©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Activation of Type 1 and Type 2 Plasminogen by Type I and Type II Tissue Plasminogen Activator (*)

(Received for publication, October 4, 1994; and in revised form, November 16, 1994)

Kazuya Mori (1)(§) Raymond A. Dwek (1)(¶) A. Kristina Downing (2) Ghislain Opdenakker (1) (3)(**) Pauline M. Rudd (1)

From the  (1)Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, the (2)Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, and the (3)Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D* (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the K value for activation by all tPA variants; N-glycosylation of type I tPA at Asn decreased the k (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D* tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.


INTRODUCTION

Plasminogen activation is a key control mechanism in fibrinolysis (1) and extracellular matrix remodelling(2, 3) . Since the discovery by Astrup and Permin(4) , two activators have been well characterized at the molecular level: urokinase and tissue plasminogen activator (tPA)(^1)(5) .

tPA was first purified from uterine tissue(6) ; however, tumor cell cultures have proved a more convenient source for studying the biochemical properties of tPA variants(7) . Einarsson and co-workers (8) were the first to describe differences in the specific activities of type I and type II tPA (8) while treatment with alpha-mannosidase was shown to increase the activity of Bowes melanoma tPA(9) . Many studies have shown that the structural differences between these two types are in the carbohydrates(10, 11, 12) . Type I tPA has N-linked oligosaccharides at sites 117, 184, and 448, while type II is glycosylated only at sites 117 and 448. Both contain an O-linked fucose residue at Thr 61(13, 14) . The role of the carbohydrates in modulating the catalytic properties of tPA has been confirmed(15, 16, 17, 18) .

The natural substrate of tPA, plasminogen, is a mixture of two major glycoforms (19) that have the same amino acid sequence(20) . Both are O-glycosylated at Thr, and the sequence contains an N-glycosylation site at Asn-Arg-Thr, which is occupied in type 1, but not in type 2 plasminogen(21, 22, 23) . Glu-plasminogen is the full-length native molecule (93,000 kDa). During activation of plasminogen to plasmin, Glu-plasminogen is converted to Lys plasminogen (84,000 kDa) as a result of proteolytic degradation by plasmin, which cleaves amino acids from the N terminus leaving a terminal lysine residue.

Evidence that the glycosylation of plasminogen modulates the functional activity of the molecule was provided by Davidson and Castellino (24) who demonstrated that differently glycosylated forms of plasminogen showed different kinetic parameters for activation by urokinase. In addition, Edelberg et al.(25) showed that human neonatal plasminogen, which is more extensively glycosylated than the adult form, had a lower activation rate by tPA. Glycosylation also modulates the binding of plasminogen to U937 cells(26) ; type 2 plasminogen binds 10 times better to the receptor than type 1. Enhancement of the activation of plasminogen by tPA on the cell surface of rat hepatocytes was greater with type 2 plasminogen than type 1(27) .

In this study, we have investigated the role of glycosylation in the modulation of plasminogen activation. In addition, we have noted the existence of a small percentage of a high molecular weight variant of tPA (type D), derived from a Bowes melanoma cell culture, which has increased fibrinolytic activity. The combinatorial effect of differential glycosylation of both tPA and plasminogen was probed by measuring the kinetic constants for the fibrin-dependent and independent interactions of combinations of type I, II, and D* tPA with type 1 and type 2 plasminogen.


MATERIALS AND METHODS

Bowes melanoma cell-derived tissue plasminogen activator, human plasmin, and the enzyme-linked immunosorbent assay kit for tPA were purchased from American Diagnostica Inc. Lys-Sepharose, concanavalin A-Sepharose, and alkaline phosphatase-conjugated anti-rabbit IgG were from Sigma. Chromogenic substrate S-2288 and S-2251, human fibrinogen and human Glu-plasminogen were purchased from Kabi Vitrum. Isoelectricfocusing gels and molecular weight markers were obtained from Amersham Corp. Endo-beta-N-acetylglucosaminidase H (Streptomyces plicatus) was obtained from Boehringer Mannheim, and peptide N-glycosidase F (Flavobacterium meningosepticum) was obtained from Oxford GlycoSystems. Purified human Glu-plasminogen type 1 and 2 was a gift from Dr J. Marshall (John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom) and anti-human tPA polyclonal IgG and HT 1080-derived type 1 plasminogen activator inhibitor (PAI-1) were provided by Dr Yoichi Sakata (Jichi Medical School, Tochigi, Japan).

