列6拷贝短肽,经分离纯化后检测其生物学活性.方法:运用基因重组技术,将骨桥蛋白RGD黏附序列的核酸片段首尾相连,与携带GST编码序列的原核表达载体连接构建融合蛋白表达质粒pGEX-3X-RGD.将重组质粒转化宿主菌后,对诱导融合蛋白表达的条件进行优化.表达产物GST-RGD经谷胱甘肽-亲和层析纯化后,分别检测其对骨桥蛋白诱导的血管平滑肌细胞黏附和迁移的影响.结果:所构建的含有6个拷贝短肽的GST-RGD融合蛋白可在大肠杆菌中以包含体的形式进行表达.用十二烷基肌氨酸钠变性溶解包含体及透析复性后,经亲和层析可得到高纯度的GST-RGD(6)融合蛋白.GST-RGD(6)融合蛋白能特异性的抑制骨桥蛋白诱导的血管平滑肌细胞的黏附和迁移.结论:骨桥蛋白RGD黏附序列6拷贝短肽可在大肠杆菌中高效表达,纯化的GST-RGD融合蛋白具有抑制血管平滑肌细胞黏附和迁移的活性.     

以整合素αvβ3为靶点的RGD配体在抗肿瘤血管新生中的应用

整合素αvβ3是一种能特异性识别RGD序列的膜受体蛋白,其与含RGD（Arg-Gly-Asp）模体的蛋白质结合的特异性在肿瘤细胞的粘附、迁移、浸润及肿瘤血管新生中起重要作用.由于整合素αvβ3在多种肿瘤细胞表面高表达而在正常细胞中低表达或不表达,因此其成为肿瘤治疗的理想靶点.肿瘤新生血管为肿瘤的生长提供营养,因此近年来抑制肿瘤血管新生也成为肿瘤治疗的重要途径.有研究显示,几种RGD毒素蛋白不但以整合素αvβ3为靶点靶向结合到肿瘤部位从而具有直接抗肿瘤细胞增殖、黏附、迁移及浸润功能,而且它们还具有抗肿瘤血管新生的作用,因此RGD毒素蛋白可从上述两方面抑制肿瘤生长与转移.本文就整合素αvβ3为靶向的RGD配体结构特点及其在肿瘤治疗中的靶向治疗和抗血管新生应用及前景加以综述.     

局部力学刺激对黄瓜系统抗病性的诱导

病原真菌在侵入植物细胞过程中,除了分泌化学物质外还通过物理挤压细胞产生力学作用.用压应力作为力学信号,研究了局部力学刺激对黄瓜系统抗病性的诱导.结果表明,力学刺激可以诱导黄瓜系统抗病性的产生.当细胞壁与质膜间的黏附被Arg-Gly-Asp(RGD)阻断后,力学刺激对黄瓜系统抗病性的诱导几乎完全被减除.通过薄层色谱和液相色谱分析,发现力学刺激可以使植保素含量明显增加.这表明黄瓜植保素的积累可能是力学刺激诱导其产生抗性的原因之一.而细胞壁与质膜间的黏附被RGD阻断后,力学刺激只能诱导植保素的部分积累.即力学刺激对植保素积累的诱导依赖于细胞膜与细胞壁间的黏附.     

含RGD模体的人白细胞介素18(IL-18)突变体的酵母表达及其抗血小板聚集活性

利用定点突变及DNA重组技术,在人白细胞介素18(IL18)cDNA序列中插入GGC序列,使IL18第39位精氨酸残基和第40位天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体.此重组的cDNA序列构建入表达质粒pPIC9K,并转化Pichiapastoris酵母GS115,利用表达系统进行了高效表达.用SephadexG100凝胶过滤纯化表达产物,获得初步纯化的蛋白.对该蛋白进行了血小板聚集抑制实验和对GPⅡbⅢa与Fn结合的抑制实验.含RGD模体的重组IL18(IL18RGD)显示了较强的体外抑制血小板聚集活性,IC50=8.8μmolL;并具有与GPⅡbⅢa的竞争结合活性,IC50=8.0μmolL.该含有RGD模体的重组IL18仍保存对PBMC诱导产生IFNγ能力.结果表明,此IL18RGD嵌合体在具有抗炎,抗感染的同时增添了新的抑制血小板聚集功能.     

