Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides.

The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60-414 or SK56-414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2- and 2.5-fold, respectively, of that generated by equimolar mixtures of native SK and HPG. Although in the absence of SK1-59 equimolar mixtures of SK56-414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N-terminal region lay mainly in imparting in SK56-414 an enhanced ability for PG activation. The ability of various synthetic peptides derived from the amino-terminal region (SK16-51, SK16-45, SK37-59, SK1-36, SK16-36, and SK37-51) to (1) complement equimolar mixtures of SK56-414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56-414 and HPG by SK16-59, and (3) directly inhibit PG activation by the 1:1 SK-HPG activator complex was tested. Apart from SK16-59, SK16-51, and 16-45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56-414 survived even in the smaller SK-peptides, viz., SK37-59 and SK37-51. However, this ability was abolished upon specifically mutating the sequence -LTSRP-, present at position 42-46 in native SK. Although SK16-51 retained virtually complete ability for potentiation of PG activation in comparison to SK16-59 or SK1-59, this ability was reduced by approximately fourfold in the case of SK16-45, and completely abolished upon further truncation of the C-terminal residues to SK16-36 or SK1-36. Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG. This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25-59) in which the sequence corresponding to this putative locus (residues 16-36) was truncated at the middle. On the other hand, fragments SK37-51 and SK37-59 did not show any inhibition of the PG activation by native activator complex. Taken together, these findings strongly support a model of SK action wherein the HPG binding site resident in the region 37-51 helps in anchoring the N-terminal domain to the strong intermolecular complex formed between HPG and the region 60-414. In contrast, the site located between residues 16 and 36 is qualitatively more similar to the previously reported PG interacting site (SK254-273) present in the core region of SK, in being involved in the relatively low-affinity enzyme-substrate interactions of the activator complex with PG during the catalytic cycle.     

Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the beta domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK(del254-262)). A kinetic analysis of SK(del254-262) revealed that its low HPG activator activity arose from a 5-6-fold increase in K(m) for HPG as substrate, with little alteration in k(cat) rates. This increase in the K(m) for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK(del254-262). The involvement of kringles was further indicated by a hypersusceptibility of the SK(del254-262).plasmin activator complex to epsilon-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK(del254-262).HPG was reduced by about 3-fold in comparison with that of nSK.HPG . Overall, these observations identify the 250 loop in the beta domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process.     

Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis

The possible role of the central beta-domain (residues 151-287) of streptokinase (SK) was probed by site-specifically altering two charged residues at a time to alanines in a region (residues 230-290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation. These mutants were then screened for altered ability to activate equimolar "partner" human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine-linker mutants of SK, one mutant, viz. SK(KK256.257AA) (SK-D1), showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli (nSK). Five other mutants were as active as nSK, with two [SK(RE248.249AA) and SK(EK281.282AA), referred to as SK(C) and SK(H), respectively] showing specific activities approximately one-half and two-thirds, respectively, that of nSK. Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK(KK256.257EE) [SK(D2)]. This mutant showed a PG activator activity approximately 10-fold less than that of SK(D1). Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a "burst" of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate. The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once "supplied" with a mature active site, the Km for PG was increased nearly eightfold in comparison to that of nSK-PN. SK mutants carrying simultaneous two- and three-site charge-cluster alterations, viz., SK(RE24249AA:EK281.282AA) [SK(CH)], SK(EK272.273AA;EK281.282AA) [SK(FH)], and SK(RE248.249AA;EK272.273AA:EK281.282AA+ ++) [SK(CFH)], showed additive/synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective "single-site" mutants, with the "triple-site" mutant [SK(CFH)] showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double- and triple-charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK beta-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X-ray crystal structure of the SK-plasmin light chain complex.     

Role of the 88-97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis

The role of a prominent surface-exposed loop (residues 88-97) in the alpha domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for beta-turn formation. The mutant so formed (termed SK(88-97-Beta Turn)), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k(cat) level, with relatively minor changes in K(m) values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK-HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1-5) showed K(d) values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88-97 loop, on the one hand, and its known position at the SK-HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88-97 loop of the alpha domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme-substrate affinity per se is minimal.     

