重组apo(a) Kringle Ⅳ-10对纤溶酶原与内皮细胞结合的影响

通过研究重组apo(a)KringleⅣ 10 (KⅣ 10 )的赖氨酸结合能力对纤溶酶原与内皮细胞结合的影响 ,探讨apo(a)在抑制纤溶过程中的作用 ,为脂蛋白 (a) [lipoprotein(a) ,Lp(a) ]致动脉粥样硬化机理研究提供依据 .将含apo(a)野生型KⅣ 10 ((wild typeKⅣ 10 Trp72 ,wt KⅣ 10 Trp72 )和突变型KⅣ 10 (mutate typeKⅣ 10 Trp72 ,mut KⅣ 10 Arg72 )基因片段重组质粒 ,分别转化至E .coliDH5α菌株中并表达含这 2个重组片段的融合蛋白 ,通过Glutathione Agarosebeads亲和层析柱进行分离和提纯 ,经L Lys Sepharose 4B亲和层析柱检测其赖氨酸结合能力 .再以异硫氰酸荧光素标记的纤溶酶原为配基 ,观察这 2种基因表达片段对纤溶酶原与人脐静脉内皮细胞 (humanumbilicalveinendothe lialcells ,HUVEC)结合的影响 .结果显示 :在E .coliDH 5α菌株中表达的野生型谷胱甘肽S 转移酶(glutathioneS transferase ,GST) KⅣ 10 Trp72 (GST wt KⅣ 10 Trp72 )融合蛋白和突变型谷胱甘肽S 转移酶 (GST mut KⅣ 10 Arg72 )融合蛋白在赖氨酸结合能力上存在明显差异 .其中GST wt KⅣ 10 Trp72能有效地抑制纤溶酶原与人脐静脉内皮细胞的结合 ;而GST mut KⅣ 10 Arg72 在任一浓度范围内均无这种抑制作用 .结果     

重组人纤溶酶原Kringle1-5的制备及其

为了研究重组人纤溶酶原 Kringle1-5(K1-5)的抗血管生成活性及其对内皮细胞增殖的影响, 通过PCR扩增人纤溶酶原K1-5 cDNA,定向克隆于原核表达载体pET30a(+)中,构建重组表达载体pET-K1-5, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-PAGE 和Western 杂交检测K1-5的表达。鸡胚尿囊膜 (CAM) 实验和MTT实验分别检测重组人纤溶酶原Kringle1-5对鸡胚新生血管生成和内皮细胞的抑制作用。结果表明,IPTG诱导原核表达载体pET-K1-5在E.coli BL21(DE3)中的表达量约占菌体总蛋白量的32%, K1-5主要以包涵体形式存在,包涵体经过洗涤、溶解、Ni-spin 亲合柱层析纯化以及蛋白质复性等步骤后,获得了纯度约为96%的重组K1-5蛋白。CAM实验表明,原核表达的重组人K1-5能有效地按剂量依赖的方式抑制鸡胚新生血管的形成。MTT实验结果显示,重组人K1-5特异地抑制内皮细胞的增殖, 而对非内皮细胞无抑制作用。     

重组人纤溶酶原Kringle1-3的生物学活性鉴定

目的：检测重组人纤溶酶原Kringle1-3（K1-3）的生物学活性。方法：用含重组人纤溶酶原K1-3基因的表达载体pET21a-Angio（K1-3）转化表达宿主菌大肠杆菌BL21（DE3）后诱导表达,表达产物经溶解、复性和纯化后,进行SDS-PAGE,计算其相对分子质量;用BCA法测蛋白浓度,用细胞抑制实验（MTT法）和鸡胚绒毛膜尿囊膜（CAM）实验鉴定纯化产物对血管内皮细胞增殖和血管生成的抑制效果。结果：表达产物的相对分子质量为38000,与预期值一致;细胞抑制实验和CAM实验结果表明表达产物具有特异抑制血管内皮细胞增殖、血管生成的功能。结论：所纯化的重组人纤溶酶原K1-3具有抑制血管内皮细胞的生物学活性,为该蛋白在病理性血管疾病等方面的应用研究提供了材料。     