日本七鳃鳗RGD毒素肽Lj-RGD2的克隆、表达及其抗血管新生活性

日本七鳃鳗口腔腺分泌蛋白Lj-RGD2毒素肽分子质量为8 kD,是含有2个RGD（Arg-Gly-Asp）模体序列的多肽.为了探讨其是否具有RGD毒素蛋白的功能,对其基因进行了克隆和表达,并对其重组蛋白进行了抗血管新生活性研究.从日本七鳃鳗口腔腺中提取mRNA,以自行设计的引物进行RT-PCR扩增,以获得Lj-RGD2基因;将获取的目的基因与pET23b载体连接,经转化、克隆、阳性转化子的筛选鉴定;将含组氨酸标签的pET23b-Lj-RGD2转化BL21菌中诱导表达;经亲和层析纯化,获得可溶性重组rLj-RGD2蛋白,并对重组rLj-RGD2蛋白的抗血管新生功能进行了研究.结果显示,rLj-RGD2蛋白的IC50为1.77μmol/L,并以剂量依赖方式抑制人脐静脉内皮细胞ECV304的增殖;rLj-RGD2蛋白还能抑制ECV304细胞对人工基质膜Matrigel的黏附、以BFGF（basic fibroblast growth factor）为趋化剂的迁移及侵袭.鸡胚绒毛尿囊膜CAM（chick chorioallantoic membrane）血管新生实验结果表明,rLj-RGD2蛋白能以剂量依赖方式抑制BFGF诱导的CAM血管新生.由此可见,rLj RGD2蛋白具有RGD毒素肽的活性特征,其有望成为一种抗血管新生基因工程新药. 关键词日本七鳃鳗; RGD毒素蛋白; 细胞黏附; 迁移及侵袭; 血管新生     

细胞黏附分子互作网络的构建与分析

借助网络分析可对基因调控、蛋白质互作和信号转导等细胞活动进行全局和局部性质分析.以细胞黏附的蛋白质相互作用为对象,通过数据挖掘和可视化软件构建了整合蛋白介导的黏附分子互作网络,该分子互作网络由156种蛋白质通过690种相互作用相连,其平均节点度为8.66、平均聚集系数为0.24,平均路径长度为2.6.黏附分子互作网络中包含数个功能模块,这些模块涉及网络内部多种分子相互作用的启动与停止,并进一步影响细胞的黏附、迁移和骨架组织.对黏附分子网络进行模体筛选和比较,发现一些数量相对较少、以三元复合物为主要结构的关键模体,同时对各网络模块和模体对细胞黏附的调控作用进行了探讨.     

QCM 监测自组装膜上心肌细胞黏附及其与心血管药物作用

目的:本试验采用石英晶体微天平(QCM)实时监测大鼠心肌细胞(H9C2)在含有细胞黏附识别多肽RGD自组装膜上的动态黏附过程及随后与两种心血管药物(一种正性肌力、另一种负性肌力)相互作用。方法:在金电极表面自组装3-巯基丙酸(MPA)单层膜,并经酰胺化共价耦合细胞黏附分子KRGD,形成对大鼠心肌细胞有特异性黏附的致密分子自组装膜。QCM以动态持续的方式实时监测MPA/RGD自组装及其不同浓度梯度H9C2细胞在自组装膜金电极上的细胞黏附过程。此外,选用20,000个H9C2细胞和正性肌力药物异丙肾上腺素、负性肌力药物维拉帕米,用QCM评估了细胞-心血管药物的相互作用。结果:与裸金电极相比,MPA/RGD修饰金电极增大了H9C2细胞黏附所引起的QCM频移(△f)与动态电阻变化(△R)响应。在所试H9C2浓度范围(5×10~4-4×10~5 cells/m L),△f与H9C2浓度呈线性关系,△R与H9C2浓度呈幂函数关系。我们用细胞粘弹性指数(CVI=△R/△f)来表征细胞的粘弹性。H9C2在异丙肾上腺素作用下,△f与△R增加、细胞-QCM表面黏附加强,细胞变硬;在维拉帕米作用下,△f与△R降低、细胞QCM表面黏附减弱,细胞变软。结论:QCM可用于不同浓度大鼠心肌细胞的动态细胞黏附监测,并可基于其细胞黏附与细胞黏弹性测定能力区分正性与负性肌力药物而可望用于心血管药物的筛选。     

细菌黏附素序列模体分析

目的：认识细菌黏附素序列保守的特征,更好地理解细菌黏附的机理。方法：利用InterProScan、MEME等分析工具对实验确认的155条细菌黏附素序列进行模体搜索。结果：用InterProScan在155个黏附素序列中搜索到119个模体,通过MEME分析发现了50个模体。结论：发现了一些与黏附功能相关的模体,为在细菌基因组内搜索黏附素序列奠定了基础。     

将RGD模体改变为RGDNM可减弱Echistatin对血小板聚集的抑制但增强血管新生的抑制作用

为了研究去整合素echistatin RGD(Arg-Gly-Asp) 模体周边氨基酸突变后对其生物学功能的影响,根据echistatin序列设计合成6个片段,利用重叠延伸PCR法合成cDNA,使echistatin RGD模体周边序列变为ARGDNM (D27→N),与载体pTXB1连接后转化E.coli BL21(DE3),建立echistatin (ARGDNM)的表达体系．工程菌经IPTG诱导后融合蛋白表达量占菌体总蛋白约30%,几丁质亲和纯化后,DTT裂解释放目的蛋白分子量约5.4 kD．体外血小板聚集和体内鸡胚绒毛尿囊膜 (chick chorioallantoic membrane, CAM)血管新生实验结果表明,echistatin (ARGDNM)抑制血小板聚集的作用减弱,而其抑制血管新生的作用增强．RGD模体周围氨基酸的改变影响了去整合素的生物学功能,echistatin (ARGDNM)增强了与αⅤβ3结合的特异性,本工作为研究特异性更强的去整合素药物奠定了基础．     

