(Received for publication, October 28, 1996, and in revised form, November 6, 1996)
From The Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037
In stark contrast to most other members of the chymotrypsin family of serine proteases, tissue type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. Instead, single-chain t-PA exhibits very significant catalytic activity. Consequently, the zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 3-9 for t-PA. Both we and others have previously proposed that Lys156 may contribute directly to this exceptional property of t-PA by forming interactions that selectively stabilize the active conformation of the single-chain enzyme. To test this hypothesis we created variants of t-PA in which Lys156 was replaced by a tyrosine residue. As predicted, the K156Y mutation selectively suppressed the activity of the single-chain enzyme and thereby substantially enhanced the enzyme's zymogenicity. In addition, however, this mutation produced a very dramatic increase in the ability of single-chain t-PA to discriminate among distinct fibrin co-factors. Compared with wild type t-PA, one of the variants characterized in this study, t-PA/R15E,K156Y, possessed substantially enhanced response to and selectivity among fibrin co-factors, resistance to inhibition by plasminogen activator inhibitor type 1, and significantly increased zymogenicity. The combination of these properties, and the maintenance of full activity in the presence of fibrin, suggest that the R15E,K156Y mutations may extend the therapeutic range of t-PA.
Proteases are normally synthesized as inactive precursors or zymogens that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, varies widely among individual members of the (chymo)trypsin family but, in almost all cases, is dramatic. For example, strong zymogens such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive with measured zymogenicities of 104 to 106 (1, 2). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (3), and the catalytic efficiency of urokinase is 250-fold greater than that of prourokinase (4). By contrast, the catalytic activities of single- and two-chain t-PA1 vary by a factor of only 3-9 (5, 6, 7, 8, 9).
We have suggested previously that the unusually high catalytic activity of single-chain t-PA results both from the absence of interactions, present in typical zymogens, that stabilize (an) inactive conformation(s) of the zymogen and the presence of interactions, absent in typical zymogens, that stabilize an active conformation of the single-chain enzyme (8, 9, 10). Recent studies have provided substantial support for this hypothesis. We demonstrated that the absence of the zymogen triad contributes to the enzymatic activity of single-chain t-PA (8, 9), and two groups have suggested that Lys156 2 stabilizes an active conformation of single-chain t-PA (11, 12). Consistent with the latter suggestion, we have demonstrated that conversion of His144 of t-PA to an acidic residue, which we predicted would alter the conformation of Lys156, selectively suppressed the catalytic activity of single-chain t-PA (10).
Although a definitive demonstration requires structural investigations, existing evidence strongly suggests that Lys156 and Asp194 interact directly in single-chain t-PA and that this interaction stabilizes the active conformation of the enzyme (10, 11, 12). To test these hypotheses, we utilized oligonucleotide directed site specific mutagenesis to replace Lys156 of t-PA with a tyrosine residue. As predicted, this mutation selectively suppressed the activity of the single-chain enzyme and thereby substantially enhanced the zymogenicity of t-PA. In a standard direct chromogenic assay the observed zymogenicity of the wild type enzyme was approximately 2.5. By contrast, the zymogenicity measured for a variant carrying the K156Y mutation was approximately 117.
Oligonucleotide-directed site-specific
mutagenesis was performed by the method of Zoller and Smith (13) as
modified by Kunkel (14). The K156Y mutation was introduced into the
290-bp SacI-SmaI fragment of cDNA encoding
t-PA that had been previously subcloned into bacteriophage M13mp18. The
mutagenic primer had the following nucleotide sequence:
5-CGGAGCGGCTGTATGAGGCTCATGT-3
.
Following mutagenesis, ssDNA corresponding to the entire 290-bp SacI-SmaI fragment was fully sequenced to assure the presence of the desired mutation and the absence of any additional mutations. Replicative form (RF) DNA was prepared for appropriate phage, and the mutated 290-bp SacI-SmaI fragment was recovered after digestion of RF DNA with SacI and SmaI and electrophoresis of the digestion products on an agarose gel. The isolated, mutated SacI-SmaI fragment was used to replace the corresponding fragment in full-length cDNAs encoding wild type t-PA or t-PA/R15E to yield new, full-length cDNAs encoding t-PA/K156Y and t-PA/R15E,K156Y.
Expression of Enzymes by Transient Transfection of COS Cells cDNAs encoding t-PA, t-PA/R15E, t-PA/K156Y, and
t-PA/R15E,K156Y were ligated into the transient expression vector pSVT7
(15) and then introduced into COS cells by electroporation using a Bio-Rad Gene Pulser. 20 µg of cDNA, 100 µg of carrier DNA, and approximately 107 COS cells were placed into a 0.4-cm
cuvette, and electroporation was performed at 320 V, 960 microfarads,
and =
. Following electroporation, cells were incubated
overnight at 37 °C in DMEM containing 10% fetal calf serum and 5 mM sodium butyrate. Cells were then washed with serum free
medium and incubated in DMEM for 48 h at 37 °C. After the
incubation with serum-free media, conditioned media were collected and
enzyme concentrations were determined by enzyme-linked immunosorbent
assay.