Lysine-Sepharose Chromatography

560 µg of single chain tPA at 1.25 mg/ml in 1 M ammonium hydrogen carbonate was diluted with equilibration buffer (10 mM sodium phosphate, pH 8.0, containing 0.15 M KSCN, 0.001% Tween 80. and 25 KIU of aprotinin/ml) to 800 µg/ml and applied to a Lys-Sepharose column (0.9 cm times 31 cm), equilibrated at 4 °C. The column was washed with 24 ml of the same buffer at a flow rate of 5.4 ml/h, and fractions of 1.5 ml were collected. tPA types were eluted successively with a linear gradient of 0-0.25 ML-arginine followed by a wash of 0.25 ML-arginine and a final step of 0.5 ML-arginine. All fractions were monitored for amidolytic activity toward the chromogenic substrate S-2288. 300 µl of each fifth fraction was dialyzed against 50 mM Tris buffer, pH 7.5, containing 0.15 M NaCl and 0.05% Tween 80. The fractions were measured for tPA antigen level in the direct (S-2288) amidolytic assay and in the indirect (S-2251) amidolytic assay with and without human fibrinogen fragments (FCB) (see below). Type I, II, and D* tPA fractions were pooled and stored at -20 °C for further analysis.

SDS-PAGE and Immunoblotting

SDS-polyacrylamide gel electrophoresis was performed according to the standard method. After electrophoresis, the separated samples in the gel were transfered to blotting membrane (Immobilon or nitrocellulose, Millipore) by electroblotting. The tPA bands were developed using rabbit anti-tPA polyclonal antibody and alkaline phosphatase or peroxidase conjugated to anti-rabbit IgG (28

Casein was used as the plasmin substrate in the direct zymographic analysis(29) .

tPAbulletPAI-1 complex

PAI-1 from HT1019 cells was activated by 4 M guanidine HCl, pH 7.4, with 0.02% Tween 80 for 30 min at room temperature. The sample was immediately dialyzed against 50 mM sodium acetate buffer, pH 5.5, with 0.02% Tween 80 at 4 °C. The dialyzed sample was stored at -80 °C. The amount of active PAI-1 was assessed by measuring the inhibition of a predetermined amount of urokinase. 1 µg/ml of active PAI-1 was incubated with 2 µg/ml of unfractionated tPA at 37 °C for 30 min. The sample was stored at -20 °C or prepared for SDS-PAGE. For reducing conditions, the samples were made 2% in beta-mercaptoethanol.

Assays of tPA Activity

All assays were performed in 96-well microtiter plates (Costar) at 37 °C. The enzyme activities were monitored using a Molecular Devices kinetic microplate reader. Each well in the 96-well microtiter plate was coated with 1% of bovine serum albumin in phosphate buffer, pH 8.0, for 16 h at 25 °C. The well was washed twice with water and then dried at room temperature. tPA antigen levels were determined in all assays using the polyclonal antibody based immunoassay.

1) Direct Amidolytic Assay

The direct amidolytic assay was performed using the broad spectrum protease chromogenic substrate S-2288. The reaction mixture consisted of 1 mM S-2288 in 0.05 M Tris-HCl buffer, pH 7.5, containing 0.05% Tween 80 (v/v), 0.1% bovine serum albumin, and 0.15 M NaCl. The tPA activity was monitored by measuring the absorbance of the reaction mixture at 405 nm. Results are expressed as the change in absorbance/min (mOD/min)/µg/ml in a 100-µl reaction mixture.

