From the
Departments of Pathology and
¶Pediatrics, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, March 27, 2003 , and in revised form, April 29, 2003.
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ABSTRACT |
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INTRODUCTION |
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EXPERIMENTAL PROCEDURES |
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ProteinsHuman Pg was resolved into its isoforms, Pg 1 and 2
(17,
18). Pg 2 was digested with
elastase and fractionated by gel and affinity chromatography to obtain
mini-Pg, followed by digestion of mini-Pg with pepsin to obtain K5
(10,
19,
20). Gel electrophoresis
(1020% gradient gel, nonreducing conditions) identified a doublet of
12 kDa, which was identified by mass spectrometry as K5. Amino-terminal
sequence analysis yielded the sequence LPTVETPSEE, corresponding to Pg
residues 450459, confirming the identification of K5
(21). Reduction/alkylation of
K5 was performed by incubating 20 µg of K5 with 1 mM
dithiothreitol for 30 min followed by incubation with 5 mM
iodoacetamide for 30 min, both at room temperature, and removal of these
reagents by dialysis versus 10 mM Hepes, pH 7.5.
Iodination of K5 was performed with 125I-labeled Bolton-Hunter
reagent (specific activity, 500700 cpm/ng).
AntibodiesAntibodies to SK were raised in rabbits, and the IgG fraction specific against the SK sequence Glu263Lys283 was purified by immunoaffinity on a resin containing this peptide conjugated to activated carboxyhexyl-Sepharose (Amersham Biosciences). The antibodies against the 21-amino acid sequence Lys235Lys255 of VDAC1 conjugated to keyhole limpet hemocyanin (22) were prepared in rabbits by COVANCE (Denver, PA). The IgG fraction specific to VDAC1 was purified by immunoaffinity on a resin containing the VDAC1 peptide conjugated to carboxyhexyl-Sepharose. The monoclonal antibody 20B12 against human mitochondrial VDAC1 was from Molecular Probes, Inc.
Endothelial Cell Proliferation AssayHUVEC from Clonetics (San Diego, CA) were grown in Dulbecco's modified Eagle's medium containing 20% bovine serum, 100 units/ml penicillin/streptomycin, 2.5 µg/ml amphotericin B, 2 mM glutamine, 5 units/ml sodium heparin, and 200 µg/ml endothelial cell growth supplement (23). The cells were washed with phosphate-buffered saline and dispersed in a 0.05% trypsin solution. The cells were resuspended in medium (25 x 103 cells/ml) and plated in 96-well culture plates (0.2 ml/well). After 24 h at 37 °C, the medium was replaced with 0.2 ml of Dulbecco's modified Eagle's medium, 5% bovine serum, 1% antibiotics, and the test samples were applied. Cell proliferation was determined at 24 h using bromodeoxyuridine labeling and a colorimetric immunoassay (Roche Applied Science). The results were expressed as percentages of control proliferation determined in the presence of VEGF (10 ng/ml) and the absence of K5.
Flow CytometryHUVEC were detached from the culture flasks
(75 cm2) by incubation for 5 min at 37 °C with Ca2+
and Mg2+-free phosphate-buffered saline containing 4 mM
EDTA and pelleted. The cells (1 x 107/ml) were washed with
phosphate-buffered saline before resuspension in ice-cold Phenol Red-free
Hanks' balanced salt solution (HBSS), 1% BSA, 0.3 mg/ml goat IgG, and 0.01%
NaN3 (staining buffer). The cell suspensions (100 µl) were
incubated 30 min with dilutions of rabbit polyclonal anti-human SK peptide
IgG, anti-human VDAC1 peptide IgG, or the murine anti-human mitochondrial
VDAC1 monoclonal antibody. The cells were washed with ice-cold staining
buffer, pelleted, and resuspended in 100 µl of ice-cold staining buffer.
The cell suspensions were incubated in the dark with an AF488-conjugated for
30 min to goat anti-rabbit or mouse IgG from Molecular Probes, Inc. The cells
were washed twice with ice-cold staining buffer, resuspended in ice-cold 1%
paraformaldehyde, and stored in the dark at 4 °C until analysis by FACS.
