A mutant streptokinase lacking the C-terminal 42 amino acids is less reactive with preexisting antibodies in patient sera

Streptokinase (SK) is an efficacious thrombolytic drug for the treatment of myocardial infarction. Because of its immunogenicity, patients receiving SK therapy develop high anti-SK antibody (Ab) titers, which might provoke severe allergic reactions and neutralize SK activity. In this report we studied the reactivity of a synthetic 42-residue peptide resembling SKC-2 C-terminus with patient sera. SKC-2(373-414) peptide was recognized by 39 and 64% of patients, before and after SKC-2 therapy, respectively. An SKC-2 deletion mutant (mut-C42), lacking the same 42 C-terminal residues, was constructed and expressed in Escherichia coli. Recognition of mut-C42 by preexisting Abs from patient sera was 51 and 68% of reactivity to SKC-2, as assessed by direct binding and competition assays, respectively. For most of the patients, mut-C42-neutralizing activity titer (NAT) significantly decreased with respect to SKC-2-NAT. This study opens the possibility of producing a less immunogenic variant of SK, which could constitute a preferred alternative for thrombolytic therapy.     

Mapping of the plasminogen binding site of streptokinase with short synthetic peptides.

Although several recent studies employing various truncated fragments of streptokinase (SK) have demonstrated that the high-affinity interactions of this protein with human plasminogen (HPG) to form activator complex (SK-HPG) are located in the central region of SK, the exact location and nature of such HPG interacting site(s) is still unclear. In order to locate the "core" HPG binding ability in SK, we focused on the primary structure of a tryptic fragment of SK derived from the central region (SK143-293) that could bind as well as activate HPG, albeit at reduced levels in comparison to the activity of the native, full-length protein. Because this fragment was refractory to further controlled proteolysis, we took recourse to a synthetic peptide approach wherein the HPG interacting properties of 16 overlapping 20-mer peptides derived from this region of SK were examined systematically. Only four peptides from this set, viz., SK234-253, SK254-273, SK274-293, and SK263-282, together representing the contiguous sequence SK234-293, displayed HPG binding ability. This was established by a specific HPG-binding ELISA as well as by dot blot assay using 125I-labeled HPG. These results showed that the minimal sequence with HPG binding function resided between residues 234 and 293. None of the synthetic SK peptides was found to activate HPG, either individually or in combination, but, in competition experiments where each of the peptides was added prior to complex formation between SK and HPG, three of the HPG binding peptides (SK234-253, SK254-273, and SK274-293) inhibited strongly the generation of a functional activator complex by SK and HPG. This indicated that residues 234-293 in SK participate directly in intermolecular contact formation with HPG during the formation of the 1:1 SK-HPG complex. Two of the three peptides (SK234-253 and SK274-293), apart from interfering in SK-HPG complex formation, also showed inhibition of the amidolytic activity of free HPN by increasing the K(m) by approximately fivefold. A similar increase in K(m) for amidolysis by HPN as a result of complexation with SK has been interpreted previously to arise from the steric hinderance at or near the active site due to the binding of SK in this region. Thus, our results suggest that SK234-253 and SK274-293 also, like SK, bound close to the active site of HPN, an event that was reflected in the observed alteration in its substrate accessibility. By contrast, whereas the intervening peptide (SK254-273) could not inhibit amidolysis by free HPN, it showed a marked inhibition of the activation of "substrate" PG (human or bovine plasminogen) by activator complex, indicating that this particular region is intimately involved in interaction of the SK-HPG activator complex with substrate plasminogen during the catalytic cycle. This finding provides a rational explanation for one of the most intriguing aspects of SK action, i.e., the ability of the SK-HPG complex to catalyze selectively the activation of substrate molecules of PG to PN, whereas free HPN alone cannot do so. Taken together, the results presented in this paper strongly support a model of SK action in which the segment 234-293 of SK, by virtue of the epitopes present in residues 234-253 and 274-293, binds close to the active center of HPN (or, a cryptic active site, in the case of HPG) during the intermolecular association of the two proteins to form the equimolar activator complex; the segment SK254-273 present in the center of the core region then imparts an ability to the activator complex to interact selectively with substrate PG molecules during each PG activation cycle.     

