Staphylokinase Requires NH2-terminal Proteolysis for Plasminogen Activation*

(Received for publication, October 2, 1996, and in revised form, December 3, 1996)

Bernhard Schlott Dagger §, Karl-Heinz Gührs Dagger , Manfred Hartmann Dagger , Anja Röcker Dagger and Désiré Collen

From the Dagger  Institute for Molecular Biotechnology, 07745 Jena, Germany and the  Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH2-terminal sequence,
<LIM><OP><UP>S</UP></OP><UL><UP>1</UP></UL></LIM><UP>SSFDKGKY</UP><LIM><OP><UP>K</UP></OP><UL><UP>10</UP></UL></LIM><UP>KGDD</UP><LIM><OP><UP>A</UP></OP><UL><UP>15</UP></UL></LIM>
<UP>S<SC>equence</SC> 1</UP>
forms a complex with plasmin, that is endowed with plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys10-Lys11 peptide bond, but the kinetics of plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-terminal proteolysis of Sak for the induction of plasminogen activating potential was studied by mutagenesis of Lys10 and Lys11 in combination with NH2-terminal microsequence analysis of equimolar mixtures of Sak and plasminogen and determination of kinetic parameters of plasminogen activation by catalytic amounts of Sak. Substitution of Lys10 with Arg did not affect processing of the Arg10-Lys11 site nor plasminogen activation, whereas substitution with His resulted in cleavage of the Lys11-Gly12 peptide bond and abolished plasminogen activation. Substitution of Lys11 with Arg did not affect Lys10-Arg11 processing or plasminogen activation, whereas replacement with His did not prevent Lys10-His11 hydrolysis but abolished plasminogen activation. Substitution of Lys11 with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect plasminogen activation, but additional deletion of Lys11 eliminated plasminogen activation.

Thus generation of plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of Lys11 as the new NH2 terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.


INTRODUCTION

Staphylokinase (Sak),1 a 16-kDa single-chain protein secreted by certain strains of transduced Staphylococcus aureus is a potent activator of plasminogen, with therapeutic potential for coronary thrombolysis in patients with acute myocardial infarction (for references, cf. Refs. 1 and 2). Sak is not an enzyme, but rather a cofactor; it forms a 1:1 stoichiometric complex with plasminogen, that is inactive and requires conversion to Sak-plasmin to expose the active site which now has a high specificity for plasminogen activation. The Sak-plasmin complex is rapidly neutralized by alpha 2-antiplasmin, but this inhibition rate is >100-fold reduced when the lysine-binding sites of plasmin are occupied, for example, by binding to fibrin. Furthermore, staphylokinase is released from the Sak-plasmin complex following inhibition by alpha 2-antiplasmin and is recycled to other plasminogen molecules (for references, cf. Ref. 3). These molecular interactions between Sak, plasminogen, alpha 2-antiplasmin, and fibrin endow the molecule with a unique mechanism of fibrin selectivity in a plasma milieu. In the absence of fibrin, no activation of plasminogen by Sak occurs, presumably at least in part because alpha 2-antiplasmin in plasma prevents the generation of active Sak-plasmin complex. At the fibrin surface, traces of plasmin are generated which form an active Sak-plasmin complex that is bound to fibrin and protected from rapid inhibition by alpha 2-antiplasmin. After digestion of the fibrin clot, the Sak-plasmin complex is released and inhibited, and further plasminogen activation is interrupted (2).

The sak gene encodes a protein of 163 amino acids, with amino acid 28 corresponding to the NH2-terminal residue of the mature secreted protein, which consists of 136 amino acids in a single polypeptide chain without disulfide bridges. Three natural variants Sakphi C, Sak42D, and SakSTAR have been identified which differ in only three amino acids; in the mature protein amino acid 34 is Ser in SakSTAR but Gly in Sakphi C and Sak42D, amino acid 36 is Gly in SakSTAR and in Sakphi C, but Arg in Sak42D, and amino acid 43 is His in SakSTAR and in Sakphi C, but Arg in Sak42D (4-6).

During plasminogen activation, mature Sak is converted to lower molecular weight (LMW) derivatives by removal of 10 (primarily) or 6 (secondarily) NH2-terminal amino acids (7, 8). These LMW derivatives have the same specific activity (7, 8) and fibrinolytic potential in human plasma (9) as the mature high molecular weight (HMW) protein. Conversion of HMW-Sak to LMW-Sak is presumed to be mediated by plasmin and occurs in association with clot lysis (10). A structure/function analysis of the NH2-terminal region revealed that deletion of 11 NH2-terminal amino acids abolished the ability to activate plasminogen, whereas elimination of potential plasmin cleavage sites (Lys6, Lys8, Lys10, and Lys11) drastically reduced the catalytic efficiency for plasminogen activation, but did not alter the exposure of an amidolytic active site in equimolar mixtures of Sak and plasminogen (11).

