The Voltage-dependent Anion Channel Is a Receptor for Plasminogen Kringle 5 on Human Endothelial Cells

Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1–3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1–3 or 4 has been identified as cell surface-associated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltage-dependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr252–Lys283, which is homologous to the primary sequence of VDAC residues Tyr224–Lys255. Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (Kd of 28 nm) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca2+ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5. Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1–3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1–3 or 4 has been identified as cell surface-associated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltage-dependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr252–Lys283, which is homologous to the primary sequence of VDAC residues Tyr224–Lys255. Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (Kd of 28 nm) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca2+ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5. Angiogenesis is essential for tumor growth (1Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6089) Google Scholar, 3Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 4Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7217) Google Scholar). Vascular endothelial growth factor (VEGF) 1The 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. is a potent mitogen promoting endothelial cell proliferation (5Dusak B.A. Czerniak P. Sun T. Eidsvoog K. Dexter D.L. Yayon A. J. Natl. Cancer Inst. 1993; 85: 121-131Crossref PubMed Scopus (95) Google Scholar, 6Kim K.J. Li B. Winer J. Armanini M. Gillet N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3353) Google Scholar), whereas angiostatin inhibits this process in vitro and suppresses tumor growth in vivo (7O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3172) Google Scholar, 8O'Reilly M.S. Holmgren L. Chen C. Folkman J. Nat. Med. 1996; 2: 689-692Crossref PubMed Scopus (1151) Google Scholar). Angiostatin is a fragment of plasminogen (Pg) consisting of either the first three or four of its kringles (7O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3172) Google Scholar). Pg kringle 5 (K5) also suppresses growth factor-stimulated angiogenesis via cell cycle G1 arrest and induction of apoptosis (9Ji W.R. Barientos L.G. Llinas M. Gray H. Villareal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P.A. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar, 10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 11Cao Y. O'Reilly M.S. Marshall B. Flynn E. Ji R.W. Folkman J. J. Clin. Invest. 1998; 101: 1055-1063Crossref PubMed Scopus (240) Google Scholar, 12Lu H. Dhanabal M. Volk R. Waterman M.J.F. Ramchandran R. Knebelman B. Segal M. Sukhatme V.P. Biochem. Biophys. Res. Commun. 1999; 258: 668-673Crossref PubMed Scopus (72) Google Scholar); however, the cellular receptor(s) mediating these effects are unknown. K5 confers on Pg the capacity to bind to human umbilical vein endothelial cells (HUVEC) with high affinity (13Wu H.L. Wu I.S. Fang R.Y. Hau J.S. Wu D.H. Chang B.I. Lin T.M. Shi G.Y. Biochem. Biophys. Res. Commun. 1992; 188: 703-711Crossref PubMed Scopus (22) Google Scholar). K5 also mediates binding of Pg to the Pg activator streptokinase (SK) (14Nihalani D. Sahni G. Biochim. Biophys. Res. Commun. 1995; 217: 1245-1254Crossref PubMed Scopus (31) Google Scholar, 15Lin L.F. Bhoung A. Reed G.L. Biochemistry. 2000; 39: 4740-4745Crossref PubMed Scopus (33) Google Scholar). Sequence similarities between SK and the mitochondrial human voltage-dependent anion channel (VDAC1) exist; specifically, the region comprising SK residues Tyr252–Lys283 are homologous to VDAC1 residues Tyr224–Lys255 (16McCabe K.M. Wheeler D.A. Adams V. Edward R.B. Biochem. Mol. Med. 1995; 56: 176-179Crossref PubMed Scopus (1) Google Scholar). We raised antibodies against peptides contained within these regions and used them to identify VDAC1 on the HUVEC surface by flow cytometry (FACS). Receptor binding assays demonstrated that K5 binds with high affinity to sites on these cells. K5 inhibits VEGF-stimulated HUVEC proliferation and induces a decrease in cytosolic pH and an increase in the potential of isolated mitochondria. Highly purified VDAC1 binds to K5 after reconstitution of the receptor into liposomes. Our data suggest that VDAC1 is a receptor for K5 on the cell surface. Materials—Culture media were from Invitrogen. Porcine pancreatic elastase, gastric mucosa pepsin, trypsin inhibitor, and pre-formed liposomes were from Sigma. Recombinant VEGF was from Calbiochem (San Diego, CA). Endothelial cell growth supplement was from Collaborative Research Inc. (Waltham, MA). 