 |
INTRODUCTION |
Plasminogen is present in the serum at a concentration of about 2 µM and is concentrated at the endothelial cell surface by virtue of its binding to cell-surface receptors such as annexin II
tetramer (reviewed in Ref. 1). Plasminogen is converted to the active
two-chain form, plasmin, by the cleavage of
Arg561-Val562 bond by tissue plasminogen
activator or urokinase-type plasminogen activator
(uPA).1 The two polypeptide
chains of plasmin are held together by disulfide bridges, and each
polypeptide chain represents a distinct functional domain of the
molecule. The larger polypeptide chain or A-chain (Lys78-Arg561) contains five kringle domains,
and the smaller polypeptide chain or B-chain
(Val562-Asn791) contains the serine protease
domain. The kringle domains contain regions that bind to lysine
residues of target proteins such as substrates, inhibitors, and
cell-surface receptors. For example, the C-terminal lysine residues of
the p11 subunit of annexin II tetramer mediate the binding of
plasminogen to annexin II tetramer (2).
Several biologically active, kringle-containing fragments of
plasmin(ogen) have been shown to occur physiologically. It has been
suggested that either tumor cells or the tumor-infiltrating cells such
as macrophages express proteinases that are capable of cleaving
plasminogen into fragments that consist of various truncations of the
kringle-containing A-chain. Typically, these kringle-containing truncations consist of the first three (K1-3) or
four (K1-4) kringle-containing domains (3-12). Recently, it has been
reported that in addition to their production by cancer cells or
cancer-associated cells, plasminogen fragments similar to the
angiostatins can be produced in non-neoplastic conditions such as a
consequence of the inflammatory response (7, 17). Furthermore,
angiostatin and other A-chain fragments have also been
detected in the tears collected after overnight eye closure suggesting
that these fragments may also be produced under normal conditions
(18).
The biological activity of the K1-3 and K1-4 fragments are very
similar. Since these A-chain fragments can inhibit
experimental primary tumor growth as well as
angiogenesis-dependent growth of metastases in mice, they
have been referred to as angiostatins (13-15). The mechanism of the
anti-angiogenic effect of the angiostatins has been suggested to be due
to endothelial cell apoptosis (16).
Mechanistically, it is unclear how tumor cells or tumor-associated
cells produce the A-chain fragments. One postulated
mechanism involves the direct cleavage of plasminogen by extracellular
proteinases. Dong et al. (6) postulated that the release of
granulocyte-macrophage colony-stimulating factor by certain tumor cells
may cause macrophages to release MMP-12, resulting in the cleavage of
plasminogen to A-chain fragments. Alternatively, plasmin
bound to the surface of macrophages may undergo autodigestion resulting
in the generation of A-chain fragments (7). It has also been
postulated that prostrate carcinoma cells produce a K1-4 fragment by a
two-step process involving the uPA-dependent conversion of
plasminogen to plasmin followed by the reduction of plasmin disulfide
bonds by free sulfhydryl donors and plasmin autodigestion (3, 19). These authors also reported the absolute requirement of free sulfhydryl donors such as N-acetyl-L-cysteine for the
generation of the K1-4 fragment by plasmin autodigestion in
vitro. Alternatively, it has also been reported that HT1080 cells
or Chinese hamster ovary cells may release a plasmin reductase and a
novel serine proteinase that are responsible for conversion of plasmin
to a A-chain fragments consisting of K1-4 and part of K5
(4, 5). Therefore, the central dogma has been that A-chain
fragment production from plasmin(ogen) is absolutely dependent on the
presence of sulfhydryl donors and/or a plasmin reductase.
We sought to provide an independent analysis of the mechanism of
A-chain fragment formation. Contrary to other reports, we have established that plasmin autodigestion can generate an
A-chain fragment with angiostatin-like properties in the
absence of free sulfhydryl donors or plasmin reductase. The major
A-chain fragment produced by plasmin autodigestion is a
novel fragment of 61 kDa that consists primarily of the amino acid
sequence Lys78-Lys468 and is composed of
kringle domains 1-4 and part of kringle 5. We have also shown that a
similar A-chain fragment is present in human serum and in
serum from normal and tumor-bearing mice. Furthermore, nontransformed
cells such as bovine capillary endothelial cells also produce a similar
molecule. Our results provide the first evidence that anti-angiogenic
plasminogen fragments are produced in a nonpathological setting simply
as a consequence of plasmin turnover. Our data also suggest that
plasmin autodigestion not only serves to regulate the concentration of
plasma membrane-associated plasmin but also to regulate the production
of anti-angiogenic plasmin fragments.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[Glu]plasminogen was purified from outdated
pooled human plasma by affinity chromatography (20). Both isoforms of
[Glu]plasminogen were used in the experiments described herein.
[Lys78]Plasminogen and two-chain urokinase-type
plasminogen activator (uPA) were generous gifts from Dr. H. Stack
(Abbott). Antibodies were purchased from the following sources:
monoclonal anti-human plasminogen kringle 1-3 antibody from Enzyme
Research Laboratories Inc; monoclonal anti-human angiostatin antibody
(GF-47) from Oncogene Research Products; rabbit anti-mouse angiostatin
antibody from Peptides International; anti-mouse horseradish
peroxidase-conjugated secondary antibody from Santa Cruz Biotechnology.
Purified commercial human angiostatin was purchased from Calbiochem.
Cell lines were obtained from the following sources: HT1080
fibrosarcoma cells, Lewis lung carcinoma cells, and HeLa cells from
American Type Culture Collection (ATCC); bovine capillary endothelial
cells from Clonetics; human umbilical vein endothelial cells were a generous gift from Dr. Kamela Patel (University of Calgary). DMEM was
obtained from JRH Biosciences and Life Technologies, Inc. Acetonitrile,
ethyl acetate, and heptane were purchased from Burdick and Jackson.