人纤溶酶原kringle 5在大肠杆菌中克隆和分泌表达

构建能够表达分泌性的人纤溶酶原kringle 5（简称hPK-5）的大肠杆菌工程菌。用PCR方法扩增获得hPK-5基因,构建原核表达载体pET-22b（＋）/hPK-5,转化大肠杆菌BL21（DE3）,经IPTG诱导表达并鉴定其免疫学活性。成功表达14kD的重组hPK-5,且约占菌体分泌性蛋白20%以上,经Western Blot分析表明其具有hPK-5的免疫抗原活性。人纤溶酶原kringle 5在大肠杆菌中BL21（DE3）获得可分泌表达。     

纤溶酶原Kringle 5的抑制血管增生作用

纤溶酶原Kringle 5是纤溶酶原中与血管抑素相连的另一个联环结构域,它能抑制内皮细胞的增殖、迁移,并能诱导内皮细胞凋亡和细胞周期停滞,具有很强的抑制血管生成活性,是抑制活性最强的纤溶酶原水解片段。同时它具有分子量小、性质稳定、毒副作用小、特异性高等优点,是一个有潜在临床应用价值和开发前景的治疗血管增生性疾病的药物。     

人纤溶酶原Kringle5在酵母中的表达及其活性研究

抽提人肝组织总RNA,通过RT-PCR方法扩增出纤溶酶原Kringle5片段,将其和分泌表达载体pPIC9K相连,转化毕赤酵母GS115,筛选获得在酵母细胞中遗传稳定表达Kringle5重组蛋白的工程菌。经甲醇诱导表达5d后,发酵液总蛋白分泌量约120mg/L,SDS-PAGE呈现相对分子质量约为15×103特异表达带。经Ni-NTA-Agarose纯化后,重组蛋白对人脐静脉内皮细胞有明显的抑制作用,ID50约为4.0μg/ml。     

人重组t-PA突变体与β2糖蛋白Ⅰ的相互作用

利用重组DNA技术和原核表达,得到了人体组织型纤溶酶原激活剂t-PA 的缺失型突变体r-PA和Kringle2功能区,并在变性条件下通过金属螯合层析纯化得到纯度较高的r-PA和Kringle2重组蛋白.用纤维蛋白活性平板检测到复性的r-PA和Kringle2都具有体外纤溶活性,并且β 2糖蛋白Ⅰ (β2-glycoprotein Ⅰ, β2GPⅠ)对r-PA的体外纤溶活性有显著的促进作用:在β2GPⅠ浓度为1 μmol/L和4 μmol/L时,r-PA的纤溶活性分别提高1.8和2.1倍.ELISA方法检测发现, β2GPⅠ与r-PA,Kringle2均有特异性结合,并且随着β2GPⅠ浓度的增加,它与包被在酶标板上的重组蛋白r-PA和Kringle2的结合都有趋于饱和的趋势,但其饱和浓度却均远高于β2GPⅠ与t-PA结合的饱和浓度.     

可溶型三聚体血管生长抑制因子Kringle5的克隆,表达,纯化及活性研究

血管生长抑制因子Kringle 5 是目前发现的抑制血管内皮细胞增殖和肿瘤生长的活性最强的纤溶酶原片段,特异性高而毒副作用小,在肿瘤的治疗方面具有潜在的巨大价值和广阔的应用前景。根据K5基因的序列设计PCR引物,通过PCR从已有的克隆载体扩增出人纤维蛋白溶酶原的K5部分基因,将K5基因克隆入原核表达载体pET15b,经序列测定,成功构建了pET15b-K5非融合表达载体。将重组载体导入大肠杆菌中IPTG诱导表达,SDS-PAGE分析目的蛋白主要以可溶形式存在于菌体中,破碎后上清通过阳离子交换层析,纯化获得纯度大于95%的目标蛋白,脱盐后对分子量测定推测形成了三聚体。通过鸡胚绒毛尿囊膜法证明蛋白产物对鸡胚绒毛尿囊膜血管增生有一定的抑制作用。     

t-PA K1区的同源模建及Kringle区与赖氨酸相互作用的研究

利用同源模建方法预测了t-PAK1区的三维结构。通过结构叠合确定了t-PAK1、K2区,纤溶酶原K1、K4区及UKK区的赖氨酸结合口袋。静电势计算及疏水性分析表明,在t-PAK2区以及纤溶酶原K1、K4区与纤维蛋白裸露的赖氨酸之间存在明显的静电势互补和疏水面契合。确定了影响Kringle区结合口袋与赖氨酸亲和的重要氨基酸,分析了t-PAK1区、UKK区不能结合赖氨酸的原因,由此设计了具有赖氨酸亲和力的新型t-PAK1区及UKK区突变体。利用模拟残基突变技术预测了突变体的结构变化,分析了突变后t-PAK1区及UKK区与赖氨酸亲和力的变化,初步在理论上肯定了设计方案的合理性。     