Molecular analysis of the interaction between cardosin A and phospholipase D(alpha). Identification of RGD/KGE sequences as binding motifs for C2 domains

Here we report the identification of phospholipase Dalpha as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase Dalpha constructs were performed. Results revealed that the C2 domain of phospholipase Dalpha contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dalpha can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains.     

RGD-dependent mechanotransduction of suspension cultured Taxus cell in response to shear stress

Plant cells cultured in bioreactors are strongly influenced by mechanical forces. However, the molecular mechanism of plant cell mechanoreception has maintained unclear. In animal cells, the Arg-Gly-Asp (RGD) motif can be found in proteins of the extracellular matrix. Integrins link the intracellular cytoskeleton of cells with the extracellular matrix by recognizing this RGD motif. Integrin has been demonstrated to function as an apparatus not only for adhesion but also for mechanotransduction. In plant cells, the molecules that mediate the structural continuity between wall and membrane are unknown. Here, we found that synthetic RGD peptide could dramatically reduce the level of phosphorylation of MAPK-like cascades that are activated by shear stress and reduce the alkalinization response, production of reactive oxygen species (ROS) and accumulation of phenolics by Taxus cuspidata cells during shear stress. These results implicate that a RGD recognition system may exist in Taxus cells and play an important role in signal transduction of shear stress. Although the Arabidopsis genome database shows that the plant seems to lack a homologue of animal integrin, plant cells may use other RGD-binding proteins to recognize the RGD motif. The correlative mechanism is discussed.     

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.     

The coxsackievirus A9 RGD motif is not essential for virus viability.

An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization.     

Role of the arginyl-glycyl-aspartic motif in the action of Ptr ToxA produced by Pyrenophora tritici-repentis

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused by Pyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coli expressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mM RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 microg mL(-1)) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.     

Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.     

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1

The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5β1 and αVβ3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL-αVβ3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVβ3. Furthermore, structure-activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVβ3 are proposed.     

Deletion of penton RGD motifs affects the efficiency of both the internalization and the endosome escape of viral particles containing adenovirus serotype 5 or 35 fiber knobs

Adenovirus (Ad) vectors are widely used for gene delivery in vitro and in vivo. A solid understanding of the biology of this virus is imperative for the development of novel, effective, and safe vectors. For the group C adenovirus serotypes 2 and 5 that use CAR as a primary attachment receptor, it is known that the penton base RGD motifs interact with cellular integrins and that this interaction promotes virus internalization. However, the RGD motif's impact on the efficiency of postinternalization steps, such as the escape of the virus particle from the endosome, is less defined. Furthermore, the role of penton-integrin interactions remains unknown for new vectors possessing group B Ad fiber knobs that use CD46 as a primary virus attachment receptor. In this study, we used vectors with the RGD motif deleted that contained Ad5 and B-group Ad35 fiber knobs and long fiber shafts and studied the role of RGD-integrin interactions in virus internalization and endosome escape. The deletion of the RGD motif in the penton base did not affect virus attachment, regardless of the type of cellular receptor used for attachment. RGD motif deletion, however, significantly reduced the rate of virus internalization for both the Ad5 and Ad35 fiber knob-containing vectors. This study also demonstrates the role of penton RGD motifs in facilitating the endosome escape step of virus infection and indicates that penton-integrin interactions are involved in internalization of capsid-chimeric CD46-interacting Ads with long fiber shafts.     

Plasma membrane-cell wall adhesion is required for expression of plant defense responses during fungal penetration

Fungal pathogens almost invariably trigger cell wall-associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane-cell wall adhesion as shown by the loss of the thin plasma membrane-cell wall connections known as Hechtian strands, reduced the expression of cell wall-associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall-associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane-cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall-associated defenses, there was no change, or an increase, in plasma membrane-cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall-associated defenses and a localized decrease in plasma membrane-cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane-cell wall adhesion resulted in reduced penetration by the rust fungus and induction of cell wall-associated defenses. These results suggest that rust fungi may induce a decrease in plasma membrane-cell wall adhesion as a means of disrupting the expression of nonspecific defense responses during penetration of host cells.     

alphavbeta5 integrin sustains growth of human pre-B cells through an RGD-independent interaction with a basic domain of the CD23 protein

CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the alphavbeta5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg(172), Lys(173), and Cys(174) (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind alphavbeta5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the beta subunit of the alphavbeta5 integrin. The interaction between alphavbeta5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to alphavbeta5.