In the present study, the requirement of NH2-terminal processing of Sak for the induction of plasminogen activating potential was investigated by studying the effect of substitution of Lys10 and Lys11 on the NH2-terminal processing, as revealed by microsequencing of the Sak component in equimolar mixtures of Sak and plasminogen, and on plasminogen activator activity as revealed by Lineweaver-Burk analysis of plasminogen activation by catalytic amounts of complexes of plasmin(ogen) with Sak. The results indicate that plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of a charged NH2-terminal amino acid (Lys, Arg, or aminoethylated Cys) is a prerequisite to generate Sak derivatives with plasminogen activating potential.


EXPERIMENTAL PROCEDURES

Materials

Enzymes and other reagents used for gene construction experiments were purchased from Boehringer (Mannheim, FRG). Sequenase was supplied by Amersham (Amersham Buchler, Braunschweig, FRG). The protein mixture for molecular weight calibration in SDS-PAGE was supplied by Life Technologies, Inc. (Eggenstein, FRG). The protein mixture used in IEF analysis was purchased from Pharmacia (Freiburg, FRG). Other components were of the highest quality commercially available. Oligonucleotides were synthesized using the phosphoamidite method with a DNA/RNA synthesizer model 394 (Applied Biosystems, Foster City, CA). Semiphosphorylated linker cassettes were obtained by chemical phosphorylation of the respective oligonucleotides at the end of the synthesis (Glen Research, Sterling, VA).

Native human plasminogen was purified from human plasma according to Deutsch and Mertz (12). The synthetic plasminogen substrate S-2251 was purchased from Chromogenix (Essen, FRG). Recombinant Glu-plasminogen with the active Ser741 replaced by Ala, rPlg(S741A), was obtained and characterized as described elsewhere (13).

Construction of Expression Plasmids Encoding Sak42D Variants

The present study was carried out with variants of Sak42D (5), derived from the expression plasmid pMEX602Sak42D (14). Variants Sak42DDelta N10 and Sak42DDelta N11 were generated as described elsewhere (11). The Sak42D gene, truncated at the 5' end was isolated as a BstUI-HindIII (containing codons 14/15 to 136 of the mature gene) fragment from plasmid pMEX602Sak42D. Synthetic oligonucleotides were used to introduce translation start signals and to reconstitute the 5'-coding sequences of the truncated Sak42DDelta N10 and Sak42DDelta N11, respectively (Table I). The appropriate linker pairs were phosphorylated at the EcoRI end and ligated to the EcoRI linearized expression vector pMEX6 (Medac, Hamburg, FRG). After HindIII digestion, the linearized vector DNA was purified by agarose gel electrophoresis. After recircularization using T4 DNA ligase, the expression plasmid containing the truncated sak gene was inserted into the vectors carrying linker cassettes. Finally the ligation mixtures were transformed into competent Escherichia coli TG1 cells (14).

Table I.

Plasmid sequences after deletion or insertion of oligodeoxynucleotide linker cassettes into SfuI and MluI sites of Sak42D variant genes


                    1    2   3   4    5   6   7    8   9   10   11  12  13   14  15   16
                 MetSerSerSerPheAspLysGlyLysTyrLysLysGlyAspAspAlaSer
Wild-type sequence aattcaggaggcctcatatgtcaagttcattCGACAAAGGAAAATATAAAAAAGGCGATGAcgcgtca
ttaagtcctccggagtatacagttcaagtaagcTGTTTCCTTTTATATTTTTTCCGCTACTGCGCagt
                                 His
Sak42DK10H                                | CAC |
                                 | GTG |
                                 Arg
Sak42DK10R                                | CGA |
                                 | GTT |
                                 His
Sak42DK11H                                | CAG |
                                 | GTG |
                                  Arg
Sak42DK11R                                | CGC |
                                 | CGG |
                                   Delta
Sak42DDelta N10                    |-------------------------------|
                                   Delta
Sak42DDelta N11                    |-------------------------------|

Sak42DK10H, Sak42DK10R, Sak42DK11H, and Sak42DK11R were constructed by insertion of oligonucleotides into the acceptor plasmid pMEXSak42D(Delta 5-13), derived from the pMEX602Sak42D expression plasmid by deletion of codons 5 to 13, substitution of codon 16 by TCA, and insertion of the spacer sequence GAA (encoding Glu) to produce the following construct with unique SfuI and MluI restriction sites.
3  4     14 15 16
<UP>SerPhe</UP>   <UP>AspAlaSer</UP>
<UP>tcatt‖cgaaga‖cgcgtca</UP>
ü÷ÿ¿ð

ì
<UP>agt<UNL>aagc‖tt</UNL>c<UNL>tgcgc‖a</UNL>gt</UP>
     Sfu<UP>I</UP>  Mlu<UP>I</UP>
<UP>S<SC>tructure</SC></UP> 1
The synthetic linker cassettes described in Table I were inserted into pMEXSak42D(Delta 5-13) to generate genes encoding the Sak42D variants mentioned above. To this end, the pMEXSak42D(Delta 5-13) acceptor plasmid was linearized with SfuI, and the 5'-phosphorylated linker pairs were annealed and ligated to the SfuI ends. The ligation mixture was precipitated with isopropyl alcohol, and the pellets were resolubilized in appropriate buffer and digested with MluI. Recombinant linearized plasmids were isolated by the glass milk method, recircularized by T4 DNA ligase,and transformed into E. coli (14). Mutant clones were preselected by SfuI restriction (deletion of the SfuI site) and identified by DNA sequencing using the standard protocol of the Sequenase kit.