The mean relative fluorescence after excitation at = 495 nm was
determined for each sample on a FACSVantage SE flow cytometer (BD Biosciences)
and analyzed with CELLQUEST® software (BD Biosciences).
Ligand Binding AnalysisThe cells were grown in tissue culture plates until the monolayers were confluent. The cells were washed in HBSS. The binding assays were performed at 4 °C in RPMI 1640 containing 2% BSA. Increasing concentrations of 125I-K5 were incubated with cells for 60 min in 96-well strip plates. Free and bound ligand were separated by aspirating the incubation mixture and washing the cell monolayers rapidly thrice with RPMI 1640 containing 2% BSA. The wells were stripped from the plates and radioactivity determined. The bound ligand was calculated after subtraction of nonspecific binding measured in the presence of 50 mM p-aminobenzamidine. The Kd and Bmax of K5 were determined by fitting the data directly to the Langmuir isotherm using the statistical program SYStat® for Windows.
Antibody Binding StudiesThe binding assays were performed in HUVEC grown in 96-well strip plates. The cells were washed in HBSS and incubated with increasing concentrations of 125I-labeled anti-human VDAC1 peptide IgG for 90 min at 25 °C in RPMI 1640 containing 2% BSA. The cells were rinsed with RPMI 1640, and the wells stripped from the plates were inserted in plastic tubes to determine radioactivity. IgG bound was calculated after subtraction of nonspecific binding measured in the presence of 50 µM nonlabeled IgG. The Bmax of the anti-VDAC1 IgG was then calculated.
Measurements of Intracellular Free Ca2+ Concentration and Cytosolic pHiHUVEC [Ca2+]i was measured by digital imaging microscopy using the fluorescent indicator Fura-2/AM (24). For measurements of pHi, HUVEC were incubated overnight in Dulbecco's modified Eagle's medium on glass coverslips and then washed with HBSS with 0.1 M sodium bicarbonate, pH 7.1. The cells were incubated for 20 min with 2 µM 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in HBSS, rinsed with buffer thrice, and placed on the fluorescent microscope stage. Intracellular pH (pHi) was measured by a digital video imaging technique in cells stimulated by the ligands, which were added after obtaining a stable base line (25).
Gel ElectrophoresisElectrophoresis was performed in 0.1% SDS employing a discontinuous Laemmli buffer system (26). The gels were stained with 0.25% Coomassie Brilliant Blue R-250. Transfer to nitrocellulose membranes was carried out by the Western blot method (27). The dye-conjugated Mr markers (Bio-Rad) used were of Mr 38,100, 28,400, 18,200, 9,200, and 4,300.
Purification of VDAC1 From 1-LN CellsIt is difficult to obtain large numbers of cultured HUVEC; however, we found that 1-LN cells are a good source of this protein. 1-LN cells were grown in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin G, and 100 ng/ml streptomycin in 20 culture flasks (150 cm2). After detaching with 10 mM EDTA in HBSS and pelleting, the cells were suspended in 10 ml of 20 mM Hepes, pH 7.2, 0.25 M sucrose containing the proteinase inhibitors (each at 0.5 mg/ml) antipapain, bestatin, chymostatin, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), leupeptin, pepstatin, o-phenanthroline, and aprotinin. The cells were lysed by sonication on ice (five 10-s bursts with 30-s intervals). The homogenate was centrifuged at 800 x g for 15 min, followed by centrifugation at 50,000 x g for 1 h. The pellet containing cell membranes was resuspended in 20 mM Tris-HCl, pH 8.0, containing 1% (v/v) Triton X-100 to solubilize membranes and centrifuged at 50,000 x g for 30 min to remove insoluble materials. VDAC was sequentially purified to homogeneity using gel filtration on Sephadex G-150 and immunoaffinity chromatography with an anti-VDAC peptide IgG conjugated to Sepharose 4-B (see Fig. 1C).