Diphenyl chlorophosphate, potassium thiocyanate, pyridine,
pentanesulfonic acid, phosphoric acid, and all other organic solvents
were purchased from Aldrich. Reagent 4R and the amino acid
thiohydantoin standard mixture for the HP G1000A were purchased from
Hewlett-Packard.
Angiostatin Generation--
[Glu]plasminogen (40 µM) was incubated in the presence or absence of 1 mM N-acetyl-L-cysteine (NAC) and
buffer containing 50 mM Tris-HCl (pH 9.0), 20 mM L-lysine, 100 mM NaCl, 1 mM EDTA, and 0.17 µM two-chain uPA. The
reaction mixture was incubated overnight at 37 °C, diluted 4-fold
with 20 mM Hepes (pH 7.4) and 140 mM NaCl,
adjusted to 1 mM DIFP, and applied to a
L-lysine-Sepharose column (previously equilibrated with 20 mM Hepes (pH 7.4) and 140 mM NaCl). After a
5-column volume wash with the equilibration buffer, the column was
subjected to a linear gradient of 0-125 mM
-ACA, and a
single protein peak was eluted, pooled, and concentrated by
ultrafiltration. Application of this protein peak to Sephacryl S-100
column (previously equilibrated with 20 mM Hepes (pH 7.4) and 140 mM NaCl) resulted in the elution of a single
protein peak that was pooled and frozen at
80 °C in small
aliquots. Reduced SDS-PAGE established that this protein peak was
composed of a doublet consisting of a major and minor Coomassie
Blue-stained protein bands of 61 and 64 kDa, respectively (Fig.
1A). Therefore, these A-chain fragments were
named A61. The concentration of A61 was
determined using an
1%,280 nm 13.6, and a molecular mass of 61 kDa. Typically, 51 mg of A61 was recovered from
100 mg of [Glu]plasminogen. A61 was also formed with a
comparable yield when [Lys]plasminogen was substituted for
[Glu]plasminogen.
Immediately before use in the endothelial cell proliferation assay,
aliquots of A61 were applied to a Detoxi-Gel column
(Pierce), and endotoxin was removed according to the manufacturer's
instructions. Endotoxin contamination of the purified angiostatin was
analyzed by Pyrotell Limulus amebocyte lysate assay
(Associates of Cape Cod Inc, Falmouth, MA) and was less than 45 pg of
endotoxin/ml (0.4 enzyme units/ml).
Cell-mediated Generation of Angiostatin--
HT1080 cells were
maintained in DMEM (Life Technologies, Inc.) supplemented with 10%
heat-inactivated fetal bovine serum, 2 mM
L-glutamine, 10 units/ml penicillin G, and 10 µM streptomycin sulfate. Approximately 1 × 105 cells in 1 ml were added to each well of 24-well tissue
culture plates and incubated at 37 °C for 24 h. The cell
monolayers were then washed three times with PBS (PBS,
phosphate-buffered saline (137 mM NaCl, 8 mM
Na2HPO4, 1.4 mM KH2PO4,
2.7 mM KCl, pH 8.0)), and 0.5 to 8 µM
[Glu]plasminogen in DMEM was added to each well. After overnight
incubation, the media were removed and diluted with SDS-PAGE sample
buffer with or without
-mercaptoethanol and subjected to SDS-PAGE
and Western blotting with the monoclonal anti-human plasminogen kringle
1-3 antibody as indicated below.
To isolate sufficient cell generated A61, HT1080 or BCE
cells, cultured in T75 tissue culture flasks, were incubated overnight with DMEM containing 2.7 µM human [Glu] plasminogen.
The culture supernatants (about 100 ml) were centrifuged at 2000 × g at 4 °C for 30 min to remove cell debris. The
supernatants were then applied to a L-lysine-Sepharose
column previously equilibrated with 20 mM Hepes (pH 7.4)
and 140 mM NaCl. The column was subsequently washed with 5 column volumes of the equilibration buffer, and the bound proteins were
eluted with a 0-0.125 M
-ACA gradient. The fractions
were analyzed by SDS-PAGE, and the peak fractions containing
A61 were pooled and concentrated. The proteins were desalted using a Sephadex PD-10 column (Amersham Pharmacia Biotech) into endotoxin-free PBS (Life Technologies, Inc.), filtered, and frozen
in small aliquots at
80 °C.
Endothelial Cell Proliferation Assay--
Bovine capillary
endothelial cells were maintained in DMEM (JRH Biosciences)
supplemented with 10% calf serum, 2 mM
L-glutamine, 10 units/ml penicillin G, 10 µM
streptomycin sulfate, and 3 ng/ml bFGF (Calbiochem). BCE cells between
passages 3 and 5 were plated into 24-well tissue culture plates (3,000 cells/well) and incubated at 37 °C for 24 h. The medium was
replaced with fresh DMEM containing 5% calf serum in the presence or
absence of A61. After a 30-min incubation, bFGF was added
to a final concentration of 1 ng/ml, and cells were further incubated
for 72 h. The cells were trypsinized, resuspended in Isoton II
(Beckman), and counted with a Coulter counter.
Analysis of Angiostatin in Serum--
200 µl of mouse or human
serum was incubated at room temperature for 30 min with 50 µl of a
1:1 suspension of L-lysine-Sepharose matrix (previously
equilibrated with 20 mM Hepes (pH 7.4), 140 mM
NaCl). The matrix was subsequently washed extensively with 5 volumes of
the same buffer. The bound proteins were eluted by boiling the resin
with SDS-PAGE sample buffer, and 20 µl of each sample was subjected
to nonreduced SDS-PAGE and Western blotting (below).