Expression and Purification of Sak42D Proteins

The recombinant Sak42D variants were expressed in transformed E. coli TG1 as described previously (14). Disruption of the E. coli cells collected from 400-ml culture volume was accomplished by three cycles of repeated freezing and thawing in extraction buffer (0.04 M Tris, 0.01 M EDTA, 0.01 M EGTA buffer, pH 7.5, containing 5 mM beta -mercaptoethanol). The cleared supernatants were concentrated by (NH4)2SO4 precipitation (85% saturation),and the pellets were dissolved in 2 ml of 0.25 M NaCl, 0.02 M phosphate buffer, pH 7.4. In a first step, the clarified 2-ml sample was subjected to gel filtration on a Superdex 75 XK 26/60 column (Pharmacia, Freiburg, FRG) at a flow rate of 2 ml/min at 4 °C. Sak-containing fractions, localized by SDS-PAGE, were pooled, and solid NaCl was added to a concentration of 2.5 M. The resulting samples (approximately 10 ml) were applied on a Phenyl-Superose HR 5/5 column (Pharmacia, Freiburg, FRG) and eluted with a reversed salt gradient at a flow rate of 0.4 ml/min, at 4 °C. Fractions containing Sak were localized by SDS-PAGE and pooled. The final materials consisted of main single protein components with apparent molecular masses of 14.5 to 16 kDa on SDS gel electrophoresis (Fig. 1). Protein concentrations were calculated from absorbance reading at 280 nm using the molar extinction coefficients provided by the pcgene software program (Intelligenetics Inc./Genofit SA).


Fig. 1. SDS-PAGE of Sak42D variants and of their equimolar mixtures (4 µM) with plasminogen. Lane 1, Sak42D; lane 3, Sak42DK10H; lane 5, Sak42DK10R; lane 7, Sak42DK11H; lane 9, Sak42DK11R; lanes 2, 4, 6, 8, and 10, corresponding equimolar mixtures of variant in preceding lane with plasminogen; lane 11, molecular mass calibration; mixtures consisting of a protein ladder with 10-kDa steps.
[View Larger Version of this Image (70K GIF file)]


Ancillary Sak42D Variants

The following Sak42D variants were additionally constructed, expressed, and purified for ancillary and control experiments, using the cassette methodology described above and corresponding linker oligodeoxynucleotides: Sak42DK6H, Sak42DK8H, Sak42DK10Q, Sak42DK11X (with X = F, C, E, P), Sak42DG12K, and Sak42DG12A. Sak42DDelta N14 was constructed by Sak42D gene truncation and religation with oligodeoxynucleotides, reconstituting the translation start signal and deleting the NH2-terminal 14 amino acids, essentially as described above.

Processed Sak42D (Sak42Dproc) was prepared by incubating solutions of Sak42D variants (concentration 2.0 mg/ml) in 0.1 M phosphate buffer, pH 7.4, containing 0.01% Tween 20, with plasminogen immobilized on CNBr-Sepharose CL-4B (Pharmacia, Freiburg, FRG). The immobilization procedure was carried out according to the recommendations of the manufacturer.

S-Aminoethylation of Sak42D Variants

Samples of Sak42D and Sak42DK11C were modified by S-aminoethylation essentially as described previously (15). Briefly, lyophilized Sak moieties were dissolved in 150 µl of 6 M guanidinium hydrochloride solution to a protein concentration of 0.2-0.4 µM. For the reduction of the protein samples, Tris, EDTA, and beta -mercaptoethanol were added yielding final concentrations of 0.6 mM, 0.25 mM, and 15 µM, respectively. The probes were incubated at room temperature under an atmosphere of argon for 4 h. A 4-fold molar excess of bromoethylamine (Sigma, Deisenhofen, FRG) over beta -mercaptoethanol was added in three portions at 10-min intervals. The samples were allowed to incubate overnight at room temperature. Finally, the protein moieties were separated from the reagents using NAP 5 columns (Pharmacia, Freiburg, FRG) equilibrated with 20 mM phosphate buffer, pH 6.0, containing 100 mM NaCl.

Analytical Methods

Isoelectric focusing was carried out using the PhastSystemTM, Dry IEFTM gels, and the IEF protein standard supplied by Pharmacia (Freiburg, FRG). The gels were rehydrated in 8 M urea containing 2.5% Pharmalyte 3-10TM (Pharmacia, Freiburg, FRG) yielding a pH gradient from 3 to 10.

