Novel Interactions between Urokinase and Its Receptor
2000; Elsevier BV; Volume: 275; Issue: 32 Linguagem: Inglês
10.1074/jbc.m002024200
ISSN1083-351X
AutoresOri Shliom, Mingdong Huang, Bruce S. Sachais, Alice A. Kuo, John W. Weisel, Chandrasekaran Nagaswami, Taher Nassar, Khalil Bdeir, Edna Hiss, Susan L. Gawlak, Scott Harris, Andrew P. Mazar, Abd Al‐Roof Higazi,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoUrokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K d of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K d. Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35–60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K d and the theoretical saturation levels (10–80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction. Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K d of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K d. Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35–60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K d and the theoretical saturation levels (10–80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction. urokinase-type plasminogen activator uPA receptor single-chain uPA two-chain uPA amino-terminal fragment of urokinase low molecular weight recombinant soluble uPA 4-morpholineethanesulfonic acid high pressure liquid chromatography electron microscopy polyacrylamide gel electrophoresis phosphate-buffered saline size exclusion chromatography Urokinase-type plasminogen activator (uPA)1 has long been implicated in fibrinolysis. uPA−/− mice show a tendency toward spontaneous thrombosis (1Carmeliet P. Schoonjans L. Kieckens L. Ream B. Degan J. Bronson R. De Vos R. van den Oord J.J. Collen D. Mulligan R.C. Nature. 1994; 369: 419-424Crossref Scopus (912) Google Scholar, 2Carmeliet P. Mulligan R.C. Collen D. J. Intern. Med. 1994; 236: 455-459Crossref PubMed Scopus (9) Google Scholar, 3Shapiro R.L. Duquette J.G. Nunes I. Roses D.F. Harris M.N. Wilson E.L. Rifkin D. Am. J. Pathol. 1997; 150: 359-369PubMed Google Scholar) and are more prone to form thrombi when exposed to endotoxin (1Carmeliet P. Schoonjans L. Kieckens L. Ream B. Degan J. Bronson R. De Vos R. van den Oord J.J. Collen D. Mulligan R.C. Nature. 1994; 369: 419-424Crossref Scopus (912) Google Scholar) or hypoxia (4Pinsky D.J. Liao H. Lawson C.A. Yan S.-F. Chen J. Carmeliet P. Loskutoff D.J. Stern D.M. J. Clin. Invest. 1998; 102: 919-928Crossref PubMed Scopus (161) Google Scholar) or when the uPA gene is disrupted in otherwise healthy tissue-type plasminogen activator−/− mice (1Carmeliet P. Schoonjans L. Kieckens L. Ream B. Degan J. Bronson R. De Vos R. van den Oord J.J. Collen D. Mulligan R.C. Nature. 1994; 369: 419-424Crossref Scopus (912) Google Scholar). Down-regulation of uPA expression in wild-type mice also correlates with fibrin deposition in response to endotoxin (5Yamamoto K. Loskutoff D.J. J. Clin. 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Del Rosso M. Biochemistry. 1997; 36: 3076-3083Crossref PubMed Scopus (16) Google Scholar). uPA binds to its receptor (uPAR) with a Kd of about 1 nm (25Roldan A.L. Cubellis M.V. Masucci M.T. Behrendt N. Lund L.R. Dano K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (541) Google Scholar, 26Barnathan E.S. Kuo A. Rosenfeld L. Kariko K. Leski M. Robbiati F. Nolli M.L. Henkin J. Cines D.B. J. Biol. Chem. 1990; 265: 2865-2872Abstract Full Text PDF PubMed Google Scholar), forming a stable complex with an off-rate of several hours. The resultant complex has been shown to be involved in two separate biological cascades: (a) plasminogen activation, which results in proteolytic activity, and (b) signal transduction, which results in cell adhesion and mitogenesis. Plasminogen activation by the complex between uPA and its receptor is apparent at concentrations close to the determinedK d (27Manchanda N. Schwartz B.S. J. Biol. Chem. 1991; 266: 14580-14584Abstract Full Text PDF PubMed Google Scholar, 28Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29Wang J. Mazar A. Quan N. Schneider A. Henkin J. Eur. J. Biochem. 1997; 247: 256-261Crossref PubMed Scopus (16) Google Scholar), whereas signal transduction is apparent only at much higher concentrations (35–65 nm) (30Koopman J.L. Slomp J. de Bart A.C. Quax P. Verheijen J.H. J. Biol. Chem. 1998; 273: 33267-33272Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 31Dumler I. Petri T. Schleuning W.