Murine Tumor Model--
Lewis lung carcinoma cells were grown,
harvested at log phase, and resuspended in PBS. Approximately
106 cells were injected subcutaneously in the middle dorsum
of 6-8-week-old C57BL/6 male mice. When tumors reached 1500 mm3 in size (~14 days after implantation), the mice were
randomly separated into two groups. The first group underwent surgical removal of the tumor, and the second group was subjected to a sham
surgical procedure in which tumors were manipulated but were left
intact. Animals from the tumor-resected group were randomly placed into
test and control groups. The test group of mice received daily
intraperitoneal injections of A61 in PBS (dose = 2.5 mg/kg/day), whereas the control group received PBS alone. Every 3rd day
after tumor resection, mice were sacrificed, and the lungs were weighed.
N- and C-terminal Sequencing of Angiostatin--
N-terminal
sequence analysis was performed by the University of Calgary Sequence
Analysis Facility and Alberta Peptide Institute (Edmonton, Canada).
C-terminal sequence analysis was performed by the Beckman Research
Institute of the City of Hope (Duarte, CA). A61 was
sequenced using a Hewlett-Packard HP G1000A C-terminal protein
sequencer and an Applied Biosystems 477C Procise C C-terminal sequencer. Consistent results were obtained with both sequencers. The
Hewlett-Packard HP G1000A, C-terminal protein sequencer utilized chemistry version 2.0 using diphenylphosphorylisothiocyanate as the
activating agent (21) and a modified cleavage system using lithium
ethiolate as the cleavage agent (48). Applied Biosystems 477C
Procise C C-terminal sequencer was running standard chemistry as
described (22).
Electrophoresis and Western Blotting--
Samples were diluted
with SDS-PAGE sample buffer in the presence (reduced SDS-PAGE) or
absence (nonreduced SDS-PAGE) of
-mercaptoethanol and subjected to
SDS-PAGE (23). Proteins were transferred to 0.45-µm pore size
nitrocellulose membrane using a Bio-Rad transblot apparatus at 4 °C
for 1 h for mini-gels or overnight for larger gels. The membrane
was blocked in TPBS (where TPBS is phosphate-buffered saline
containing 137 mM NaCl, 8 mM
Na2HPO4, 1.4 mM
KH2PO4, 2.7 mM KCl (pH 8.0), and
0.1% Tween 20) containing 5% skim milk at room temperature for 1 h and then incubated with primary antibody in TPBS containing 1% skim
milk at 4 °C overnight. The following dilutions of primary antibody
were used: 1:5000 dilution of 1 mg/ml monoclonal anti-human plasminogen
kringle 1-3 antibody; 1:500 dilution of 0.1 mg/ml monoclonal
anti-human angiostatin antibody (GF-47); 1:500 dilution of polyclonal
anti-mouse angiostatin antibody. The blot was then washed with TPBS at
least 6 times (10 min each) at room temperature and then incubated in
1:1000 dilution of anti-mouse or anti-rabbit horseradish
peroxidase-conjugated secondary antibody in TPBS containing 1% skim
milk at 37 °C for 1 h. The membrane was extensively washed with
TPBS at least six times (10 min each), developed with the Supersignal
reagent (Pierce), and visualized by chemiluminescence.
Circular Dichroism--
Circular dichroism (CD) measurements
were performed with a Jasco J-810 spectropolarimeter. The
spectropolarimeter was calibrated with an aqueous solution of
recrystallized ammonium d-10-camphorsulfonate. A61
(0.4-0.6 mg/ml) was incubated in 10 mM Tris-HCl (pH 7.5)
and 150 mM NaCl in the presence or absence of ligand for 20 min at room temperature. Samples (0.1 ml) were scanned in a quartz
cuvette (0.5-mm path length) from 178 to 260 nm at a rate of 20 nm/min, using a bandwidth of 1 nm and a response time of 4 s. CD spectra of proteins were obtained by averaging three wavelength scans and were
corrected by subtracting buffer scans or, where appropriate, scans of
ligand in buffer. Results are expressed as

(M
1·cm
1).
The A61 secondary structure content was assessed with the
program CDsstr version 1.8 (24).
Intrinsic Fluorescence Measurements--
Excitation and emission
spectra were collected with a PerkinElmer Life Sciences LS 50B
fluorescence spectrometer equipped with a constant temperature cell
holder. The excitation and emission slit widths were set to 5 and 10 nm, respectively. The spectra were collected at 25 °C with
A61 (3.7 µM) in a buffer consisting of 20 mM Hepes (pH 7.4) and 140 mM NaCl and in the
presence or absence of ligand. The data were corrected for the slight
dilution consequent to ligand additions.
Measurement of Molecular Mass by Mass Spectrometry--
Mass
spectrometry was performed by the Beckman Research Institute of the
City of Hope. Matrix-assisted laser desorption ionization (MALDI) mass
spectra were obtained using a Voyager DE-STR (PE Biosystems) MALDI
time-of-flight mass spectrometer. The analyte solutions were
individually loaded onto pre-equilibrated ZipTips (Millipore Corp.) in
four 4-µl increments, for de-salting and pre-concentration. The
analytes were each eluted from their Tip with 2 µl of recrystallized
Sinapinic (3,5-dimethoxy-4-hydroxycinnamic acid) matrix solution, 1 µl at a time, onto a 100-well stainless steel sample slide. After
introduction of the slide into the mass spectrometer, a linear MALDI
method was employed to obtain sample spectra. The method employed a
20-kV accelerating voltage, a nitrogen laser (337 nm, UV wavelength),
and a 350-ns extraction delay time. Each data set was noise-filtered,
smoothed twice, entroided, and de-isotoped. This processing removes the
confusion of isoform mass overlap by removing the isotope contributions
from the centroided-isoform spectrum.