The NH2-terminal amino acid sequences of all purified Sak42D variants were determined on the Applied Biosystems model 476A (Applied Biosystems). For the Sak species generated in equimolar (4 µM) mixtures with plasminogen after 5-min incubation at 37 °C, high resolution electrophoresis was carried out with the Mini-Protean II system (Bio-Rad, Munich, FRG) using 16%T, 3%C gels in the discontinuous Tris-Tricine buffer system according to Schagger and von Jagow (16). Samples were applied to the gels after reduction by heating at 100 °C for 5 min in the presence of 1% SDS and 0.4% beta -mercaptoethanol. Following gel separation, the proteins were blotted onto polyvinylidene difluoride membranes (Millipore, Eschborn, FRG) and then subjected to amino acid sequencing.

Generation of Amidolytic Activity in Equimolar Mixtures of Sak42D Variants and Plasminogen

Amidolytic activity was quantitated with the chromogenic plasmin substrate S-2251 (final concentration 1 mM) and monitored at 405 nm for up to 12 min using a Spectronic 401 (Milton Bay, Analis Belgium) spectrophotometer or a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA). Plasminogen (final concentration 3.6 µM) was incubated with the Sak moieties (final concentration 4 µM) at 37 °C in 100 mM phosphate buffer, pH 7.4, containing 0.01% Tween 80 (activation buffer). Samples were withdrawn at 2-minute intervals and diluted 40-fold in buffer containing chromogenic substrate S-2251, and the change in absorbance at 405 nm (Delta A405) was recorded.

Activation of Plasminogen by Catalytic Amounts of Sak42D Variants

The activation of plasminogen (final concentration 1.5 µM) by Sak42D moieties (final concentration 5 nM) was assayed at 37 °C in activation buffer. At different time intervals up to 30 min, generated plasmin was measured with S-2251 substrate.

Kinetic Constants of Plasminogen Activation by Catalytic Amounts of Preformed Equimolar Complexes of Sak42D Variants with Plasmin

The kinetic constants of plasminogen activation by Sak-plasmin complexes were derived from Lineweaver-Burk plots. For this purpose, equimolar mixtures of Sak42D variants and plasminogen were preincubated for 5 min in activation buffer containing 25% glycerol (v/v) and kept on ice. For most mixtures, this preincubation resulted in complete conversion of single-chain plasminogen to two-chain plasmin (see Fig. 1). These preformed activator complexes (final concentration 10-40 nM) were then mixed with plasminogen (final concentration 0.25-10 µM), and generated plasmin was measured at 37 °C from Delta A405 with S-2251.

Binding of Sak42D Variants to Plasminogen

Association rate constants (ka) and dissociation rate constants (kd) for the interaction between Glu-Plg or rPlg(S741A) and the Sak variants were determined by real-time biospecific interaction analysis using the BIAcore instrument (Pharmacia) (17). Glu-Plg or rPlg(S741A) were immobilized on the surface of sensor chip CM5 using the amine coupling kit (Pharmacia), as recommended by the manufacturer. This procedure links primary amino groups in the ligand to the carboxymethylated dextran surface of the sensor chip (18). Immobilization was performed from protein solutions at 10 µg/ml in 10 mM sodium acetate at pH 5.0, at a flow of 5 µl/min during 6 min, resulting in covalent attachment of 1000 to 5000 resonance units corresponding to approximately 0.07 pmol/mm2 for both ligands (19). The Sak42D variants were injected in solution over the sensor. The concentration of free analyte was kept constant by maintaining a continuous flow of solution at 20 °C past the sensor surface. At least three concentrations of each analyte (25-100 nM or 2-6 µM) in 10 mM Hepes, 3.4 mM EDTA, 0.15 M NaCl, and 0.005% surfactant P20, pH 7.2, were injected at a flow rate of 20 µl/min during 2 min in the association phase. After equilibrium was reached, sample was replaced by buffer, also at a flow rate of 20 µl/min during 2 min. After each cycle, the surface of the sensor chip was regenerated by injection of 10 µl of 5 mM HCl. Association (ka) and dissociation (kd) rate constants were derived from the observed response curves (resonance/time) as described in detail in the BIAevaluation 2.0 Software Handbook (Pharmacia Biosensor AB).


RESULTS

Production and Characteristics of Sak42D Variants

The Sak42D variants were purified from E. coli TG1 cells with yields of 70 to 500 µg/liter culture, representing average recoveries of 22%. SDS gel electrophoresis (Fig. 1) displayed single bands with relative migrations corresponding to the expected molecular masses. NH2-terminal amino acid sequencing revealed homogeneous sequences as illustrated in Table II, which confirmed that all Sak moieties, except Sak42DDelta N10, were correctly processed in the E. coli expression system with removal of the NH2-terminal Met.

Table II.