Wild type and mutated variants of t-PA were purified using an FPLC system and a 1-ml HiTrap chelating column (Pharmacia Biotech Inc.). The column was charged with 0.1 M CuSO4 solution, washed with 5-10 ml of distilled water, and equilibrated with start buffer (0.02 M NaHPO4, pH 7.2, 1 M NaCl, and 1 mM imidizole). Conditioned medium containing wild type or variants of t-PA was adjusted to 1 M NaCl and injected into the column with a 50-ml superloop (Pharmacia Biotech Inc.). The column was then washed with 10 column volumes of start buffer and eluted using a 0-0.32 M linear gradient of imidizole in the same buffer. Peak fractions were collected and pooled. Purified t-PA samples were concentrated, and buffer was exchanged to 25 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, using a centriplus 30 concentrator (Amicon).
Kinetic Analysis of t-PA Activity Using a Small, Synthetic SubstrateThe direct chromogenic assay utilized the substrate methylsulfonyl-D-cyclohexyltyrosyl-glycyl-arginine-p-nitroaniline (Spectrozyme t-PA, American Diagnostica) and was performed as described previously (16, 17).
Kinetic Analysis of Plasminogen Activation Using Indirect Chromogenic AssaysIndirect chromogenic assays of t-PA utilized the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) and were performed as described previously (15, 18, 19). Assays were performed both in the presence and absence of the co-factor DESAFIB (American Diagnostica).
Indirect Chromogenic Assays in the Presence of Various Fibrin Co-factorsStandard indirect chromogenic assays were performed as described previously (15, 18, 19). Briefly, 0.25-1 ng of enzyme, 0.2 µM Lys-plasminogen and 0.62 mM Spectrozyme PL were present in a total volume of 100 µl. Assays were performed either in the presence of buffer, 25 µg/ml DESAFIB, 100 µg/ml fibrinogen, 100 µg/ml cyanogen bromide fragments of fibrinogen (American Diagnostica), or 100 µg/ml of the stimulatory, 13-amino acid peptide P368. P368 was kindly provided by Marshall Runge (University of Texas Medical Center, Galveston, TX). Assays were performed in microtiter plates, and the optical density at 405 nm was read every 30 s for 1 h in a Molecular Devices Thermomax. Reactions were performed at 37 °C.
Measurement of Second Order Rate Constants for Inhibition by PAI-1Second order rate constants for the inhibition of wild type
and mutated t-PA were measured under pseudo-first order conditions as
described previously (18, 19, 20, 21). Briefly, enzyme and inhibitor were
preincubated at 23 °C for periods of time varying from 0 to 30 min.
Following preincubation, the mixtures were diluted, and the residual
enzymatic activity was measured in a standard indirect chromogenic
assay. For each enzyme, the concentrations of enzyme and inhibitor and
the times of preincubation were chosen to yield several data points for
which the residual enzymatic activity varied between 20 and 80% of the
initial activity. Data were analyzed by plotting ln (residual
activity/initial activity) versus time of preincubation and
measuring the resulting slopes. Division of this slope by [I], with
[I] representing the concentration of PAI-1 during the preincubation,
yielded the second order rate constants shown.
We used oligonucleotide-directed site-specific mutagenesis to test the hypothesis that mutation of Lys156 of t-PA would selectively suppress the catalytic activity of single-chain t-PA. Lysine 156 was replaced by a tyrosine residue to yield t-PA/K156Y. Accurate measurement of the enzymatic activity toward plasminogen of the single-chain form of this variant proved difficult, however, because the plasmin produced in this assay rapidly converted the single-chain enzyme into its mature, two-chain form by cleaving the Arg15-Ile16 peptide bond. Consequently, to overcome this technical difficulty, we also constructed a noncleavable form of the mutated enzyme by introducing the additional mutation R15E into the existing mutant to yield t-PA/R15E,K156Y.
Wild type t-PA, t-PA/R15E, t-PA/K156Y, and t-PA/R15E, K156Y were expressed by transient expression of COS cells. Since this procedure yielded predominantly single-chain enzymes, two-chain t-PAs were generated by treating the enzyme preparations with plasmin-Sepharose (5). Quantitative conversion of the enzymes into their mature, two-chain form was confirmed by SDS-polyacrylamide gel electrophoresis. As demonstrated previously (7), variants containing the mutation R15E were synthesized and secreted exclusively as single-chain enzymes and were not cleaved by plasmin-Sepharose (data not shown).