Properties of streptokinase incorporated into polyethylene glycol microcapsules

Thrombolytic therapy with high doses of streptokinase (SK), which are required due to its rapid clearance from the bloodstream, is accompanied by side effects. In this work, the SK was incorporated into water-soluble polyethylene glycol (PEG) microcapsules with the double emulsification method in order to increase its lifetime in bloodstream and decrease side effects. Four preparations of SK*PEG-microcapsules with a high degree of the SK enclosure (～90–91%) and total retention of fibrinolytic activity were produced under varying emulsification conditions (PEG molecular mass 20 or 40 kDa and PEG/SK ratio 12 or 8 mg of PEG/1000 IU SK). SK was released from the PEG-microcapsules at different rates: the time of complete release varied from 45 to 90 min (pH 7.4, 37°C). Comparative in vitro study of thrombolytic activity and side effects of the SK in a free and encapsulated state was conducted. It was found that the rate of human plasma clot lysis under the action of encapsulated SK preparations is equal (with exception of a short lag-period) to the rate of lysis induced by the free SK, provided that the doses were equal (500 IU/mL). Furthermore, the SK*PEG-microcapsules caused the reduced exhaustion of plasminogen and fibrinogen in plasma when compared with the free SK.     

Anti-human cardiac myosin autoantibodies in Kawasaki syndrome.

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.     

Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation

The bacterial protein streptokinase (SK) activates human plasminogen (Pg) into the fibrinolytic protease plasmin (Pm). Roughly 40 residues from the SK C-terminal domain are mobile in the crystal structure of SK complexed with the catalytic domain of Pm, and the functions of this C-tail remain elusive. To better define its roles in Pg activation, we constructed and characterized three C-terminal truncation mutants containing SK residues 1-378, 1-386, and 1-401, respectively. They exhibit gradually reduced amidolytic activity and Pg-activator activity, as well as marginally decreased binding affinity toward Pg, as more of the C-terminus is deleted. As compared with full-length SK, the shortest construct, SK(1-378), exhibits an 80% decrease in amidolytic activity (k(cat)/K(M)), an 80% decrease in Pg-activator activity, and a 30% increase in the dissociation constant toward the Pg catalytic domain. The C-terminal truncation mutations did not attenuate the resistance of the SK-Pm complex to alpha(2)-antiplasmin. Attempts at using a purified C-tail peptide to rescue the activity loss of the truncation mutants failed, suggesting that the integrity of the SK C-terminal peptide is important for the full function of SK.     

Infection of human cytomegalovirus in cultured human gingival tissue

Background

Matrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs.

Results

We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 μg/ml) even after two consecutive infections but increased to ~50 μg/ml after the third infection. Competition with free M2e peptide indicated that ~20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a ≥ 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 μg/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV.

Conclusion

The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.     

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

Differential immunogenicity of two peptides isolated by high molecular weight-melanoma-associated antigen-specific monoclonal antibodies with different affinities

Peptide mimics isolated from phage display peptide libraries by panning with self-tumor-associated Ag (TAA)-specific mAbs are being evaluated as immunogens to implement active specific immunotherapy. Although TAA-specific mAb are commonly used to isolate peptide mimics, no information is available regarding the Ab characteristics required to isolate immunogenic TAA peptide mimics. To address this question, we have used mAb 763.74 and mAb GH786, which recognize the same or spatially close antigenic determinant(s) of the human high m.w.-melanoma-associated Ag (HMW-MAA), although with different affinity. mAb 763.74 affinity is higher than that of mAb GH786. Panning of phage display peptide libraries with mAb 763.74 and mAb GH786 resulted in the isolation of peptides P763.74 and PGH786, respectively. When compared for their ability to induce HMW-MAA-specific immune responses in BALB/c mice, HMW-MAA-specific Ab titers were significantly higher in mice immunized with P763.74 than in those immunized with PGH786. The HMW-MAA-specific Ab titers were markedly increased by a booster with HMW-MAA-bearing melanoma cells, an effect that was significantly higher in mice primed with P763.74 than in those primed with PGH786. Lastly, P763.74, but not PGH786, induced a delayed-type hypersensitivity response to HMW-MAA-bearing melanoma cells. These findings suggest that affinity for TAA is a variable to take into account when selecting mAb to isolate peptide mimics from a phage display peptide library.     

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.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

p185, an immunodominant epitope, is an autoantigen mimotope

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.     