NH2-terminal processing and catalytic efficiency for plasminogen activation of Sak variants


Variant NH2-terminal sequencea
Maximal amidolytic activity (% Sak42D) Catalytic efficiency kcat/KM Binding to plasminogen Ka (×106)
Starting materialb Processed material Glu-Plg rPlg(S741A)

µM-1·s-1
Sak42D SSSFDKGKYKKGDDA           KGDDA 100 0.08 0.44 350
Sak42DK10H SSSFDKGKYHKGDDA            GDDA 190 <0.001 0.39 340
Sak42DK10R SSSFDKGKYRKGDDA           KGDDA 120 0.04 0.68 220
        YRKGDDA
Sak42DDelta N10          MKGDDA          MKGDDA 150 0.03 0.64 240
Sak42DK11H SSSFDKGKYKHGDDA           HGDDA 150 <0.001 0.30 280
Sak42DK11R SSSFDKGKYKRGDDA           RGDDA 220 0.18 0.35 270
Sak42DDelta N11            GDDA            GDDA 160 0.002 0.30 320
Sak42DK11Fc SSSFDKGKYKFGDDA           FGDDA 240 <0.001 0.32 260
Sak42DG12K SSSFDKGKYKKKDDA SSSFDKGKYKKKDDA 62 <0.001 0.71 320
          KKDDA
Sak42DG12A SSSFDKGKYKKADDA           KADDA 130 0.05 0.50 340

a  The sequences are aligned with Ala15(1) of the mature protein.
b  The mutagenized amino acid is boldface.
c  Sak42DK11E and Sak42DK11P yielded very similar results.

Similar purities and expected NH2-terminal sequences were obtained with the variants Sak42DK6H, Sak42DK8H, Sak42DK10Q, Sak42DK11E, Sak42DK11P, Sak42DK11C, Sak42DK11F, Sak42DG12A, Sak42DG12K, Sak42DDelta N14, and Sak42Dproc (not shown).

Functional Characterization of the Sak42D Variants

SDS gel electrophoresis of equimolar mixtures of plasminogen and Sak variants (Fig. 1) revealed processing of Sak (faster migration) and plasminogen activation (conversion of single-chain plasminogen to two-chain plasmin) with all substitution variants. This was confirmed by NH2-terminal sequencing of the Sak components, as summarized in Table II.

Sak42D was exclusively processed at the Lys10-Lys11 peptide bond with exposure of Lys11 as the new NH2-terminal amino acid. The variants Sak42DK10R, Sak42DK11H, and Sak42DK11R were also hydrolyzed at the Lys10-X11 peptide bond (Sak42DK10R with some alternative processing at Lys8-Tyr9), whereas Sak42DK10H was hydrolyzed at the Lys11-Gly12 bond. The variants Sak42DK6H, Sak42DK8H, Sak42DG12A, and Sak42DG12K were also processed at the Lys10-Lys11 peptide bond, whereas Sak42DK10Q was hydrolyzed at the Lys11-Gly12 bond (not shown). Sak42DG12K showed a "partial processing" at the Lys10-Lys11 bond yielding a mixture of two components present in approximately equal amounts: the unprocessed protein and a processed form with two lysines at the NH2-terminal positions.

Generation of maximal amidolytic activity occurred within 4 to 6 min (Fig. 2). The maximal amidolytic activity of equimolar mixtures of plasminogen with variants Sak42DK10H, Sak42DK10R, Sak42DDelta N10, Sak42DK11H, Sak42DK11R, and Sak42DDelta N11 was at least equal to and up to 2-fold higher than that of Sak42D.


Fig. 2. Generation of plasmin in equimolar (4 µM) mixtures of plasminogen by Sak42D variants. black-square, Sak 42D; open circle , Sak42DDelta N10; down-triangle, Sak42DK10H; triangle , Sak42DK10R; black-down-triangle , Sak42DK11H; black-triangle, Sak42DK11R; bullet , Sak42DDelta N11.
[View Larger Version of this Image (25K GIF file)]


Sak processing and plasminogen activation with generation of amidolytic activity similar to or higher than that obtained with Sak42D was observed with Sak42Dproc, Sak42DK10Q, Sak42DK11F, Sak42DK11C, Sak42DG12A, and Sak42DDelta N14. Reduced amidolytic activity (<= 70% of Sak42D) was generated with Sak42DK6H, Sak42DK8H, Sak42DK11E, and Sak42DK11P (not shown).

Activation of Plasminogen by Catalytic Amounts of Sak Variants

Catalytic amounts of Sak42D induced progressive activation of plasminogen to plasmin (Fig. 3), with a lag phase of 5 min, reaching a maximal rate (determined from the slope at the inflection point of these curves) after 15 to 20 min. Plasminogen activation was similar to that of Sak42D with Sak42DDelta N10, Sak42DK10R, and Sak42DK11R, but was virtually absent with Sak42DK10H, Sak42DK11H, and Sak42DDelta N11. In addition, plasminogen activation was comparable to that of Sak42D with Sak42DK6H, Sak42DK8H, Sak42Dproc, and Sak42DG12A, but was virtually absent with Sak42DK10Q, Sak42DK11E, Sak42DK11P, Sak42DK11F, Sak42DK11C, Sak42DG12K, and Sak42DDelta N14 (not shown).