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Incorporation of VDAC1 into Liposomes and Binding of K5 to the
Reconstituted ReceptorPurified VDAC1 was reconstituted into
liposomes (28,
29) as follows. 50 µl of a
suspension of liposomes (8 µM L--phosphatidylcholine, 8
µM phosphatidylethanolamine, 8 µM
-oleoyl-
-palmitate and 6.9 µM cholesterol) in 5%
Me2SO were mixed with VDAC1 (5 µg) and incubated with agitation
for 30 min at room temperature. The concentration of Me2SO was
reduced to 0.5% with 50 mM Tris-HCl, pH 7.4. After the addition of
125I-K5 (10 nM) and incubation for another 30 min at
room temperature, the mixture was filtered through a Sephadex G-75 column (55
x 2 cm). To study inhibition of K5 binding to VDAC1 reconstituted into
liposomes, the mixture was incubated with the specific anti-VDAC1 IgG for 30
min at room temperature before the addition of 125I-K5.
The kinetic parameters of K5 binding to VDAC1 on reconstituted liposomes
were performed on large unilamellar liposomes (0.4 µm in diameter) prepared
by extrusion of multilamellar vesicles through 0.4-µm defined polycarbonate
filters (Nucleopore, Pleasanton, CA)
(30). For these experiments,
proteoliposomes containing VDAC1 or BSA were prepared by mixing the proteins
(50 µg) in 2.5 mM Hepes, pH 7.4, 145 mM NaCl, and 0.3
mM N-dodecyl--D-maltopyranoside with
N-dodecyl-
-D-maltopyranoside saturated (0.6
mM) liposomes at a 1:3 volume ratio of protein preparations to
liposomes (31). The detergent
was removed after three 2-h incubations at 4 °C of the proteoliposomes
with 10 mg of Biobeads SM2 (Bio-Rad) followed by three 30-min centrifugations
at 100,0000 x g. Phospholipid phosphate was then determined
(32). To assess the
orientation and amount of VDAC1 incorporated, the liposomes (10
µM phospholipid) in 2.5 mM Hepes, pH 7.4, were
incubated with increasing amounts of 125I-labeled anti-VDAC1 IgG
for 1 h at room temperature followed by filtration over 0.1-µm pore size
VM-MultiScreen filters (Millipore Corp., Bedford, MA). After three rinses with
200 µl of 2.5 mM Hepes, pH 7.4, the filters were stripped from
the plates, and radioactivity was determined. Increasing concentrations of
125I-K5 were incubated for 1 h at room temperature with VDAC1
proteoliposomes, BSA proteoliposomes, or empty liposomes (10 µM
phospholipid) in 2.5 mM Hepes, pH 7.4, containing 145 mM
NaCl. Filtration and determination of kinetic parameters were carried out as
described above.
Preparation of MitochondriaMitochondria from 1-LN cells were isolated (33), and the protein levels were estimated using the bicinchonic acid method (34).
Mitochondrial Membrane PotentialMembrane potential
() was determined at room temperature using DSMP+, a fluorescent
indicator of membrane potential
(35). The assay consisted of a
final volume of 2 ml containing sucrose (250 mM), Hepes (10
mM), EGTA (2.5 mM) pH 7.4, mitochondria (0.2 mg)
cellular protein, DSPM + 2 (nmol), rotenone (1 µg), sodium succinate (10
µM), pH 7.4, and increasing concentrations of K5. The mixture
was incubated for 20 min with K5 prior to addition of DSPM+. The fluorescence
intensity was measured (excitation
= 489 nm, emission
= 566
nm) in a Shimadzu RF-5301PC spectrofluorometer (Shimadzu Corporation, Kyoto,
Japan). Maximal fluorescence in the absence of K5 was obtained with
mitochondria incubated with DSPM+ alone. The results are the means of
fluorescence determinations from three experiments.