-D. FEBS Lett. 1993; 322: 37-40Crossref PubMed Scopus (92) Google Scholar). The lack of agreement between the concentrations required for signal transduction and the K d has led to the assumption that the uPA receptor is not involved in uPA-induced signal transduction (30Koopman J.L. Slomp J. de Bart A.C. Quax P. Verheijen J.H. J. Biol. Chem. 1998; 273: 33267-33272Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). An alternative explanation for the lack of agreement between the knownK d and the uPA concentration needed to induce signal transduction is the existence of additional binding epitopes in both uPA and uPAR, which interact with lower affinity. The possible presence of more than one kind of interaction is supported by our data (32Higazi A.A.-R. Mazar A. Wang J. Quan N. Griffin R. Reilly R. Henkin J. Cines D.B. J. Biol. Chem. 1997; 272: 5348-5353Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and the data of others (33Behrendt N. Ronne E. Dano K. J. Biol. Chem. 1996; 271: 22885-22894Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), which indicate that uPAR contains several binding epitopes that participate in the binding of uPA to uPAR. In the present paper we demonstrate the existence of such low affinity interactions. These novel interactions correlate with a modification of the activity of the resultant scuPA/suPAR complex and further activation of tcuPA by suPAR. Furthermore, the low affinity interaction stems from the capacity of suPAR to form dimers and oligomers that bind scuPA with relatively lower affinity and dissociate by the addition of an equimolar concentration scuPA. scuPA, the amino-terminal fragment of scuPA (amino acids 1–135; ATF), low molecular weight scuPA (amino acids 145–411; LMWscuPA), and low molecular weight tcuPA, scuPA, and suPAR (in part) were all gifts of Dr. Jack Henkin (Abbott Laboratories, Abbott Park, IL) and were characterized as described in previous publications (32Higazi A.A.-R. Mazar A. Wang J. Quan N. Griffin R. Reilly R. Henkin J. Cines D.B. J. Biol. Chem. 1997; 272: 5348-5353Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 34Higazi A.A.-R. Cines D.B. Thromb. Res. 1996; 84: 243-252Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Neutralizing polyclonal anti-uPAR antibodies were a gift of Dr. Douglas Cines from the University of Pennsylvania. Human thrombin was obtained from Sigma. tcuPA, Glu-plasminogen, and the plasmin substrate Spectrozyme PL were purchased from American Diagnostica (Greenwich, CT). Plasma was obtained from the Hadassah Hospital Blood Bank (Jerusalem). Blood used to obtain plasma was drawn from healthy volunteers. 450 ml of blood were collected in bags produced by Travenol Laboratories (Ashdod, Israel), containing 63 ml of citrate-phosphate-dextrose (CPD) solution (1.66 g of sodium citrate (hydrous), 61 g of dextrose, 206 mg of citric acid, and 140 mg of monobasic sodium phosphate). Plasma was separated by centrifugation at 2500 rpm for 7.5 min and at 4500 rpm for an additional 5 min to remove platelets. cDNA encoding soluble uPAR (amino acids 1–277), was generated by polymerase chain reaction using pTracer-uPAR as templates. The fragments were digested withBglII and Xho and sub-cloned into the expression vector (pMT/BiP/V5, Invitrogen). Wild-type suPARs was expressed inDrosophila Schneider S2 cells (DES system, Invitrogen) as described by the manufacturer and purified from the media using a polyclonal anti-uPAR antibody affinity column. SuPAR from Abbott Laboratories (see “Materials”) was in use too. The plasminogen activator activity was measured in the presence of plasminogen and the plasmin substrate Spectrozyme PL (28Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Increasing concentrations of scuPA in the absence or presence of 5 nm suPAR were incubated with 20 nmGlu-plasminogen and 0.5 mm Spectroyme PL in phosphate-buffered saline (PBS), pH 7.4, for 20 min. The reaction was monitored at 405 nm. In another set of experiments, 5 nmscuPA was incubated in the absence or presence of different concentrations of suPAR. In control experiments, a 10-fold molar excess of ATF or 100 nm anti-suPAR antibodies were added to suPAR before its addition to the reaction mixture. Kinetic analysis of the plasminogen activation was performed as described ((28Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 35Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar). Briefly, an equimolar concentration of the scuPA/suPAR complex (5 nm) was incubated in the presence or absence of an additional 5 nm suPAR and increasing concentrations of Glu-plasminogen and Spectrozyme PL in PBS, pH 7.4, at 37 °C. The concentration of generated plasmin was calculated during each time interval from the rate of change at 405 nm using a standard curve made with known concentrations of plasmin. The kinetic parameters of plasmin generation were then plotted using a double-reciprocal plot of the rates of plasmin generationversus plasminogen concentration. Purified fibrinogen was radiolabeled with 125I and resuspended in plasma to a specific activity of 30,000 cpm/ml. Clots were formed in 16-mm diameter tissue culture wells (Costar, Cambridge, MA) by adding 0.4 NIH units to 0.4 ml of plasma. Fibrinolysis was measured as described previously (41Higazi A.A.