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• W2000268063 abstract "Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN347–361(the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (τ = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (τ = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN347–361, demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions. Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN347–361(the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (τ = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (τ = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN347–361, demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions. vitronectin amino acid(s) synthetic polysulfonated carboxymethyldextran Cascade Blue® phosphate-buffered saline environment-dependent fluorescence intensity fluorescence resonance energy transfer arbitrary fluorescence units Vitronectin (VN),1 an abundant, multifunctional glycoprotein of plasma and extracellular matrix (1Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar, 2Tomasini B.R. Mosher D.F. Prog. Hemost. Thromb. 1991; 10: 269-305PubMed Google Scholar), exists in a monomeric and a multimeric form. VN monomers are synthesized in the liver and secreted into the plasma. Circulating VN is essentially monomeric, while VN in the extravascular space is essentially multimeric (1Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar). VN avidly binds glycosaminoglycans (GAGs;e.g. heparin (2Tomasini B.R. Mosher D.F. Prog. Hemost. Thromb. 1991; 10: 269-305PubMed Google Scholar)); however, the role and mechanisms of GAG binding by VN remains poorly understood. (i) What is the physiological role of GAG binding? A role in VN activation and VN deposition in tissues has been hypothesized (2Tomasini B.R. Mosher D.F. Prog. Hemost. Thromb. 1991; 10: 269-305PubMed Google Scholar); however, currently only little experimental evidence concerning these hypotheses is available. There is evidence that the heparin-binding to VN allosterically effects ligand binding to other domains of VN (3Seiffert D. J. Biol. Chem. 1997; 272: 9971-9978Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Note, however, that the heparin-binding domain of VN does not only interact with glycosaminoglycans but also directly interacts with proteins, including other serum and extracellular matrix proteins (e.g.complement components (4Lim B.L. Reid K.B. Ghebrehiwet B. Peerschke E.I. Leigh L.A. Preissner K.T. J. Biol. Chem. 1996; 271: 26739-26744Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and osteonectin (5Rosenblatt S. Bassuk J.A. Alpers C.E. Sage E.H. Timpl R. Preissner K.T. Biochem. J. 1997; 324: 311-319Crossref PubMed Scopus (84) Google Scholar)) as well as with microbial proteins (e.g. Staphylococcus aureus(6Chhatwal G.S. Preissner G. Müller-Berghaus Blobel H. Infect. Immun. 1987; 55: 1878-1883Crossref PubMed Google Scholar, 7Liang O.D. Maccarana M. Flock J.-I. Paulsson M. Preissner K.T. Wadström T. Biochim. Biophys. Acta. 1993; 1225: 57-63Crossref PubMed Scopus (35) Google Scholar, 8Liang O.D. Flock J.-I. Wadström T. J. Biochem. (Tokyo). 1994; 116: 457-463Crossref PubMed Scopus (18) Google Scholar)). (ii) Is the heparin-binding site exposed in monomeric VN? Several studies have suggested that only multimeric and not monomeric VN is able to bind heparin (1Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar, 9Kost C. Stuber W. Ehrlich H.J. Pannekoek H. Preissner K.T. J. Biol. Chem. 1992; 267: 12098-12105Abstract Full Text PDF PubMed Google Scholar, 10Tschopp J. Masson D. Schäfer S. Peitsch M. Preissner K.T. Biochemistry. 1988; 27: 4103-4109Crossref PubMed Scopus (90) Google Scholar). This was attributed to a heparin-binding sequence, which is cryptic in monomeric but exposed in multimeric heparin (1Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar, 11Stockmann A. Hess S. Declerck P. Timpl R. Preissner K.T. J. Biol. Chem. 1993; 268: 22874-22882Abstract Full Text PDF PubMed Google Scholar). A recent study has challenged this point of view, suggesting that monomeric and multimeric VN exhibit similar affinity for heparin and that the differences in heparin binding properties induced upon denaturation are due to self-association in a multivalent form (12Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). (iii) Which regions of the VN molecule are involved in heparin binding? Several studies have suggested a role of a highly basic VN region (aa 347–361) to be the heparin-binding site (1Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar,11Stockmann A. Hess S. Declerck P. Timpl R. Preissner K.T. J. Biol. Chem. 1993; 268: 22874-22882Abstract Full Text PDF PubMed Google Scholar, 13Tomasini B.R. Mosher D.F. Blood. 