 |
RESULTS |
Identification of the Plasmin(ogen) Fragments Produced by Plasmin
Autodigestion in Vitro--
Gately et al. (19) observed
that a K1-4-containing plasminogen cleavage product of 50 kDa
(nonreduced SDS-PAGE) was generated in a cell-free system consisting of
plasminogen, uPA, and N-acetyl-L-cysteine. Interestingly, it was also reported that the formation of the K1-4
plasminogen A-chain fragment was absolutely dependent on the
presence of a sulfhydryl donor such as
N-acetyl-L-cysteine. However, in a previous
report we observed that plasmin autodigestion resulted in the
generation of a similar A-chain fragment in the absence of
sulfhydryl donors (25). To re-examine the issue of the requirement of
sulfhydryl donors for the generation of A-chain fragments,
we incubated human plasminogen with uPA overnight without any
sulfhydryl donor. The reaction products were then purified by
L-lysine-Sepharose affinity and gel permeation
chromatography. We found that the overnight incubation of plasminogen
with uPA resulted in the generation of a doublet of apparent mass of 61 and 64 kDa, respectively, on a Coomassie-stained, denaturing, reduced
SDS-PAGE (Fig. 1A). The two
fragments produced a diffuse band of an apparent mass of 50 kDa on
Coomassie-stained, denaturing, nonreduced SDS-PAGE (Fig.
1B). These plasminogen fragments reacted with a monoclonal
antibody against the K1-3 fragment of plasminogen, confirming that the
plasminogen fragments isolated by our procedure were A-chain
fragments (Fig. 1C). Since the major A-chain
fragment produced in our cell-free system was 61 kDa on reduced
SDS-PAGE, we have named this plasminogen fragment A61.
Typically, we recovered 51 mg of A61 from 100 mg of
plasminogen.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of plasminogen fragments
produced in a cell-free system. [Glu]plasminogen (40 µM) was incubated in buffer containing 50 mM
Tris-HCl (pH 9.0), 20 mM L-lysine, 100 mM NaCl, 1 mM EDTA, and 0.17 µM
two-chain uPA (lane 1). The reaction mixture was incubated
at 37 °C overnight (lane 2). The mixture was diluted
4-fold with 20 mM Hepes (pH 7.4) and 140 mM
NaCl, adjusted to 1 mM DIFP, and applied to a
lysine-Sepharose (Lys-Seph) column (previously equilibrated with 20 mM Hepes (pH 7.4) and 140 mM NaCl). After a 5-column volume wash with the
equilibration buffer, the column was subjected to a linear gradient of
0-125 mM -ACA gradient. Fractions containing 61- and
64-kDa plasminogen fragments were pooled (lane 3),
concentrated by ultrafiltration, and applied to a Sephacryl gel
permeation (Gel Perm) chromatography column previously
equilibrated with 20 mM Hepes (pH 7.4) and 140 mM NaCl. The protein peak was then pooled (lane
4). Aliquots of pooled fractions were analyzed by reduced
(A) and nonreduced SDS-PAGE (B) and stained with
Coomassie Blue. Alternatively, aliquots were analyzed by nonreduced
SDS-PAGE, transferred to nitrocellulose, and Western blotted using the
monoclonal anti-human plasminogen kringle 1-3 antibody
(C).
|
|
The A61 could have been produced in our cell-free system as
a result of the cleavage of plasminogen to plasmin by the uPA followed
by the autodigestion of this plasmin. Alternatively, the direct
cleavage of plasminogen by plasmin could have been responsible for the
generation of A61.
To examine further the mechanism of A61 production, we
examined the time course of generation of A61 from
plasminogen and uPA. As shown in Fig.
2A, 5 min after initiation of
the reaction, the plasminogen was completely converted to plasmin, and
the plasmin heavy and light chains were visualized by reduced SDS-PAGE.
However, at this point in the reaction, A61 was not
present. After the reaction progressed for about 1 h, degradation
of the plasmin heavy and light chains was apparent. At 2-4 h the
plasmin light chain had disappeared, whereas little change in the
plasmin heavy chain was apparent. However, analysis of the 2-4-h
reaction by nonreduced SDS-PAGE clearly showed that during this period
A61 was generated (Fig. 2B). We also observed
that a recombinant human plasminogen (S741C) that is catalytically
inactive when converted to plasmin was not converted to A61
(data not shown). These results therefore suggest that plasminogen is
cleaved by uPA to form plasmin which then undergoes autodigestion
resulting in the formation of A61.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 2.
Time course analysis of plasminogen cleavage
to A61. [Glu]plasminogen (Pg) (8.7 µM) was incubated at 37 °C in a buffer containing 50 mM Tris (pH 9.0), 100 mM NaCl, 1 mM
EDTA, and 20 mM L-lysine. The reaction was
initiated by addition of 39 nM uPA, and aliquots of the
reaction were removed at the indicated times, subjected to reduced
(A) and nonreduced-SDS-PAGE (B), and stained with
Coomassie Blue. PmH, plasmin heavy chain;
PmL, plasmin light chain.
|
|
Characterization of A61--
The large scale
purification of A61 from plasminogen allowed a detailed
characterization of the structure of the molecule. The N-terminal
sequence of the A61 was determined as
Lys78-Glu83. Major and minor C-terminal
sequences were identified as Lys468-Gly465 and
Arg471-Gly467, respectively (Table
I). Thus the N- and C-terminal sequencing establishes that the autoproteolytic cleavage sites of plasmin were
Lys77-Lys78,
Lys468-Gly469, and
Arg471-Gly472. Therefore, the major species of
A61 has the primary amino acid sequence of
Lys78-Lys468, whereas the minor species is
Lys78-Arg471. The average molecular mass
measured from this amino acid sequence is 44.1 kDa. Mass spectrometry
of A61 revealed a broad cluster of features of about 1-kDa
width. The dominant species in the cluster smoothed into a
Mr of 46,616 when the cluster was centroided and
de-isotoped. The 44.1-kDa fragment derived from the analysis of
the primary structure is significantly smaller than the average apparent mass of 46.6 kDa measured by mass spectrometry. This difference is probably due to the glycosylation of the molecule (26,
27).
