From the
A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It
is a single chain glycoprotein with an apparent molecular weight of
33,000 and an isoelectric point of pH 5.2. It specifically activates
plasminogen through an enzymatic reaction. The activation of human
native Glu-plasminogen by TSV-PA is due to a single cleavage of the
molecule at the peptide bond Arg
Oligonucleotide primers designed on the basis of the N-terminal and
the internal peptide sequences of TSV-PA were used for the
amplification of cDNA fragments by polymerase chain reaction. This
allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA
library prepared from the venom glands. The deduced complete amino acid
sequence of TSV-PA indicates that the mature TSV-PA protein is composed
of 234 amino acids and contains a single potential
N-glycosylation site at Asn
The native form of human plasminogen present in blood and in
many tissue fluids is a single-chain glycoprotein with a molecular
weight of 93,000, characterized by a N-terminal glutamic acid
(Glu-plasminogen).
In
all mammalian species so far investigated, two distinct plasminogen
activators have been identified, urokinase-type plasminogen activator
(u-PA, M
Envenomations of mammals, including humans, by snakes, especially by
those belonging to the Crotalidae and Viperidae families, are characterized by an inhibition of blood coagulation
due to the action of fibrino(geno)lytic enzymes. This might be also
caused by the activation of plasminogen into plasmin by venom
components; however, none of the proteases from snake venoms that have
been purified and studied until now are able to activate plasminogen
in vitro (Stocker, 1990). In a previous study, it was observed
that Trimeresurus stejnegeri venom significantly activates
human plasminogen in vitro (Zhang et al., 1993). This
report precisely describes the purification of a plasminogen activator
(TSV-PA) which is responsible for this activity from T. stejnegeri venom. The major molecular and physiological properties of TSV-PA
were characterized. The cDNA sequence encoding TSV-PA and the deduced
amino acid sequence were also obtained and are reported here. This
plasminogen activator is the first snake venom protease which
specifically activates plasminogen.
Materials T. stejnegeri venom was from the stock of the Kunming Institute
of Zoology, Chinese Academy of Sciences. The products for gel
filtration and FPLC, Sephadex G-75 (Superfine), and Mono-Q 16/10 column
were from LKB Pharmacia. p-Nitroanilide chromogenic
substrates, H-DPhe-Pip-Arg- pNA (S-2238),
H-DVal-Leu-Lys- pNA (S-2251),
H-DPro-Phe-Arg- pNA (S-2302),
Bz-Ile-Glu-Gly-Arg- pNA (S-2222), and
H-DVal-Leu-Arg- pNA (S-2266), were obtained from Kabi
Vitrum (Stockholm, Sweden); H-DLys(Cbo)-Pro-Arg- pNA
(CBS-6525) was from Diagnostica Stago (Asnières, France). Sodium
dodecyl sulfate (SDS), PMSF,
p-nitrophenyl- p-guanidinobenzoate (NPGB),
iodoacetamide, benzamidine, p-aminobenzamidine, aprotinin,
soybean trypsin inhibitor (type I-S), and bovine serum albumin
(fatty-acid free) were purchased from Sigma. PAGE gels and reagents for
Phastsystem (fast PAGE system) were obtained from LKB Pharmacia. All
other reagents were of the highest purity available from Merck or
Prolabo (Paris, France).
From the cDNA
fragment sequence obtained from the first PCR amplification, two TSV-PA
specific primers, P
All oligonucleotide primers were
synthetized by Genset Laboratory (Paris, France). PCR products were
subcloned by the dideoxythymidine-tailed vector method into a pGEM
vector (Promega). DNA sequencing was performed by the dideoxy
termination method using a T
The bacterium library was titered.
The culture was diluted with LB broth/100 µg
Computer analysis of
protein sequences and nucleotide acid sequences were performed with the
Clustal V sequence software package.
Gel
filtration of T. stejnegeri venom in Sephadex G-75
resulted in the separation of seven protein peaks
(Fig. 1 A). The plasminogen activation activity was
predominantly associated with fraction IV and was separated from
procoagulant and phospholipase A
Peak 7, obtained from the Mono-Q column run at pH 7.8,
was pooled, lyophilized, dissolved in water, and dialyzed for 24 h
against 20 mM Tris-HCl, pH 8.8. The sample was again applied
to a Mono-Q preparative column (HR 16/10), eluted with a shallow linear
NaCl gradient. Four peaks were thus obtained. The plasminogen
activation activity was only found in the largest one, peak 4
(Fig. 1 C), associated again with amidolytic activities
on S-2238 and S-2251. This protein fraction is referred to as TSV-PA.
On the other hand, peak 1 had no proteolytic activity, while peaks 2
and 3 possessed amidolytic activity on S-2238, but not on S-2251.
The purification procedure of TSV-PA is summarized in .
TSV-PA was purified 200-fold from crude venom, with a yield higher than
40%. It represents a minor component of the venom, about 0.5% of total
venom proteins.
In general, the
amidolytic activities of u-PA and t-PA are much lower than that of
TSV-PA on different chromogenic substrates (data not shown). For
example, whereas TSV-PA hydrolyzed S-2251 significantly, u-PA had
undetectable activity on this substrate in our assay system.
In addition to
S-2251, fibrinogen was used as a substrate for activated plasminogen.
First, it was observed that unlike most other venom proteases, TSV-PA
has no direct fibrinogenolytic activity in the absence of plasminogen
(Fig. 4 A). SDS-PAGE analysis revealed that in the
absence of plasminogen and after long incubation times (150-180
min) at 37 °C, there was no significant degradation of fibrinogen,
TSV-PA producing only traces of fragments derived from the A
The
specific oligonucleotide primers P
Snake venoms contain numerous proteases which act at almost
all steps of the blood coagulation cascade, either by specific
proteolytic activation or by nonspecific degradation of blood factors
(for a review, see Stoker (1990)). But the existence of components that
could activate plasminogen, a major zymogen responsible for
fibrinolysis in vivo, had not been previously
reported. Such a protease that catalyzes the direct activation of
plasminogen in vitro has been purified from the venom of
T. stejnegeri. This plasminogen activator, designated
as TSV-PA, represents about 0.5% of the proteins in a venom pool
collected from a large number of the snakes. In fact, TSV-PA appears to
be a constant component of individual venoms: all the individual
samples collected from 15 living snakes of T. stejnegeri (8 from Anhwei Province in the central part of China and 7 from
Yunnan Province in South West China) had similar plasminogen activation
activity in vitro.