1998; 72: 903-912Crossref Google Scholar). However, recent studies have identified novel heparin-binding domains of VN (14Liang O.D. Rosenblatt S. Chhatwal G.S. Preissner K.T. FEBS Lett. 1997; 407: 169-172Crossref PubMed Scopus (43) Google Scholar). (iv) what are the kinetics parameters of heparin binding to VN? Since most previous studies on heparin-VN interaction were done with discontinuous binding assays, no analysis of the kinetic parameters of the heparin-VN interaction has been performed so far. In this study, we analyze the kinetics of the interaction between heparin and multimeric VN and characterize VN interaction with soluble and endothelial cell surface glycosaminoglycans. Chemicals were purchased from Merck (Basel, Switzerland) or Sigma (Fluka Chemie AG, Buchs, Switzerland). If not stated otherwise, experiments were performed in phosphate-buffered saline (Life Technologies, Inc., Basel, Switzerland). RPMI 1640 was from Life Technologies, Inc.). Chondroitinase was from Sigma, and heparitinase was from Seikogaku Corp. (Tokyo, Japan). Heparin (meanM r 10,000) and CMDBS (a synthetic polysulfonated carboxymethyldextran, mean M r 45,000; see also Refs. 15Jozefonvicz J. Jozefowicz M. J. Biomater. Sci. Polym. Ed. 1990; 1: 147-165Crossref PubMed Scopus (65) Google Scholar and 16Jozefonvicz J. Jozefowicz M. Pure Appl. Chem. 1992; 64: 1783-1788Crossref Scopus (17) Google Scholar) were kindly provided by J. Jozefonvicz (LRM, University Paris Nord Villetaneuse, France). Cascade Blue® covalently coupled to heparin was obtained from Molecular Probes, Inc. (Eugene, OR). Concentrations of fluorescent heparin were assayed using the carbazole method (17Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5218) Google Scholar). Briefly, the fluorescent heparin was hydrolyzed in H2SO4, and after the addition of carbazole reagent, a colored component presenting a maximum absorbency at 530 nm was yielded. A solution of heparin and a solution of gluconolactone were used as standard. Considering the biological activity of our heparin, we used a previously described assay to evaluate the effect of heparin or derivatives on bacterial adherence to fibronectin-coated surface (18Vaudaux P. Avramoglou T. Letourneur D. Lew D.P. Jozefonvicz J. J. Biomater. Sci. Polym. Ed. 1992; 4: 89-97Crossref PubMed Scopus (22) Google Scholar). This assay revealed that both heparins, either the H108 or the Cascade Blue®(CB)-heparin exhibited similar inhibition efficacy on S. aureus adhesion to fibronectin-coated PMMA coverslips (not shown). The anticoagulant property was also checked using the inactivation rate of factor Xa (19Colliec S. Fischer A.M. Tapon-Bretaudiere J. Boisson C. Durand P. Jozefonvicz J. Thromb. Res. 1991; 64: 143-154Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Heparin H108 showed an activity of 75 IU/mg, whereas the activity of CB-heparin was 83 IU/mg. Both tests confirmed that the coupling procedure does not affect the biological properties of the molecule. Monomeric VN was purified from human plasma as described previously (1Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar, 20Preissner K.T. Wassmuth R. Muller-Berghaus G. Biochem. J. 1985; 231: 349-355Crossref PubMed Scopus (114) Google Scholar, 21Preissner K.T. Muller-Berghaus G. J. Biol. Chem. 1987; 262: 12247-12253Abstract Full Text PDF PubMed Google Scholar) and stored at −70 °C. Multimeric VN was generated by incubation of the monomeric form for 1 h at 37 °C in 8 m urea, followed by extensive dialysis (11Stockmann A. Hess S. Declerck P. Timpl R. Preissner K.T. J. Biol. Chem. 1993; 268: 22874-22882Abstract Full Text PDF PubMed Google Scholar). Previous studies have shown that this protocol leads to VN multimers containing 3–18 molecules of the protein (11Stockmann A. Hess S. Declerck P. Timpl R. Preissner K.T. J. Biol. Chem. 1993; 268: 22874-22882Abstract Full Text PDF PubMed Google Scholar, 13Tomasini B.R. Mosher D.F. Blood. 