Note: Descriptions are shown in the official language in which they were submitted.
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THROMBOLYTIC AGENTS DERIVED FROM STREPTOKINASE
Background of the Invention
The present invention is generally in the field of thrombolytic agents
and more particularly directed to thrombolytic agents derived from
S streptokina.se.
Plasminogen (SEQ ID NO:1) is the principal serine protease
zymogen in the extracellular fluids of vertebrates, and its active form,
plasmin, is implicated in pericellular proteolysis associated with a wide
range
of physiological and pathological processes, including the hydrolysis of
fibrin into soluble degradation products and the suppression of tumors by
angiogenesis inhibition (Gately, Proceedings of the National Academy of
Sciences, ZISA 1997; 94: 10868-10872). In general, plasminogen expression
is fairly stable and the regulation of the activity of the fibrinolytic system
occurs mainly via up- and down- regulation of the expression of plasminogen
activators and the inhibitors of these activators. Activation of plasminogen
is
a consequence of cleavage of the Arg56~-Va15s2 bond, to form a two-chain,
disulfide linked plasmin. The two known physiological plasminogen
activators are the serine proteases tissue-type plasminogen activator (t-PA)
(SEQ ID N0:2) and urokinase (u-PA or UK) (SEQ ID N0:3), both of which
directly catalyze the hydrolysis of the activation bond. However,
plasminogen can also be activated by another completely different
mechanism, which requires formation of an activator complex with a
molecule such as streptokinase. When streptokinase is complexed with
plasminogen, plasminogen spontaneously converts into plasmin. This
complexed plasmin is then able to activate free plasminogen. Plasmin on its
own cannot activate plasminogen.
Streptokinase (SK) (SEQ ID N0:4) is a single-peptide secretory
protein of 414 amino acid residues produced by various strains of hemolytic
Streptococcus (Jackson and Tang, Biochemistry 1982; 21: 6620-6625;
Malke et al., Gene 1985; 34: 357-361). SK does not contain cysteine or
carbohydrate. Proteolytic digestion, NMR and other biochemical-
biophysical studies indicate that SK is a flexible mufti-domain protein
CA 02327526 2000-11-06
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(Conejero-Lara et al., Protein Science 1996; 5: 2583-2591; Medved et al.,
European Journal ofBiochemistry 1996; 239: 333-339; Parrado et al.,
Protein Science 1996; 5: 693-704; Rodriguez et al., European Journal of
Biochemistry 1995; 229: 83-90). SK and human plasminogen can form an
equimolar activator complex that catalyzes the conversion of plasminogen
from different mammalian species to plasmin. This property renders SK a
potent clinical agent for the treatment of blood clotting disorders, such as
acute myocardial infarction and stroke (Coleman et al., Hemostasis and
Thrombosis. Basic Principles and Clinical Practice J. B. Lippincott Co.:
Philadelphia, 1994). The activation of human plasminogen by SK involves
the formation of a streptokinase-plasminogen (SK-Plg) complex that alters
the conformation of the catalytic domain of the zymogen to complete its
enzyme-active center. The SK-Plg complex converts to a streptokinase-
plasmin (SK-Plm) complex spontaneously. Both the SK-Plg and the SK-Plm
complexes catalyze the hydrolysis of the specific activation bond, Argssi-
Va1562, of the substrate plasminogen, resulting in the formation of plasmin.
However, plasmin alone is not a plasminogen activator.
A native plasminogen molecule contains at least seven structural
domains, including the N-terminal 77-residue pre-activation peptide, five
'kringles' and a C-terminal trypsinogen-like zymogen domain (Sottrup-
Jensen et al., Program of Chemical Fibrinolysis Thrombolysis 1978; 3: 191-
209). An isolated catalytic domain of plasmin(ogen) is called micro-
plasmin(ogen) (p,Plm/~t.Plg). Human plasminogen contains 24 disulfide
bonds. Human plasminogen is glycosylated at two positions that are located
within the third kringle and between the third and fourth kringles,
respectively. Many isolated SK and plasminogen fragments, obtained via
proteolytic reaction or recombinant methods, have been used to identify the
regions involved in the interaction between the two molecules (Rodriguez et
al., European Journal of Biochemistry 1995; 229: 83-90; Shi, et al., Journal
ofBiological Chemistry 1988; 263: 17071-17075; Young et al., Journal of
Biological Chemistry 1998; 273: 3110-3116).
Accordingly, it would be useful to have a crystalline structure of the
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streptokinase-micro plasmin(ogen) (SK-p,Plg) complex. Such a structure
would make it possible to predict the portions of SK that complex with
plasminogen and to design modified streptokinases, such as a streptokinase
having less antigenicity but which is still able to complex plasminogen and
lead to activation of plasminogen.
It is an object of the invention to provide a structure for
streptokinase-micro plasminogen complex and identify the plasminogen
complexation site and the streptokinase portions that are not essential for
plasminogen complexation or activation.
It is an object of the present invention to provide streptokinase
derived thrombolytic agents.
It is an object of the present invention to provide a method of making
thrombolytic agents derived from streptokinase.
Summary of the Invention
Structural information about the streptokinase-micro plasminogen
complex has been used to identify the part of the streptokinase structure not
involved in plasminogen cornplexation or activation. These nonessential
portions can be modified to reduce antigenicity, for example, by changing
the nonessential portions of streptokinase to more human-like polypeptide
portions ("humanization of streptokinase"). One way this can be done is to
compare the nonessential portion to a structural database of human proteins
to find similar structures. Then the streptokinase nonessential structural
part
is replaced with the human structural part such as by genetic engineering of a
mutant encoding the individual streptokinases, which is then expressed in a
bacterial host such as E. toll. Alternatively, the nonessential portions can
be
removed or truncated to simplify streptokinase to a smaller molecule which
retains plasminogen activation activity. Such smaller proteins should have
reduced antigenicity and be cheaper and easier to produce. The modified
streptokinases are useful in treating clotting disorders.
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Description of the Drawings
Figure 1 is a stereo view of the activation pocket of the plasmin
catalytic domain. A 2.9 A resolution 2IFobsI-IF~~,~l electron density map,
phased with the final refined model and contoured at 1.0 a, is superimposed
S on the refined model.
Figure 2 is a stereo view of the crystal structure of the complex of
human micro plasmin (p.Plm) and streptokinase. The pPlm molecule is in
the middle of the complex. The a-domain of SK is at the top, left side of the
complex. The (3-domain of SK is to the right side of the complex. The ~y-
domain of SK is at the bottom of the complex. Only the Ca traces are
shown.
Figures 3(a)- (c) illustrate ribbon diagrams of the three domains of
streptokinase. Figure 3(a) is a ribbon diagram of the a-domain; Figure 3(b)
is a ribbon diagram of the ~i-domain; and Figure 3(c) is a ribbon diagram of
1 S the y-domain. The domains are oriented to illustrate the similarity in
their
overall (3-grasp folding.
Figure 4(a) is a stereo view of the interaction between human micro
plasmin and the a-domain of streptokinase. The micro plasmin molecule is
at the bottom of the complex. Figure 4(b) is a stereo view of the interaction
between human micro plasmin and the y-domain of streptokinase. The micro
plasmin molecule is at the top of the complex. The side chains which are
involved in the interaction are displayed along with the Ca backbones.
Figure 5 is a stereo view of the superposition of the catalytic domains
of human plasmin and human two-chain tissue-type plasminogen activator (t-
PA) (Lamba et al., Journal ofMolecularBiodogy 1996; 258: 117-135).
Selected positions are numbered accordingly. Only the Ca traces are shown.
Some commonly used loop names for trypsin-like proteases are also included
(Renatus, et al., EMBO Journal 1997; 16: 4797-4805).
Figure 6(a) is modeled view of the substrate binding site of the micro
plasminogen-streptokinase complex showing the molecular surface of the
complex. The orientation of the complex is similar to the orientation of the
4
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WO 99/57251 PCT/US99/10086
complex in Figure 1. Figure 6(b) is a stereo view illustrating docking of a
substrate micro-plasminogen into the substrate binding site. The enzyme
micro-plasmin is to the left of the complex. The streptokinase molecule is in
the center of the complex, from top to bottom. The substrate micro-
plasminogen is to the right of the complex.