2) Unstimulated Indirect Amidolytic Assay

This assay measured the activation of plasminogen in a reaction mixture containing 0.13 mM human Glu-plasminogen, 0.7 mM chromogenic plasmin substrate S-2251, and 5-10 ng/ml tPA sample in 0.05 M Tris-HCl buffer, pH 7.5, containing 0.05% Tween 80, 0.1% bovine serum albumin, and 0.15 M NaCl. The production of plasmin was monitored each 3 min up to 60 min by measuring the absorbance of the reaction mixture at 405-490 nm. Subtraction of the absorbance at 490 nm was to allow for any turbidity resulting from the reaction that might have contributed to the reading at 405 nm. The absorbance was plotted against square of each measuring time (min^2). The slope of the plot (dA/dt^2) is proportional to tPA activity. Results are expressed as the slope of the plot of OD at 405-490 nm versus the square of the reaction time (mOD/min^2) after normalizing to a tPA concentration (µg/ml). This slope was given by the method of least squares.

3) Stimulated Indirect Amidolytic Assay

Human fibrinogen fragments obtained by cleavage of fibrinogen with CNBr (FCB) were used as a stimulator(30) , and the assay was performed as described previously(16) . The reaction mixture consisted of 0.13 mM Glu-plasminogen, 0.7 mM chromogenic plasmin substrate S-2251, 0.6 mM soluble fragments of human fibrinogen, and 5-10 ng/ml of tPA sample in 0.05 M Tris-HCl buffer, pH 7.5, containing 0.05% Tween 80, 0.1% bovine serum albumin, and 0.15 M NaCl. A constant rate of plasminogen activation results in a constant increase in plasmin activity, as indicated by a constant increase in the rate of absorbance change at 405 nm (dOD/dt). To calculate dOD/dt, the absorbance of the reaction mixture was read at 3-min intervals, and the dOD/dt values were replotted against the reaction time. The dmOD/dt/min (mOD/min^2) values were calculated from linear region of the graph. The result is expressed as mOD/min^2 after normalizing to a tPA concentration (µg/ml) or IU/µg(15) .

Determination of Kinetic Parameters

Kinetic parameters for the activation of Glu-plasminogen type 1 and type 2 by tPA in the stimulated indirect amidolytic assay were determined by varying the concentration of each type of plasminogen with the tPA concentration held constant about 10 ng/ml. Concentrations of plasminogen were determined by measuring of absorbance at 280 nm (Extinction coefficient = 16.1). V(max) and K(m) were determined using a plot of the [substrate] (s)/velocity (v) versuss(31) . To determine the k (the catalytic rate constant), the amidolytic activity of a known amount of plasmin toward S-2251 was measured in the presence of the same amount of FCB as in the tPA assay. V(max) values (mOD/min^2) were converted to rate of formation of plasmin (nM/min). Molecular weights of 93,000, 91,000, 80,000, and 67,000 for Glu-plasminogen type 1, Glu-plasminogen type 2, plasmin, and tPA, respectively, were used to calculate molar protein concentrations.


RESULTS

Separation and Characterization of tPA Variants by Lysine-Sepharose Chromatography

The glycosylated variants of Bowes melanoma tPA were separated by lysine-Sepharose affinity chromatography using an L-arginine (L-Arg) gradient (Fig. 1A and Fig. 2A). Fractions were assayed for protease activity (direct assay) using the artificial chromogenic substrate S-2288 (Fig. 1A and 2A). Consistent with previous data, type I and type II tPA eluted as the two major peaks. Additionally, a fraction (D*) with high binding affinity was recovered, which is shown below to consist of a stable mixture of type II tPA and a high molecular weight fraction designated type D. Protein concentration was measured by an enzyme-linked immunosorbent assay system using a rabbit anti-tPA polyclonal antibody (Fig. 1C). Each tPA fraction eluted from the column showed similar protease activity. The maximum difference was about 20% (SD ± 10%) from the average, although most differences were considerably less than this (Fig. 1A). The activity in the S-2288 assay of each fraction correlated with the tPA antigen level. This shows that the specific activity of each fraction was the same and that the protease activity of tPA toward the S-2288 chromogenic substrate is independent of Lys-affinity and glycosylation.