Fig. 3. Plasminogen activation by catalytic amounts of Sak42D variants. black-square, Sak 42D; open circle , Sak42DDelta N10; down-triangle, Sak42DK10H; triangle , Sak42DK10R; black-down-triangle , Sak42DK11H; black-triangle, Sak42DK11R; bullet , Sak42DDelta N11.
[View Larger Version of this Image (17K GIF file)]


Activation of plasminogen by preformed Sak-plasmin complexes obeyed Michaelis-Menten kinetics, as revealed by linear double-reciprocal plots of the initial activation rate versus the plasminogen concentration (not shown). The catalytic efficiency kcat/Km ranged between 0.03 and 0.18 nM-1·s-1 for Sak42D, Sak42DK10R, Sak42DK11R, and Sak42DDelta N10 but was <= 0.002 nM-1·s-1 for Sak42DK10H, Sak42DK11H, and Sak42DDelta N11 (Table II). Catalytic efficiencies were within the normal range with Sak42DK6H, Sak42DK8H, Sak42Dproc, and Sak42DG12A, but were <0.002 nM-1·s-1 with Sak42DK10Q, Sak42DK11E, Sak42DK11P, Sak42DK11F, Sak42DK11C, Sak42DG12K, and Sak42DDelta N14 (not shown).

Binding of Sak Variants to Plasmin(ogen)

The apparent affinity equilibrium constants (Ka) for binding of Sak42D moieties to native Glu-Plg in the presence of excess plasmin inhibitor or to rPlg(S741A), are summarized in Table II. The Ka values of all Sak42D variants studied were very similar to those of wild-type Sak42D. The affinity constants for binding to native Glu-Plg were approximately 1000-fold lower than those for binding to rPlg(S741A), as has previously been observed (20).

Effect of S-Aminoethylation of Sak42DK11C on Plasminogen Activation

The extent of plasminogen activation within 30 min by catalytic amounts of Sak42DK11C was unmeasurable (Fig. 4), whereas its catalytic efficiency for plasminogen activation was <= 0.001 nM-1·s-1, both before and after processing on insolubilized plasminogen (Table III). S-Aminoethylation, which introduces a positive charge into the side chain of cysteine, increased the extent of plasminogen activation within 30 min to that observed with Sak42D and raised the catalytic efficiency of Sak42DK11C-plasmin complex to 0.13 nM-1·s-1, whereas wild-type Sak42D was not markedly affected by treatment with the aminoethylating reagent.


Fig. 4. Plasminogen activation by catalytic amounts of Sak42D and Sak42DK11C. Effect of S-aminoethylation and processing with insolubilized plasminogen. Closed symbols, Sak42D; open symbols, Sak42DK11C; squares, basal conditions; circles, after processing; diamonds, after S-aminoethylation; inverted triangles, after processing and subsequent S-aminoethylation; triangles, after S-aminoethylation and subsequent processing.
[View Larger Version of this Image (18K GIF file)]


Table III.

Effect of S-aminoethylation on plasminogen activation with Sak42DK11C


Plg activation (% wild-type)
kcat/KM
Binding to plasminogen Ka (×106)
Sak42D Sak42DK11C Sak42D Sak42DK11C Glu-Plg
rPlg(S741A)
Sak42D Sak42DK11C Sak42D Sak42DK11C

nM-1·s-1
Baseline 100 0 0.08 0.001 0.44 0.29 350 260
Aminoethylated 65 88 0.04 0.13 0.30 0.27 250 220
Processed 69 0 0.11 <0.001   NDa 1.20   NDa 370
Processed then aminoethylated   ND a 98   NDa 0.08   ND a   NDa   NDa   NDa

a  ND, not determined.

Isoelectric focusing (Fig. 5) revealed that the isoelectric point of Sak42D was not affected by S-aminoethylation. Sak42DK11C had a lower isoelectric point than Sak42D which was increased by S-aminoethylation. Processing with insolubilized plasminogen decreased the isoelectric points of Sak42D and Sak42DK11C to a similar extent (Fig. 5).


Fig. 5. Isoelectric focusing (IEF) (pH range 3-10) of Sak42D and Sak42DK11C. Effects of S-aminoethylation (AE) and processing with insolubilized plasminogen (proc). Lane 1, IEF-standard: 1, trypsinogen, 9.30; 2, lentil lectin, 8.65; 3, lentil lectin, 8.15; 4, horse myoglobin, 7.35; 5, horse myoglobin, 6.85; 6, human carbonic anhydrase B, 6.55; 7, beta -lactoglobulin, 5.20; 8, soybean trypsin inhibitor, 4.55; 9, amyloglycosidase, 3.50; lane 2, Sak42D; lane 3, Sak42D(AE); lane 4, Sak42DK11C; lane 5, Sak42DK11C(AE); lane 6, Sak42D(AE, proc); lane 7, Sak42DK11C(AE, proc); lane 8, Sak42DK11C(proc, AE).
[View Larger Version of this Image (116K GIF file)]