1998; 72: 903-912Crossref Google Scholar). All indicated VN concentrations are based on the molecular weight of the monomer (M r 72,000). Since nondenaturing gel electrophoresis demonstrated the spontaneous formation of VN dimers within the monomer preparation, we designed a simple protocol to remove the dimers; 100-μl fractions of plasma VN (1.4 mg/ml) were mixed with 10 μl of a 50% slurry of heparin-Sepharose 6B in PBS. After 2 h of incubation with constant shaking, each sample was centrifuged for 5 min at 2000 × g. The purity of the preparation was assessed by nondenaturing polyacrylamide gel electrophoresis (see below). VN-derived peptides VN347–361 and VN371–383 were synthesized through standard procedures. A polyclonal α-VN antibody was obtained by immunization of rabbit with the synthetic peptide (VN347–361) (13Tomasini B.R. Mosher D.F. Blood. 1998; 72: 903-912Crossref Google Scholar). The IgG fraction of the antiserum recognized VN multimers but not the native protein (Fig. 3 A). Antibody was then desalted on G-25 gel filtration column equilibrated in PBS. Overnight cultures of S. aureus 6850 and Staphylococcus epidermidis KH11 were grown in chemically defined medium (2 liters) (22Hussain M. Wilcox M.H. White P.J. Faulkner M.K. Spencer R.C. J. Hosp. Infect. 1992; 20: 173-184Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 23Heilmann C. Hussain M. Peters G. Götz F. Mol. Microbiol. 1997; 24: 1013-1024Crossref PubMed Scopus (574) Google Scholar) and washed in PBS. Cells were then suspended in 1 ml of PBS containing 2% SDS. Cells were heated for 5 min at 95 °C. After cooling, SDS was removed by exhaustive dialysis against PBS. Protein content was assayed using a commercially available kit (Pierce method; Socochim, Lausanne, Switzerland) and subjected to SDS-polyacrylamide gel electrophoresis followed by a silver staining procedure. Extracts showed the characteristic pattern of staphylococcal proteins with a repartition of molecular weight ranging from 20,000 to 140,000. Human endothelial cord cells (ECV304, ATCC:CRL-1998) were cultured in RPMI 1640 supplemented with 10% fetal calf serum at 37 °C in an atmosphere containing 5% CO2, in 75-cm2 culture dishes, containing at confluence approximately 107 cells. Adherent cells were detached using EDTA 2.5 mm for 5 min. After rinsing twice in PBS, one half of the suspension was suspended in culture medium, whereas the other half was suspended in PBS plus 0.1% albumin (PBSA) containing 50 mIU/ml and 0.5 IU/ml of heparitinase (Seikagaku Kogyo, Tokyo) and chondroitinase ABC (Sigma). Both suspensions were incubated for 3 h at 37 °C with constant and gentle shaking. This treatment has been shown to lead to an almost total degradation of cellular glycosaminoglycans (24de Agostini A.I. Ramus M.A. Rosenberg R.D. J. Cell. Biochem. 1994; 54: 174-185Crossref PubMed Scopus (13) Google Scholar, 25de Agostini A.I. Watkins S.C. Slayter H.S. Youssoufian H. Rosenberg R.D. J. Cell Biol. 1990; 111: 1293-1304Crossref PubMed Scopus (200) Google Scholar, 26Ueda H. Fujimori O. J. Vet. Med. Sci. 1998; 60: 1169-1174Crossref PubMed Scopus (3) Google Scholar). After glycosaminoglycan removal, cells were washed with PBSA, resuspended in 100 μl of PBSA, and subjected to VN-binding experiments. Plasma VN (2 μg) or a mixture of plasma VN and heparin H108 (2 or 5 μg) was mixed with 1.5 × 106 of enzymatically treated or untreated cells. After an incubation period of 60 min at 4 °C, each sample was centrifuged for 2 min at 1000 rpm. The supernatants were then collected in new tubes. In parallel, 2 μg of plasma VN was incubated in the same condition as above but in PBSA or in PBSA enriched with heparin. Protein samples were subjected to gel electrophoresis (8% acrylamide) under nondenaturing, nonreducing conditions as described (13Tomasini B.R. Mosher D.F. Blood. 