Figure 1: Separation of tPA by lysine-Sepharose chromatography and the enzyme activities of the fractions. Bowes melanoma-derived tPA was applied to a lysine-Sepharose affinity column and eluted with an L-arginine gradient (0-0.25 M) followed by a wash of 0.25 ML-arginine and a final step of 0.5 ML-arginine. Every fifth fraction was dialyzed against buffer, and both the reactivity to anti-tPA antibody and the enzyme activity were measured. A, times, conductivity of arginine in the gradient (mS/cm2); circle, tPA activity in the direct assay (mOD/min, vertical scale) using chromogenic substrate (S-2288); bullet, antigen level of tPA measured by polyclonal antibody based enzyme-linked immunosorbent assay (µg/ml). B, circle, tPA activity in direct assay (same data as A); box, tPA activity in the indirect amidolytic assay without FCB (mOD/(min^2/µg/ml)); , tPA activity in indirect amidolytic assay with FCB (mOD/(min^2/µg/ml) times 10). The fractions 52-63, 70-83, and 110-123 were pooled as types I, II, and D*, respectively. C, Western blot analysis of the fractions eluted from the lysine-Sepharose column. Each fraction was separated by SDS-PAGE (9%) and then transferred to a blotting membrane. tPA was detected using polyclonal rabbit anti-tPA IgG and alkaline phosphatase-conjugated polyclonal anti-rabbit IgG. I and II indicate positions of type I or II tPA. The high molecular weight form of tPA (type D) is present in fractions 85-120.




Figure 2: Separation of tPA by lysine-Sepharose chromatography and the casein zymography of the fractions. Bowes melanoma-derived tPA was applied to a lysine-Sepharose affinity column and eluted with an L-arginine gradient (0-0.25 M) (times) followed by a wash of 0.25 ML-arginine. Individual fractions were monitored by the direct amidolytic assay using the artificial chromogen, S-2288 (continuous line) (panel A). Five separate pools (A-E) were normalized to the same protease activity after analysis with S-2288. Following separation by SDS-PAGE, the five pools were also analyzed by zymography using casein as the plasmin substrate (Fig. 1B). (ArrowheadsI and II indicate type I and II tPA, respectively.) The high molecular weight complex (Type D tPA) was present in fraction E together with type II. The mixture of type II and type D has been designated D*.



To test for tPA activity, pooled fractions (Fig. 2A) were analyzed by casein zymography (29) (Fig. 2B). Most of the overall activity resided in types I and II. The high molecular weight complex, present as a minor component, also possessed caseinolytic activity in the presence of plasminogen, indicating that it is able to activate plasminogen or that it has itself caseinolytic activity.

Analysis of Plasminogen Activation Activity

The protein concentration of tPA in every fifth fraction eluted from the Lys-Sepharose column was normalized using a polyclonal antibody to tPA (Fig. 1A). The activity with human Glu-plasminogen and a chromogenic plasmin substrate, S-2251, was measured in the presence or absence of cyanogen bromide fibrinogen fragments (Fig. 1B). Consistent with earlier findings(15) , the FCB-dependent activity of type II tPA fractions was higher than type I tPA fractions. After the type II tPA fractions had been eluted, the FCB dependent activity continued to increase with Lys affinity. Consistent with earlier findings in this laboratory(15, 16) , the highest activity was found in the tPA fractions that bound most tightly to the Lys-Sepharose and were eluted by 0.5 ML-Arg. In the absence of FCB, the plasminogen activation was very low and there was no significant difference between the type I and type II fractions. However, for fractions eluting later than type II, the activity increased with Lys affinity. From these results it can be concluded that the high affinity tPA, designated type D, is a highly active tPA variant.