Figure 7 is a stereo view illustrating a putative active-zymogen form
of plasminogen, compared with plasmin. The cleaved activation loop of
plasmin, shown in a thicker tube, is at the center of the complex, as is the
side chain of Lys69g in plasmin. The corresponding parts of the active-
zymogen are towards the right. The rest of plasmin(ogen) is towards the top
of the complex and possesses an active conformation around the active site,
particularly the peptide segment (shown in a thicker tube) upstream of the
nucleophile Ser'4'. ~i-strands in the surrounding region are shown as arrows.
The salt bridge distance between the tips of Lys69g and Asp'4° in the
active-
zymogen form is approximately the same as that between the free amino
group of Valssz and Asp'4° in plasmin. Upstream of Lys698 is the
binding site
to the y-domain of streptokinase.
Figure 8 is a stereo view of the a-domain of streptokinase (SK)
superimposed on staphylokinase (SAK) (SEQ ID NO:S) (Rabijns, et al.,
Nature Structural Biology 1997; 4: 357-360). In addition to the Ca traces,
the side chains of SK Val'9 (SAK Met26) and SK G1u39 (SAK G1u46) are
plotted.
Detailed Description
The flexibility and multidomain nature of both SK and plasminogen
have heretofore prevented the crystallization and determination of the crystal
structures of plasmin(ogen), SK and the SK-Plg complex. The rapid
autolysis of the SK-Plg complex renders the crystallization of the wild-type
SK-Plg complex impractical.
It is known that the catalytic domain of human plasminogen can bind
and be activated by SK (Shi et al., Nature Structural Biology 1990; 4: 357-
360; Wang and Reich, Protein Science 1995; 4: 1758-1767). A recombinant
5
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human microplasminogen (wPlg) was constructed, containing an alanine
residue substituted for the active-site serine residue. The streptokinase-
microplasminogen (SK-p.Plg) complex spontaneously, but slowly, converts
to a streptokinase-microplasmin (SK-p,Plm) complex. However, it does
crystallize and the crystal diffracts to atomic resolution. The three-
dimensional structure of the SK-p,Plm complex is disclosed herein.
1. Streptokinase Structure
Streptokinase Portions Complexed With Pdasmin(ogen)
Streptokinase complexation portions refers to single amino acid
residues or polypeptides of streptokinase required for the complexation of
plasminogen and plasmin to streptokinase and for the activation of
plasminogen to plasmin.
ii. Streptokinase Portions Involved In Substrate Spec~city
Streptokinase substrate specificity portions refers to single amino
acid residues or polypeptides required to impart substrate specificity upon
plasmin complexed to streptokinase as compared to plasmin alone.
iii. Nonessential Portions of Sireptokinase
Nonessential portions as used herein refers to those portions of
streptokinase that can be modified, such as by being removed or replaced,
without destroying the ability of the streptokina.se to complex with
plasminogen and without destroying the ability of the SK-Plm complex to
activate plasminogen. Moreover, the nonessential portions include portions
that can be modified, such as by being removed or replaced, or by
substituting or deleting one or more of the amino acids, without destroying
the ability of streptokinase to impart substrate specificity to plasmin when
complexed as SK-Plm compared to plasmin alone. The modified
streptokinase proteins disclosed and claimed can have one or more ofahese
nonessential portions removed or replaced.
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2. Design and Methods of Makin;~ the Modified Streptokinase
i. Design
Native streptokinase can induce formation of anti- streptokinase
antibodies following administration of a single dose. Subsequent doses are
then attacked by these anti-streptokinase antibodies, making subsequent
doses ineffective.
To form a humanized chimeric streptokinase, the nonessential
portions are compared against a database of human proteins to identify
human proteins or portions thereof which are structurally similar to the
nonessential streptokinase portions. A chimeric humanized streptokinase
mutant can then be made in which the nonessential portions) are replaced
with the human protein portions. Alternatively, or in addition, a truncated
protein can be made in which one or more nonessential portions, or one or
more amino acids therein, have been removed.
Preferably, the human protein or portion thereof has a high degree of
structural similarity to the streptokinase portion. However, the human
portion does not have to be structurally identical to the streptokinase
portion.
Preferably, the human portion does not retain any of the function of the
native human protein from which it is derived. Of course, the humanized or
truncated protein should retain substantially all or a substantial fraction of
its
ability to complex and activate plasminogen.
ii. Methods of making the modified Streptokinase
The humanized or truncated proteins may be made using methods
known to those of skill in the art. These include chemical synthesis,
modif cations of existing proteins, and expression of humanized proteins or
truncated proteins using recombinant DNA methodology. The humanized
protein can be made as a single polypeptide or the human protein portion can
be attached to the base streptokinase polypeptide after separate synthesis of
the two component polypeptides.
Where the protein is relatively short (i.e. less than about 50 amino
acids) the protein may be synthesized using standard chemical peptide
synthesis techniques. Solid phase synthesis in which the C-terminal amino
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acid of the sequence is attached to an insoluble support followed by
sequential additional of the remaining amino acids in the sequence is the
preferred method for the chemical synthesis of the proteins described herein.
Chemical synthesis produces a single stranded oligonucleotide. This may be
S converted into a double stranded DNA by hybridization with a
complementary sequence, or by polymerization with a DNA polymerase
using the single strand as a template. One of skill would recognize that while
current methods for chemical synthesis of DNA are limited to preparing
sequences of about 100 bases, longer sequences may be obtained by the
ligation of shorter sequences. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-
284 in The Peptides; Analysis, Synthesis, Biology Vol. 2. Special Methods
in Peptide Synthesis, Part A, Merrifield, et al., J. Am. Chem. Soc. 1963; 85:
2149-2156, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed Pierce
Chem. Co.: Rockford, Ill., 1984.
Alternatively, the protein may be made by chemically modifying a
native protein. Generally, this requires cleaving the native protein at one or
more sites and then annealing desired polypeptides onto the newly formed
termini. The desired cleaved peptides can be isolated by any protein
purification technique that purifies on the basis of size (e.g. by size
exclusion
chromatography or electrophoresis). Alternatively, various sites in the
protein may be protected from hydrolysis by chemical modification of the
amino acid side chains which may interfere with enzyme binding, or by
chemical blocking of the vulnerable groups participating in the peptide bond.
In the preferred embodiment, the humanized or truncated proteins
will be synthesized using recombinant methodology. Generally, this
involves creating a polynucleotide sequence that encodes the protein, placing
the polynucleotide in an expression cassette under the control of a suitable
expression promoter, expressing the protein in a host, isolating the expressed
protein and, if required, renaturing the protein. Additional proteins can be
made separately and then ligated to the modified streptokinase, or the
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polynucleotide sequence can encode the streptokinase in phase with anther
protein.
DNA encoding the protein can be prepared by any suitable method,
including, for example, cloning and restriction of appropriate sequences or
S direct chemical synthesis by methods such as the phosphotriester method of
Narang et al., Meth. Enzymol. 1979; 68: 90-99; the phosphodiester method of
Brown et al., Meth. Enzymol. 1979; 68: 109-151; the diethylphosphoramidite
method of Beaucage et al., Tetra. Lett. 1981; 22: 1859-1862; and the solid
support method of U.S. Patent No. 4,458,066.
Alternatively, partial length sequences may be cloned and the
appropriate partial length sequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired DNA
sequence.
In a preferred embodiment, DNA encoding the protein will be
produced using DNA amplification methods, for example polymerise chain
reaction (PCR).
The proteins may be expressed in a variety of host cells, including E.
coli or other bacterial hosts, yeast, and various higher eukaryotic cells,
such
as the COS, CHO and HeLa cells lines, insect cells, and myeloma cell lines.
The recombinant protein gene is operably linked to appropriate expression
control sequences for each host. The plasmids encoding the protein can be
transferred into the chosen host cell by well-known methods such as calcium
chloride transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the plasmids can
be selected by resistance to antibiotics conferred by genes contained on the
plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the protein can be purified according to standard
procedures such as ammonium sulfate precipitation, affinity columns,
column chromatography, or gel electrophoresis. Substantially pure
compositions of at least about 90 to 95% homogeneity are preferred, and 98
to 99% or more homogeneity are most preferred for pharmaceutical uses.
One of skill in the art would recognize that after chemical synthesis,
9
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biological expression, or purification, the protein may possess a
conformation substantially different than the native protein. In this case, it
may be necessary to denature and reduce the protein and then to cause the
protein to re-fold into the preferred conformation. Methods of reducing and
S denaturing the protein and inducing re-folding are well known to those of
skill in the art. For example, the expressed, purified protein may be
denatured in urea or guanidium chloride and renatured by slow dialysis.