Inhibition of TSV-PA by various
inhibitors and sequence comparisons established that it is a serine
protease, belonging to the family of venom serine proteases. The
homology of TSV-PA with venom and mammalian serine proteases indicates
that the amino acid residues which form the catalytic triad are
His
Although trypsin could also
activate plasminogen in vitro (Wohl et al., 1980),
TSV-PA can be readily distinguished from trypsin by its highly specific
substrate specificity and its insensitivity to soybean trypsin
inhibitor.
The sequence of TSV-PA exhibits a high degree of identity
(60-66%) with other snake venom proteases, such as RVV-V from
V. russelli venom (Tokunaga et al., 1988),
batroxobin from B. atrox venom (Itoh et al.,
1987), and ACC-C from A. contortrix contortrix venom
(McMullen et al., 1989). However, these venom serine proteases
present considerable difference in their substrate specificity. In
particular, and at variance with the other snake venom proteases,
TSV-PA did not clot nor degrade fibrinogen and did not activate nor
degrade factor X, prothrombin, protein C. Sequence alignment of venom
serine proteases reveals several variable regions which may explain
their different substrate specificity. Particularly, in the region
43-50, TSV-PA lacks the positively charged arginine residues
which are conserved in other venom proteases. This might explain why,
at variance with other venom proteases, fibrinogen is a poor substrate
for TSV-PA. In addition, the presence of Lys
The comparison of 5`- and 3`-noncoding
regions of cDNAs encoding the venom serine proteases, TSV-PA,
batroxobin, and a serine protease from Calloselasma rhodostoma venom (Au et al., 1993), surprisingly indicates that
their untranslated regions are more conserved than coding regions, with
a degree of identity of about 85% compared with 60-65%,
respectively. A similar observation has already been reported in the
case of snake venom phospholipase A
TSV-PA
also shares a weak sequence identity with the serine protease domain of
t-PA (23%) and u-PA (21%). In addition to the catalytic domain, t-PA
and u-PA have been shown to contain one or two kringle domains, one
epidermal growth factor domain, and a fibronectin finger-like domain
(Pennica et al., 1983; Gunzler et al., 1982), whereas
TSV-PA consists only of a serine protease domain. Unlike t-PA and u-PA,
TSV-PA has a relatively strong activity on small synthetic peptide
substrates, like S-2238 or S-2251, but its plasminogen activation
activity is about 50-fold lower than that of t-PA in the presence of
fibrin fragments in the experimental conditions of our in vitro assay system. Binding to and stimulation by fibrin are hallmarks
of t-PA, but the molecular details of these two important and
distinctive processes, however, remain unclear (Madison, 1994). The
fibronectin finger-like and kringle 2 domains of t-PA have been
proposed to be responsible for its binding to fibrin (Horrevoets et
al., 1994). In contrast with t-PA, which possesses a selectivity
for fibrin clots (Hoylaerts et al., 1982; Fischer and Will,
1990), the activating activity of TSV-PA is not increased by the
presence of fibrin fragments. The molecular structure of TSV-PA, which
does not contain any kringle or fibronectin finger-like domains, does
in fact suggest the involvement of these regions in the interaction of
t-PA with fibrin.
Another important feature of plasminogen
activators is their circulating half-life in vivo.
Site-specific mutagenesis studies suggested that critical determinants
recognized by hepatic receptors may be present in the finger, epidermal
growth factor, and kringle 1 domains. For example, the epidermal growth
factor-like domain has been implicated in the uptake of t-PA by hepatic
cells (Collen et al., 1988). Deletion of this domain in t-PA
markedly prolonged its half-time in blood, from less than 5 min for
natural t-PA to 15 min for the deleted mutant molecule. As other snake
venom proteases, TSV-PA may be expected to have an even lower clearance
rate: for example, a half-life of 6.2 h was observed for defibrinase,
the thrombin-like enzyme from Agkistrodon acutus venom
(Fukutake and Ukita, 1988).
The most important endogenous inhibitor
of t-PA and u-PA is endothelial cell PAI-1, a member of the serpin
family. The fast inactivation of t-PA in vivo is in fact
mainly due to its interaction with PAI-1. The interaction site of PAI-1
with t-PA and u-PA has been demonstrated to be localized in the
N-terminal part of the serine protease domain, which is rich in
positively charged amino acids, including the sequences KHRR (t-PA) and
RRHR (u-PA). Deletion or charge reversal of these basic residues in
t-PA resulted in a more than 1000-fold reduction in the inhibition rate
by PAI-1 (Madison et al., 1989; Bennett et al., 1991;
Keijer et al., 1991). The sequence of TSV-PA indicates that it
lacks these positively charged residues, the corresponding region being
absent in this molecule. It is therefore expected that TSV-PA will be
insensitive to PAI-1.
In conclusion, a serine protease plasminogen
activator, TSV-PA, showing a high specificity for plasminogen has been
characterized for the first time in snake venom. Such an activator
should play an important role in the mechanism of snake envenomations
through fibrinogenolysis and hemorrhage, which are common aspects of
such envenomations (Stocker, 1990). On the other hand, TSV-PA appears
to be an interesting pharmacological tool to investigate fibrinolysis
in vitro as well as in vivo. Studies are in progress
to precisely investigate its mechanism of action. TSV-PA might also be
useful in the design of new generations of thrombolytic agents by
protein engineering. Recently, it has been shown that the fibrin
specificity of t-PA can be substantially increased by mutations at
several sites in the protease domain (Bennett et al., 1991;
Paoni et al., 1993). The sequence of TSV-PA, which possesses a
high selectivity for plasminogen, suggests additional mutations which
might prove to be interesting in this line. On the other hand, it might
be possible to improve the efficiency of TSV-PA by adding a fibrin
binding domain to this molecule, to direct it to fibrin clots.