Activity of Type D* tPA

The activities of pooled fractions from the type I tPA peak, the type II tPA peak, and the type D* tPA in three different assays are summarized in Table 1. A polyclonal antibody-based enzyme-linked immunosorbent assay was used to normalize the tPA concentration. In the direct amidolytic assay using S-2288, these 3 types of tPA had similar specific protease activity. In the unstimulated (without FCB) indirect amidolytic assay, using S-2251, type D+II (D*) tPA showed a 5.8 and 4.2 times higher activity than type I or type II tPA, respectively. In the presence of FCB, type D* tPA had 3.9 and 2.4 times higher activity than type I and type II tPA, respectively. By implication, the values for type D were even higher. Interestingly, the activity of type D tPA was significantly less stimulated by FCB than were type I and type II tPA (1:1.6:1.7, respectively).



Analysis of Type D tPA

Fig. 1C shows an immunoblot analysis of the fractions from tPA separated by lysine-Sepharose (Fig. 1B) and separated on a nonreducing 9% acrylamide gel. In addition to monomeric type II tPA (64-69 kDa), the tPA-fractions 85-120 (D*) contained material migrating at 120 kDa (type D tPA). The reducing gel and immunoblots of type I, II, and D* tPA, run separately and also as a mixture (Fig. 3A), showed that this 120-kDa material could be reduced by beta-mercaptoethanol to a single band migrating at the position of type II tPA. Fig. 3A also shows that lanes2, 4, and 5 contain additional minor bands (<10% of the material). Such bands have been noted in previous studies (28) and appear to be the result of processing rather than glycosylation or other post-translational modifications or clipping. To test the possibility that the sample might contain unglycosylated tPA (which would be expected to bind to lysine-Sepharose with high affinity), the tPA in lane5 (type D*) was subjected to concanavalin A-Sepharose affinity chromatography (data not shown). There was no detectable tPA in the flow-through, indicating that all of the tPA is glycosylated.


Figure 3: A, Western blot analysis of types I, II, and D tPA eluted from lysine-Sepharose after reduction. All samples were reduced with 2% 2-mercaptoethanol before applying to the SDS-gel (9%). The immunodetection was carried out using rabbit anti-tPA polyclonal antibody and alkaline phosphatase-conjugated anti-rabbit IgG. Track1, mixture of type I, II, and D tPA; 2, unfractionated tPA; 3, type I tPA; 4, type II tPA; 5, type D* tPA. B, Western blot analysis comparing tPAbulletPAI-1 complex with type D tPA before and after reduction. Tracks1 and 3, tPAbulletPAI-1 complex; tracks2 and 4, type D* tPA. The samples were separated by SDS-PAGE (9%) and transfered to the blotting membrane. tPA was detected by rabbit anti-tPA IgG and alkaline phosphatase-conjugated polyclonal anti-rabbit IgG.



Type D tPA Is a Dimer

Silver staining of SDS gels of type D tPA run under reducing conditions did not reveal the presence of any other molecular weight species other than that migrating to the same position as type II tPA (data not shown). However, the possibility remains that the 120-kDa protein is a complex of tPA with some other molecule of a similar molecular weight. For example, tPA can be isolated as a complex with plasminogen activator inhibitor, PAI-1. The molecular mass of this complex (110 kDa) is close to that of a tPA dimer (120 kDa). The data in Fig. 3B eliminate the possibility that type D tPA may be such a complex of tPA and PAI-1.

Fig. 3B, track1, shows the immunobloting by anti-tPA antibody of unfractionated tPA preincubated with PAI-1 and run under nonreducing conditions. Track2 contains type D* tPA (a mixture of type II tPA monomers and dimers) run under nonreducing conditions. Tracks3 and 4 contain the same samples run under reducing conditions. Type D tPA has a molecular mass consistent with a tPA dimer (120 kDa) and does not migrate to the same position as either monomeric tPA (64-69 kDa) or tPAbulletPAI-1 complex. Under reducing conditions the tPAbulletPAI-1 complex is not cleaved and continues to run at 110 kDa (track3). Type D tPA, on the other hand, is reduced and migrates to the same position as type II tPA (track4). In lane2 there is some high molecular mass material. This was reduced with beta-mercaptoethanol (lane4) and may consist of larger aggregates of tPA formed when the sample was concentrated.