1998; 72: 903-912Crossref Google Scholar). After separation, proteins were transferred to polyvinylidene difluoride membranes using a liquid transblot system (Bio-Rad) in a 20 mm phosphate buffer, pH 6.5, for 2 h under constant voltage (15 V). Membranes were blocked in PBS containing 2.5% BSA and 0.1% Tween 20. All VN forms were detected using a monoclonal antibody against human VN (1:1000; Life Technologies, Inc., Basel, Switzerland), followed by incubation with anti-mouse IgG coupled to peroxidase (1:10,000). Western immunoblots were also performed using the rabbit anti-VN347–361 peptide (1:50), followed by incubation with anti-rabbit IgG coupled to peroxidase (1:10,000). Detection was performed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Films showing immunoreactive bands were scanned (Molecular Dynamics, Inc., Sunnyvale, CA) and analyzed with an imaging system (Molecular Dynamics ImageQuant Software), and the ratio of dimeric Vn/monomeric Vn was calculated. All fluorescence measurements were performed on a Perkin-Elmer LS-3 fluorescence spectrometer, coupled to a computerized data acquisition program (Daqsys, University of Geneva). The sampling rate was 50 ms (standard measurements) or 30 ms (kinetic analysis). Recorded traces were analyzed by the package Origin (MicroCal Software Inc., Northampton, MA) using a PC/AT 486 computer. All fluorescence resonance energy transfer (FRET) experiments were monitored in solution with λexcitation = 280 nm and λemission = 426 nm. All environment-dependent fluorescence intensity (EDFI) experiments were monitored in solution with λexcitation = 380 nm and λemission = 426 nm. For displacement curves, data were fitted by a logistic equation (y = y max/(1 + (IC50/x) n) (see also Ref. 27Barlow R. Blake J.F. Trends. Pharmacol. Sci. 1989; 10: 440-441Abstract Full Text PDF PubMed Scopus (53) Google Scholar), wherex is the concentration of the tested compound, yis the observed fluorescence signal, y max is the maximal fluorescence signal observed, and n is the Hill coefficient (also referred to as slope factor). For kinetic analysis, data were fitted by single and double exponential decay functions. The quality of the fit was assessed by χ2 test. It is important to note that a χ2 test does not allow assessing whether the degree of freedom of the function to be fitted is appropriately chosen. We therefore used an F test (28Schrenzel J. Demaurex N. Foti M. Van Delden C. Jacquet J. Mayr G. Lew D.P. Krause K.H. Biophys. J. 1995; 69: 2378-2391Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 29Patel J.K. Kapadia C.H. Owen D.B. Handbook of Statistical Distributions. Marcel Dekker, Inc., New York1976Google Scholar) to determine whether a single or a double exponential fit was appropriate for the data. For the statistic in Table I, a relativeF value was calculated by dividing the F value obtained when fitting a curve with a single-exponential decay through the F value obtained when fitting the same curve with a double exponential decay. A relative F value below 0.5 indicates that a double exponential fit is more appropriate than a single exponential fit. If not otherwise indicated, the indicated values are means of three or four different experiments.Table IKinetic properties of heparin interaction with full-length VN and with VN 347–361 peptidePropertiesFull-length VNVN347–361RelativeF test for binding (single/double exponential fit)10 ± 2.57.5 ± 2RelativeF test for unbinding (single/double exponential fit)0.2 ± 0.10.32 ± 0.1On rate0.4 ± 0.10.3 ± 0.1τ of conversion to stable complex180 ± 47205 ± 22Off rate 11.4 ± 0.22.4 ± 0.1Off rate 214 ± 322 ± 4 Open table in a new tab The fluorescence intensity of CB-heparin (10 nm) was compared with the fluorescence intensity of its parental compound CB-acetylazide (10 nm). The ratio was found to be 1.85 molecules, indicating that the CB-heparin had 1–2 mol of CB bound per mole of heparin. The excitation maximum of CB-heparin was around 380 nm. When elicited with an excitation wavelength of 380 nm, maximal emission was between 420 and 430 nm. The fluorescence intensity of CB-heparin was high in a polar solvent (PBS) and decreased as the hydrophobicity of the solvent increased. These fluorescent properties are similar to those of the parental compound CB-acetylazide (30Corbett E.F. Oikawa K. François P. Tessier D.C. Kay C. Bergeron J.J. Thomas D.Y. Krause K.H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Changes in fluorescence intensity as a function of the hydrophobicity of the environment have been observed previously (12Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 30Corbett E.F. Oikawa K. François P. Tessier D.C. Kay C. Bergeron J.J. Thomas D.Y. Krause K.H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 31Berger W. Prinz H. Striessnig J. Kang H.C. Haugland R. Glossmann H. Biochemistry. 