The nucleotide sequence(s)
reported in this paper has been submitted to the GenBank/EMBL Data
Bank with accession number(s) U21903.
We gratefully acknowledge Dr. Eduardo Angles-Cano for
his helpful advice, Dr. David Ojcius (Biologie des Interactions
Cellulaires, Institut Pasteur, Paris) for critical reading of the
manuscript, and Dr. Jacques d'Alayer (Laboratoire de
microséquence, Institut Pasteur, Paris) for performing the amino
acid sequence analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Val
.
Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide
p-nitroanilide substrates, does not activate nor degrade
prothrombin, factor X, or protein C and does not clot fibrinogen nor
show fibrino(geno)lytic activity in the absence of plasminogen. The
activity of TSV-PA was readily inhibited by phenylmethanesulfonyl
fluoride and by p-nitrophenyl- p-guanidinobenzoate.
. The sequence of
TSV-PA exhibits a high degree of sequence identity with other snake
venom proteases: 66% with the protein C activator from Agkistrodon
contortrix contortrix venom, 63% with batroxobin, and 60% with the
factor V activator from Russell's viper venom. On the other hand,
TSV-PA shows only 21-23% sequence similarity with the catalytic
domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site
that has been demonstrated to be responsible for the interaction of
t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type
1.
(
)
A modified form,
Lys-plasminogen, is generated by plasmin by cleavage of the
Lys
-Lys
or Lys
-Val
bond of Glu-plasminogen (Robbins et al., 1967). The main
physiological function of plasmin is fibrinolysis. Conversion of
plasminogen to plasmin, however, also plays a role in various other
processes, such as ovulation, spermatogenesis, embryonic development,
involution of mammary glands, prohormone processing, and tumor cell
invasion (Dano et al., 1985; Saksela and Rifkin, 1988).
55,000) and tissue-type plasminogen
activator (t-PA, M
65,000). They are both serine
proteases with similar structural and catalytic properties. Both
enzymes cleave plasminogen, either Glu-plasminogen or Lys-plasminogen,
at the Arg
-Val
bond and generate plasmin, a
serine protease with broad proteolytic activity. However, important
functional differences exist between the two plasminogen activators.
Whereas t-PA binds to fibrin, an interaction that dramatically
increases its catalytic activity for clot-bound plasminogen (Hoylaerts
et al., 1982), u-PA binds to specific membrane receptors of a
variety of normal and transformed cell types (Blasi et al.,
1987; Del Rosso et al., 1985; Dano et al., 1994). The
activity of u-PA is thus localized at the cell surface, while that of
t-PA is mainly directed against fibrin. Accordingly, the two
plasminogen activators have distinct physiological roles.
Proteins
Proteins were from human origin except when
indicated. Prothrombin, Lys-plasminogen, urokinase (two-chain form,
M 33,000), plasmin, anti-thrombin III, factor X
(bovine), and streptokinase ( Streptococcus) were obtained from
Sigma. Purified Glu-plasminogen was a gift from Dr. Eduardo Angles-Cano
(INSERM U.143, le Kremlin Bictre, France). Protein C, t-PA, and fibrin
CNBr fragments (t-PA stimulator) were obtained from Diagnostica Stago.
Fibrinogen (grade L) from Kabi Vitrum was pretreated with diisopropyl
fluorophosphate according to the instructions of the manufacturer. The
absence of plasminogen was confirmed by incubating the fibrinogen for 6
h at 37 °C with streptokinase (1000 units
mg
fibrinogen): no evidence of degradation was found by SDS-PAGE.
The concentration of plasminogen was determined by measuring the
absorbance at 280 nm, using E
= 16.8
(Wallén and Wiman, 1972). Methods
Chromogenic Assays
The amidolytic activity of the
enzymes was measured with a Kontron spectrophotometer in 1-cm path
length plastic cuvettes. Assays were performed in 50 mM
Tris-HCl, pH 7.8, 0.01% Tween 80 in a total volume of 500 µl. The
reactions were initiated by the addition of the enzyme at a final
concentration of 18 nM and the formation of
p-nitroaniline was monitored continuously at 405 nm. The
amount of substrate hydrolyzed was calculated from the absorbance at
405 nm by using a molar extinction coefficient of 10,000
Mcm
for free
p-nitroaniline. The kinetic parameters
K
and k
were
determined by analysis of double-reciprocal plots of the initial
velocity as a function of substrate concentration.
Electrophoretic Studies
SDS-PAGE, native-PAGE, and
isoelectric focusing were performed in a Phastsystem apparatus (LKB
Pharmacia) following the manufacturer's directions and
recommended separation methods. For SDS-PAGE, samples were pretreated
in 2.5% SDS alone (nonreducing conditions) or in 2.5% SDS and 5%
-mercaptoethanol (reducing conditions) at 100 °C for 10 min.
Gels were stained with 0.1% Coomassie Brilliant Blue R in
methanol/acetic acid/water (3/1/6) and destained in the same solution.
The molecular weights were estimated by interpolation from a linear
semilogarithmic plot of relative molecular mass versus distance of migration, using the following proteins as standards:
phosphorylase b (94, 0) , bovine serum albumin
(67, 0) , ovalbumin
(43, 0) , carbonic
anhydrase
(30, 0) , soybean trypsin inhibitor
(20, 100) , and
-lactalbumin
(14, 400) . For isoelectric focusing, protein markers
were used for calibration of the gel (isoelectric focusing calibration
kit, Pharmacia). Gels were fixed with 20% trichloroacetic acid, stained
in methanol/acetic acid/water (3/1/6) containing 0.02% Coomassie
Brilliant Blue R and 0.1% CuSO
, and destained as described
above.
Plasminogen Activation Assays
The initial rate of
plasminogen activation was measured with a Kontron spectrophotometer.