To determine which proteins are preferred, the proteins should be
assayed for biological activity. Such assays, well known to those of skill in
the art, generally fall into two categories; those that measure the binding
affinity of the protein to a particular target, and those that measure the
biological activity of the protein.
Methods Of Using Modified Streptokinase
1 S The modified streptokinase molecules can be administered for
treatment of blood clot disorders, such as in heart attacks, as known in the
art
for administration of native streptokinase and tissue-type plasminogen
activator (t-PA) and urokinase (u-PA or UK).
The compounds are preferably administered intravenously in
appropriate Garners. The appropriate dosages will depend upon the route of
administration and the treatment indicated, and can be readily determined by
one skilled in the art. Dosages are generally initiated at lower levels and
increased until desired effects are achieved.
The present invention is further described by the following
nonlimiting examples.
Example 1: Proteinpreparation and crystallization
Recombinant streptokinase and S741A mutant of human ~,Plg were
constructed. The proteins were expressed in E. coli as inclusion bodies
which were washed, dissolved in 8 M urea and combined to be refolded
together by rapid dilution. The 1:1 complex between SK and p,Plg was
purified further using S300-chromatography. The protein sample was stored
CA 02327526 2000-11-06
WO 99/5?251 PCT/US99/10086
at 0°C for more than two months before being used for a successful
crystallization. The initial crystallization condition was determined by the
use of sparse-matrix screens from Hampton Research. Crystals were grown
at 20°C from sitting drops with wells containing 1.0 M sodium citrate,
0.2 M
HEPES (pH 8.0), 1 mM magnesium chloride. The protein concentration
used was 40 mg/ml. Crystals appeared in about two weeks and had typical
dimensions of 0.1 x 0.1 x 0.5 mm. The crystal belongs to space group P21
with cell parameters of a=80.0 A, b=125.1 A, c=86.8 A and ~i=105.4°.
One
crystallographic asymmetric unit contains two SK-p,Plm complexes with
VM=2.9 A3/Da (Matthews, Journal of Molecular Biology 1968; 3 3 : 491-
497).
To confirm the crystal content, selected crystals were dissolved in
H20 and analyzed by SDS PAGE. The results showed that the p.Plg was
converted to p.Plm and SK was digested at two positions to different extents.
1 S The proteolytic cleavage in ~Plg renders the complex changed from SK-pPlg
to SK-N.PIm. The low proteolytic activity in the sample may come from
trace-amount protease contamination, and/or the Ser~4' to Ala mutant leaking
back to the "wild-type" pPlg during the E. coli expression.
Example 2: C sry tallogr_aphic methods and data~rocessing
Data collection and heavy-atom derivative screen was conducted at
room temperature on a Siemens area detector. The program SAINT was used
to process the data. Molecular replacement searches for the ~Plm molecules
were carried out with the program MRX (Zhang and Matthews, Acta.
Crystallographica 1994; DSO, 675-686). The solution clearly showed a local
two fold symmetry between the two SK-ltPlm complexes. However the
overall quality of the electron density based on this molecular replacement
solution alone was strongly biased by the model used and was not useful for
obtaining interpretable electron density beyond the region of the search
model.
The phases of the crystal structure of the SK-p,Plm complex were
11
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WO 99/57251 PCTNS99/10086
solved by the use of multiple isomorphous replacement (Mm) techniques,
using platinum, mercury and iridium derivatives. The program packages
SOLVE (Terwilliger and Berendzen, Acta. Crystallographica 1996; D52:
749-757) and MLPHARE (Otwinowsky, Proceedings of the CCP4 Study
Weekend, 25-2b January, (W. Wolf, P. R. Evans and A. G. W. Leslie, eds.),
pp 80-86, Daresbury Laboratory: SERC, 1991) were used to identify and
refine the heavy atom parameters. The statistics of the X-ray data collection
and MlZt phasing are summarized in Table 1.
12
CA 02327526 2000-11-06
WO 99/57251 PCT/US99/10086
x
et M O~ ~h N M
' ~ C/~
~r
V)
b
00 v1 d' ~1 00 O o0 N
t~ 00 00 00 00 00 Ov
~ O O O O O O O ~ ~ .-~-~ O
0.i ~ l~ l~ OWE 00 l'~ Q\ E~ O ~ ~ O
O O O O O O O M O
U
O N M
Mp~pM b N
('~ 0~0 I'~ ~ ~ OM1 ~ O
O O O O O O O
~i ~ i w ~ w w. W y.., 00 '~ O
N M ~ ~ O ~O N ~. ~ ~ .-~ N
.-i .-~ Cj .-. .... r-i p M r. '~T
' O
4,
v1 a1 'd' ~ O
b
~ OO V1 ~ 'd: vl ~O h ~ ~ N .C'
O 'd' 00 ~O ~ ~O 00 M V1
N N ~-~ N N N ~
dp O 'C
~ v~ Ov .y
NNoo~M~MN ~M~ e~ ~ r,;
A-i '~ ai vi O Q\ d' ~t Ov 00 r.r 'b at
NNNNN,~!NN ~ '" ,O
ie3 M ~O O~ Ov Ow1 O~ 00 ~ ' ~ U
V1 l~- 00 ~ 00 O I~ t~ U
'd ~--i ~-~ Owe V'~
ccS ~,
,rj d' ~ ~ ~ V
V N ~ b ~ ~i,
_ O .o N
No~O~t'~'N~v~'1~
~ N N N oo pv ~ 00 WO 'd'
O M 00 O 00 N ~ ~ O ~ M ~ U
M ,_, M .--~ N N M N f~ ~ ~ C1. ~ U
O y 4~
O o ~ I~ tt ~O o0 VD 00 W p pip ,n O ~ ~ cUC
..., TJ
V \ cG N ~-~ I~ N N O Ov t~ ,~ ,..., ~ U
U .;~ ~ O ~ OwD V Ov O ~O ~ t~ N
=, ~'' 00 00 l~ 00 I~ 00 00 h O ~, O
~O\Od'~V~1~0~ '
V M V~ ~-~ M N OW t v~ N ~U-' w O
U ~-mt ~ M N dv N O N Ov 00 ø' ~ v ~*"'
Ov CT Q\ Q\ oo Ov Ov O~ N ~
A Qi b
O O ~ ~ ~ Q,
a~n 01 v0 O vD N et O 'fit ~ U O
4, '.r N M M M M M M M
~ o,
a)
~' .c7 p ' ~ W
'N z ~ ~ U cd b °)
r '~ C! ~ ~ ~ O x p O
H ~ ~. O ~ ~ Z ,~ . ° ~; ~ oo Q, v~ w v
.y U ~x x ~U V ~ ob
w a. x ~ a. o,
a zxx~~,xxx ~z° ~ .~ ~ .~ v
13
CA 02327526 2000-11-06
WO 99/57251 PCT/US99/10086
The initial MIR phases at 3.0 A resolution were confirmed by the MR
solution of the p,Plm part of the complex. The phases were further improved by
electron density averaging over two-fold non-crystallographic symmetry and
solvent flatting. The initial mask for the electron density averaging was
derived
from the MR solution. The electron density improvement was carried out with
the program DM (Cowtan and Main, Acta. Crystallographica 1996; D52: 43-
48).
From the improved electron density map, the SK molecule was seen in
three distinct domains adjacent to the region of the p,Plm molecule. The
initial
model of p,Plm was built using the crystal structure of chymotrypsinogen (Wang
et al., Journal ofMolecular Biology 1985; 185: 595-624) as a template. The
structure of SK was built directly from the electron density map. Model
building was performed with the program O (Jones et al., Acta.
Crystallographica 1991; A47: I 10-119). Iterative refinement and model
building were used to improve the model gradually. SigmaA-weighted maps
were calculated with the program SIGMAA (Read, Acta. Crystallographica
1986; A42: 140-149) and used in the initial model building.
Refinement was carried out with the program XPLOR (Brunger et al.,
Science 1987; 235: 458-460) and TNT (Tronrud et al., Acta. Crystallographica
1987; A43 : 489-501 ). None-crystallographic-symmetry constrains were used
throughout the refinement; in the final model, the two copies of the
crystallographic independent SK-p,Plm complexes are practically identical. All
of the chemically expected 250 residues of the pPlm molecule were included in
the final model at 2.9 A resolution; and of the 414 residues of the SK, 322
were
modeled. A representing region of the electron density map of p,Plm is shown
in
Figure 1. The final R factor is 20.3% over the 8.0-2.9 A resolution shell
(28,600
reflections), and the free R (Brunger, Nature 1992; 355: 472-474) is 30%
(3,150
reflections). Bond and angle deviations are 0.01 A and 1.8°,
respectively, as
determined by XPLOR using Engh and Huber parameters (Engh and Huber,
Acta. Crystallographica 1991; A47: 392-400). Structural superposition and
14
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WO 99/57251 PCT/US99/10086
solvent accessible surface calculation were carried out with EDPDB (Zhang and
Matthews, Journal ofApplied Crystallography 1995; 28: 624-630). Figures
were created by usingMOLSCRIPT (Kraulis, J. Appl. Cryst. 1991; 24: 96-950),
RASTER3D (Merntt and Murphy, Acta. Cryst. 1994; DSO: 869-873) and GRASP
(Nicholls et al., Proteins 1991; 11: 281-296).
