Purification and Characterization of A61

AN ANGIOSTATIN-LIKE PLASMINOGEN FRAGMENT PRODUCED BY PLASMIN AUTODIGESTION IN THE ABSENCE OF SULFHYDRYL DONORS*

Geetha Kassam, Mijung Kwon, Chang-Soon Yoon, Kenneth S. GrahamDagger , Mary K. YoungDagger , Stefan Gluck, and David M. Waisman§

From the § Cancer Biology Research Group, Departments of Biochemistry and Molecular Biology and Oncology, University of Calgary, Calgary, Alberta T2N 4N1, Canada and Dagger  Beckman Research Institute of the City of Hope, Duarte, California 91010

Received for publication, October 4, 2000, and in revised form, December 8, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000)Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys78-Lys468. To avoid confusion with the plasmin(ogen) fragment, angiostatin® (Lys78-Ala440), we have named this protein A61. Unlike angiostatin, A61 was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A61 bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A61 was composed of 21% beta -strand, 14% beta -turn, 18% 31-helix and 8% 310-helix. A61 was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A61 by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A61 was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a nonpathological setting.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 epsilon -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 epsilon 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 beta -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 epsilon -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 beta -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 Delta epsilon (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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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 epsilon -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.


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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).

                              
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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.

                              
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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% beta -strand, 14% beta -turn, 18% 31-helix, 8% 310-helix, and 40% unordered. Similar to other kringle-containing structures, A61 does not contain any alpha -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.


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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 epsilon -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 epsilon -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.

                              
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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).


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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.


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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.

                              
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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').


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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.


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

    FOOTNOTES

* This work was supported by a grant from the Medical Research Council of Canada and NSF Grant DBI 9977623, for financial support given to the BRI/City of Hope, U. S. A.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Depts. of Biochemistry and Molecular Biology and Oncology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3022; Fax: 403-283-4841; E-mail: waisman@ucalgary.ca.

Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M009071200

    ABBREVIATIONS

The abbreviations used are: uPA, two-chain urokinase-type plasminogen activator; PAGE, polyacrylamide gel electrophoresis; BCE cells, bovine capillary endothelial cells; HUVEC, human umbilical vein endothelial cells; AMCHA, trans-4-aminomethylcyclohexanecarboxylic acid; A61, plasminogen fragment defined by the amino acid sequence Lys78-Lys468 NAC-N-acetyl-L-cysteine; DIFP, diisopropylfluorophosphate; epsilon -ACA, epsilon -amino-n-caproic acid; DMEM, Dulbecco's modified Eagle's media; bFGF, bovine fibroblast growth factor; MALDI, matrix-assisted laser desorption ionization.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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