Glu-plasminogen or Lys-plasminogen was incubated at a final
concentration of 1.3 µM in 50 mM Tris-HCl, pH
7.8, 0.01% Tween 80. The reaction was initiated by the addition of
TSV-PA at a final concentration of 15-120 nM, in a total
reaction volume of 250 µl. Aliquots (50 µl) were taken at
various time intervals and assayed for plasmin activity. They were
introduced into plastic cuvettes which contained 450 µl of S-2251
(0.3 mM) in 50 mM Tris-HCl, pH 7.8, 0.01% Tween 80
and the formation of p-nitroaniline was monitored at 405 nm.
One unit of activity was defined as the amount of TSV-PA which,
activating plasminogen during 1 min under the conditions above,
produced a plasmin amidolytic activity of 1 milliabsorbance unit per
min.
Fibrinogenolytic Assays with or without
Plasminogen
Fibrinogen was incubated in 50 mM Tris-HCl,
pH 7.8, at a final concentration of 5 mgml
with
TSV-PA (final concentration 6-120 nM), in the presence
or absence of Lys-plasminogen (final concentration 1.3
µM), at 37 °C for different time intervals. Thrombin
(final concentration 2.5 NIH units
ml
) was then
added and incubation was continued at room temperature for 15 min to
allow the formation of the fibrin clot. The clot was centrifuged at
3,200
g for 20 min and the protein content of the
supernatant was determined by the method of Folin (Lowry et
al., 1951), using bovine serum albumin as a standard.
Plasminogen Cleavage by TSV-PA and
u-PA
Glu-plasminogen (11 µM) was incubated with
TSV-PA (1.2 µM) or urokinase (2.4 µM) in 50
mM Tris-HCI, pH 7.8, in the presence of soybean trypsin
inhibitor (7.1 µM, in the case of TSV-PA) or aprotinin (10
TIUml
, in the case of u-PA) at 37 °C for
various times. Aliquots (5 µl) were taken to determine the
amidolytic activity on S-2251 in 500 µl (final volume) of 50
mM Tris-HCI, pH 7.8, containing 0.01% Tween 80. Samples were
then analyzed by SDS-PAGE.
Inhibitor Studies
The effects of various protease
inhibitors specific for different classes of proteases were examined.
Stock solutions of inhibitors were freshly prepared as follows: NPGB
and PMSF were dissolved in dimethyl sulfoxide, hirudin, antithrombin
III, soybean trypsin inhibitor, aprotinin, p-aminobenzamidine,
iodoacetamide, benzamidine, and EDTA were dissolved in 50 mM
Tris-HCl, pH 7.8, 0.1 M NaCl. TSV-PA (18 nM) was
incubated 30 min at room temperature in 50 mM Tris-HCl, pH
7.8, in the absence or presence of the inhibitors at the indicated
concentrations. The initial rate of amidolysis was measured after
addition of 50 µl of 2 mM S-2238. In the case of NPGB,
PMSF, L-cysteine, and iodoacetamide, mixtures containing 0.6
µM TSV-PA in the absence or presence of the indicated
concentrations of the inhibitors were incubated for 30 min at 37
°C, then 15-µl aliquots were taken to determine amidolytic
activity on S-2238.
Effect of TSV-PA on Blood Coagulation
Factors
Assays for activation of prothrombin and factor X were
performed according to the methods described by Hofmann and Bon (1987a,
1987b). Purified prothrombin (0.7 µM) was incubated at 37
°C in 50 mM Tris-HCI, pH 7.8, 0.1 M NaCl with
TSV-PA (60 nM). Aliquots (50 µl) were taken at various
times and their amidolytic activity was assayed in 500 µl (final
volume) of the same buffer containing 0.2 mM S-2238. Purified
factor X (bovine) (0.43 µM) was incubated at 37 °C in
50 mM Tris-HCl, pH 7.8, 0.1 M NaCl, containing 5
mM CaCl with TSV-PA (60 nM). Aliquots (50
µl) were taken at various times and their amidolytic activity was
immediately assayed in 500 µl of 50 mM Tris-HCl, pH 7.8,
0.1 M NaCl, containing 0.2 mM S-2222. After
incubating prothrombin or factor X (bovine) with TSV-PA for 3 h at 37
°C and at a molar ratio of 50/1, samples were analyzed by SDS-PAGE
both under reducing and nonreducing conditions. In these experiments,
Bothrops atrox venom was used as a positive control for
activation and cleavage (Hofmann and Bon 1987a, 1987b). Protein C
activation was assayed by the method described by Orthner et al. (1988): protein C (80 nM) was incubated with TSV-PA (60
nM) in 50 mM Tris-HCl, pH 7.8, containing 1
mg
ml
polyethylene glycol and 5 mM EDTA
at 37 °C. At various times, aliquots (50 µl) were taken to
determine the amidolytic activity on CBS65-25 (final
concentration 0.3 mM). In parallel, A. contortrix
contortrix venom was used as a positive activation control (Kisiel
et al., 1987).
Determination of Partial Amino Acid Sequence of
TSV-PA
Sequence determinations of proteins and peptides were
performed by Edman degradation with a 470A gas-phase sequencer.
Phenylthiohydantoin-derivatives were separated and identified by
on-line reverse phase high pressure liquid chromatography in an
RP column, with an Applied Biosystem 120-A analyzer. For
internal amino acid sequence of TSV-PA, the purified protein was
digested with trypsin, and the resultant digested fragments were
separated by high pressure liquid chromatography in a C
column. For determination of the cleavage site of plasminogen by
TSV-PA, plasmin chains generated by TSV-PA were first submitted to
SDS-PAGE and electrotransferred to an Immobilon membrane. The protein
bands were analyzed with the protein sequencer.