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: Critical contribution of kringle-4 revealed by the mutagenesis approaches

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein-protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1-K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3-K5CD (K3K4K5CD), K2-K5CD (K2K3K4K5CD) and K1-K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1-2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the ‘addition’ of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the “long-range” enzyme-substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme-substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.     

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: critical contribution of kringle-4 revealed by the mutagenesis approaches

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein-protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1-K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3-K5CD (K3K4K5CD), K2-K5CD (K2K3K4K5CD) and K1-K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1-2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the 'addition' of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the "long-range" enzyme-substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme-substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.     

Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.     

Amino-Terminal Fusion of Epidermal Growth Factor 4,5,6 Domains of Human Thrombomodulin on Streptokinase Confers Anti-Reocclusion Characteristics along with Plasmin-Mediated Clot Specificity

Streptokinase (SK) is a potent clot dissolver but lacks fibrin clot specificity as it activates human plasminogen (HPG) into human plasmin (HPN) throughout the system leading to increased risk of bleeding. Another major drawback associated with all thrombolytics, including tissue plasminogen activator, is the generation of transient thrombin and release of clot-bound thrombin that promotes reformation of clots. In order to obtain anti-thrombotic as well as clot-specificity properties in SK, cDNAs encoding the EGF 4,5,6 domains of human thrombomodulin were fused with that of streptokinase, either at its N- or C-termini, and expressed these in Pichia pastoris followed by purification and structural-functional characterization, including plasminogen activation, thrombin inhibition, and Protein C activation characteristics. Interestingly, the N-terminal EGF fusion construct (EGF-SK) showed plasmin-mediated plasminogen activation, whereas the C-terminal (SK-EGF) fusion construct exhibited ‘spontaneous’ plasminogen activation which is quite similar to SK i.e. direct activation of systemic HPG in absence of free HPN. Since HPN is normally absent in free circulation due to rapid serpin-based inactivation (such as alpha-2-antiplasmin and alpha-2-Macroglobin), but selectively present in clots, a plasmin-dependent mode of HPG activation is expected to lead to a desirable fibrin clot-specific response by the thrombolytic. Both the N- and C-terminal fusion constructs showed strong thrombin inhibition and Protein C activation properties as well, and significantly prevented re-occlusion in a specially designed assay. The EGF-SK construct exhibited fibrin clot dissolution properties with much-lowered levels of fibrinogenolysis, suggesting unmistakable promise in clot dissolver therapy with reduced hemorrhage and re-occlusion risks.     

Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation

The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.     

Molecular recognition sites on factor Xa which participate in the prothrombinase complex.

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions. To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276 could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.     

Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation

Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancing and/or stabilizing the interaction of free PG.     

alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.     

Enhanced plasminogen activation by staphylokinase in the presence of streptokinase beta/betagamma domains: plasminogen kringles play a role

Presence of isolated beta or betagamma domains of streptokinase (SK) increased the catalytic activity of staphylokinase (SAK)-plasmin (Pm) complex up to 60%. In contrast, fusion of SK beta or betagamma domains with the C-terminal end of SAK drastically reduced the catalytic activity of the activator complex. The enhancement effect mediated by beta or betagamma domain on Pg activator activity of SAK-Pm complex was reduced greatly (45%) in the presence of isolated kringles of Pg, whereas, kringles did not change cofactor activity of SAK fusion proteins (carrying beta or betagamma domains) significantly. When catalytic activity of SAK-microPm (catalytic domain of Pm lacking kringle domains) complex was examined in the presence of isolated beta and betagamma domains, no enhancement effect on Pg activation was observed, whereas, enzyme complex formed between microplasmin and SAK fusion proteins (SAKbeta and SAKbetagamma) displayed 50-70% reduction in their catalytic activity. The present study, thus, suggests that the exogenously present beta and betagamma interact with Pg/Pm via kringle domains and elevate catalytic activity of SAK-Pm activator complex resulting in enhanced substrate Pg activation. Fusion of beta or betagamma domains with SAK might alter these intermolecular interactions resulting in attenuated functional activity of SAK.     