Antibody cross-reactivity between myelin oligodendrocyte glycoprotein and the milk protein butyrophilin in multiple sclerosis

The etiology of multiple sclerosis (MS) is believed to involve environmental factors, but their identity and mode of action are unknown. In this study, we demonstrate that Ab specific for the extracellular Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) cross-reacts with a homologous N-terminal domain of the bovine milk protein butyrophilin (BTN). Analysis of paired samples of MS sera and cerebrospinal fluid (CSF) identified a BTN-specific Ab response in the CNS that differed in its epitope specificity from that in the periphery. This effect was statistically significant for the Ab response to BTN(76-100) (p = 0.0026), which cosequestered in the CSF compartment with Ab to the homologous MOG peptide MOG(76-100) in 34% of MS patients (n = 35). These observations suggested that intratheccal synthesis of Ab recognizing BTN peptide epitopes in the CNS was sustained by molecular mimicry with MOG. Formal evidence of molecular mimicry between the two proteins was obtained by analyzing MOG-specific autoantibodies immunopurified from MS sera. The MOG-specific Ab repertoire cross-reacts with multiple BTN peptide epitopes including a MOG/BTN(76-100)-specific component that occurred at a higher frequency in MS patients than in seropositive healthy controls, as well as responses to epitopes within MOG/BTN(1-39) that occur at similar frequencies in both groups. The demonstration of molecular mimicry between MOG and BTN, along with sequestration of BTN-reactive Ab in CSF suggests that exposure to this common dietary Ag may influence the composition and function of the MOG-specific autoimmune repertoire during the course of MS.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.     

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.     

Isolation and characteristics of antistreptokinase antibodies from blood serum of myocardial infarct patients treated with streptokinase

Antistreptokinase IgG (antiSK IgG) from blood of 8 patients with acute myocardial infarction that were treated by streptokinase (SK) has been investigated. AntiSK IgG contained 1.8% of total serum IgG. They had high affinity to SK (K50% approximately 10 nM) and inhibited activation of plasminogen (Pg) by SK with K50% approximately 6 nM. AntiSK IgG were bound with chymothriptic fragments of SK with affinity decreased in the set of fragments: 47 > 36 > 30 > 17 > or = 11 > 7 kDa. 11 linear epitopes of antiSK IgG were localized in I1-S12, T43-M70, G139-Q152, T163-I190, T193-S222, F241-Y252, Y275-P286, T315-L336, I365-E376, S379-T390 and Y397-N410 sites of SK primary structure using SK decapeptides. 70% of antibodies were bound with T43-M70 (38.3%), T315-L336 (13.2%) and Y397-N410 (17.7%) SK sequences located in alpha and gamma SK domains. By depletion of antiSK IgG on Pg-SK complex it was shown that 80-85% of antiSK IgG bound to Pg-SK complex, 47.9% of that contained antibodies with epitopes located in I1-S12, T43-M70, T193-S222 and S379-T390 SK sequences, and rest of IgG had probably spatial epitopes. Unbound with Pg-SK complex antibodies inhibited activation of Pg by SK with higher affinity (Ki approximately 1.2 nM) in comparison with total antiSK IgG fraction. The role of different sites in antigenity of SK and in Pg-SK complex formation is discussed.     

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.     

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance.

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 degrees C and its stability is largely independent of the presence of the other domains. The high-temperature transition in intact SK (at approximately 63 degrees C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.     

Conformational properties of streptokinase

The conformational properties of streptokinase (SK) have been assessed by the techniques of differential scanning calorimetry, circular dichroism (CD), and through a combinational approach employing several algorithms which are predictive of secondary structural characteristics. In low ionic strength buffers, SK undergoes a reversible two-state thermal transition with a temperature of maximum heat capacity (Tm) of 46.1 +/- 0.9, a delta Hcal of 98 +/- 11 kcal/mol and a delta Hcal/delta HvH of approximately 1. In high ionic strength buffers, similar calorimetric properties were obtained with the exception that the delta Hcal/delta HvH values were considerably less than 1, indicating the existence of an additional irreversible thermally induced alteration in the molecule, most likely resulting in its aggregation. The effect of pH on the thermal unfolding properties of SK was determined. The results demonstrated that single two-state thermal transitions were obtained, with progressively decreasing Tm values, as the pH was reduced from 6.4 to 3.4, indicating a destabilization of the entire molecule at reduced pH. In the alkaline region, between pH 8.4 and 9.4, stabilization of a separate region of the molecule was obtained, as evidenced by an increase in the delta Hcal/delta HvH to values approximating 2. CD analysis was performed in order to estimate secondary structural characteristics of SK. The best fit of secondary structural parameters to the experimental CD spectrum provided estimates of 17% helices, 28% beta-sheet, 21% beta-turns, and 34% disordered structures. Both the intensity of the spectral band at 208 nm and the level of antiparallel beta-sheet strongly suggest that SK is an alpha + beta protein.