PCR and RACE-RT-PCR
On the basis of the determined
N-terminal and internal peptide sequences of TSV-PA, and the analysis
of genetic code usage for the published venom protease cDNA sequences,
corresponding to batroxobin (Itoh et al., 1987) and ancrod (Au
et al., 1993), two oligonucleotide primers, P and
P
, were designed for the amplification of a cDNA internal
fragment by PCR. Primer P
(5`-CTTGTAGTCTTGTTCAA(CT)TCTAACGG-3`) is oriented in the sense
direction and corresponds to the N-terminal residues 15-23 of
TSV-PA. Primer P
(5`-ACAGATGAGGGGTCCCCCAGAGTC-3`) is
oriented in the antisense direction and corresponds to TSV-PA residues
179-186. Total RNA was prepared from T. stejnegeri venom
glands according to the method of Chomczynski and Sacchi (1987).
Single-stranded cDNAs were prepared from the mRNAs contained in total
RNA (5 µg) by reverse transcriptase (Life Technologies, Inc.),
using an oligo-d(T)
primer. A first amplification by PCR
with the primers P
and P
was carried out using
Taq polymerase (Promega), under the following conditions: a
first step of 5 min at 94 °C was followed by 30 cycles of 1 min at
94 °C, 1 min at 60 °C, and 1 min at 72 °C.
(5`-CAGTTGCTGTTTGGTGTG-3`) and P
(5`-CACACCAAACAGCAACTG-3`), oriented in the sense (P
)
or the antisense (P
) direction and corresponding to TSV-PA
residues 48-53, were defined. They were used in 5` and 3`
RACE-RT-PCR to obtain the full-length cDNA sequence encoding TSV-PA, as
essentially described by Frohman (1990) with the following
modifications. In the case of 5` RACE-RT-PCR amplification, the reverse
transcription was performed by using primer P
, and an
oligonucleotide T (5`-GATCCCCTATAGTGAGTCG-3`) was ligated to the 3` end
of the first cDNA strand. Then a first PCR amplification was carried
out by using primers R
(5`-AAGGATCCGTCGACATC-3`) and
P
in the presence of an adaptor primer E
(5`-AAGGATCCGTCGACATCGATAATACGACTCACTATAGGG-3`) which contains the
sequences of primers R
, R
(5`-GACATCGATAATACGAC-3`), and the reverse complementary sequence
of oligonucleotide T. This PCR amplification was followed by a second
one carried out using primers R
and P
. For
amplification of the 3`-end, the reverse transcription was performed by
using oligonucleotide primer E with an additional 3` poly(T)
tail. Two consecutive PCR amplifications were then carried out
using primers R
and P
, and primers R
and P
.
sequencing kit (LKB
Pharmacia).
Construction and Screening of a cDNA Library
mRNAs
were prepared from the total RNA of T. stejnegeri venom glands by oligo(dT) cellulose chromatography. A directional
cDNA library was constructed with a plasmid cloning kit (SuperScript
plasmid system, Life Technologies, Inc.) with some modifications.
Size-selected cDNAs were ligated to pTT
D
EcoRI/ NotI/BAP Phagemid vector arms (LKB Pharmacia)
and used to transform MAX efficiency DH10B competent cells, producing a
library of about 4
10
independent colonies. A
PCR-based method for high stringency screening of DNA libraries
(Israel, 1993) was used for screening and isolating TSV-PA clones. Two
TSV-PA specific primers, P
as described previously and
P
(5`-GCCACCTGCCGCCATGAATAAGC-3`) corresponding to TSV-PA
residues 153-160 in the antisense orientation, and a vector
T
promoter primer located 5` of the cloned insert, were
used in PCR reactions. The presence of an insert encoding TSV-PA and
its size were examined by PCR analysis between P
-P
and T
-P
, under the following conditions:
5 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min
at 55 °C, 1 min at 72 °C.
ml
ampicilline, plated in an 8
8 matrix (64 wells total, 100
µl/well) in a 96-well multiwell plate (Costar), and amplified at 37
°C overnight. Titers of approximately 500 and 30 bacteria/well were
used for the first round screening and the second round screening,
respectively. Aliquots of amplified bacteria from 8 wells across a row
or 8 wells down a column were pooled. The matrix of 64 wells was thus
reduced to 16 pools which were used as templates for PCR analysis, as
described above. Bacteria from positive wells in the second round
screening were plated out. Single colonies were picked out and analyzed
by PCR to obtain individual positive clones.
Purification of TSV-PA
The progress of the
purification was followed by determining the following for each
fraction: activation of Lys-plasminogen, amidolytic activities on
S-2238 and S-2251, fibrinogen clotting activity, fibrinogenolytic
activity, procoagulant and anti-coagulant activities, activation and
inhibition of platelet aggregation, and finally phospholipase A activity. The methods used to assay platelet aggregation and
phospholipase A
were those as described by Born (1962) and
Lôbo de Araujo and Radvanyi (1987), respectively.
activities. Furthermore,
no plasminogen activation activity was found in other fractions.
Phospholipase A
activity and inhibition of the platelet
aggregation induced by thrombin were concentrated in fractions V to
VII; fibrinogen clotting activity was found in fraction II, and
fraction III showed a strong fibrinogenolytic activity (not shown in
Fig. 1A for clarity).
Figure 1:
Purification of TSV-PA. A, gel
filtration of T. stejnegeri venom in a Sephadex G-75
column. Lyophilized T. stejnegeri venom (2 g) was
dissolved in 20 ml of 50 mM Tris-HCl buffer, pH 7.8,
containing 0.1 M NaCl. The solution was centrifuged, and the
small pellet was discarded. The supernatant was applied to a Sephadex
G-75 column (5 100 cm), and equilibrated at 4 °C with the
same buffer. The elution was achieved with the same buffer at a flow
rate of about 24 ml
h
, collecting fractions of
8 ml. The protein concentration was estimated from the absorbance at
280 nm (
). Plasminogen activating activity (
), and
amidolytic activity on S-2238 (
) and S-2251 (
) were
determined as described under ``Experimental Procedures.''