Peptide N-glycosidase F, which cleaves the N-glycosidic linkage between the N-linked oligosaccharides and the protein, digested type I, II, and D tPA to the same molecular size on reducing gels (data not shown), suggesting that the molecular size differences between the type I and type II and D populations are due to glycosylation.

Effect of Variable Glycosylation of Both Glu-Plasminogen and tPA on Kinetic Parameters of Plasminogen Activation

The kinetic constants for the fibrin-stimulated activation of plasminogen 1 and 2 by tPA I, II, and D* are shown in Table 2. The K(m) values are similar for the activation of type 1 plasminogen by all of the tPA variants. Likewise the K(m) values for the activation of type 2 plasminogen by each of the tPA variants are similar. However the K(m) values for interactions involving type 2 plasminogen were always lower than for type 1. We can speculate that N-glycosylation at kringle 3 in type 1 plasminogen may interfere in some way with the association of tPA and Glu-plasminogen.



The k values were always larger for type II tPA compared with type I tPA acting on the same plasminogen variant. This suggests that the turnover rate (k) is faster for the less glycosylated type II tPA variant. Using native Glu-plasminogen, Howard et al.(18) showed that the glycosylation at Asn on tPA kringle 2 affected only the k. Using the separated plasminogen glycosylation variants, we have confirmed this. In addition, these data show that the K(m) of type II tPA with type 2 plasminogen is lower than for type I tPA with type 1 plasminogen. The other combinations of the types of tPA and plasminogen fall in between these extremes. The overall combined effect of variations in the glycosylation of tPA and of plasminogen is a 2.3-fold activity span in which the two fully glycosylated molecules have a FCB-dependent k/K(m) value that is 43% of the k/K(m) values resulting from the interaction of the two least glycosylated variants. A representative experiment is shown in Fig. 4.


Figure 4: The kinetics of the activation of type 1 and 2 plasminogen by tPA variants I, II, and D* in the presence of fibrinogen fragment. The concentrations of tPA and plasminogen in the assay were adjusted to 10 ng/ml and 0.1 µM, respectively. Generation of plasmin was monitored by measuring the absorbance at 405-490 nm at 3-min intervals. circle, type I tPA and type 1 plasminogen; bullet, type I tPA and type 2 plasminogen; box, type II tPA and type 1 plasminogen; , type II tPA and type 2 plasminogen; up triangle, type D* tpA and type 1 plasminogen; , type D* tPA and type 2 plasminogen.



The K(m) values for the interaction of type D* tPA with plasminogen are of the same order as those obtained from the interactions of type I and type II tPA, suggesting that the association of tPA and plasminogen in the presence of FCB is independent of the type of tPA. It also suggests that the type II dimer (type D tPA) reacts in a kinetically similar manner to the monomer in this step. However, the k values for type D* tPA with type 1 and type 2 plasminogen are significantly greater than for either type I or type II tPA. This suggests that the pure dimers of type II tPA (type D) have an increased turnover rate. We should note that the rate constants are for type D diluted with type II; the absolute value for D will be even higher than that shown in Table 1and Table 2.

The k/K(m) value for the interaction of type D* tPA with type 2 plasminogen is more than 4 times as large as the value for type I tPA and type 1 plasminogen. The FCB-dependent catalytic activity of tPA therefore spans at least a 4-fold range and is dependent on both the glycosylation of tPA and plasminogen and the dimers of type II tPA. The K(m) values, on the other hand, are modulated only by glycosylation at site 288 on kringle 3 of plasminogen.