1994; 33: 11875-11883Crossref PubMed Scopus (30) Google Scholar). Since the binding of a ligand to a protein leads to a change in the hydrophobicity of the environment (in general, an increase in hydrophobicity), this approach can be used to monitor directly the kinetics of ligand-protein interaction. We will refer to this approach as EDFI. Note that, depending on the fluorescent probe, binding of the ligand to the protein can either lead to a decrease of fluorescence (e.g. CB-coupled ligand in this study) or to an increase in fluorescence (e.g. coumarin-labeled ligand in a previous study) (12Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Due to aromatic amino acids, most proteins display autofluorescence with absorption maximum around 280 nm and an emission maximum around 380 nm. The superimposition of the excitation spectrum of CB-heparin and emission spectrum of VN showed an overlapping, confirming that an interaction between both compounds might yield to FRET. The question of whether heparin binding is restricted to multimeric VN (13Tomasini B.R. Mosher D.F. Blood. 1998; 72: 903-912Crossref Google Scholar) or also occurs with monomeric VN (12Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Liang O.D. Rosenblatt S. Chhatwal G.S. Preissner K.T. FEBS Lett. 1997; 407: 169-172Crossref PubMed Scopus (43) Google Scholar) remains controversial. To investigate this question, we compared the heparin binding to both forms of VN. Continuous FRET recordings were performed with an excitation wavelength of 280 nm and an emission wavelength of 426 nm. Under these conditions, the addition of multimeric VN (Fig. 1 B) led to a fluorescence increase. The fluorescence increase was due to specific binding, since it could be completely reversed by the addition of an excess of nonfluorescent heparin. When the same amount of nonfluorescent heparin was added to CB-heparin in the absence of VN, no effect on the fluorescence was observed (not shown). As expected from the decreased CB-heparin fluorescence in a hydrophobic environment, EDFI recording of the fluorescence showed a decrease upon the addition of multimeric VN to the probe (Fig. 1 E). The fluorescence decrease was due to specific binding, since it was completely reversed by the addition of an excess of nonfluorescent heparin. When the same experimental protocol was performed with monomeric VN, the signal with FRET was very low to undetectable, while EDFI gave a small signal (∼10% of the signal seen with multimeric VN), similar to what has been previously observed (12Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The latter observation has been used to challenge the concept that the heparin-binding site is cryptic in monomeric VN and is only exposed in the multimeric form of the protein (12Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We considered an alternative possibility, namely spontaneous formation of a heparin-binding form within the monomeric VN preparation. Indeed, nondenaturing gel electrophoresis clearly showed that in addition to the band of monomeric VN of about M r 72,000, a second band of approximately M r 150,000, presumably corresponding to a VN dimer, was present (Fig. 1 A, lane 2). To investigate whether this dimer could be responsible for the small proportion of heparin binding observed with the monomer preparation, we absorbed the monomer preparation using heparin-Sepharose (see “Experimental Procedures”). Nondenaturing gel electrophoresis (Fig. 1 A,lane 3) revealed that this treatment removed theM r 150,000 band. In contrast, the intensity of the M r 72,000 band was not diminished by the heparin-Sepharose absorption. Indeed, densitometric quantification demonstrated that the intensity of the M r 72,000 band was 106 ± 10% (mean ± range, n = 2) of control in the heparin-Sepharose-treated preparation. When the remaining monomeric VN was tested in the fluorescent binding assays, strictly no binding was detected (Fig. 1, D andG). Thus, our results demonstrate that specific high affinity binding to VN is restricted to the multimeric form of the protein. These results, however, do not exclude additional low affinity binding sites on the protein (14Liang O.D. Rosenblatt S. Chhatwal G.S. Preissner K.T. FEBS Lett. 1997; 407: 169-172Crossref PubMed Scopus (43) Google Scholar). To study further the properties of heparin binding to multimeric VN, we studied the displacement of CB-heparin (as evidenced by a decrease in FRET) by standard heparin