Example 3 : Overall Structure of human uPlm
The crystal structure of SK-p,Plm complex was determined at 2.9 ~
resolution using X-ray crystallography. There are two SK-~,Plm complexes per
crystallographic asymmetric unit, which are practically identical with each
other. Figure 2 shows the Ca trace of one SK-pPlm complex. The ~Plm
component of the complex contains the region from residue Alasa2 to the C-
terminal residue Asn~91 of plasmin. The dimensions of the N.PIm molecule are
about 40 x 45 x 50 A. Resembling the architecture of many other trypsin-like
serine proteases, p.Plm consists of two domains, each of a six-stranded (3-
barrel.
The C-terminus of pPlm ends with an a-helix packing against the N-terminal (3-
barrel. The catalytic residues, His6o3, Aspsas~ ~d Ser~4' position confirmed
the
Ser to Ala substitution.
Consistent with the high sequence homology (i.e. 39% identity), the
coordinate root mean square deviation (rmsd) between ~,Plm and chymotrypsin
(Harel et al., Biochemistry 1991; 30: 5217-5225) is 0.7 A for 193 Ca atoms,
using a 1.5 A cutoff. Compared to both chymotrypsin and chymotrypsinogen
crystal structures (Harel et al., Biochemistry 1991; 30: 5217-5225; Wang et
al.,
Journal ofMolecular Biology 1985; 185: 595-624), the active site conformation
of the catalytic domain of plasmin is indeed in its enzyme form. The
activation
bond, Argss'-Valssz, has been cleaved as indicated by SDS PAGE and N-
terminal sequencing of the crystal contents (data not shown). The new C-
terminus ofthe cleaved loop, containing Pross9, Glysso~ ~d ~gs6y is mobile in
the crystal and can be seen in the electron density only at a low contour
level.
The newly liberated N-terminus (WGG) (amino acids 562-565 of SEQ m
CA 02327526 2000-11-06
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NO:1) enters the activation pocket that is designed precisely to fit both the
main-
chain atoms and the divaline side chains. Its stability and proper positioning
is
reinforced by the solvent inaccessible salt bridge linking the terminal amino
group to the carboxylate group of Asp'4°. This salt bridge ensures that
the loop
immediately upstream of nucleophile Ser'41 changes its conformation to an
active form. Consequently, the oxyanion hole is formed by the amide groups of
residues 738-740, and the S1 specificity pocket is properly formed.
Six disulfide bonds stabilize the structure of p.Plg. Three of them,
CyS58R-CyS604' Cyssso-Cys7a7~ and C s737-C s~65
y y , are within six-residue ranges of
the catalytic triad residues and function to maintain the platform of the
catalytic
triad. Another one, CysSSa-Cys566~ w~ch is absent in many other typsin-like
proteases, flanks the activation cleavage site. In the zymogen form, this
disulfide bond likely constrains the conformation of the short activation loop
such that the Arg561-V~562 bond is confined to be readily cleaved by
plasminogen activators. The two new termini liberated by the activation
cleavage are also constrained by the disulfide bond.
Immediately beneath the imidazole ring of His6o3 is residue Ala6°'
which
sits in a pocket perfect for its methyl group side chain. This residue was
found
to be mutated to a threonine residue in the plasmin of a group of patients
with a
predisposition of thrombosis (Ichinose et al., Proceedings of the National
Academy of Sciences, U.S.A. 1991; 88: 115-119). Such an Ala-to-Thr mutation
disrupts the hydrogen bond between His6°3 and Asp6a6 ~d impairs the
charge
delay network of the catalytic triad.
Example 4: Overall structure of streptokinase
In the crystal structure of the SK-pPlm complex, SK appears as a three
domain protein with several segments in the primary sequence disordered in the
crystal lattice. The three domains are linked with each other by coiled coil
peptides and are likely to fold independently in solution. They are denoted as
a-
16
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(i-, and y-domains hereafter along the peptide chain from the N-terminus to
the
C-terminus (see Figure 2).
The a-domain begins at residue Asnls and ends at about residue Pro~4s.
Residues 1-11 and two extra residues (Gly-Ser) adopted from the cloning vector
are disordered in the electron density map. Similarly the region of residues
45-
70 has a lack of interpretable electron density. A proteolytic cleavage that
occurs at the bond between Lyss9 and Ser6° is located in this
disordered region.
The ~i-domain begins at residue Alalss and ends at residue Pro2g3. This domain
also contains a cleavage site, Lys25'-Serz58, which is cleaved only in a
portion of
total SK-pPlm complexes as shown in our SDS PAGE analysis. No preference
of SK molecules, cleaved and non-cleaved, is seen at this site in the two
crystallographically independent SK-p,Plm complexes. The y-domain starts at
residue Asp2g5 and becomes invisible beyond residue Arg3'2, although SDS
PAGE suggests that the C-terminal 40 or so residues are attached. The domain
boundaries found in the crystal structure are consistent with the results from
various protease mediated SK-degradations (Parrado et al., Protein Science
1996; 5: 693-704). It is also consistent with the previous observations that
the
N-terminal 16 residues and the C-terminal 40 residues of SK are functionally
dispensable for plasminogen activation (Kim et al., Biochemical and
Biophysical Research Communications 1996; 40: 939-945; Young et al.,
Journal of Biological Chemistry 1995; 270: 29601-29606). In fact, the N-
terminal 16 residues of SK play a role in the secretion of this protein from
the
host cell (Pratap et al., Biochem. Biophys. Res. Commun. 1996; 227: 303-310).
There is also evidence showing that some fragments in the region of residues
45-70, which is disordered in the complex structure, exist in an inherently
flexible state (Nihalani et al., Protein Science 1998; 7: 637-648).
Roughly speaking, every one of the three domains of SK belongs to the
(3-grasp folding class (Murzin et al., Journal of Molecular Biology 1995; 247:
536-540), but with some noticeable differences (see Figure 3). Like a typical
(i-
grasp protein, the SK a-domain contains a single a-helix packing against a
17
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mixed five-stranded (3-sheet. In addition, there is a short two-stranded (3-
sheet
on the same side of the major (3-sheet as the a-helix. The (3-strands forming
the
major (3-sheet are "~i2, "(3~, "(3~, "/34, and "(3s. The topology of this ~3-
sheet
(Richardson et al., Journal ofMolecular Biology 1976; 102: 221-235) is (+1,-
3x,-1,2x). The hydrogen bond network of the major (3-sheet is disrupted at the
middle of the "~3z strand by a bulge at position 36. The a-helix is located
between "~i3 and "(34 and is thus named "a3,4. Between the major ~i-sheet and
the
a-helix is the hydrophobic core of the a-domain. Disturbing this hydrophobic
core is likely to result in a dysfunctional SK as shown by a G1y24 to His
mutation
(Lee et al., Biochemical and Biophysical Research Communications 1989; 165:
1085-1090). The SK (3-domain shares the same overall folding with the SK a-
domain. The coordinate rmsd between the two domains is 1.7 A for 81 residues,
using a 4.0 A cutoff. Some corresponding loops between the two domains,
however, have different lengths. The SK y-domain contains a four-stranded
major (3-sheet and a short two-stranded ~i-sheet. The major ~3-sheet has a
topology similar to that of the major (3-sheet of the a-domain without "/3s.
Between Y(32 and y~33 are some coiled coil loops. The qualities of the
electron
density in the a- and y-domains of SK are significantly better than that of
the (3-
domain region. Correspondingly, the average temperature factors of the a-, ~3-
,
and y-domains are 43, 80, and 39 A2, respectively. These differences appear
correlated with the interactions of each domain of SK with the ~Plm molecule
in
the complex.