DISCUSSION

The aim of the present study was to investigate the role of NH2-terminal processing of staphylokinase (Sak) in plasminogen activation. Although conversion of native Sak with NH2-terminal Ser-Ser-Ser- to a proteolytic derivative with NH2-terminal Lys-Gly-Asp-, by plasmin-mediated removal of the NH2-terminal 10 amino acids, was found not to be a rate-limiting step (10), deletion or substitution of Lys11 greatly reduced the plasminogen activator properties of the Sak variants (11). The requirement of NH2-terminal processing of Sak for the induction of plasminogen activating potential was established by site-specific mutagenesis of Lys10 and Lys11 (P1-P'1 positions according to Berger and Schechter (21)). Substitution of either amino acid with Arg did not affect hydrolysis of the P1-P'1 peptide bond nor plasminogen activation, replacement of Lys10 with His resulted in cleavage of the Lys11-Gly12 (P'1-P'2) peptide bond and loss of plasminogen activating potential, whereas exchange of Lys11 with His produced cleavage of the Lys10-His11 (P1-P'1) peptide bond with generation of an inactive derivative. Furthermore, deletion of the 10 NH2-terminal amino acids yielded a fully active Sak derivative, but additional deletion of Lys11 inactivated the molecule.

In aggregate, these data are compatible with the interpretation that Sak requires hydrolysis in the NH2-terminal region with exposure of a positively charged amino acid at the new NH2 terminus. This was confirmed using variant Sak42DK11C in combination with S-aminoethylation with 2-bromoethylamine, which introduces a positive charge in the side chain of cysteine converting it to "pseudolysine" (15). As anticipated, hydrolysis of the Lys10-Cys11 (P1-P'1) peptide bond with insolubilized plasmin(ogen) yielded an inactive derivative, which could, however, be fully activated by S-aminoethylation. Conversely, S-aminoethylation before processing yielded a derivative which was processed to a fully active product.

The apparent discrepancy between the low plasminogen activating potential of catalytic amounts of processed variants lacking a positively charged NH2-terminal amino acid and the full conversion of plasminogen to plasmin in equimolar mixtures can be explained by the fact that the affinity of the Sak42D variants for plasminogen is unaltered and that Sak-plasmin complex is a more potent activator of plasminogen bound to Sak than of free plasminogen. Consequently, even Sak42D variants with very poor plasminogen activating potential will rapidly convert plasminogen to plasmin in equimolar mixtures. The larger than expected variability in maximal amidolytic activity of equimolar mixtures may be explained by the fact that the amidolytic activity of free plasmin is 2- to 3-fold higher than that Sak-plasmin complexes with plasminogen activating potential (23). Thus, mixtures with higher amidolytic activity may predominantly contain Sak-plasmin complexes in which the active site of plasmin is not converted into the plasminogen activator configuration with a reduced amidolytic capacity.

Determination of the association equilibrium constants for the binding of the Sak42D variants to plasminogen revealed no significant differences with Sak42D, with respect neither to the previously observed low affinity binding to native Glu-Plg in the presence of excess plasmin inhibitor, nor to the high affinity binding to rPlg(S741A) (20). In the absence of altered binding of Sak42D variants to plasminogen, the mutations that reduce the efficiency of Sak-plasmin complexes to activate plasminogen must affect the conversion of the active site of plasmin into the plasminogen activator configuration.

NH2-processing of the Lys10-Lys11 (P1-P'1) peptide bond with exposure of a positively charged NH2-terminal amino acid is important but neither necessary nor sufficient to endow the Sak molecule with plasminogen activating potential. Indeed Sak42DDelta N10, from which the NH2-terminal Met is not removed in the E. coli expression system, has NH2-terminal Met-Lys, but generates a complex with plasmin with (nearly) intact plasminogen activating potential. Alternatively, Sak42DG12K is normally although slowly processed at the Lys10-Lys11 site, but generates a derivative with NH2-terminal Lys-Lys which has a very poor catalytic efficiency for plasminogen activation. Apparently the presence of an uncharged amino acid in position 12 is also important for the plasminogen activating potential of Sak. Finally, preliminary experiments revealed that Sak42DDelta N14, which does not generate a plasminogen activating complex with plasmin, can be "rescued" by substituting its NH2-terminal Ala with Lys yielding an active derivative with NH2-terminal Met-Lys (data not shown).

In the absence of the three-dimensional structures of Sak and plasmin, interpretation of the present results in terms of the submolecular configuration of the active site of the plasminogen activating complex as compared to that of free plasmin, remains speculative. The present observations, however, provide a structural mechanism for the plasmin-mediated priming of plasminogen activation by Sak and the fibrin selectivity of clot lysis with Sak in a plasma milieu (22). In circulating blood, traces of generated plasmin are rapidly inhibited by alpha 2-antiplasmin, whereby Sak cannot be processed to the plasminogen activating derivative. In contrast, at the fibrin surface, plasmin is protected from rapid inactivation by alpha 2-antiplasmin (23) and has an increased affinity for Sak (20), allowing efficient local plasminogen activation. This mechanism may account for the high fibrin specificity and thrombolytic efficacy of Sak in patients with acute myocardial infarction (24) or peripheral arterial occlusion (25).