人纤溶酶原饼环区5基因的原核表达及活性测定

 人纤溶酶原饼环区 5 (hPgnK5 )是新的血管生成抑制因子 .PCR法改造hPgnK5基因 ,所得hPgnK5基因包括编码人纤溶酶原C4 62 到P54 4共 83个氨基酸残基 ,而且在 5′ ,3′分别引入了EcoRⅠ和BamHⅠ位点 .以pThiohisA构建hPgnK5基因原核表达载体 ,在大肠杆菌TOP10中表达该蛋白 .通过柱层析法获得hPgnK5纯化蛋白 .免疫印迹反应表明该蛋白具有hPgnK5免疫活性 .体外实验表明 ,该蛋白具有抑制血管内皮细胞增殖活性 .提示hPgnK5可能具有良好应用前景     

High efficacy and minimal peptide required for the anti‐angiogenic and anti‐hepatocarcinoma activities of plasminogen K5

Kringle 5(K5) is the fifth kringle domain of human plasminogen and its anti‐angiogenic activity is more potent than angiostatin that includes the first four kringle fragment of plasminogen. Our recent study demonstrated that K5 suppressed hepatocarcinoma growth by anti‐angiogenesis. To find high efficacy and minimal peptide sequence required for the anti‐angiogenic and anti‐tumour activities of K5, two deletion mutants of K5 were generated. The amino acid residues outside kringle domain of intact K5 (Pro452‐Ala542) were deleted to form K5mut1(Cys462‐Cys541). The residue Cys462 was deleted again to form K5mut2(Met463‐Cys541). K5mut1 specifically inhibited proliferation, migration and induced apoptosis of endothelial cells, with an apparent two‐fold enhanced activity than K5. Intraperitoneal injection of K5mut1 resulted in more potent tumour growth inhibition and microvessel density reduction than K5 both in HepA‐grafted and Bel7402‐xenografted hepatocarcinoma mouse models. These results suggested that K5mut1 has more potent anti‐angiogenic activity than intact K5. K5mut2, which lacks only the amino terminal cysteine of K5mut1, completely lost the activity, suggesting that the kringle domain is essential for the activity of K5. The activity was enhanced to K5mut1 level when five acidic amino acids of K5 in NH₂ terminal outside kringle domain were replaced by five serine residues (K5mut3). The shielding effect of acidic amino acids may explain why K5mut1 has higher activity. K5, K5mut1 and K5mut3 held characteristic β‐sheet spectrum while K5mut2 adopted random coil structure. These results suggest that K5mut1 with high efficacy is the minimal active peptide sequence of K5 and may have therapeutic potential in liver cancer.     

Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin

The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.     

Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.     

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.     

Role of disulfide bonds in biologic activity of human interleukin-6.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.     

Kringle 5 peptide-albumin conjugates with anti-migratory activity

Three peptide fragments of the kringle 5 region of plasminogen and their respective N- and C-terminus maleimido derivatives conjugated to Cys34 of human serum albumin were evaluated in vitro using a human umbilical vein endothelial cell (HUVEC) migration assay and a human plasma stability assay. The N-terminus maleimido derivative of the 64 to 74 segment of kringle 5 conjugated to human serum albumin possessed remarkable anti-migratory activity.     

Functional structure of the somatomedin B domain of vitronectin

The N-terminal somatomedin B domain (SMB) of vitronectin binds PAI-1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the crystal structure of the PAI-1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5-Cys21, Cys9-Cys39, Cys19-Cys32, Cys25-Cys31), notably claiming that the first disulfide is Cys5-Cys9 rather than the Cys5-Cys21 bonding shown in the structure. To test if the claimed Cys5-Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N-terminal peptide (residue 1-14 or 1-24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI-1.     

Energetics of structural domains in alpha-lactalbumin.

alpha-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in alpha-helix (the alpha-domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in beta-sheet (the beta-domain), has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine alpha-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three-disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact alpha-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. the two-disulfide form of alpha-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact alpha-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, alpha-lactalbumin retains its calcium-binding beta-domain, whereas the alpha-domain is unfolded. It appears that the beta-domain does not require alpha-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded alpha-domain.     

Defining the native disulfide topology in the somatomedin B domain of human vitronectin

The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).     

Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a

The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.