The enzymatic activity of the single- and two-chain forms of wild type and mutated t-PAs toward a small synthetic substrate is listed in Table I. Mutation of lysine 156 had little effect on the activity of two-chain t-PA. Two-chain t-PA/K156Y displayed approximately 90% of the activity of the two-chain, wild type enzyme in this assay. By contrast, the K156Y mutation had a very substantial effect on the activity of single-chain t-PA. Single-chain t-PA/R15E,K156Y exhibited approximately 2% of the activity of single-chain t-PA/R15E. In this assay, the "zymogenicity," or ratio of the activities of the two and single-chain form of a particular enzyme, was approximately 2.5 for wild type t-PA. By contrast, for variants containing the K156Y mutation, this ratio increased to approximately 117 (Table I).
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In the absence of a co-factor, the K156Y mutation had little effect on the activity of two-chain t-PA toward plasminogen; however, this mutation significantly reduced the catalytic efficiency of single-chain t-PA (Table I). Compared with that of single-chain t-PA/R15E, the activity of single-chain t-PA/R15E,K156Y was reduced by a factor of 17. By contrast, the activities of two-chain t-PA and t-PA/K156Y differed by a factor of only 1.2.
All of the variants analyzed in this study maintained reasonably high enzymatic activity toward the natural substrate plasminogen in the presence of the co-factor fibrin (Table I). The single-chain form of variants containing the K156Y mutation were, however, affected to a slightly greater extent than the corresponding mature, two-chain enzymes. Two-chain t-PA/K156Y possessed approximately 75% of the activity of two-chain t-PA, while single-chain t-PA/R15E,K156Y exhibited approximately 40% of the activity of single-chain t-PA/R15E.
Molecular details of the stimulation of t-PA by fibrin, a complex process that almost certainly involves multiple points of contact between the two proteins, remain obscure (22, 23, 24, 25, 26, 27). Fibrin stimulation of two-chain t-PA may occur through a single mechanism; stimulation of single-chain t-PA by fibrin co-factors, however, appears to utilize at least two distinct mechanisms. First, fibrin apparently stimulates both single- and two-chain t-PA through a templating mechanism, resulting in formation of a ternary complex that greatly augments the local concentration of enzyme and substrate. Second, because single- and two-chain t-PA have equivalent activity in the presence but not the absence of fibrin (5, 7, 9, 28), it seems likely that binding to fibrin induces a conformational change in the activation domain of single-chain t-PA. Induction of this conformational change in the absence of activation cleavage and concomitant generation of the mature amino terminus is particularly intriguing but not unprecedented. Similar activation of plasminogen upon binding to streptokinase (29, 30, 31, 32, 33, 34), as well as activation of prothrombin by binding to staphylocoagulase (35, 36), have been described previously. Although the mechanism of this nonclassical, nonproteolytic activation of serine protease zymogens remains completely unclear, the behavior of single-chain t-PA/R15E,K156Y suggests that Lys156 does not play an essential role in this process. The properties of two-chain t-PA/K156Y suggest, in addition, that Lys156 does not play an essential role during zymogen activation of t-PA through the classical, proteolytic mechanism.
As predicted, the primary effect of the K156Y mutation was a selective reduction of the activity of single-chain t-PA in the absence of fibrin and, consequently, an increase in the zymogenicity of the enzyme. At the molecular level this effect seems likely to be mediated by disruption of a key, buried salt bridge formed by Lys156 and Asp194. After performing either molecular modeling studies (12) or structural studies of two-chain t-PA (11), two groups have predicted the existence of this salt bridge in single-chain t-PA and have suggested that it plays an important role in stabilizing the active conformation of the single-chain enzyme. Although definitive proof of the existence of this salt bridge will require structural analysis of single-chain t-PA, it seems very likely, based upon the existing evidence, that the proposed interaction between Lys156 and Asp194 does exist in single-chain t-PA. Moreover, as observed in this study, disruption of a salt bridge between Lys156 and Asp194 would be expected to selectively suppress the activity of single-chain t-PA because Lys156 does not interact with Asp194 in the two-chain enzyme. Instead, in two-chain t-PA, as in other mature chymotrypsin like enzymes, the mature amino terminus inserts into the activation pocket and plays this role (11). Consequently, as observed, two-chain t-PA/K156Y is expected to maintain high catalytic activity. Variants of t-PA containing a tyrosine residue at position 156, therefore, exhibit significantly enhanced zymogenicity.
