Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.     

On the mechanism of fibrin-specific plasminogen activation by staphylokinase

The mechanism of plasminogen activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with plasminogen that activates plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface.     

Induction conditions for maximizing recombinant staphylokinase expression in Escherichia coli

Staphylokinase, a profibrinolytic bacterial protein, was cloned into Escherichia coli, following the amplification of its gene via PCR. The amplificated gene was inserted in a pKK223-3 plasmid vector. The recombinant protein (STAR), expressed from a tac promoter, was obtained in the periplasmic space when IPTG was added to the culture medium. Both the concentration of the inducer as well as the growth phase of recombinant cells at which it was added affected the final yield of periplasmic STAR. The protein was purified by a one-step procedure in an acylated-plasminogen Sepharose coupled column.     

Staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism

Alpha-defensins are peptides secreted by polymorphonuclear cells and provide antimicrobial protection mediated by disruption of the integrity of bacterial cell walls. Staphylokinase is an exoprotein produced by Staphylococcus aureus, which activates host plasminogen. In this study, we analyzed the impact of interaction between alpha-defensins and staphylokinase on staphylococcal growth. We observed that staphylokinase induced extracellular release of alpha-defensins from polymorphonuclear cells. Moreover, a direct binding between alpha-defensins and staphylokinase was shown to result in a complex formation. The biological consequence of this interaction was an almost complete inhibition of the bactericidal effect of alpha-defensins. Notably, staphylokinase with blocked plasminogen binding site still retained its ability to neutralize the bactericidal effect of alpha-defensins. In contrast, a single mutation of a staphylokinase molecule at position 74, substituting lysine for alanine, resulted in a 50% reduction of its alpha-defensin-neutralizing properties. The bactericidal properties of alpha-defensins were tested in 19 S. aureus strains in vitro and in a murine model of S. aureus arthritis. Staphylococcal strains producing staphylokinase were protected against the bactericidal effect of alpha-defensins. When staphylokinase was added to staphylokinase-negative S. aureus cultures, it almost totally abrogated the effect of alpha-defensins. Finally, human neutrophil peptide 2 injected intra-articularly along with bacteria alleviated joint destruction. In this study, we report a new property of staphylokinase, its ability to induce secretion of defensins, to complex bind them and to neutralize their bactericidal effect. Staphylokinase production may therefore be responsible in vivo for defensin resistance during S. aureus infections.     

葡激酶及其化学修饰研究进展

葡激酶(SAK)是一种由金黄色葡萄球菌分泌的含有136个氨基酸残基的蛋白质,大量研究证明它具有潜在的溶血栓特性。但葡激酶是一种外源蛋白,注入体内会引起不良反应,解决这些问题的重要途径就是对蛋白质进行化学修饰。本综述SAK的结构、洛栓机理、化学修饰及临床研究等。     

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.     

Kinetic and stoichiometric analysis of the modification process for N-terminal PEGylation of staphylokinase

Staphylokinase (SAK) is a therapeutic protein with promise for thrombolytic therapy of acute myocardial infarction. In this study, polyethylene glycol (PEG) aldehyde was used for N-terminal PEGylation of SAK to improve the pharmacological profiles of SAK. Due to the presence of the competitive PEGylation between the N terminus and the Lys residues, kinetic and stoichiometric analysis was carried out to investigate the process for the N-terminal PEGylation of SAK. To achieve this objective, size exclusion chromatography and tryptic peptide mapping were used to measure the PEGylation extent of SAK molecule and its specific amino acid residues, respectively.     

Enhancement of secretion and extracellular stability of staphylokinase in Bacillus subtilis by wprA gene disruption

Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.     

Proteolytic modification of staphylokinase

There are three types of staphylokinase of different isoelectric points (6.7, 6.1 and 5.7). Staphylokinase of pI 6.7 was converted to that of pI 6.1 and then to that of pI 5.7 by the treatment with trypsin.Heterogeneity of staphylokinase might be the result of post-translational modification by proteolytic enzyme.     

Intermolecular interactions in staphylokinase-plasmin(ogen) bimolecular complex: function of His43 and Tyr44

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with human plasmin (Pm) and switches its substrate specificity to generate a plasminogen (Pg) activator complex. Site-directed mutagenesis of SAKHis43 and SAKTyr44 demonstrated the crucial requirement of a positively charged and an aromatic residue, respectively, at these positions for optimal functioning of SAK-Pm activator complex. Molecular modeling studies further revealed the role of these residues in making cation-pi and pi-pi interactions with Trp215 of Pm and thus establishing the crucial intermolecular contacts within the active site cleft of the activator complex for the cofactor activity of SAK.     

Physical and conformational properties of staphylokinase in solution

The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy. Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm. The sedimentation coefficient is 1.71 S. These physical parameters indicate that the shape of staphylokinase is very elongated. The protein molecule consists of two folded domains of similar size. The mean distance of the centres of gravity of the domains is 3.7 nm. The mutual positions of the two domains are variable in solution. Thus, the molecule is shaped like a flexible dumbbell. About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in β-sheets and 20% form turns.     

Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43-Tyr44 pair and specificity of staphylokinase-plasmin activator complex

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with plasmin (Pm) and changes its substrate specificity to create a plasminogen (Pg) activator complex. The His(43)-Tyr(44) pair of SAK resides within the active site cleft of the partner Pm and generates intermolecular contacts to confer Pg activator ability to the SAK-Pm bimolecular complex. Site-directed mutagenesis and molecular modeling studies unravelled that mutation at 42nd or 45th positions of SAK specifically disrupts cation-pi interaction of His(43) with Trp(215) of partner Pm within the active site, whereas pi-pi interaction of Tyr(44) with Trp(215) remain energetically favoured.     