The fractions were pooled as indicated. B, purification of
TSV-PA by FPLC on a Mono-Q preparative column (HR16/10). Fraction IV of
Sephadex G-75 (158 mg, 1.5 mg
ml
) was dialyzed
against 20 mM Tris-HCl, pH 7.8, for 24 h and applied to a
Mono-Q (16/10) preparative column (40 mg of proteins were loaded in
each run). The elution was performed at a flow rate of 5.5
ml
min
with the indicated NaCl gradient. The
absorbance of the eluant was monitored at 280 nm. The plasminogen
activating activity was mainly associated with protein peak 7,
indicated by an arrow in the figure. C, peak 7 of the
first Mono-Q column, pH 7.8, was lyophilized, dissolved in 10 ml of
distilled water, and dialyzed against 20 mM Tris-HCI, pH 8.8,
for 24 h. A total of 11 mg of proteins in 12 ml was obtained and loaded
again on the Mono-Q HR16/10 preparative column previously equilibrated
with the same buffer. Elution was performed at a flow rate of 5.5
ml
min
with the NaCl gradient, as shown in the
figure. The plasminogen activation activity was found in peak
4.
Fraction IV, containing the
activity for plasminogen activation, was dialyzed against 20
mM Tris-HCl, pH 7.8, for 24 h and applied on a FPLC Mono-Q
preparative column (HR 16/10) equilibrated with the same buffer.
Elution was achieved with a linear NaCl gradient, yielding 12 protein
peaks (Fig. 1 B). The plasminogen activation activity was
concentrated in peak 7, associated with amidolytic activities on S-2238
and S-2251. SDS-PAGE revealed that fraction 7 contained a major
polypeptide of M 30,000 ± 3000, in both
reducing and nonreducing conditions. PAGE carried out in native
conditions revealed, however, the presence of other small protein
components.
Characterization of TSV-PA
Fig. 2A shows the SDS-PAGE analysis of the samples from the various steps
of the purification. The purified TSV-PA appeared as a single band and
therefore consisted of a single polypeptide chain of 33,000. Purified
TSV-PA was also analyzed by PAGE in native conditions
(Fig. 2 B) and by isoelectric focusing
(Fig. 2 C). It appeared as a homogeneous protein in both
conditions, with an apparent isoelectric point of 5.2. PAGE, combined
with Schiff's reagent staining (Van-Seuningen and Davril, 1992),
showed that TSV-PA is a glycoprotein.
Figure 2:
PAGE
and isoelectric focusing of fractions during purification of TSV-PA.
A, SDS-PAGE (20% acrylamide): lane 1, crude venom;
lane 2, fraction IV of Sephadex G-75 column; lane 3,
fraction 7 of the first Mono-Q column, pH 7.8; lanes 4 and
5, TSV-PA fraction of the second Mono-Q column. Minus
() and plus (+) indicate that SDS-PAGE was performed under
nonreducing conditions and reducing conditions (5%
-mercaptoethanol), respectively. Proteins were stained with
Coomassie Brilliant Blue. The polarity of the electrodes is indicated
by + and
. B, PAGE in native conditions at pH 8.5
(20% acrylamide): lane 1, crude venom; lane 2,
fraction IV of Sephadex G-75 column; lane 3, fraction 7 of the
first Mono-Q column; lane 4, purified TSV-PA. C,
isoelectric focusing of TSV-PA in the pH 3-9 range. Lane
1, TSV-PA; lane 2, LKB Pharmacia isoelectric focusing
calibration kit pH 3-10.
Amidolytic Activity of TSV-PA
Purified TSV-PA
possesses amidolytic activity toward several synthetic substrates. A
detailed investigation was carried out by measuring the reaction
velocities at different substrate concentrations and determining the
Kand k
parameters
of TSV-PA by Lineweaver-Burk plots for the following synthetic
substrates: S-2238, S-2251, and S-2302. Among the compounds tested,
S-2238 was the best substrate for TSV-PA (). It can be
seen from these studies that TSV-PA has a preference for arginine over
lysine at the position that is cleaved. But TSV-PA also differentiates
among substrates possessing an arginine at the cleavage site, since its
activity was 30-fold lower with S-2302 than with S-2238, whereas its
amidolytic activity on S-2222, which is very sensitive to trypsin, was
250-fold lower than on S-2238 (data not shown).
Activation of Plasminogen by TSV-PA
The activation
of human Glu- and Lys-plasminogen by TSV-PA was determined by measuring
the amidolytic activity of activated plasminogen on S-2251.
Fig. 3A shows that the initial rate of activation of
human Glu- and Lys-plasminogen by TSV-PA was proportional to the
concentration of the venom activator. In addition, the activation of
Lys-plasminogen was 2-3-fold higher than that of Glu-plasminogen
at the same concentration of TSV-PA. This difference was also observed
in the case of plasminogen activation by t-PA (Fig. 3 B).
The addition of CNBr fibrin fragments to the assay medium increased the
rate of plasminogen activation of t-PA about 5-fold, as reported by
Hoylaerts et al. (1982), but did not significantly modify the
activation catalyzed by TSV-PA (Fig. 3 A). While the
amidolytic activity of TSV-PA toward small peptide substrates is higher
than that of t-PA, activation of plasminogen by TSV-PA was about
50-fold lower than that observed with t-PA stimulated by fibrin
fragments.
Figure 3:
Plasminogen activation by TSV-PA.
Activation of purified human Glu-plasminogen and Lys-plasminogen (1.3
µM) by TSV-PA (120 nM) ( A) and t-PA (50
IUml
, 2.8 nM) ( B) was
determined in the absence ( open symbols) or in the presence
( closed symbols) of fibrin CNBr fragments (250
µg
ml
).
,
: Glu-plasminogen;
,
: Lys-plasminogen. Inset, initial rate of
plasminogen activation at different concentrations of the activator.
, Glu-plasminogen;
, Lys-plasminogen. C,
purified human Glu-plasminogen (11 µM) was incubated at 37
°C in 50 mM Tris-HCl, pH 7.8, with TSV-PA (1.2
µM) and soybean trypsin inhibitor (7.1 µM) or
with urokinase (2.4 µM) and aprotinin (10
TIU
ml
). Samples removed at the indicated
incubation times were analyzed by SDS-PAGE (20% acrylamide) under
nonreducing ( left part) or reducing ( right part)
conditions. C, lanes 7, plasminogen control:
Glu-plasminogen incubated in the buffer without activators at 37 °C
for 3 h.