and related compounds. Standard heparin (M r 10,000) displaced CB-heparin with an IC50 of 43 nm (Fig. 2 A). Other heparin-like compounds able to displace CB-heparin were CMDBS (15Jozefonvicz J. Jozefowicz M. J. Biomater. Sci. Polym. Ed. 1990; 1: 147-165Crossref PubMed Scopus (65) Google Scholar) (IC50= 150 nm) and chondroitin sulfate (IC50 = 1.6 μm) (32Hedman K.S. Johansson T. Vartio L. Kjellen A. Vaheri A. Höök M. Cell. 1983; 28: 663-671Abstract Full Text PDF Scopus (142) Google Scholar) (Fig. 2 C). Dermatan sulfate, de-N-sulfated heparin, or dextran T70 (Fig. 2 A) did not displace CB-heparin. Thus, the rank order of affinity of various heparin-like compounds for the VN-binding site is as follows: heparin > CMDBS > chondroitin sulfate [tmt] dermatan sulfate = de-N-sulfated heparin = dextran T70. It has been previously suggested that the binding sites of VN forS. aureus and for heparin are, at least partially, overlapping (8Liang O.D. Flock J.-I. Wadström T. J. Biochem. (Tokyo). 1994; 116: 457-463Crossref PubMed Scopus (18) Google Scholar). Binding of VN to S. epidermidis has also been suggested (23Heilmann C. Hussain M. Peters G. Götz F. Mol. Microbiol. 1997; 24: 1013-1024Crossref PubMed Scopus (574) Google Scholar, 33Paulsson M. Ljungh A. Wadstrom T. J. Clin. Microbiol. 1992; 30: 2006-2012Crossref PubMed Google Scholar); however, its relationship to the heparin-binding site is undefined. Using preparations of extracts from the two staphylococcal strains in the FRET assay, S. aureusbut not S. epidermidis proteins displaced CB-heparin from VN (Fig. 2 A). The high affinity heparin-binding site of VN is thought to lie within the C-terminal region of the protein (aa 347–361; VN347–361) (11Stockmann A. Hess S. Declerck P. Timpl R. Preissner K.T. J. Biol. Chem. 1993; 268: 22874-22882Abstract Full Text PDF PubMed Google Scholar). However, additional regions of VN might bind heparin as suggested recently (14Liang O.D. Rosenblatt S. Chhatwal G.S. Preissner K.T. FEBS Lett. 1997; 407: 169-172Crossref PubMed Scopus (43) Google Scholar, 21Preissner K.T. Muller-Berghaus G. J. Biol. Chem. 1987; 262: 12247-12253Abstract Full Text PDF PubMed Google Scholar). To investigate the identity of the FRET-generating heparin-binding site, we studied the effect of a polyclonal antibody raised against VN347–361. Western blot performed in native conditions revealed that this antibody recognizes dimeric or higher molecular forms of VN but does not recognize monomeric VN, which is the most abundant VN form in the sample (Fig. 3 A). The addition of 4 μg/ml of the antibody decreased the FRET signal by approximately 50%. The subsequent addition of free heparin completely abolished the FRET signal (Fig. 3 B). Repetitive addition of the antibody almost completely abolished the FRET signal, and the final addition of heparin led only to a minor decrease of the fluorescence signal (Fig. 3 C). To further investigate the role of VN347–361 in heparin binding by VN, we directly analyzed heparin binding to the peptide. No significant FRET signal could be obtained with the VN347–361 peptide. This is expected, since there is only one aromatic amino acid contained within this sequence (Phe352), while in the full-length protein there are several flanking aromatic amino acids (Trp320, Trp382, Trp405, and Trp450). This is also illustrated by the absence of detectable autofluorescence of the VN347–361 peptide (not shown). As opposed to FRET, EDFI does not require the presence of aromatic amino acid, and consequently this assay clearly gave a positive signal with the peptide. The addition of the VN347–361 peptide to CB-heparin led to a rapid decrease in fluorescence (Fig. 4 A). The fluorescence decrease was completely reversed by the addition of unlabeled heparin, demonstrating the specificity of the binding. A cont" @default.
• W2000268063 created "2016-06-24" @default.
• W2000268063 creator A5010055797 @default.
• W2000268063 creator A5025329558 @default.
• W2000268063 creator A5039627923 @default.
• W2000268063 creator A5048716653 @default.
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• W2000268063 date "1999-12-01" @default.
• W2000268063 modified "2023-10-02" @default.
• W2000268063 title "Vitronectin Interaction with Glycosaminoglycans" @default.
• W2000268063 cites W1480134646 @default.
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