Enhancement of Secretion and Extracellular Stability of Staphylokinase in Bacillus subtilis by wprA Gene Disruption

Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.     

High-level expression of non-glycosylated and active staphylokinase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (~1 g/l) of extracellular glycosylated rSAK (~18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (~15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.     

Staphylokinase-specific cell-mediated immunity in humans.

Staphylokinase is a highly fibrin-specific clot-dissolving agent that constitutes a promising drug for clinical development. It is of bacterial origin, and the majority of patients develop neutralizing Ab after its administration. Several antigenic regions, recognized by these Ab, have been identified, but the underlying immunogenic features of staphylokinase remain unknown. In this study, we show that staphylokinase is a T cell-dependent Ag, and that an immunological memory may be acquired, even without administration of staphylokinase. Thrombolysis with staphylokinase provokes the proliferation of staphylokinase-specific T lymphocytes, which remain elevated over 10 mo posttreatment. Interestingly, analysis of a large number of staphylokinase-specific T cell clones isolated from 10 unrelated donors revealed only six distinct immunogenic regions in the molecule. Moreover, five of the six regions are recognized by T lymphocytes from several individuals, indicating that these regions are not restricted to a single HLA-DR allele. Therefore, these new insights can guide the design of variants with a lower immunogenic profile in humans.     

Staphylococcus aureus: Staphylokinase

Staphylokinase is a 136 aa long bacteriophage encoded protein expressed by lysogenic strains of Staphylococcus aureus. Present understanding of the role of staphylokinase during bacterial infection is based on its interaction with the host proteins, alpha-defensins and plasminogen. alpha-Defensins are bactericidal peptides originating from human neutrophils. Binding of staphylokinase to alpha-defensins abolishes their bactericidal properties, which makes staphylokinase a vital tool for staphylococcal resistance to host innate immunity. Complex binding between staphylokinase and plasminogen results in the formation of active plasmin, a broad-spectrum proteolytic enzyme facilitating bacterial penetration into the surrounding tissues. We have recently shown high levels of staphylokinase expression in clinical isolates of skin and mucosal origin and relative low levels in isolates invading internal organs. These findings are supported by sepsis studies using isogenic S. aureus strains demonstrating increased bacterial load in the absence of staphylokinase production. Our observations indicate that staphylokinase favours symbiosis of staphylococci with the host that makes it an important colonization factor.     

Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli

Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.     

RGD-葡激酶突变体(K130T,K135R)的制备与活性分析

以葡激酶突变体质粒mSAK(K130T ,K135R)-pBV220为模板,PCR重叠引物延伸法引入突变位点,并将该片段克隆至载体pBV220 ,构建了RGD-mSAK-pBV220质粒,转化大肠杆菌后热激诱导获得了高效表达,表达产物占菌体总蛋白的50%以上,且主要以可溶性形式存在,所获蛋白依次用Q SepharoseHP柱、SephaycrylS200HR柱和SP柱进行纯化,纯化的蛋白的纯度可达98%以上,纤维蛋白溶圈法体外溶栓活性测定结果表明,所获RGD-mSAK蛋白溶栓活性与野生型葡激酶相当,豚鼠体内免疫试验证明突变体的免疫原性也有所降低,血小板聚集试验分析突变体蛋白的抗血小板聚集能力,RGD 葡激酶突变体具有一定的抗血小板聚集能力。     

Laboratory-scale permeabilization of Escherichia coli cells for recovery of a small recombinant protein – Staphylokinase

The recovery of recombinant proteins includes a purification process that has to be compressed to a minimum of steps in order to get high yields with a low cost expenditure. A selective liberation of recombinant proteins by cell permeabilization leads to both a high product purity just in the beginning of the recovery process and to a simplification of the cell residue separation compared to the mechanical cell disruption. In case of the purification of the bacterial plasminogen activator Staphylokinase from E. coli cells, yields of 82% with a purity of 46% were attained by utilization of permeabilization by biomass freezing, resuspension in a Tris/EDTA-buffer and following micro-diafiltration. A recovery process without interruption (freezing) is possible due to the addition of guanidine-HCl and Triton X100 to the buffer. These methods were developed on a laboratory-scale.     

NMR secondary structure of the plasminogen activator protein staphylokinase

Staphylokinase (Sak) is a 15.5 kDa protein secreted by several strains of Staphylococcusaureus. Due to its ability to convert plasminogen, the inactive proenzyme of the fibrinolyticsystem, into plasmin, Sak is presently undergoing clinical trials for blood clot lysis in thetreatment of thrombovascular disorders. With a view to developing a better understanding ofthe mode of action of Sak, we have initiated a structural investigation of Sak viamultidimensional heteronuclear NMR spectroscopy employing uniformly 15N- and 15N,13C-labelled Sak. Sequence-specific resonance assignments have been made employing 15N-editedTOCSY and NOE experiments and from HNCACB, CBCA(CO)NH, HBHA(CBCACO)NHand CC(CO)NH sets of experiments. From an analysis of the chemical shifts,3JHNH scalar coupling constants, NOEs and HN exchange data, the secondary structural elements of Sakhave been characterized.