The catalytic cleavage of the human Glu-plasminogen
polypeptide by TSV-PA was further investigated by SDS-PAGE. In these
experiments, soybean trypsin inhibitor was added to the activation
medium in order to prevent the proteolytic action of the plasmin
generated, since the inhibitor blocks plasmin but not TSV-PA activity
(I). Electrophoretic analysis of the samples removed
during incubation of Glu-plasminogen with TSV-PA in the presence of
soybean trypsin inhibitor showed no modification in the molecular
weight of Glu-plasminogen under nonreducing conditions
(Fig. 3 C, left). Under reducing conditions, the
polypeptide band of apparent molecular weight of 94,000 corresponding
to Glu-plasminogen progressively disappeared, with the concomitant
appearance of two bands of apparent molecular weight of 68,000 and
27,000 corresponding to Glu-plasmin (Fig. 3 C,
right). This pattern of activation is identical to that
observed with u-PA (Fig. 3 C, right). Analysis
of the N-terminal sequences of the plasmin chains generated by TSV-PA
demonstrated that TSV-PA cleaved plasminogen at the
Arg-Val
bond, located in a loop formed by a
disulfide bridge. The position of cleavage is identical to that
occurring upon activation by u-PA and t-PA (Robbins et al.,
1967), therefore forming two chain Glu-plasmin.
chain
of fibrinogen (Fig. 4 B). Recent studies showed that
physiological plasminogen activators (t-PA and u-PA) not only hydrolyze
a specific arginine-valine bond in plasminogen, but also cleave other
proteins such as fibronectin and directly degrade fibrinogen (Weitz
et al., 1988; Weitz and Leslie 1990). When plasminogen was
incubated together with TSV-PA and fibrinogen (Fig. 4, A and C), the content of clottable protein greatly
decreased after a short incubation time. PAGE analysis showed that the
A
, B
, and also
chains of human fibrinogen disappeared
within 30 min, and were replaced by degradation fragments which were
similar to those obtained with plasmin in control experiments. This
result was consistent with the assay of clottable protein content
(Fig. 4 A), which showed that the clottable protein
completely disappeared in the presence of plasminogen within 30 min.
The results indicate that TSV-PA converted plasminogen to its activated
form and that activated plasminogen rapidly degraded fibrinogen.
Figure 4:
Analysis of direct and indirect
fibrinogenolytic activity of TSV-PA. A, human fibrinogen (15
µM) was incubated at 37 °C with TSV-PA 6 nM
(,
), 24 nM (
,
), or 120 nM
(
,
) in the presence ( closed symbols) or absence
( open symbols) of human plasminogen (final concentration 1.3
µM) in 50 mM Tris-HCl, pH 7.8, 0.1 M
NaCl. The clottable protein remaining at the indicated time was assayed
on aliquots as described under ``Experimental Procedures.''
B and C, human fibrinogen samples incubated with
TSV-PA (120 nM) in the absence of plasminogen ( B) or
in the presence of plasminogen ( C) were also analyzed by
SDS-PAGE in reducing conditions. B, lane 7, control
in the absence of TSV-PA. C, lanes 6 and 7,
positive control: fibrinogen incubated with human plasmin (1
unit
ml
). C, lane 8, negative
control: fibrinogen incubated with plasminogen at 37 °C for 120
min.
Effect of Protease Inhibitors on
TSV-PA
I shows the effects of reversible and
irreversible protease inhibitors on the activity of TSV-PA. The
activity of TSV-PA was not affected by a metal chelator (EDTA), nor by
thiol group reagents. In contrast, NPGB, a specific alkylating agent of
the active-site serine residue of serine proteases, completely
inhibited the amidolytic activity of TSV-PA (I) and its
plasminogen activation activity (data not shown). The amidolytic
activity of TSV-PA was also inhibited by the irreversible serine
protease inhibitor PMSF, while the reversible inhibitor aprotinin
partially inhibited its activity, and soybean trypsin inhibitor had
little or no effect (I). Antithrombin III/heparin or
hirudin were also ineffective. The amidolytic activity of TSV-PA was
inhibited by -aminobenzamidine and benzamidine. These observations
show that TSV-PA is not a thiol protease nor a metalloprotease and
indicate that it is a serine protease.
Effect of TSV-PA on Various Coagulation
Factors
The possible actions of TSV-PA on prothrombin, factor X,
and protein C were examined. Blood coagulation factors were incubated
with TSV-PA (60 nM) at 37 °C for different time intervals
with bovine factor X (0.43 µM, 5 mM
CaCl), human prothrombin (0.7 µM), or human
protein C (80 nM). In contrast with positive controls carried
out with A. contortrix contortrix venom for
activation of protein C (Kisiel et al., 1987) and with
B. atrox venom for activation of prothrombin and
factor X (Hofmann and Bon, 1987a, 1987b), no activation was observed
with any of these proteins. Furthermore, after incubation with TSV-PA,
prothrombin, and factor X could still be activated by B.
atrox venom and achieved full activation (results not shown).
As expected, SDS-PAGE analysis, both under reducing and nonreducing
conditions, showed that prothrombin or factor X were not cleaved after
a 3-h incubation with TSV-PA, at a substrate/enzyme ratio of 50/1
(results not shown).
Molecular Cloning and Sequence Analysis of
TSV-PA
The purified protein was subjected to amino acid
sequencing. The N-terminal amino acid sequence and internal peptide
sequences of tryptic fragments of TSV-PA, obtained by Edman
degradation, showed high similarity with the sequence of snake venom
serine proteases such as batroxobin, a thrombin-like enzyme from
B. atrox venom (Itoh et al., 1987).
(in the sense direction
and corresponding to TSV-PA N-terminal residues 15-23) and
P
(in the antisense direction and corresponding to
residues 179-186, in a region conserved among serine proteases)
were designed to amplify by PCR a partial cDNA sequence of 510 bases.