View this table:
[in this window]
[in a new window]
|
Table I
Amino- and carboxyl-terminal sequence of human A61
Human A61 was generated from human plasminogen and uPA
in the cell-free system as described under "Experimental
Procedures." The molar ratio of A61 C-sequence A and B was
2:5.
|
|
A comparison of the amino acid sequence of A61 with other
A-chain fragments is presented in Table
II. These data establish that
A61 is a novel A-chain fragment. The sequence of
the molecule suggests that A61 is similar to other
A-chain fragments such as K1-4 angiostatin. However, unlike
the majority of angiostatins, A61 consists of the first
four kringles of plasminogen plus the K4-K5 linker region and seven
residues of the fifth kringle.
View this table:
[in this window]
[in a new window]
|
Table II
Comparison of various A-chain fragments
The abbreviations used are: K, kringle; r, amino acid residues; MME,
macrophage metalloelastase. Numbering is based on the sequence of human
plasminogen (791 residues) excluding the 19 amino acid signal peptide
that ends at Met19 and beginning at Glu1 (47). The
angiostatin activity of the A-chain fragments, the
IC50, was determined as the concentration of A-chain
fragment required to half-maximally inhibit the proliferation of bovine
capillary endothelial cells. To allow a direct comparison of the
angiostatin activity, results were pooled from several reports (14).
|
|
We used circular dichroism to examine the secondary structure of
A61 (Fig. 3A). The
far-UV circular dichroism spectrum of A61 exhibited a
strong negative band at 202 nm and a weak positive band at about 227 nm. This spectrum is similar to that reported for other kringle
containing A-chain fragments such as K4 (28-30). Analysis
of the secondary structure content from CD spectra yields about 21%
-strand, 14%
-turn, 18% 31-helix, 8%
310-helix, and 40% unordered. Similar to other
kringle-containing structures, A61 does not contain any
-helix structure. These results established that the A61
generated in the cell-free system in the absence of a sulfhydryl donor
did not result in the denaturation of the molecule.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Spectroscopic analysis of
A61. A, CD spectrum of A61.
Wavelength scans were conducted at 20 °C in 10 mM Tris
(pH 7.5), 150 mM NaCl. The protein concentration of
A61 was 8.5 µM. The line through the
points represents the best fit for the data reconstructed from the
average of the calculated combinations of secondary structure content.
B, intrinsic fluorescence spectra of A61 in the
absence and presence of AMCHA. The excitation (Ex) and emission
spectra (Em) of ligand-free (solid line) and
AMCHA-saturated A61 (dotted line) are presented.
The spectrum was measured at 20 °C in 20 mM HEPES (pH
7.4) and 140 mM NaCl. The concentration of A61
was 3.7 µM.
|
|
The kringles of plasminogen play a critical role in substrate
recognition and in determining the conformation of plasminogen. The
binding of lysine-type zwitterions such as
-ACA to the kringles of
plasminogen results in an increase of about 7% in the intrinsic protein fluorescence (31). Fig. 3B presents the excitation
and emission spectra for A61. The excitation and emission
maxima were 283 and 342 nm, respectively. The binding of lysine and
other lysine analogues such as
-ACA and
N-acetyl-L-lysine (which mimics the structure of
a C-terminal lysine) caused a significant increase in the intrinsic
fluorescence emission spectra. AMCHA caused the largest increase (27%)
in fluorescence intensity of the emission spectra. However,
N-acetyl-L-lysine methyl ester, a lysine
analogue that mimics the structure of an internal lysine residue, did
not cause a significant change in the fluorescence emission spectra. These data are summarized in Table III.
These results suggest that the kringles of A61 are
functionally active and can bind to free lysine residues or C-terminal
lysine residues but not to internal lysine residues.
View this table:
[in this window]
[in a new window]
|
Table III
Effect of lysine analogues on the intrinsic fluorescence emission
spectrum of A61
A61 was incubated at 20 °C in 20 mM HEPES (pH
7.4) and 140 mM NaCl at a concentration of 3.7 µM. The excitation wavelength was 283 nm, and the
emission wavelength was 342 nm. Saturating concentrations of lysine
analogues were used in these experiments. The percent stimulation was
calculated as follows: % stimulation = (EEM 342 nm (A61 + ligand) EEM 342 nm
(A61)/EEM 342 nm (A61)) × 100.
|
|
Biological Activity of A61--
To investigate whether
A61 had angiostatin-like activity, we examined the ability
of A61 to inhibit endothelial cell proliferation. Purified
A61 was incubated with BCE cells that were stimulated with
1 ng/ml FGF. A61 inhibited BCE cell growth in a
dose-dependent manner (Fig.
4A). The concentration of
A61 required for 50% inhibition was about 35 ± 10 nM (mean ± S.D., n = 4) for
A61 prepared in the absence of NAC. Maximum inhibition of
proliferation of the BCE cells was observed at an A61
concentration of about 200 nM. The potency of
A61 prepared in the absence or presence of NAC was
identical. Consistent with other reports, we also found that concentrations of A61 as high as 2 µM did not
inhibit the proliferation of several nonendothelial cell lines such as
HT1080 fibrosarcoma cells, HeLa cells, and 293 cells (data not
shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Biological activity of A61.
A, inhibition of bovine capillary endothelial cell
proliferation. BCE cells were incubated with 1 ng/ml bFGF and various
concentrations of A61 which was prepared in the presence
(open circles) or absence (closed circles) of
NAC. After 72 h of incubation, the cells were trypsinized,
resuspended in Isoton II solution, and counted with a Coulter counter.
B, inhibition of metastatic tumor growth in vivo.
Mice were injected intraperitoneally with PBS or A61 (in
PBS) (2.5 mg/kg/day) immediately after removal of the primary tumor.