链激酶研究概况及其进展

链激酶(Streptok inase,SK)是世界上最早发现的纤维蛋白酶原激活剂,也是最早作为临床药品治疗血栓性疾病的溶栓酶。它是由A,C,G群链球菌中β-溶血性链球菌分泌的胞外非酶蛋白质,能和纤溶酶原结合,将纤溶酶原激活为纤溶酶,具有溶解血栓的作用。本文详细综述了该酶的性质、在溶栓酶中的地位、研究历史、作用机理等。此外,由于它有半衰期短、不具有纤维蛋白特异性、治疗后出血和血栓易复发的缺点,所以有必要用基因工程的手段进行改造,以达到更好的治疗效果。     

Characterization of Lys-698-to-Met substitution in human plasminogen catalytic domain

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys-698 of human Pg was hypothesized to participate in triggering activation in the SK-Pg complex. Here, we report a study of the Lys-698 to Met substitution in the catalytic domain of Pg (microPg) containing the proteolytic activation-resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant (K(D)), the recombinant protein shows a nearly 60-fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 A crystal structure of this mutant microPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys-698 of human Pg plays a functional role in the so-called N-terminal insertion activation mechanism by SK.     

人NDRG2启动子的克隆与活性鉴定

目的:克隆人NDRG2的启动子,并进行启动子的活性鉴定。方法:用Advantage-GC Taq酶,采用PCR方法,以BCA克隆R-998D1为模板,克隆人NDRG2(-1455/ 274)的启动子。分别构建NDRG2(-1131/ 274)、NDRG2(-273/ 274)、NDRG2(-135/ 274)、NDRG2(-79/ 274)和NDRG2(-79/ 57)等不同长度的截短体,并分别亚克隆入pGL3-basic报告基因载体,测序鉴定。分别转染HEK293和HeLa细胞后,运用双荧光报告基因系统进行启动子活性分析,从而判断核心启动子的位置。结果:人NDRG2的启动子克隆成功,并构建了不同长度的截短体,DNA测序结果与报道一致。报告基因分析结果将人NDRG2启动子的核心区域定位于NDRG2(-79/ 57)。结论:随着人NDRG2的启动子的长度逐渐被截短,启动子的基础活性也逐渐降低,可将人NDRG2启动子的核心区域定位于NDRG2(-79/ 57)。     

Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-AMP-incorporated peptides

The photoaffinity inhibitor analog [2-3H]8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase. The reaction site(s) of [2-3H]8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog. Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label. The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity. A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region. This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-[14C]glucose (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido-AMP were also identified by chemical characterization. One region, containing 20% of the covalently bound label, was composed of residues 11-68. This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure.     

pH-dependent Interactions of the carboxyl-terminal helix of steroidogenic acute regulatory protein with synthetic membranes

Steroidogenic acute regulatory (StAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259-282; at pH 6.5 this region was partially digested into 254-272, 254-273, and 254-274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal alpha4 helix and that residues Leu(275), Thr(263), and Arg(272) in alpha4 form stabilizing interactions with Gln(128), Asp(150), and Asp(106) in adjacent loops. CD spectroscopy of a 37-mer model of alpha4 (residues 247-287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was alpha-helical and achieved maximal alpha-helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal alpha4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the alpha-helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane.     

Analysis of the interactions between streptokinase domains and human plasminogen.

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.     

Fibrin‐targeted plasminogen activation by plasminogen activator,PadA, from Streptococcus dysgalactiae

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three‐domain streptokinase (SK) and two‐domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2‐antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single‐domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single‐domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single‐domain plasminogen activator that activates bovine plasminogen in a fibrin‐targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin‐specific plasminogen activators for thrombolytic therapy.