DISCUSSION

We have provided experimental evidence for different specific activities of type 1 and type 2 plasminogen with types I, II, and D tPA. These differences are associated with plasminogen glycosylation as well as the glycosylation of tPA and show that the rate of production of plasmin is greatest when the least glycosylated variants of both tPA and plasminogen are involved. The variable glycosylation of tPA and plasminogen, as well as the dimerization of tPA, contribute to the k/K(m) values, which range from 1.5 to 6.4. While the type of tPA had little effect on the K(m) values, the lack of glycosylation at Asn on plasminogen type 2 was associated with a decrease in the K(m) values for activation by tPA. Together these data suggest that only the glycosylation of Glu-plasminogen affects the association of the ternary complex formed by tPA, plasminogen and fibrin. k, however, depended on the tPA type rather than on the glycosylation status of plasminogen, and the turn over rate of tPA was increased in type II tPA and type D*.

The three variants of tPA were probed in two further assays. First, in a direct amidolytic assay, using the chromogenic substrate S-2288 to probe the interaction of a small peptide with the active site in the tPA serine protease domain, all three types of tPA showed a similar specific activity (activity/monomer). This suggests that the proteolytic cleavage site is unaffected by either glycosylation or dimerization. In contrast, in an indirect unstimulated amidolytic assay, which probed the interaction of tPA variants with native unfractionated human Glu-plasminogen substrate, activities were in the ratio 6:1:1.2 for type D* tPA/type I/type II tPA. This suggests that dimerization of tPA results in an increased rate of plasminogen activation in the absence of fibrin.

The Effects of Glycosylation at Asn on Plasminogen

The influence of the N-linked sugar chain on kringle 3 (K3) of plasminogen has been investigated by Davidson and Castellino,(24) . In the presence of urokinase, types 1 and 2 had similar activation rates, suggesting that the interaction of plasminogen with the active site is not affected by glycosylation. Here we show that, in the presence of tPA, N-glycosylation increases the K(m) values. Taken together, these data suggest that the glycosylation of K3 affects either the interaction between tPA A chain and plasminogen or between fibrin and plasminogen but not the interaction of plasminogen with the active site. Zamarron et al.(32) showed that N-glycosylation of intact Glu-plasminogen had no effect on fibrin binding(32) . However the glycosylation decreases lysine binding affinity suggesting that after cleavage of fibrin by plasmin, which exposes new plasminogen binding sites, (33) the N-linked glycan may decrease the binding to terminal lysine.

Interestingly, glycosylation at Asn does not significantly affect the binding constants of ligands for the two allosteric sites (K4 and K5) nor the forward rate of alpha- to beta-conformational change. It does, however, reduce the rate of the reverse beta- to alpha-conformational change, which is from an open conformation to a compact form, and this may be important in the formation and rearrangement of the ternary complex with fibrin and tPA (35

Formation of a tPA Dimer

This work describes a new tPA variant (type D) from Bowes melanoma cells, which has a high specific plasminogen activation activity and high affinity for lysine. Evidence is provided that this is a dimer of type II tPA. A similar fraction has been observed previously in this laboratory in kinetic studies on tPA from other cell lines including murine C127 and human colon fibroblast fractionated by lysine-Sepharose chromatography(15, 16) . (^2)