The lungs were removed at specific intervals, and the lung weights were
compared.
|
|
We also examined the anti-angiogenic activity of A61
in vivo. C57 BL/6 mice were inoculated with Lewis Lung carcinoma
cells, and after 14 days the tumor was resected. One group of mice
received daily intraperitoneal injections of 2.5 mg of
A61/kg, whereas the control group received PBS (Fig.
4B). The lung weight of the PBS-treated, tumor-resected mice
increased over time. Eighteen days after the tumor resection, lung
weight, which correlated with total tumor burden, reached 770 ± 42 mg (n = 4). In contrast, the average lung weight of
the mice that received daily doses of A61 increased only to
250 ± 14 mg (n = 4). The average weights of lungs
of normal and 18-day tumor-bearing mice were 191 ± 25 (n = 4) and 199 ± 20 mg (n = 4),
respectively. The increase in the weight of the lungs in the
tumor-resected mice corresponded with an increase in the metastatic
foci that were observable (data not shown). These results establish
that A61 is a potent anti-angiogenic protein.
Identification of Cell-generated Plasminogen Fragments--
The
ability of many cells to bind plasminogen at the cell surface has been
well established. Whereas plasminogen can be converted to plasmin at
the cell surface by the action of plasminogen activators such as uPA,
it has been recently shown that other plasminogen fragments are also
produced at the cell surface (3, 32, 33). To compare the plasminogen
fragments produced on the cell surface with A61, HT1080
fibrosarcoma cells were incubated with plasminogen, and the
cell-produced plasminogen fragments were analyzed by reduced (Fig.
5A) or nonreduced (Fig.
5B) SDS-PAGE and Western blotting. When [Glu]plasminogen
was incubated overnight in DMEM in the absence of cells, an
A61-like plasminogen fragment was faintly detected with the
monoclonal anti-human plasminogen kringle 1-3 antibody. However, in the presence of HT1080 cells, a plasminogen fragment of
comparable molecular mass to A61 was easily detected. The
concentration of this plasminogen fragment increased with increasing
plasminogen concentrations.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Comparison of A61 with
cell-generated plasminogen (Pg) fragments. HT1080
cells were incubated with DMEM containing the indicated concentrations
of [Glu]plasminogen. After an overnight incubation, the media were
analyzed by 15% reduced (A) and nonreduced (B)
SDS-PAGE followed by Western blotting with monoclonal anti-human
plasminogen kringle 1-3 antibody: [Glu]plasminogen standard
(lane 1); [Glu]plasminogen (8 µM) after
overnight incubation in the absence of cells (lane 2). The
HT1080 cells were incubated with the following concentrations of [Glu]plasminogen; 0.5 µM (lane 3); 1 µM (lane
4); 2 µM (lane 5); 4 µM
(lane 6); 8 µM [Glu]plasminogen (lane
7); A61 standard (lane 8). Human
[Glu]plasminogen (8 µM) in DMEM was incubated overnight
with HT1080, HeLa, BCE, or HUVEC (C). HT-1080
(lane 3), HeLa (lane 4), BCE (lane 5),
or HUVEC (lane 6). After an overnight incubation the media
were subjected to 12.5% reduced SDS-PAGE followed by Western blotting
with the monoclonal anti-human plasminogen kringle 1-3 antibody. The
plasminogen standard (lane 1) and A61 standard
(lane 2) are also shown. Human [Glu]plasminogen (2.7 µM) was incubated with HT1080 or BCE cells overnight
(D). The A61 was purified from the culture media
of the HT1080 (closed circles) and BCE cells (open
circles) by affinity chromatography with
L-lysine-Sepharose. Various concentrations of the
cell-generated A61 were added to BCE cells, and the extent
of inhibition of BCE cell growth was determined as indicated under
"Experimental Procedures."
|
|
Several other cell lines were compared with HT1080 cells for their
ability to produce plasminogen fragments. Although plasminogen fragments were barely detected in the media obtained from HeLa and
HUVEC, we found that the media from BCE cells contained
A61-like plasminogen fragment (Fig. 5C). This
result demonstrated that normal cells and not just cancer cells could
produce A61-like plasminogen fragments.
Although our data established that normal and cancerous cells produce
an A61-like plasminogen fragment, it does not establish if
these fragments are biologically active. Therefore, we incubated HT1080
and BCE cells with plasminogen and purified the A61-like plasminogen fragment from the conditioned media by affinity
chromatography. As shown in Fig. 5D, the
A61-like plasminogen fragment released into the media by
HT1080 or BCE cells inhibited the proliferation of bovine capillary
endothelial cells with similar potency and was therefore biologically
active. The concentration of A61-like plasminogen fragment
produced by HT1080 and BCE cells necessary for half-maximal inhibition
was about 45 nM.
The A61-like plasminogen fragments that were isolated from
the conditioned media of the HT1080 or BCE cells were also subjected to
N-terminal analysis. These data showed that the N-terminal amino acid
sequence of the A61-like plasminogen fragment obtained from
the media of the HT1080 or BCE cells were identical (Table IV). Furthermore, the N-terminal sequence
of the cell-produced protein was identical to the N-terminal sequence
of the A61 protein produced by our cell-free system.
View this table:
[in this window]
[in a new window]
|
Table IV
N-terminal sequence of cell-generated A61
Human A61 was generated from human plasminogen and uPA in the
cell-free system as described under "Experimental Procedures."