125I-Labeled Bolton-Hunter reagent was obtained from PerkinElmer Life Sciences. The 21-amino acid peptides EINNTDLISLEYKYVLKKGEK (Glu263–Lys283) of SK and KVNNSSLIGLGYTQTLKPGIK (Lys235–Lys255) of VDAC1 were from Research Genetics (Huntsville, AL). Fura-2/AM and bis(carboxyethyl)-carbonyl fluorescein and the 2-(dimethylaminostyryl)-1-methyl-pyridinium ion (DSPM+) were purchased from Molecular Probes, Inc. (Eugene, OR). Proteins—Human Pg was resolved into its isoforms, Pg 1 and 2 (17Deutsch D. Mertz B.T. Science. 1970; 170: 1095-1096Crossref PubMed Scopus (1670) Google Scholar, 18Gonzalez-Gronow M. Robbins K.C. Biochemistry. 1984; 23: 190-196Crossref PubMed Scopus (35) Google Scholar). 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 (10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 19Sottrup-Jensen L. Claeys H. Zajdel M. Petersen T.E. Magnusson S. Prog. Chem. Fibrinol. Thrombol. 1978; 3: 191-209Google Scholar, 20Thewes T. Ramesh V. Simplaceanu E.L. Llinas M. Biochim. Biophys. Acta. 1987; 912: 254-269Crossref PubMed Scopus (42) Google Scholar). Gel electrophoresis (10–20% 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 450–459, confirming the identification of K5 (21Petersen T.E. Martzen M.R. Ichinose A. Davie E.W. J. Biol. Chem. 1990; 265: 6104-6109Abstract Full Text PDF PubMed Google Scholar). 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, 500–700 cpm/ng). Antibodies—Antibodies to SK were raised in rabbits, and the IgG fraction specific against the SK sequence Glu263–Lys283 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 Lys235–Lys255 of VDAC1 conjugated to keyhole limpet hemocyanin (22Kagen A. Glick M. Jaffe B.B. Behrman H.R. Methods of Hormone Radioimmunoassay. Academic Press, New York1979: 328-329Google Scholar) 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 Assay—HUVEC 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 (23Morales D.E. McGowan K.A. Grant D.S. Mashewari S. Bhartiya D. Cid M.C. Kleinman H.K. Schnaper H.W. Circulation. 1995; 91: 755-763Crossref PubMed Google Scholar). The cells were washed with phosphate-buffered saline and dispersed in a 0.05% trypsin solution. The cells were resuspended in medium (25 × 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 Cytometry—HUVEC 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 × 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 and The cell were 30 min with of SK peptide VDAC1 peptide or the mitochondrial VDAC1 monoclonal The cells were washed with ice-cold and resuspended in 100 of ice-cold The cell were in the with an for 30 min to goat or IgG from Molecular Probes, Inc. The cells were washed with ice-cold resuspended in ice-cold 1% and in the at 4 °C analysis by The fluorescence after at was determined for on a flow and with Biosciences). cells were grown in culture plates the were The cells were washed in The binding assays were performed at 4 °C in containing of were with cells for min in 96-well and were by the incubation and the cell with containing The were from the plates and The was after of binding in the presence of mm The and of K5 were determined by the data to the using the for binding assays were performed in HUVEC grown in 96-well The cells were washed in and with of 125I-labeled VDAC1 peptide IgG for min at °C in containing The cells were with and the from the plates were in to IgG was after of binding in the presence of The of the IgG was of Ca2+ and was by using the Fura-2/AM M. G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of pHi, HUVEC were in Dulbecco's modified Eagle's medium on and washed with with sodium pH The cells were for 20 min with 2 in HBSS, with and on the pH was by a in cells by the which were after a V. G. B. Adams Science. PubMed Scopus Google Scholar). Gel was performed in a Nature. 