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RESULTS |
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Binding of K5 to VDAC1 Incorporated into LiposomesThe experiments described above demonstrate a significant impact on the ability of purified receptor to bind to K5; therefore, the purified VDAC1 was incorporated into liposomes and gel filtration on Sephadex G-75 employed to identify and separate the reactants (Fig. 2). 125I-K5 eluted at a column volume of 100120 ml (Fig. 2A). When 125I-K5 was incubated with solubilized VDAC1 (2 µg), the radiolabeled material eluted in the same fractions as above, suggesting no reactivity between K5 and solubilized receptor (Fig. 2B). When VDAC1 was incorporated into liposomes and then reacted with K5, the radiolabeled material eluted from the column as two peaks, one of them corresponding to the void volume where VDAC1 elutes and the other corresponding to the elution volume of unreacted K5 (Fig. 2C). These data indicate that K5 binds to VDAC1 when this receptor is incorporated into a lipid membrane. Binding of K5 to membrane-incorporated VDAC1 was inhibited by anti-VDAC1 (peptide Lys235Lys255) IgG, demonstrating that this is the region responsible for binding to K5 (Fig. 2D).
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K5 binds to VDAC1 proteoliposomes (Fig. 3A) in a dose-dependent manner with high affinity (Kd of 22 ± 3.1 nM). The binding is specific for VDAC1 because control proteoliposomes prepared with BSA or empty liposomes show little specific binding (Fig. 3A). Binding of 125I-K5 to VDAC1 proteoliposomes is inhibited by unlabeled K5 or anti-VDAC1 IgG (Fig. 3B), suggesting that VDAC1 is a receptor for K5.
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Analyses of VDAC1 on the Cell Surface of HUVEC by Flow CytometryAs determined by FACS, HUVEC reacted with an antibody against the SK peptide (Fig. 4A) as well as an antibody against the VDAC1 peptide (Fig. 4B) or a murine antibody against human mitochondrial VDAC1 (Fig. 4C), as expected because mitochondrial and plasma membrane VDAC1 share the same primary structure (35). FACS analysis of HUVEC reacted with K5 (0.1 µM) prior to reaction with antibody against the VDAC1 peptide (Fig. 4D) shows inhibition of binding of this antibody, suggesting that both K5 or the IgG compete for the same binding site. Taken together, these experiments show that VDAC1 is not only expressed on the surface of HUVEC but also establishes the structural relationship between SK and VDAC1 hypothesized by McCabe et al. (16).
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Inhibition of Endothelial Cell Proliferation by K5K5 inhibited VEGF-dependent HUVEC proliferation in a dose-dependent manner (Fig. 5A). As observed previously (10), the antiendothelial cell proliferation of K5 was abolished after reduction/alkylation of the protein, suggesting that the formation of appropriate disulfide bridges is essential to maintain its activity.
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Binding of K5 to HUVECK5 binds to these cells in a
dose-dependent manner with high affinity (Kd of 28
± 1.37 nM) and to a large number of sites (12.6 ±
0.56 x 105 binding sites/cell)
(Fig. 5B). The value
of the Kd is comparable with that determined for binding
of K5 to VDAC1 reconstituted in proteoliposomes. Electrophoretic separation of
proteins in a HUVEC lysate followed by a blot binding assay with a rabbit
anti-VDAC1 IgG (Fig.
5B, inset) shows only one band of
Mr 32,000. Binding of K5 to HUVEC is inhibited by Pg,
Pg peptides containing K5, or by an IgG fraction against VDAC1 peptide showing
structural relatedness to SK (Fig.
5C).
Binding of Anti-VDAC1 Peptide IgG to HUVEC125I-Labeled anti-VDAC1 IgG bind to HUVEC in a dose-dependent manner to a large number of sites (Bmax of 11.6 x 105 binding sites/cells) (Fig. 5D). This value is comparable with that determined for the binding of K5 to HUVEC, suggesting VDAC1 as a unique receptor for K5 on the cell surface.
Effect of K5 Binding on HUVEC [Ca2+]i and pHiWe also investigated whether K5 binding to HUVEC produced changes in [Ca2+]i or pHi and compared these changes with those produced by Pg 2. Pg 2 (100 nM) added to HUVEC induces a transient rise in [Ca2+]i lasting about for 90 s before returning to base line (Fig. 6A). Pg 2 also induced a rise in pHi, which was continuous for 400 s (Fig. 6B). A similar concentration (100 nM) of K5 induced a small a rise in [Ca2+]i (Fig. 6C) and produced a continuous decrease in pHi during the same time period (Fig. 6D). Incubation of HUVEC with K5 followed by Pg 2 shows a decreased stimulation in [Ca2+]i (Fig. 6E); however, the decrease in pHi induced by K5 is abolished after the addition of Pg (Fig. 6F). Incubation of HUVEC with anti-VDAC1 peptide IgG prior to the addition of K5 causes no change in [Ca2+]i (Fig. 6G) or pHi (Fig. 6H and Table I).