The data suggest that type D tPA may be composed of a particular subset of type II tPA glycoforms. It is not the result of an equilibrium involving monomers since, after the initial separation on lysine-Sepharose, it does not reappear in the major fraction of type II tPA even after concentration. In addition, the data (Fig. 3B, lanes2 and 4) indicate that type D tPA is disulfide bonded and that type D* is a stable mixture of type II monomers and dimers. In proposing a possible site for linking two tPA type II monomers to form the more active type D tPA, there are a number of considerations to be taken into account. First, type D tPA has a higher activity than other types of tPA both in the presence and absence of fibrin. This suggests that dimerization does not interfere with the access of plasminogen to the active site (the catalytic triad, His-Ser-Asp) in the serine protease domain. Second, the increased FCB-dependent activity of type D tPA suggests that fibrin binding sites in the finger domain, and in kringle 2, where the lysine binding site binds degraded fibrin, (36) remain accessible. Third, type D tPA has a similar activity to type I and type II tPA in the direct assay using the artificial plasminogen substrate, S-2288 (a short conjugated peptide), to measure protease activity. This is consistent with the previous data, which suggest that type D has a similar structure to type I and II at the active site in the serine protease domain, which is the primary binding site for plasminogen. However, since type D has a >9-fold increase in activity over types I and II tPA in the unstimulated indirect assay using S-2251, it is possible that dimerization may cause a conformational change that affects a secondary binding site to plasminogen. Such a secondary site has been identified by Geppert and Binder (37) and is in the A-chain. Finally, the dimer is dissociated into monomers by reducing agents, and this suggests that a disulfide bond is involved in its formation. The primary structure of tPA includes a number of cysteine residues that are believed to be cross-linked. In addition there is a free cysteine residue at site 83, and the possibility exists that, within a subpopulation of type II tPA, this may interact with the equivalent cysteine on a second tPA molecule to form an intermolecular disulfide bond producing a homodimer. Examination of the tPA molecule by molecular modelling suggests that the formation of a bond between free cysteine 83 residues in the epidermal growth factor domains of two adjacent tPA molecules is a possibility (Fig. 5). One of the clearance mechanisms for tPA involves the recognition of the O-linked fucose residue at Thr by a high affinity receptor on hepatocytes(38) . This residue is close to Cys; the exact nature of the dimer and any consequences for the interaction of the fucose residue with the hepatocyte receptor remains to be explored. Although the dimer is a minor species of glycosylated tPA, it has a significantly greater activity, and in view of the use of tPA in the clinic it is an interesting finding. Just as the N-linked glycans of tPA and plasminogen play a role in modulating the activity of tPA, so does the dimerization.


Figure 5: Two views of a schematic model of a dimer of type II tPA. Each tPA monomer (one shown in blue, the other in magenta) is composed of five domains: a fibronectin type I finger module, an epidermal growth factor-like module, two kringles, and a serine protease domain. This model was constructed using the coordinates of the finger and growth factor pair (B. O. Smith, A. K. Downing, and I. D. Campbell, manuscript in preparation) and kringle 2 (39) from human tPA. Kringle 1 and the serine protease domains were modelled by homology. In A, the oligomannose carbohydrate at site 117 and fucose at site 61 are yellow or green. The disulfide cross-link at Cys is shown in red. The complex structures at site 448 (also yellow or green) are shown in B where the molecule has been rotated through 180° relative to the orientation shown in A.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Performed this work at the Glycobiology Institute while on leave from Nippon Shinyaku Co. Ltd., Nishiohji Hachijo, Minamiku, Kyoto, 601 Japan.

To whom correspondence should be addressed. Tel.: 44-865-275344; Fax: 44-865-275216.

**
Senior Research Associate of the Belgian National Fund for Scientific Research and Research Director of the Cancer Foundation of the General Savings and Retirement Fund (ASLK)/CGER/Belgium.

(^1)
The abbreviations used are: tPA, tissue plasminogen activator; K, kringle; FCB, cyanogen bromide fibrinogen fragments; Type D tPA, tPA type II dimers; Type D* tPA, stable mixture of type II monomers and dimers; PAI-1, plasminogen activator inhibitor, type 1; PAGE, polyacrylamide gel electrophoresis.

(^2)
P. M. Rudd, G. Opdenakker, and R. A. Dwek, unpublished data.


ACKNOWLEDGEMENTS

We thank Professor Iain Campbell for many helpful discussions regarding the structure of tissue plasminogen activator and Dr. Yoshiaki Yoshikuni and Dr. Kiyoshi Kimura (Nippon Shinyaku Co. Ltd.) for support. We also thank Dr. Yoichi Sakata (Jichi Medical School) for providing tPA antibody and PAI-1, Dr. Julian Marshall (The John Radcliffe Hospital) for providing type 1 and type 2 plasminogen, and Dr. Christopher Ponting for useful discussions.


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