1970; PubMed Scopus Google Scholar). The were with to was by the H. T. J. Natl. S. A. PubMed Scopus Google Scholar). The used were of and of VDAC1 is to obtain of however, that cells are a of this cells were grown in with bovine serum, 100 units/ml and 100 in 20 culture flasks After with 10 mm EDTA in and the cells were in 10 ml of 20 mm Hepes, pH containing the at and The cells were by on with The was at × for min, followed by at × for 1 The containing cell was resuspended in 20 mm pH containing 1% to and at × for 30 min to VDAC was purified to using gel on and immunoaffinity chromatography with an peptide IgG conjugated to of VDAC1 into and of K5 to the VDAC1 was reconstituted into liposomes D. Y. Glick D. Methods in New Scholar, Y. S. D. Press, Scholar) as of a of liposomes and in 5% were with VDAC1 and with for 30 min at room The of was to with mm pH After the of (10 nm) and incubation for 30 min at room temperature, the was a × 2 of K5 binding to VDAC1 reconstituted into the was with the specific IgG for 30 min at room before the of The of K5 binding to VDAC1 on reconstituted liposomes were performed on liposomes in prepared by of CA) Biochim. Biophys. Acta. PubMed Scopus Google Scholar). these containing VDAC1 or were prepared by the proteins in 2.5 mm Hepes, pH mm and 0.3 mm with liposomes at a of protein to liposomes B. D. Biochim. Biophys. Acta. 1995; PubMed Scopus Google Scholar). The was after three at 4 °C of the with 10 of followed by three at × was determined C. Acta. Scopus Google Scholar). the and of VDAC1 the liposomes (10 in 2.5 mm Hepes, pH were with of 125I-labeled IgG for 1 h at room followed by MA). After three with 200 of 2.5 mm Hepes, pH the were from the and was of were for 1 h at room with VDAC1 or liposomes (10 in 2.5 mm Hepes, pH containing mm and of were as of from cells were isolated D. a Press, Scholar), and the protein were using the acid Biochem. PubMed Scopus Google Scholar). potential was determined at room using a of membrane potential H.W. J. PubMed Scopus Google Scholar). The of a of 2 ml containing (10 pH (0.2 cellular 2 (1 sodium (10 pH and of K5. The was for 20 min with K5 to of The fluorescence was nm) in a fluorescence in the absence of K5 was obtained with with The results are the of fluorescence from three of Sequence between SK and Human regions of sequence between streptokinase and human VDAC1 were identified by the by the of The for proteins was with of the also by the of and a the mitochondrial membrane VDAC1 residues We raised antibodies to the SK peptide and the VDAC1 peptide A of the purified VDAC a of A binding with a IgG with this protein the purified VDAC1 with the IgG confirming the structural between VDAC1 and the purified VDAC1 not with when to a membrane of K5 to VDAC1 into a on the of purified receptor to bind to the purified VDAC1 was into liposomes and gel on to identify and the at a of ml was with VDAC1 the in the as between K5 and receptor VDAC1 was into liposomes and with K5, the from the as of them corresponding to the VDAC1 and the corresponding to the of K5 These data that K5 binds to VDAC1 when this receptor is into a membrane. of K5 to VDAC1 was inhibited by that this is the region for binding to K5 K5 binds to VDAC1 in a with high affinity (Kd of The binding is specific for VDAC1 control prepared with or liposomes specific binding of to VDAC1 is inhibited by K5 or IgG that VDAC1 is a receptor for K5. of VDAC1 on the Cell of HUVEC by Flow determined by HUVEC with an antibody against the SK peptide as as an antibody against the VDAC1 peptide or a antibody against human mitochondrial VDAC1 as mitochondrial and plasma membrane VDAC1 the primary H.W. J. PubMed Scopus Google Scholar). analysis of HUVEC with K5 to with antibody against the VDAC1 peptide of binding of this that both K5 or the IgG for the binding these that VDAC1 is not only expressed on the surface of HUVEC but also the structural relationship between SK and VDAC1 by (16McCabe K.M. Wheeler D.A. Adams V. Edward R.B. Biochem. Mol. Med. 1995; 56: 176-179Crossref PubMed Scopus (1) Google Scholar). of Endothelial Cell Proliferation by inhibited HUVEC proliferation in a (10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. 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