Example S: Interaction between streptokinase and pPlm
The SK molecule has extensive interactions with the uPlm molecule,
mostly through the SK /3- and y-domains. The values of buried molecular
surface area are 1,650 AZ , 950 AZ and 1,500 A2 between ~Plm and the a, ~3 and
'y-domains of SK, respectively.
The SK a-domain is located near the catalytic triad of ~,Plm. There are
18
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three major contact regions between the SK a-domain and p,Plm (see Figure 4a).
The first region contains the interaction between the major (3-sheet,
particularly
the strands of °'(3, and °'(i2, of SK and the loop region of
residues 713-721 of
p,Plm. In this contact region, Arg"9 of plasmin (SEQ ID NO:1) forms salt
bridges with both G1u39 and Glu'34 of SK (SEQ ID N0:4), and it also has van de
Waals interaction with SK Val'9. The uncharged alkyl group side-chain of
residue 19 of SK has been shown to be important for plasminogen activation
(Lee et al., Biochemistry and Molecular Biology International 1997; 41: 199-
207). Arg"9 of plasminogen has also been identified as an important residue
involved in the SK-Plg complex formation (Dawson and Pontin, Biochemistry
1994; 33: 12042-12047). The second contact region contains the interaction
between the bulge region in the °'~i2 strand of SK and the 643-645
region of
~Plg, which is the upstream region of the catalytic residue Asp6a6. The
positively charged side chain of pPlm Lys6aa also protrudes towards the C-
terminus of the a-helix, °'a3,4, of SK, presuming a helix dipole-charge
interaction. The third contact region is between the loop following the a-
helix,
aa3,4, of the SK a-domain and the 606-609 region of p,Plm. The latter is the
down stream region of the catalytic residue His6os_ These close interactions
between the SK a-domain and the catalytic triad of p,Plm are likely to
contribute
to the substrate specificity difference between plasmin and the SK-plasmin
complex. The mode of interaction between the SK a-domain and N,PIm is
clearly different from that of some other (i-grasp folding proteins. For
example,
the Ras-binding protein, C-raft, binds to its target protein, Rap 1 a, by
forming an
extended ~3-sheet between the edges of their existing (3-sheets (Nassar et
al.,
Nature Structural Biology 1996; 3 : 723-729).
The SK y-domain binds to p.Plm near the activation cleavage site of
plasmin (see Figure 4b). On the pPlm part, this interaction mainly involves
two
loop regions: p,Plm(622-628) in the so called calcium-binding loop and
p,Plm(692-695) in the so called autolysis loop. The interactions include a
salt-
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bridge between SK Lys3sa and p,Plm Glu6z3, hydrogen bonds (e.g. SK Glu3" and
p.Plm G1n622), and hydrophobic interactions. The amino acid sequence of region
622-628 in human plasmin(ogen) is "QEVNLEP" (amino acids 622-628 of SEQ
» NO:1), and in bovine plasmin(ogen) is "NEKVREQ" (amino acids 643-649
of SEQ >D N0:6). Since this region of p,Plm is involved in the SK binding, the
sequence difference shown above may provide explanation why the catalytic
domain of bovine plasminogen binds with SK significantly weaker than human
plasminogen does (Young et al., Journal of Biological Chemistry 1998; 273
3110-3116). On the other hand, the only close interaction of SK with the
activation loop region of ~tPlm (i.e. around p,Plm(558-566)) is that between
SK
Ala3az and p,plm Valsb'. Therefore human plasminogen activation by SK is
unlikely to require direct contact of SK with the activation loop of
plasminogen.
Furthermore, the observed C-terminus of the SK y-domain is on the side
opposite to these l,~Plm binding regions. Hence it is probable that the last
40 or
so residues, which are disordered in the crystal, have nothing to do with the
SK-
Plg complex formation.
Although there is no kringle domain present in the complex crystal,
kringle domains have been shown to be involved in plasminogen activation by
SK. Since the N-terminus of the catalytic domain of plasmin(ogen) is on the
hemisphere opposite to the SK binding sites, the extension of kringle S from
the
catalytic domain is unlikely to disturb the observed interactions between SK
and
p,Plm. Also, it has been shown previously (Rodriguez et al.. European Journal
of Biochemistry 1995; 229: 83-90) that the complex of plasmin with the
fragment SK(143-293) (i.e. the (3-domain) or SK(143-386) (i.e. the ~3- and y-
domains) is very rapidly inhibited by a2-antiplasmin, whereas the complex with
intact SK is resistant to inhibition. Furthermore in the case of SK(143-386),
inhibition by a2-antiplasmin results in dissociation of the SK-Plm complex;
SK(143-293), in contrast, remains associated with the a2-antiplasmin-plasmin
complex. The results suggest that the a2-antiplasmin-plasmin interface
overlaps
with the SK a-domain binding site and partially overlaps with the SK y-domain
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binding site on the plasmin surface, while the SK [3-domain binding site may
have nothing to do with the a2-antiplasmin binding site.
Example 6: Putative substrate bindine site of the SK-Plm complex
Although it is active against fibrin, plasmin alone can not convert
plasminogen to plasmin. This substrate binding specificity can be explained by
a comparison of the crystal structure of p,Plm with that of the catalytic
domain
of human t-PA (Renatus et al., EMBO Journal 1997; 16: 4797-4805}. While
both are trypsin-like proteases, human t-PA has high specificity towards
activation of plasminogen. The overall structures of these two domains are
similar (see Figure 5). The coordinate rmsd between the catalytic domain of
human two-chain t-PA (Lamba et al., Journal ofMolecular Biology 1996; 258:
117-135) and human p,Plm is 0.74 A for 177 Ca atoms, using a 1.5 A cutoff
However there are many structural differences on the enzyme surface around the
active site. There are at least three significant backbone differences around
the
active site. 1) Corresponding to plasmin(ogen) between residues 644 and 645, t-
PA has a six-residue insertion with three aspartate residues in a row. This
insertion in t-PA is structurally replaced by part of the SK a-domain in the
SK-
p,Plm complex. 2) The "autolysis loop" region of ~Plm(689-695), which
contacts the SK ~y-domain, is different from the corresponding region of t-PA.
3)
The region of p,Plm(711-720) is different from the corresponding region in t-
PA,
where it is called the "methionine loop". The pPlm conformation in this region
makes it possible to have a complementary contact with the SK a-domain. 4) In
the so called "37-loop" region of t-PA which interacts with the natural
inhibitor,
PAI-1, and is involved in fibrin specificity (Bennett et al., Journal
ofBiological
Chemistry 1991; 266: 5191-5201; Madison, et al., Proceedings of the National
Academy of Sciences, USA 1990; 87: 3530-3533), human plasmin(ogen) is four
residues shorter around residue 583. Some of these differences, if not all,
are
likely responsible for the substrate specificity difference between plasmin
and t-
PA. The SK-plasmin complex may change the substrate specificity of plasmin
by compensating for some of these differences. On the other hand, it has been
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shown that the isolated, synthetically prepared activation loop of plasminogen
can not be cleaved by plasminogen activator (Gams and Shaw, International
Journal of Peptide Protein Research 1982; 20: 421-428). This observation
suggests that not only the amino acid sequence of the cleavage site but also
its
conformation and the overall structure of the substrate zymogen contribute to
the specificity of the activator.
The crystal structure of the SK-~,Plm complex shows that the complex
has an opened cavity (see Figure 6a) compared with the spherical (convex)
shape of the catalytic domain of plasmin(ogen). Therefore the SK-Plg complex
should provide more substrate binding surface than the plasmin molecule alone
can. A manual molecular model to dock a model micro plasminogen molecule
into the substrate binding site of the SK-p,Plm complex is shown in Figure 6b.
In such a model, the activation bond, Arg56i-Va1562~ of the substrate (micro)
plasminogen is positioned into the active site of the catalytic (micro)
plasmin;
the N-terminus of substrate N.PIg is positioned to be closed to the disordered
region of SK(45-70). From the bottom of the substrate binding concave, p.Plm
contributes approximately 1,OSOA2 binding area, mostly from the surface of the
strand, °'(32, and the a-helix, aa3,4. Since it might be modeled close
to the kringle
domain of the substrate plasminogen, the flexible SK(45-70) region may
provide extra substrate binding surface; that would explain the observed high
amity of the SK a-domain with the kringle domains of plasminogen (Young et
al., Journal of Biological Chemistry 1998; 273 : 3110-3116) as well as the
important role played by residues 45-51 of SK in binding with plasminogen
(Nihalani et al., Protein Science 1998; 7: 637-648). For the SK ~i-domain, the
major (3-sheet forms part of the wall of substrate binding concave with its
helix
side facing outside. The (3-strand on the rim of the ~i-sheet, ~(i2,
potentially
forms hydrogen bonds with the strand of residues 625-629 of substrate
plasminogen. The SK ji-domain contributes ~550 A2 binding surface in total.