The extent of fibrin stimulation displayed by the single-chain form of t-PA/R15E,K156Y is significantly greater than that displayed by wild type t-PA. Wild type, two-chain t-PA possesses a fibrin stimulation factor, defined as the ratio of the catalytic efficiencies in the presence and absence of fibrin, of approximately 250 (Table I). The two-chain t-PA/K156Y variant displays a similar stimulation factor of 230. Single-chain wild type t-PA, with a fibrin stimulation factor of 3800, is stimulated to a substantially greater degree than the two-chain enzymes, presumably reflecting the ability of fibrin to stimulate the single-chain enzymes not only through a templating mechanism but also by inducing nonproteolytic zymogen activation. Stimulation of single-chain t-PA is further enhanced by the K156Y mutation. The fibrin stimulation factor for single-chain t-PA/R15E,K156Y is approximately 26,000. Enhanced fibrin stimulation of the variant did not result from increased activity in the presence of fibrin but rather from decreased activity in the absence of a stimulator, an observation consistent with our proposal that the effects of these mutations are mediated by disruption of a salt bridge between Lys156 and Asp194 in single-chain t-PA.
The mutated enzyme t-PA/R15E,K156Y is not only stimulated to a
significantly greater extent by soluble fibrin than t-PA (Table I), but
it is also substantially more discriminating among fibrin co-factors
than the wild type enzyme (Fig. 1). The two-chain form of both wild type t-PA and t-PA/K156Y is strongly stimulated by soluble
fibrin monomers (DESAFIB), fibrinogen, CNBr fragments of fibrinogen,
and a 13-amino acid peptide (P368). Single-chain t-PA/R15E, on the
other hand, is stimulated strongly by soluble fibrin and fibrinogen and
moderately by the CNBr fragments and peptide P368. In striking contrast
to these enzymes, single-chain t-PA/R15E,K156Y, although dramatically
stimulated by fibrin monomers, is virtually nonresponsive to
fibrinogen, CNBr fragments of fibrinogen, peptide P368.
The ratio of the specific activity of a plasminogen activator in the presence of fibrin to that in the presence of fibrinogen, or "fibrin selectivity factor," may be one indication of the "clot selectivity" an enzyme will demonstrate in vivo. An enzyme with enhanced fibrin selectivity may be able to accomplish efficient thrombolysis while displaying decreased systemic activity. Under the conditions of the assays reported here, the fibrin selectivity factor is 1.5 for two-chain t-PA, 1.5 for two-chain t-PA/K156Y, and 1.0 for single-chain t-PA/R15E. The fibrin selectivity factor for single-chain t-PA/R15E,K156Y, however, is 146. This double mutant, therefore, is approximately 2 orders of magnitude more discriminating between fibrin and fibrinogen than either single- or two-chain wild type t-PA.
The single-chain form of a zymogen-like variant of t-PA is expected to exhibit reduced activity not only toward substrates (Table I) but also toward specific inhibitors. To test this hypothesis, we measured the second order rate constant for inhibition of the single-chain form of both t-PA/R15E and t-PA/R15E,K156Y by the serpin plasminogen activator inhibitor, type 1 (PAI-1), the primary physiological inhibitor of t-PA (Table II). As expected, t-PA/R15E,K156Y exhibited resistance to inhibition by PAI-1. The second order rate constant for inhibition by PAI-1 of t-PA/R15E,K156Y was reduced by a factor of approximately 230, compared with t-PA/R15E.
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t-PA exhibits unusually high catalytic activity as a single-chain enzyme and consequently very low zymogenicity. An important finding of this study is that conversion of lysine 156 to a tyrosine residue selectively suppresses the activity of single-chain t-PA and thereby substantially enhances the zymogenicity of the enzyme. We have demonstrated, in addition, that single-chain t-PA/R15E,K156Y is significantly more fibrin-stimulated and substantially more fibrin-selective than either single- or two-chain, wild type t-PA. Single-chain t-PA/R15E,K156Y also exhibits marked resistance to inhibition by PAI-1. We propose that the effects of this mutation are mediated by disruption of a critical salt bridge formed by Lys156 and Asp194 that has been predicted to be present in single- but not two-chain t-PA. Consistent with observations of this study, the primary role of this putative salt bridge is believed to be stabilization of the active conformation of single-chain t-PA. Two-chain t-PA/K156Y, therefore, as demonstrated in this study, is expected to maintain high enzymatic activity.
Although these studies were designed to examine structure/function relationships within the activation domain of t-PA, and specifically to test the hypothesis that Lys156 and Asp194 interact directly to form a buried salt bridge that selectively stabilizes the active conformation of the single-chain enzyme, these results may also aid the design of improved thrombolytic agents. t-PA/R15E,K156Y, for example, exhibits significantly enhanced fibrin stimulation, dramatically increased discrimination among fibrin co-factors, marked resistance to inhibition by PAI-1, and substantially increased zymogenicity, a combination of properties that might enhance the therapeutic potential of the enzyme.