Conditioned media from human HT1080 fibrosarcoma and bovine capillary
endothelial cells were subjected to affinity chromatography with
L-lysine-Sepharose, and the bound proteins were subjected
to SDS-PAGE. The protein band corresponding to A61 was sliced
from the gel and subjected to N-terminal sequencing.
|
|
Identification of Plasminogen Fragments Present in Sera--
It
was originally proposed that an A-chain plasminogen fragment
of 38-43 kDa on reduced SDS-PAGE was present in the urine and serum of
mice bearing Lewis lung carcinoma tumors. This A-chain fragment was named angiostatin and was shown to be present in the serum
of tumor-bearing mice but not in the serum of normal mice or
tumor-resected mice (13). However, as shown in Fig. 6A, the serum from normal,
tumor-bearing, and tumor-resected mice all had detectable levels of an
A61-like plasminogen fragment. This mouse plasminogen
fragment ran with a mass of about 50 kDa on nonreduced SDS-PAGE and was
also detectable with a anti-human K1-3 monoclonal antibody (Fig.
6C, a'). We also examined the serum of mice for several days
after tumor resection. We did not observe an appreciable change in the
concentration of A61-like plasminogen fragment from 3 to 15 days after tumor resection (Fig. 6, B and C,
b').

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
Comparison of A61 with
plasminogen fragments present in mouse sera. Mice sera were
incubated with 50 µl of L-lysine-Sepharose (equilibrated
in 20 mM Hepes, (pH 7.4), 140 mM NaCl) for 30 min at room temperature. The matrix was washed with 5 volumes of
equilibration buffer, boiled with SDS-PAGE sample buffer, and subjected
to nonreduced SDS-PAGE. Western blotting was performed with rabbit
anti-mouse angiostatin antibody (A) or monoclonal anti-human
plasminogen kringle 1-3 antibody (B). The lanes in
A are as follows:, control mouse serum (1 and
2); mouse serum from two individual mice with 14-day old
tumor (3 and 4); mouse serum from two individual
mice 14 days after tumor resection (5 and 6). The
lanes in B are as follows: A61 standard
(1); mouse with 14-day old tumor (2); 3 days
after tumor resection (3); 6 days after tumor resection
(4); 9 days after tumor resection (5); 12 days
after tumor resection (6); 15 days after tumor resection
(7). Alternatively, the Western blot presented in
A and B were probed with human monoclonal
anti-human plasminogen kringle 1-3 antibody (C, a') or
rabbit anti-mouse angiostatin antibody (C, b'),
respectively. The results in A were consistent with our test
groups consisting of 21 normal mice, 9 tumor-bearing mice, and 29 tumor-resected mice.
|
|
The results obtained with mouse serum suggested that
A61-like plasminogen fragments were present in the serum of
normal and tumor-bearing animals. We therefore examined the serum of
healthy human volunteers and found the presence of an
A61-like plasminogen fragment in all of the subjects
examined (Fig. 7A). The human plasminogen fragment corresponded with the upper band of our
A61. We then examined the serum of patients diagnosed with
several forms of cancer. As shown in Fig. 7B, all patients
tested demonstrated the presence of an A61-like plasminogen
fragment in their serum.

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 7.
Comparison of A61 with
plasminogen fragments present in human sera. Frozen human sera
from normal volunteers (A) or cancer patients (B)
were thawed and then incubated with 50 µl of lysine-Sepharose
(equilibrated in 20 mM Hepes (pH 7.4), 140 mM
NaCl) for 30 min at room temperature. The matrix was washed with 5 volumes of equilibration buffer, boiled with SDS-PAGE sample buffer,
and subjected to nonreduced SDS-PAGE. Western blotting was performed
with monoclonal anti-human angiostatin antibody. A,
angiostatin (Calbiochem) standard (250 ng) (lane 1);
A61 standard (250 ng) (lane 2); serum from
healthy laboratory volunteers (lanes 3-11). B,
angiostatin (Calbiochem) standard (250 ng) (lane
1); A61 standard (250 ng) (lane
2); testicular cancer (lane 3); head and neck
cancer (lanes 4 and 5); testicular
cancer (lanes 6-8); head and neck cancer
(lanes 9 and 10).
|
|
 |
DISCUSSION |
Our initial studies of the mechanism of plasmin autodigestion had
shown that a major product of the plasmin autoproteolytic reaction was
an A-chain plasmin fragment of approximately 61 kDa (25).
This report contrasted with an earlier report that concluded that the
generation of an A-chain fragment with angiostatin-like activity in a cell-free system required plasminogen and uPA and was
absolutely dependent on the presence of a sulfhydryl donor (19). Here,
we sought to provide an independent analysis of the mechanism of
A-chain formation. In this communication we report that two
A-chain fragments were generated from plasminogen and uPA in
the absence of a sulfhydryl donor. Since the major protein band on
SDS-PAGE was 61 kDa, we named the A-chain fragment
A61. Since the uPA-dependent conversion of
plasminogen to plasmin precedes the formation of A61, we
have concluded that plasmin autodigestion is responsible for the
formation of A61. The availability of large quantities of
A61 has allowed characterization of the structure, function, and physiological relevance of this A-chain fragment.
N- and C-terminal sequencing has allowed the precise elucidation of the
primary structure of A61 as comprising the amino acid sequence Lys78-Lys468. Other
A-chain fragments have been reported, but the absence of
C-terminal sequencing has made the exact identification of these
fragments difficult. Plasminogen is cleaved by a variety of proteinases
including metalloelastase, matrix metalloproteinases, and serine
proteinases to generate K1-3, K1-4, and K1-4.5 fragments (Table II).
Typically these A-chain fragments terminate at amino acid
residues in the linker regions between the K3 and K4 (C-terminal Val338/354 residues for K1-3), the linker regions between
K4 and K5 (C-terminal Ala440 or Pro446/447 for
K1-4), or within residues of K5 (C-terminal Arg530 for
K1-4.5). Therefore, A61 is a novel A-chain
fragment that belongs to the K1-4.5 class of A-chain
fragments except that it contains only 7 residues of K5.
Comparison of the 46.6-kDa fragment of A61 derived
from mass spectrometric analysis with the 44.5-kDa fragment derived
from the amino acid sequence of the protein suggests that like the parent molecule, plasminogen, A61 is glycosylated.