In a second step, this cDNA sequence was used to design TSV-PA specific
primers for 5` and 3` RACE-RT-PCR to obtain the complete cDNA sequence
encoding TSV-PA. The sequences obtained by PCR greatly facilitated the
subsequent molecular cloning of TSV-PA. A cDNA library constructed from
T. stejnegeri venom glands was screened at high
stringency, as described under ``Methods,'' by an efficient
and rapid PCR-based procedure. A positive clone (clone D16), which
contained a 1.6-kilobase insert, was thus identified and isolated. Both
strands of clone D16 were sequenced. The complete nucleotide sequence
of TSV-PA cDNA and the deduced amino acid sequence are shown in
Fig. 5
.
Figure 5:
Nucleotide sequence of TSV-PA cDNA and
predicted amino acid sequence of the TSV-PA precursor protein. The
nucleotide residues are numbered in the 5` to 3` direction. The
predicted amino acid sequence is shown above; the numbering
starts at the amino-terminal amino acid of mature TSV-PA (valine).
Peptides corresponding to determined amino acid sequences are
underlined. A potential glycosylation site was
boxed.
The cDNA structure of TSV-PA is similar to that of
batroxobin. It was found to contain a coding region of 774 nucleotides,
a 5`-noncoding region of 179 nucleotides, and a 3`-noncoding region of
653 nucleotides. The encoded amino acid sequence corresponds to a
polypeptide of 258 residues. The determined N-terminal and internal
amino acid sequences were found exactly in the deduced sequence
(Fig. 5, underlined), thereby unequivocally confirming
the identity of the isolated cDNA clone. The N-terminal residue valine
is preceded by 24 amino acid residues which are strictly identical to
those reported for the signal peptide of batroxobin. This signal
peptide consists of an amino-terminal hydrophobic pre-peptide (18 amino
acids) and a hydrophilic pro-peptide (6 amino acids), as suggested in
the case of batroxobin (Itoh et al., 1987). The predicted
molecular weight of the mature TSV-PA (amino acids 1-234) is
25,609. The difference with the apparent molecular weight
(33, 0) is consistent with the presence of sugars, since TSV-PA is a
glycoprotein. Accordingly, a potential N-glycosylation site,
Asn- X-Thr, is located at amino acid residues 161-163.
Sequence Homology
The amino acid sequence of
mature TSV-PA was compared with those of venom serine proteases, rat
trypsin I, and the protease domain of human t-PA and u-PA
(Fig. 6). The amino acid sequence of TSV-PA exhibits a high
degree of sequence identity with other snake venom serine proteases:
66% with ACC-C, a protein C activator from A. contortrix
contortrix venom; 63% with batroxobin; and 60% with RVV-V, a
factor V activator from Vipera russelli venom. On the other
hand, TSV-PA shows only 23 and 21% sequence identity with the serine
protease domain of t-PA and u-PA. Unlike t-PA and u-PA, TSV-PA does not
contain any additional domains, such as the kringle and growth factor
domains. Furthermore, it is important to note that the N-terminal
region of TSV-PA does not contain the positively charged amino acid
sequences such as the motifs of t-PA (KHRR) and u-PA (RRHR) which have
been demonstrated to be involved in their interaction with plasminogen
activator inhibitor 1 (PAI-1) (Madison et al., 1989). The
homology of TSV-PA with venom and mammalian serine proteases indicates
that the amino acid residues which form the catalytic triad are
His, Asp
, and Ser
(Fig. 6).
Figure 6:
Amino acid sequence comparison of TSV-PA
with snake venom serine proteases, t-PA, u-PA, and trypsin. The
numbering starts at the amino-terminal amino acid of TSV-PA. Gaps have
been introduced to optimize the sequence homology. Identical residues
in all sequences are shown by asterisks. Sequences were from
the following sources: batroxobin, Itoh et al. (1987); RVV-V,
Tokunaga et al. (1988); ACC-C, McMullen et al. (1989); rat trypsin I, MacDonald et al. (1982), and the
protease domain of human t-PA and u-PA, Pennica et al. (1983)
and Steffens et al. (1982).
, Asp
, and Ser
(Fig. 6). The amino acid sequences surrounding these
residues are well conserved among serine proteases, including TSV-PA
(Fig. 6). Like batroxobin (Itoh et al., 1987), TSV-PA
contains 12 cysteine residues and by homology with trypsin it could be
predicted that they are paired as: Cys
-Cys
,
Cys
-Cys
, Cys
-Cys
,
Cys
-Cys
,
Cys
-Cys
, and
Cys
-Cys
.
, instead of
Gln which was conserved in trypsin and other venom serine proteases,
renders TSV-PA more similar to t-PA and u-PA, both of which have Arg
residue at this position.
isozymes (Nakashima
et al., 1993). The venom serine proteases which have been
characterized so far are regarded as a family of proteins which
specifically interact with very different proteins on which they exert
their physiological action. A phenomenon of accelerated evolution in
the protein-coding regions of venom serine proteases, allowing an
adaptive evolution of proteases, could therefore be postulated, as has
been done in the case of venom phospholipase A
.
Table: Kinetic parameters of TSV-PA for different
amide substrates
Table: Effect
of protease inhibitors on amidolytic activity of TSV-PA
-plasminogen; Lys-plasminogen, plasmin-derived
Lys
-plasminogen; u-PA, urokinase-type plasminogen
activator; t-PA, tissue-type plasminogen activator; TSV-PA,
Trimeresurus stejnegeri venom plasminogen activator; FPLC,
fast performance liquid chromatography; PMSF, phenylmethanesulfonyl
fluoride; NPGB, p-nitrophenyl- p-guanidinobenzoate;
PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain
reaction; RACE-RT-PCR, rapid amplification of cDNA ends-reverse
transcription polymerase chain reaction; RVV-V, Vipera russelli venom factor V activator; ACC-C, Agkistrodon contortrix
contortrix venom protein C activator; PAI-1, plasminogen activator
inhibitor type 1; pNA, p-nitroanilide; Bz, benozyl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.