The SK y-domain contributes some coiled coil, around residue 330, to the
substrate binding, about 150 A2 in total. Several potential salt bridges can
be
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predicted from this hypothetical model, including Arg56~ of the substrate
plasminogen (s-Plg) to Asp'35 of the catalytic plasmin (c-Phn), s-Plg Lysss'
to c-
Plm Glu6o6, and s-Plg Lys'S° to SK Asp'g. Lys55' of plasminogen was
found to
be important for plasminogen activation by t-PA (Wang and Reich, Protein
Science 1995; 4: 1769-1779) and could be explained if similar binding modes
were assumed for binding of t-PA to plasminogen. The Asp'35 of c-Plm,
interacting with the side chain of Args6~ of s-Plg, defines the substrate
specificity of the SK-Plm complex at the S 1 position.
Example 7: A possible activation mechanism of human plasmino~by SK
One of the functions of SK is to turn the zymogen plasminogen into an
active "enzyme" without cleaving the peptide chain. It appears from the
crystal
structure of the SK-pPlm complex that it is the interaction between the SK y-
domain and the catalytic domain of plasminogen that creates the enzymatic
activity of human plasminogen-SK complex.
As shown by many crystal structures of trypsin(ogen)-like proteases, one
characteristic of the proteases in this family is a buried salt bridge formed
in the
activation pocket associated with the activation. In the classical case, this
salt
bridge is formed between the carboxylate group of the aspartate residue that
is
immediately upstream of the catalytic nucleophile serine residue and the
liberated amino terminus after the activation cleavage. The formation of the
salt
bridge reorients the aspartate residue relative to the zymogen structure and
thus
restructures the active site, which includes the oxyanion hole, the catalytic
triad
and the S 1 specificity pocket (Freer et al., Biochemistry 1970; 9: 1997-
2009).
However, non-cleavage activation has also been found in some proteins in this
family, including t-PA and vampire-bat plasminogen activator (v-PA) (SEQ ID
N0:7) (Renatus et al., EMBO Journal 1997; 16: 4797-4805; Renatus et al.,
Biochemistry 1997; 36: 13483-13493). Human t-PA has significantly high
plasminogenolytic activity in its single-chain form. v-PA, on the other hand
lacks the activation cleavage site. Both of them switch between the active and
the inactive stages in response to environmental changes, including the
presence
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WO 99/57251 PCT/US99/10086
of fibrin. In their crystal structures, complexed with high affanity
inhibitors,
both plasminogen activators were frozen in the "active stage". These
structures
demonstrate that in such an intact protein, a buried lysine residue replaces
the
functional role of the released amino terminus of the protease activated by
the
cleavage. The conformation of this lysine residue, Lys'ss (numbered as Bode &
Renatus (Bode and Renatus, Current Opinion Structural Biology 1997; 7: 865-
872), unlike the newly released amino terminus which usually stays in one
form,
may switch between the active and the inactive stages depending on
environmental conditions such as the local concentration of cofactors (Lamba
et
al., Journal ofMolecularBiology 1996; 258: 117-135; Nienaber et al.,
Biochemistry 1992; 31: 3852-3861).
A similar scenario is likely to exist in the SK-Plg complex (see Figure 7).
Based on three-dimensional structural comparison and sequence alignment, it is
evident that the same lysine residue is also conserved in plasminogen and is
located at position 698. When the activation loop remains intact, and the
activation pocket is not occupied by the released amino terminus, the
plasminogen molecule is a two-stage proenzyme which predominantly stays in
its active form. The binding of SK y-domain to the "autolysis" loop region,
which is upstream of the conserved lysine residue is likely to be the trigger
for
plasminogen to switch from its inactive form to the active form. In such an
active form, Lys698 forms the critical salt bridge with Asp'4°. On the
other hand,
when the activation pocket is occupied by the released amino terminus, the
binding of SK y-domain will have little effect on the amidolytic activity of
plasmin(ogen). Some factors, which were proposed to favor the active form of
single-chain t-PA and v-PA, seem to have little effect on plasminogen
activation
by SK. For example, the so called zymogen triad, Asp'94, His4°, and
Ser32,
present in trypsinogen hut not in t-PA or v-PA, was assumed to lock Asp'94,
and
thus the oxyanion stabilizing loop, in its inactive form. These residues are
present in plasminogen as Asp'a°, Hissss~ ~d SerS'g, but they do not
prohibit the
formation of active-zymogen upon binding with SK.
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It is believed that the SK 'y-domain binds to plasminogen to create an
active-zymogen, while the binding of the SK a- and ~i-domains changes the
substrate specificity of the active-zymogen. Although the detailed mechanism
remains to be established, this model can be used to explain the following
observations. The combination of SK(220-414) and SK( 16-251 ), but not either
peptide alone, effectively activates human plasminogen (Young et al., Journal
of
Biological Chemistry 1998; 273: 3110-3116). An explanation to this
observation might simply be that the a-domain and y-domain have different
functions that compensate with each other in plasminogen activation. SK(16-
251) dose-dependently enhanced the activation of plasminogen by SK(16-414)
(Young et al., Journal of Biological Chemistry 1998; 273 : 3110-3116). SK or
SK(16-414) can convert plasminogen to plasmin; however, plasmin alone can
not convert other plasminogen to plasmin. The additional SK(16-251) peptides,
on the other hand, form complexes with the newly formed plasmin molecules
and modulate their substrate specificity such that new plasminogen activators
are formed.
Example 8: Structure comparison between the a-domain of SK and
staphylokinase
Among the (3-grasp folding family (Murzin et al., Journal ofMolecular
Biology 1995; 247: 536-540), staphylokinase (SAK) is another bacterial source
plasminogen activator, whose crystal structure has been determined recently
(Murzin et al., Journal ofMolecular Biology 1995; 247: 536-540). Like SK,
staphylokinase activates human plasminogen by forming a zymogen-activator
complex {Lijnen et al., Journal of Biological Chemistry 1991; 266: 11826-
11832). However, SK and staphylokinase do not share detectable sequence
homology. The size of staphylokinase is only one third that of streptokinase,
and its binding mode and activation mechanism to human plasminogen are
unknown. It is particularly interesting to find that staphylokinase shares a
three-
dimensional folding with the SK a-domain (see Figure 8). The coordinate rmsd
CA 02327526 2000-11-06
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between staphylokinase and the SK a-domain is 1.8 A for 91 Ca atoms, using a
4.0 A cutoff
Based on the three-dimensional structural similarity between the SK oc-
domain and staphylokinase, we propose that staphylokinase binds to
plasminogen in the same mode as the SK oc-domain. Along this line, several
observations on the staphylokinase-plasminogen interaction could be explained.
First, SAK G1u46, which corresponds to SK G1u39, was found to be important for
the formation of the SAK-Plg complex (Silence et al., Journal of Biological
Chemistry 1995; 270: 27192-27198). This residue would be located on the /32
strand and form a salt-bridge with Arg"9 of plasmin(ogen). SAK Met2~, which
corresponds to SK Val'9, was also found to be of crucial importance for the .
activation of plasminogen by staphylokinase (Schlott et al., Biochemical and
Biophysical Acta. 1994; 1204: 235-242). Since the side chain at this position
has van de Waals contact with the hydrophobic portion of the Arg"9 side chain,
disturbing such a contact would result in disturbing the complex contact
through
both van de Waals and electrostatic interactions. A few more residues of
staphylokinase that were found important for the activity of the complex of
SAK-Plg, including LysS°, G1u65, and Asp69, all would be located
on the
substrate binding surface that is similar to that we have proposed for the SK-
p,Plm complex. In such a scenario, the N-terminal fragment of staphylokinase
(i.e. residues 1-20), most of which are disordered in the crystal structure,
would
be located at a position that potentially could affect the conformation of the
active site by allosteric binding. Therefore, a one domain protein like
staphylokinase would perform multiple functions that are accomplished by
two/three domains in SK.
The teachings of the references cited herein and referenced below are
specifically incorporated herein. Modifications and variations of the present
invention will be obvious to those skilled in the art from the foregoing
detailed
description and are intended to be encompassed by the following claims.