Plasminogen contains N-linked carbohydrate residues on K3
and two O-linked carbohydrate on Ser249 and
Thr346 of the K2-K3 linker and K3-K4 linker, respectively.
The role of these glycosylations is unclear, but studies of
A-chain fragment glycoforms have suggested that the extent
of glycosylation may affect the biological activity or half-life of the
protein in circulation (34).
The availability of milligram quantities of A61 has allowed
detailed analysis of the secondary structure of the molecule. The
absence of
-helical structure in A61 was indicated from
analysis of the far-UV CD spectrum using CDsstr, a recently released
computer program (24). Overall, the secondary structure of
A61 is comparable to other kringle-containing proteins.
Recently, it was demonstrated that kringle-containing proteins contain
significant 31-helix. This structure was not reported in
earlier x-ray structures because of the difficulty in visualization.
However, the far-UV CD spectrum is uniquely sensitive to
31-helix. The presence of 31-helix in A61 is consistent with the 31-helix structures
recently reported for other kringle-containing structures such as
plasminogen (35). Since the intrinsic fluorescence emission spectrum of
A61 increased in the presence of lysine or lysine
analogues, we have concluded that the kringles of A61 are
functionally active.
A61 inhibits the proliferation of BCE cells and blocks the
growth of Lewis lung carcinoma metastatic foci in C57BL/6 mice. The
IC50 for inhibition of endothelial proliferation by
A61, prepared in the presence or absence of NAC, was
identical. However, our IC50 of 35 nM is
significantly lower than the IC50 of 300 nM
reported by Soff and co-workers (19). It is unclear why this difference in potency is observed. Interestingly, the dosage of A61
used in our experiments to inhibit the growth of Lewis lung metastatic foci in mice, 2.5 mg/kg/day, was also lower than the 15 mg/kg/day used
by Soff and co-workers. A61 is therefore as biologically active as the A-chain fragment made in the presence of
sulfhydryl donors. Since A61 blocks the growth of
metastatic foci, it is by definition an angiostatin.
The concept that plasmin is capable of autodigestion in the absence of
sulfhydryl donors is not novel. Other laboratories have shown that
plasmin autodigestion involves a bimolecular reaction in which both
heavy and light chains are proteolyzed (36-42). Two autodigestive
processes involving human plasmin have been reported by Wu and
co-workers (42). In a slightly acidic solution the light chain was
found to be cleaved faster than the heavy chain of plasmin. The
cleavage of the light chain correlated with loss of plasmin activity.
Both the heavy chain and the light chain were cleaved at pH levels
between 6.5 and 11.0. On the other hand, alkaline pH favored the
cleavage of the heavy chain. A cleaved heavy chain of molecular weight
50,000 (reduced SDS-PAGE) (36) or 58,000 was observed (42). The C
terminus of the Mr 58,000 fragment was shown to
be Arg530 (41). Based on these data it has been theorized
that because of their proximity in the plasminogen structure, the
disulfide bonds between Cys512 and Cys536 and
between Cys462 and Cys541 could be split by
hydroxyl ions (41). The cleavage of the disulfide bonds between
Cys462 and Cys541 could explain how plasmin
autodigestion results in the formation of A61 in the
absence of sulfhydryl donors or a plasmin reductase.
We also compared A61 with A-chain fragments that
were produced by cultured cells or that were present in mouse or human
serum. First, we found that cultured cells produced a plasminogen
fragment of similar Mr to that of
A61 which suggested that the predominant cell-produced
A-chain fragment was composed of at least four intact kringles. Second, the observation that BCE cells produced an
A61-like protein suggested that the formation of
A-chain fragments was not restricted to cancer cells.
Furthermore, the N-terminal sequencing of the A61-like
plasminogen fragment produced by the HT1080 or BCE cells established
that the N terminus of the cell produced A61 was identical
to the A61 produced in our cell-free system. Our
observation that the A61-like protein produced by HT1080 or BCE cells possessed potent anti-angiogenic activity further supports our notion that normal unstimulated cells can produce anti-angiogenic plasminogen fragments. Third, we also observed the presence of an
A-chain fragment similar to A61 in mouse and
human serum. The plasminogen fragment present in mouse serum was of
similar size to the lower molecular weight band of A61,
whereas the plasminogen fragment present in human serum was of similar
size to the higher molecular weight form of A61. These
observations are consistent with the size of the A-chain
fragments produced by cultured cells and suggest that the physiological
A-chain fragment is about 61 kDa. However, it is not clear
if the A61-like plasminogen fragment detected in the sera
of normal, tumor-bearing, or tumor-resected mice are biologically
active. Further experiments will be necessary to establish the amino
acid sequence and carbohydrate sequence of the physiological
A-chain fragment. Circulating plasminogen consists of two
glycoforms, and each glycoform is heterogeneous with respect to the
size of the carbohydrate side chains resulting in at least six
additional species for each glycoform. It will therefore be interesting
to see if the circulating A-chain fragment is also
heterogeneous. Fourth, we found an A61-like protein in the
serum of normal mice and humans. This result suggests that A-chain fragments are not exclusively produced in a
pathophysiological setting.
In conclusion, it has been suggested that angiostatin-like
A-chain fragments are produced by the primary tumor as a
means of inhibiting the growth of metastatic foci. Our experiments, however, have shown that this conceptually satisfying hypothesis does
not accurately reflect the situation in vivo. Rather, we propose that the A61-like A-chain fragments can
be produced merely as a function of plasmin autodigestion. Although our
data establish that normal, tumor-bearing, and tumor-resected mice or
normal or tumor-bearing humans produce the A61-like
A-chain fragment at comparable levels, we cannot at this
time establish if the A61-like A-chain fragment
detected in the sera of mice and humans is biologically active. Since
these proteins were purified by affinity chromatography with
L-lysine-Sepharose, we can conclude that the kringles of
the proteins were not functionally compromised.