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Effect of K5 on Mitochondrial Membrane PotentialThe
fluorescent dye DSPM+ was used as an indicator of a coupled membrane potential
() (35). We
observed that K5 induces a concentration-dependent increase in
with mitochondria isolated from 1-LN cells
(Fig. 7).
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DISCUSSION |
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Endothelial cell proliferation is preceded by an increase in cytosolic
pHi, leading to angiogenesis and the repair of injured
endothelial cells (43). Cell
proliferation is also dependent on modulation of
[Ca2+]i
(43). Our data suggest that K5
interferes with both cytosolic pHi and
[Ca2+]i in HUVEC. Because the most abundant
protein of the mitochondrial outer membrane is VDAC1
(44), we investigated the
interaction of K5 with isolated mitochondria. Regulation of apoptosis involves
selective mitochondrial membrane permeabilization
(45). Mitochondria are
organelles with two well defined compartments: the matrix, surrounded by the
inner membrane, and the intermembrane space, surrounded by the outer membrane.
The inner membrane is folded into numerous christae to increase its surface
area and contains the protein complexes from the electron transport chain, ATP
synthase, and the adenine nucleotide translocator. To function properly, the
inner membrane is almost impermeable under physiological conditions, thereby
allowing the respiratory chain to create an electrochemical gradient
(). The
results from the respiration-driven, electron
transport chain-mediated pumping of protons out of the inner membrane and is
indispensable for driving ATP synthase, which phosphorylates ADP to ATP. In
the aptoptotic program, mitochondrial
may be either decreased or
increased, depending on the mechanism affecting VDAC1
(45). An increase in
transfers charges to the outer membrane, resulting in the closure
of VDAC1 (46,
47). An early feature of
apoptosis is intracellular acidification, which induces hyperpolarization of
mitochondrial outer membrane
(47,
48). The intracellular
acidification induced in HUVEC by K5 via interaction with VDAC1 suggests a
mechanism by which K5 may cause apoptosis of endothelial cells
(12).
It is thought that VDAC1 is concentrated in caveolae and caveolae-related domains of the plasma membrane (39). Caveolin-1 and -2 are abundantly expressed in normal human endothelial cells (49). VEGF induces endothelial cell proliferation via a mechanism that produces a significant reduction in the expression of caveolin-1. In the absence of VEGF, angiostatin (kringles 13) does not affect endothelial cell proliferation or alter the levels of expression of caveolin-1 (50). However, in the presence of VEGF, angiostatin blocks VEGF-induced down-regulation of caveolin-1 (50). Caveolae are structures that cluster groups of fibrinolytic proteinases, thus providing a favorable environment for proteinase cooperation. The urokinase-type plasminogen activator receptor is localized in caveolae (51). Receptors for Pg such as annexin II and the ganglioside GM1 are also localized in caveolae (5254) along with metalloproteinase 2 (55). Annexin II and the ganglioside GM1 bind Pg via the L-lysine binding site in kringle 1, whereas VDAC binds Pg via a site in K5. In situ degradation of receptor-bound Pg by metalloproteinases may generate anti-angiogenic peptides (kringles 13 and K5) (56), which once generated may control angiogenesis via alternative pathways.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Pathology, Duke
University Medical Center, Box 3712-DUMC, Durham, NC 27710. Fax: 919-684-8689;
E-mail:
gonza002{at}mc.duke.edu.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; K5,
kringle five; Pg, plasminogen; SK, streptokinase; VDAC, voltage-dependent
anion channel; HUVEC, human umbilical vein endothelial cells; FACS;
fluorescence assisted cytometry scanning; pHi,
intracellular pH; HBSS, Hanks' balanced salt solution; BSA, bovine serum
albumin; DSPM+, 2-(dimethylaminostyryl)-1-methyl-pyridinium ion.
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REFERENCES |
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