(Received for publication, October 4, 1994; and in revised form, November 16, 1994)
From the
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.
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)()(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 -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.
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--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).
Casein was used as the plasmin substrate in the direct zymographic analysis(29) .
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, , conductivity of arginine
in the gradient (mS/cm2);
, tPA activity in the direct assay
(mOD/min, vertical scale) using chromogenic substrate
(S-2288);
, antigen level of tPA measured by polyclonal antibody
based enzyme-linked immunosorbent assay (µg/ml). B,
,
tPA activity in direct assay (same data as A);
, tPA
activity in the indirect amidolytic assay without FCB
(mOD/(min
/µg/ml));
, tPA activity in indirect
amidolytic assay with FCB (mOD/(min
/µg/ml)
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) () 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.
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 tPAPAI-1 complex with type D tPA before and after
reduction. Tracks1 and 3, tPA
PAI-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.
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 tPA
PAI-1 complex. Under
reducing conditions the tPA
PAI-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
-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.
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
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
value
that is 43% of the k
/K
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. , type I tPA and type 1 plasminogen;
, type I tPA
and type 2 plasminogen;
, type II tPA and type 1 plasminogen;
, type II tPA and type 2 plasminogen;
, type D* tpA and
type 1 plasminogen;
, type D* tPA and type 2
plasminogen.
The K 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
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
values, on the other hand, are modulated only by glycosylation at
site 288 on kringle 3 of plasminogen.
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
values, which range
from 1.5 to 6.4. While the type of tPA had little effect on the K
values, the lack of glycosylation at Asn
on plasminogen type 2 was associated with a decrease in the K
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.
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
- to
-conformational change. It does, however, reduce
the rate of the reverse
- to
-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
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.