FOOTNOTES

*   This work was supported by Bundes Minïsterium für Biologische Forschung Grant 0311015. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Institute for Molecular Biotechnology, 07745 Jena, Germany.
1    The abbreviations used are: Sak, staphylokinase; Sak42D, wild-type staphylokinase with Gly34, Arg36, and Arg43; Sak42DDelta N10, Sak42DDelta N11, and Sak42DDelta N14, Sak42D with deletion of the 10, 11, and 14 NH2-terminal amino acids, respectively; Sak42DAnB, Sak42D with amino acid A in position n of mature staphylokinase substituted with amino acid B; HMW-Sak, high molecular weight Sak (mature Sak with 136 amino acids); LMW-Sak, low molecular weight Sak (Sak after removal of the 10 NH2-terminal amino acids); Glu-Plg, native human plasminogen with NH2-terminal Glu; rPlg(S741A), recombinant plasminogen with the active site Ser741 mutagenized to Ala; ka, association rate constant; kd, dissociation rate constant; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; Ka, association equilibrium constant; S-2251, D-Val-Phe-Lys-p-nitroanilide.

REFERENCES

  1. Collen, D., and Lijnen, H. R. (1994) Blood 84, 680-686 [Free Full Text]
  2. Collen, D. (1996) Circulation 93, 857-865 [Free Full Text]
  3. Lijnen, H. R., and Collen, D. (1996) Fibrinolysis 10, 119-126
  4. Sako, T., and Tsuchida, N. (1983) Nucleic Acids Res. 11, 7679-7693 [Abstract]
  5. Behnke, D., and Gerlach, D. (1987) Mol. & Gen. Genet. 210, 528-534 [Medline] [Order article via Infotrieve]
  6. Collen, D., Zhao, Z. A., Holvoet, P., and Marynen, P. (1992) Fibrinolysis 6, 226-231
  7. Sako, T. (1985) Eur. J. Biochem. 149, 557-563 [Abstract]
  8. Collen, D., Silence, K., Demarsin, E., De Mol, M., and Lijnen, H. R. (1992) Fibrinolysis 6, 203-213
  9. Lijnen, H. R., Van Hoef, B., Vandenbossche, L., and Collen, D. (1992) Fibrinolysis 6, 214-225
  10. Ueshima, S., Silence, K., Collen, D., and Lijnen, H. R. (1993) Thromb. Haemostasis 70, 495-499 [Medline] [Order article via Infotrieve]
  11. Gase, A., Hartmann, M., Gührs, K. H., Röcker, A., Collen, D., Behnke, D., and Schlott, B. (1996) Thromb. Haemostasis 76, 755-760 [Medline] [Order article via Infotrieve]
  12. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1095-1096 [Medline] [Order article via Infotrieve]
  13. Busby, S. J., Mulvihill, E., Rao, D., Kunar, A. A., Lioubin, P., Heipel, M., Sprecher, C., Halfpap, L., Prunkard, D., Gambee, J., and Foster, D. C. (1991) J. Biol. Chem. 266, 15286-15292 [Abstract/Free Full Text]
  14. Schlott, B., Hartmann, M., Gührs, K. H., Birch-Hirschfeld, E., Pohl, H. D., Vanderschueren, S., Van de Werf, F., Michoel, A., Collen, D., and Behnke, D. (1994) Biotechnology 12, 185-189 [Medline] [Order article via Infotrieve]
  15. Hofmann, T. (1964) Biochemistry 3, 356-364
  16. Schagger, H., and von Jagow, G. (1987) Adv. Biochem. 166, 368-379
  17. Jönsson, U., and Malmqvist, M. (1992) Adv. Biosensors 2, 291-336
  18. Johnsson, B., Löfas, S., and Lindquist, G. (1991) Anal. Biochem. 198, 268-277 [Medline] [Order article via Infotrieve]
  19. BIAcore System Manual (1991) Chapter 5.2, Pharmacia Biosensor AB, Uppsala, Sweden
  20. Sacharov, D. V., Lijnen, H. R., and Rijken, D. C. (1996) J. Biol. Chem. 271, 27912-27918 [Abstract/Free Full Text]
  21. Berger, A., and Schechter, I. (1970) Philos. Trans. R. Soc. Lond. B Biol. Sci. 257, 249-264 [Medline] [Order article via Infotrieve]
  22. Lijnen, H. R., Van Hoef, B., De Cock, F., Okada, K., Ueshima, S., Matsuo, O., and Collen, D. (1991) J. Biol. Chem. 266, 11826-11832 [Abstract/Free Full Text]
  23. Collen, D. (1980) Thromb. Haemostasis 43, 77-89 [Medline] [Order article via Infotrieve]
  24. Vanderschueren, S., Barrios, L., Kerdsinchai, P., Van den Heuvel, P., Hermans, L., Vrolix, M., De Man, F., Benit, E., Muyldermans, L., Collen, D., and Van de Werf, F. (1995) Circulation 92, 2044-2049 [Abstract/Free Full Text]
  25. Vanderschueren, S., Stockx, L., Wilms, G., Lacroix, H., Verhaeghe, R., Vermylen, J., and Collen, D. (1995) Circulation 92, 2050-2057 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.