26
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SEQUENCE LISTING
<110> Oklahoma Medical Research Foundation
<120> Thrombolytic Agents Derived from Streptokinase
<130> 5208-213
<140>
<141>
<150> 60/084,392
<151> 1998-05-06
<160> 7
<170> PatentIn Ver. 2.0
<210> 1
<211> 791
<212> PRT
<213> Homo sapiens
<220>
<221> PEPTIDE
<222> (1)..(791)
<223> human plasminogen
<400> 1
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser
1 5 10 15
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr
65 70 75 80
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met
85 90 95
Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser
100 105 110
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu
115 120 125
Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp
130 135 140
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu
145 150 155 160
Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly
165 170 175
Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser
180 185 190
Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys
27
CA 02327526 2000-11-06
195 200 205
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro
210 215 220
Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile
225 230 235 240
Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys
245 250 255
Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val
260 265 270
Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His
275 280 285
Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr
290 295 300
Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn
305 310 315 320
Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser
325 330 335
Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr
340 345 350
Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly
355 360 365
Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser
370 375 380
Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala
385 390 395 400
Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro
405 410 415
Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu
420 425 430
Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val
435 440 445
Val Leu Leu Pro Asn Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe
450 455 460
Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly
465 470 475 480
Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile
485 490 495
Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys
500 505 510
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn
515 520 525
Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro
530 535 540
Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly
545 550 555 560
28
CA 02327526 2000-11-06
Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln
565 570 575
Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu
580 585 590
Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser
595 600 605
Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val
610 615 620
Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu
625 630 635 640
Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala
645 650 655
Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr
660 665 670
Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr
675 680 685
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val
690 695 700
Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val
705 710 715 720
Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser
725 730 735
Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys
740 745 750
Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro
755 760 765
Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile
770 775 780
Glu Gly Val Met Arg Asn Asn
785 790
<210> 2
<211> 562
<212> PRT
<213> Homo Sapiens
<220>
<221> PEPTIDE
<222> (1)..(562)
<223> human tissue plasminogen activator
<400> 2
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Gln Glu Ile His Ala Arg Phe Arg Arg
20 25 30
Gly Ala Arg Ser Tyr Gln Val Ile Cys Arg Asp Glu Lys Thr Gln Met
35 40 45
Ile Tyr Gln Gln His Gln Ser Trp Leu Arg Pro Val Leu Arg Ser Asn
29
CA 02327526 2000-11-06
50 55 60
Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gln Cys His Ser
65 70 75 80
Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr
85 90 95
Cys Gln Gln Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu
100 105 110
Gly Phe Ala Gly Lys Cys Cys Glu Ile Asp Thr Arg Ala Thr Cys Tyr
115 120 125
Glu Asp Gln Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser
130 135 140
Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gln Lys Pro
145 150 155 160
Tyr Ser Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His
165 170 175
Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val
180 185 190
Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys
195 200 205
Ser Glu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg
210 215 220
Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn
225 230 235 240
Ser Met Ile Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala
245 250 255
Gln Ala Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly
260 265 270
Asp Ala Lys Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp
275 280 285
Glu Tyr Cys Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gln Tyr
290 295 300
Ser Gln Pro Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala
305 310 315 320
Ser His Pro Trp Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro
325 330 335
Gly Glu Arg Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile
340 345 350
Leu Ser Ala Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu
355 360 365
Thr Val Ile Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu
370 375 380
Gln Lys Phe Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp
385 390 395 400
Asp Thr Tyr Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser
405 410 415
CA 02327526 2000-11-06
Ser Arg Cys Ala Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro
420 425 430
Pro Ala Asp Leu Gln Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly
435 440 445
Tyr Gly Lys His Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys
450 455 460
Glu Ala His Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gln His
465 470 475 480
Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr
485 490 495
Arg Ser Gly Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln Gly Asp
500 505 510
Ser Gly Gly Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val
515 520 525
Gly Ile Ile Ser Trp Gly Leu Gly Cys Gly Gln Lys Asp Val Pro Gly
530 535 540
Val Tyr Thr Lys Val Thr Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met
545 550 555 560
Arg Pro
<210> 3
<211> 431
<212> PRT
<213> Homo sapiens
<220>
<221> PEPTIDE
<222> (1)..(431)
<223> urokinase
<400> 3
Met Arg Ala Leu Leu Ala Arg Leu Leu Leu Cys Val Leu Val Val Ser
1 5 10 15
Asp Ser Lys Gly Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp
20 25 30
Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile
35 40 45
His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile
50 55 60
Asp Lys Ser Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly
65 70 75 80
Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser
85 90 95
Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu
100 105 110
Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg
115 120 125
Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln
31
CA 02327526 2000-11-06
130 135 140
Glu Cys Met Val His Asp Cys Ala Asp Gly Lys Lys Pro Ser Ser Pro
145 150 155 160
Pro Glu Glu Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg
165 170 175
Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp
180 185 190
Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val
195 200 205
Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His
210 215 220
Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly
225 230 235 240
Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val
245 250 255
Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His
260 265 270
His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys
275 280 285
Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr
290 295 300
Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys
305 310 315 320
Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val
325 330 335
Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr Tyr Gly
340 345 350
Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln Trp Lys
355 360 365
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Ser Leu
370 375 380
Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys
385 390 395 400
Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu
405 410 415
Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu
420 425 430
<210> 4
<211> 415
<212> PRT
<213> Streptococcus sp.
<220>
<221> PEPTIDE
<222> (1)..(415)
<223> streptokinase
32
CA 02327526 2000-11-06
<400> 4
Ile Ala Gly Pro Glu Trp Leu Leu Asp Arg Pro Ser Val Asn Asn Ser
1 5 10 15
Gln Leu Val Val Ser Val Ala Gly Thr Val Glu Gly Thr Asn Gln Asp
20 25 30
Ile Ser Leu Lys Phe Phe Glu Ile Asp Leu Thr Ser Arg Pro Ala His
35 40 45
Gly Gly Lys Thr Glu Gln Gly Leu Ser Pro Lys Ser Lys Pro Phe Ala
50 55 60
Thr Asp Ser Gly Ala Met Ser His Lys Leu Glu Lys Ala Asp Leu Leu
65 70 75 80
Lys Ala Ile Gln Glu Gln Leu Ile Ala Asn Val His Ser Asn Asp Asp
85 90 95
Tyr Phe Glu Val Ile Asp Phe Ala Ser Asp Ala Thr Ile Thr Asp Arg
100 105 110
Asn Gly Lys Val Tyr Phe Ala Asp Lys Asp Gly Ser Val Thr Leu Pro
115 120 125
Thr Gln Pro Val Gln Glu Phe Leu Leu Ser Gly His Val Arg Val Arg
130 135 140
Pro Tyr Lys Glu Lys Pro Ile Gln Asn Gln Ala Lys Ser Val Asp Val
145 150 155 160
Glu Tyr Thr Val Gln Phe Thr Pro Leu Asn Pro Asp Asp Asp Phe Arg
165 170 175
Pro Gly Leu Lys Leu Thr Lys Leu Leu Lys Thr Leu Ala Ile Gly Asp
180 185 190
Thr Ile Thr Ser Gln Glu Leu Leu Ala Gln Ala Gln Ser Ile Leu Asn
195 200 205
Lys Asn His Pro Gly Tyr Thr Ile Tyr Glu Arg Asp Ser Ser Ile Val
210 215 220
Thr His Asp Asn Asp Ile Phe Arg Thr Ile Leu Pro Met Asp Gln Glu
225 230 235 240
Phe Thr Tyr Arg Val Lys Asn Arg Glu Gln Ala Tyr Arg Ile Asn Lys
245 250 255
Lys Ser Gly Leu Asn Glu Glu Ile Asn Asn Thr Asp Leu Ile Ser Leu
260 265 270
Glu Tyr Lys Tyr Val Leu Lys Lys Gly Glu Lys Pro Tyr Asp Pro Phe
275 280 285
Asp Arg Ser His Leu Lys Leu Phe Thr Ile Lys Tyr Val Asp Val Asp
290 295 300
Thr Asn Glu Leu Leu Lys Ser Glu Gln Leu Leu Thr Ala Ser Glu Arg
305 310 315 320
Asn Leu Asp Phe Arg Asp Leu Tyr Asp Pro Arg Asp Lys Ala Lys Leu
325 330 335
Leu Tyr Asn Asn Leu Asp Ala Phe Gly Ile Met Asp Tyr Thr Leu Thr
340 345 350
33
CA 02327526 2000-11-06
Gly Lys Val Glu Asp Asn His Asp Asp Thr Asn Arg Ile Ile Thr Val
355 360 365
Tyr Met Gly Lys Arg Pro Glu Gly Glu Asn Ala Ser Tyr His Leu Ala
370 375 380
Tyr Asp Lys Asp Arg Tyr Thr Glu Glu Glu Arg Glu Val Tyr Ser Tyr
385 390 395 400
Leu Arg Tyr Thr Gly Thr Pro Ile Pro Asp Asn Pro Asp Asp Lys
405 410 415
<210> 5
<211> 136
<212> PRT
<213> Staphylococcus aureus
<220>
<221> PEPTIDE
<222> (1)..(136)
<223> staphylokinase
<400> 5
Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala Ser
1 5 10 15
Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val
20 25 30
Glu Gly Lys Glu Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro
35 40 45
Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val
50 55 60
Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu
65 70 75 80
Leu Asp Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys
85 90 95
Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val
100 105 110
Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile
115 120 125
Thr Lys Val Val Ile Glu Lys Lys
130 135
<210> 6
<211> 812
<212> PRT
<213> Bos taurus
<220>
<221> PEPTIDE
<222> (1)..(812)
<223> bovine plasminogen
<400> 6
Met Leu Pro Ala Ser Pro Lys Met Glu His Lys Ala Val Val Phe Leu
1 5 10 15
Leu Leu Leu Phe Leu Lys Ser Gly Leu Gly Asp Leu Leu Asp Asp Tyr
34
CA 02327526 2000-11-06
20 25 30
Val Asn Thr Gln Gly Ala Ser Leu Leu Ser Leu Ser Arg Lys Asn Leu
35 40 45
Ala Gly Arg Ser Val Glu Asp Cys Ala Ala Lys Cys Glu Glu Glu Thr
50 55 60
Asp Phe Val Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys
65 70 75 80
Val Val Met Ala Glu Asn Ser Lys Asn Thr Pro Val Phe Arg Met Arg
85 90 95
Asp Val Ile Leu Tyr Glu Lys Arg Ile Tyr Leu Leu Glu Cys Lys Thr
100 105 110
Gly Asn Gly Gln Thr Tyr Arg Gly Thr Thr Ala Glu Thr Lys Ser Gly
115 120 125
Val Thr Cys Gln Lys Trp Ser Ala Thr Ser Pro His Val Pro Lys Phe
130 135 140
Ser Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn Tyr Cys Arg
145 150 155 160
Asn Pro Asp Asn Asp Glu Asn Gly Pro Trp Cys Tyr Thr Thr Asp Pro
165 170 175
Asp Lys Arg Tyr Asp Tyr Cys Asp Ile Pro Glu Cys Glu Asp Lys Cys
180 185 190
Met His Cys Ser Gly Glu Asn Tyr Glu Gly Lys Ile Ala Lys Thr Met
195 200 205
Ser Gly Arg Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His
210 215 220
Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Met Asn Tyr
225 230 235 240
Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr Asp
245 250 255
Pro Gln Lys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys Thr Thr Pro
260 265 270
Pro Pro Ser Ser Gly Pro Lys Tyr Gln Cys Leu Lys Gly Thr Gly Lys
275 280 285
Asn Tyr Gly Gly Thr Val Ala Val Thr Glu Ser Gly His Thr Cys Gln
290 295 300
Arg Trp Ser Glu Gln Thr Pro His Lys His Asn Arg Thr Pro Glu Asn
305 310 315 320
Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asn Gly
325 330 335
Glu Lys Ala Pro Trp Cys Tyr Thr Thr Asn Ser Glu Val Arg Trp Glu
340 345 350
Tyr Cys Thr Ile Pro Ser Cys Glu Ser Ser Pro Leu Ser Thr Glu Arg
355 360 365
Met Asp Val Pro Val Pro Pro Glu Gln Thr Pro Val Pro Gln Asp Cys
370 375 380
CA 02327526 2000-11-06
Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile
385 390 395 400
Thr Gly Arg Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His
405 410 415
Leu Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr
420 425 430
Cys Arg Asn Pro Asp Ala Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp
435 440 445
Pro Arg Val Arg Trp Glu Phe Cys Asn Leu Lys Lys Cys Ser Glu Thr
450 455 460
Pro Glu Gln Val Pro Ala Ala Pro Gln Ala Pro Gly Val Glu Asn Pro
465 470 475 480
Pro Glu Ala Asp Cys Met Ile Gly Thr Gly Lys Ser Tyr Arg Gly Lys
485 490 495
Lys Ala Thr Thr Val Ala Gly Val Pro Cys Gln Glu Trp Ala Ala Gln
500 505 510
Glu Pro His Gln His Ser Ile Phe Thr Pro Glu Thr Asn Pro Gln Ser
515 520 525
Gly Leu Glu Arg Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Asn Gly
530 535 540
Pro Trp Cys Tyr Thr Met Asn Pro Arg Lys Pro Phe Asp Tyr Cys Asp
545 550 555 560
Val Pro Gln Cys Glu Ser Ser Phe Asp Cys Gly Lys Pro Lys Val Glu
565 570 575
Pro Lys Lys Cys Ser Gly Arg Ile Val Gly Gly Cys Val Ser Lys Pro
580 585 590
His Ser Trp Pro Trp Gln Val Ser Leu Arg Arg Ser Ser Arg His Phe
595 600 605
Cys Gly Gly Thr Leu Ile Ser Pro Lys Trp Val Leu Thr Ala Ala His
610 615 620
Cys Leu Asp Asn Ile Leu Ala Leu Ser Phe Tyr Lys Val Ile Leu Gly
625 630 635 640
Ala His Asn Glu Lys Val Arg Glu Gln Ser Val Gln Glu Ile Pro Val
645 650 655
Ser Arg Leu Phe Arg Glu Pro Ser Gln Ala Asp Ile Ala Leu Leu Lys
660 665 670
Leu Ser Arg Pro Ala Ile Ile Thr Lys Glu Val Ile Pro Ala Cys Leu
675 680 685
Pro Pro Pro Asn Tyr Met Val Ala Ala Arg Thr Glu Cys Tyr Ile Thr
690 695 700
Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Glu Gly Leu Leu Lys Glu
705 710 715 720
Ala His Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Asn Glu Tyr
725 730 735
Leu Asp Gly Arg Val Lys Pro Thr Glu Leu Cys Ala Gly His Leu Ile
36
CA 02327526 2000-11-06
740 745 750
Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys
755 760 765
Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu
770 775 780
Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Pro
785 790 795 800
Tyr Val Pro Trp Ile Glu Glu Thr Met Arg Arg Asn
805 810
<210> 7
<211> 265
<212> PRT
<213> Desmodus rotundus
<220>
<221> PEPTIDE
<222> (1)..(265)
<223> vampire bat saliva plasminogen activator
<400> 7
Thr Cys Gly Leu Arg Lys Tyr Lys Glu Pro Gln Leu His Ser Thr Gly
1 5 10 15
Gly Leu Phe Thr Asp Ile Thr Ser His Pro Trp Gln Ala Ala Ile Phe
20 25 30
Ala Gln Asn Arg Arg Ser Ser Gly Glu Arg Phe Leu Cys Gly Gly Ile
35 40 45
Leu Ile Ser Ser Cys Trp Val Leu Thr Ala Ala His Cys Phe Gln Glu
50 55 60
Ser Tyr Leu Pro Asp Gln Leu Lys Val Val Leu Gly Arg Thr Tyr Arg
65 70 75 80
Val Lys Pro Gly Glu Glu Glu Gln Thr Phe Lys Val Lys Lys Tyr Ile
85 90 95
Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asn Asn Asp Ile Ala Leu
100 105 110
Leu Gln Leu Lys Ser Asp Ser Pro Gln Cys Ala Gln Glu Ser Asp Ser
115 120 125
Val Arg Ala Ile Cys Leu Pro Glu Ala Asn Leu Gln Leu Pro Asp Trp
130 135 140
Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Lys Ser Ser Ser Pro
145 150 155 160
Phe Tyr Ser Glu Gln Leu Lys Glu Gly His Val Arg Leu Tyr Pro Ser
165 170 175
Ser Arg Cys Ala Pro Lys Phe Leu Phe Asn Lys Thr Val Thr Asn Asn
180 185 190
Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Glu Ile Tyr Pro Asn Val
195 200 205
His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asn
210 215 220
37
CA 02327526 2000-11-06
Asp Asn His Met Thr Leu Leu Gly Ile Ile Ser Trp Gly Val Gly Cys
225 230 235 240
Gly Glu Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr Leu
245 250 255
Gly Trp Ile Arg Asp Asn Met His Leu
260 265
38