-R. Bdeir K. Hiss E. Arad S. Kuo A. Barghouti I. Cines D.B. Blood. 1998; 92: 2075-2083Crossref PubMed Google Scholar). Briefly, radiolabeled fibrin clots enriched with 100 nm plasminogen were overlaid for 1 h at 37 °C with 0.4 ml of serum containing 25 nmplasminogen activator (scuPA or tcuPA, in the absence or presence of increasing concentrations of suPAR), and the release of radiolabeled soluble fibrin degradation products was measured. A TSK3000SW SEC column (Beckman) was equilibrated with PBS. Samples (0.1 ml total volume) were injected using a 0.2-ml loop, and the column was developed with PBS at a flow rate of 1 ml/min. Absorbance was measured at 220 nm, and data were collected using a Beckman Gold Chromatography system. Dynamic light scattering was performed with a Dynapro-801 molecular sizing instrument (Protein Solutions, Inc., Charlottesville, VA) equipped with a 20-μl micro-sampling cell at room temperature (22 °C). suPAR and scuPA were in a buffer of 20 mm Tris, pH 7, and 10 mmMES, pH 5.0, respectively, and were concentrated to 2 mg/ml. A complex of suPAR and scuPA was made at 1:1 ratio. All protein solutions were filtered through a membrane of 0.02 μl porosity to remove any dust prior adding to the micro-sampling cell. Fifteen to 20 μl of solutions was used to measure the light-scattering signal. For each sample, at least 10 light-scattering measurements were taken, and the data were processed by Protein Solution's DynaLS and DYNAMICS software, version 4.0. Rotary-shadowed samples were prepared by spraying a dilute solution of protein (final concentration about 15 μg/ml) in a volatile buffer (0.05m ammonium formate, pH 7.4) and 70% glycerol onto freshly cleaved mica and shadowing with tungsten followed by deposition of a carbon film in a vacuum evaporator (Denton Vacuum Co., Cherry Hill, NJ) (36Fowler W.E. Erickson H.P. J. Mol. Biol. 1979; 134: 214-249Crossref Scopus (201) Google Scholar, 37Veklich Y.I. Gorkun O.V. Medved L.V. Niewenhuizen W. Weisel J.W. J. Biol. Chem. 1993; 268: 13577-13585Abstract Full Text PDF PubMed Google Scholar, 38Weisel J.W. Stauffacher C.V. Bullit E. Cohen C. Science. 1985; 230: 1388-1391Crossref PubMed Scopus (223) Google Scholar). Preparations of scuPA, suPAR, and mixtures of the suPAR/scuPA were made at a molar ratio of 2.5:1. The specimens were examined in a Philips 400 electron microscope (FEI Co., Hillsboro, OR) operating at 80 kV. All experiments were repeated several times, and many micrographs were taken of randomly selected areas to ensure that the results were reproducible and representative. About 1000 particles were counted to determine the prevalence of different particles. Binding of scuPA to suPAR was measured using a BIA 3000 optical Biosensor (Biacore, AB, Sweden) (39Myszka D.G. Curr. Opin. Bio/Technol. 1997; 8: 50-57Crossref PubMed Scopus (427) Google Scholar). This method detects binding interactions in real time by measuring changes in the refractive index at a biospecific surface and enables association and dissociation rate constants to be calculated. For these studies, recombinant suPAR and recombinant scuPA were coupled to CM5 research grade sensor chip flow cells (Biacore) via standard amine coupling procedures (40Johnsson B. Lofas S. Lindquist G. Edstrom A. Muller-Hillgran R.-M. Hanson A. J. Mol. Recognit. 1995; 8: 125-131Crossref PubMed Scopus (130) Google Scholar) usingN-hydroxysuccinimide/methyl-N′-[3-(dimethylamino) propyl] carbodiimine hydrochloride (Pierce) at a level of 1000 relative units each. Sensor surfaces were coated with ligands (10 μg/ml) in 10 mm NaAc buffer, pH 5.0. Following immobilization, unreacted groups were blocked with 1 methanolamine, pH 8.5. A third flow cell, similarly activated and blocked without immobilization of protein, served as a control surface. The binding buffer was PBS, pH 7.4, 0.005% Tween 20. Binding of scuPA was measured at 25 °C at a flow rate of 30 μl/min for 6.7 min followed by 3 min of dissociation. The bulk shift due to changes in the refractive index was measured using the control surface and was subtracted from the binding signal at each condition to correct for nonspecific signals. Surfaces were regenerated with a single 30-s pulse of 1 m NaCl, pH 3.4, followed by an injection of binding buffer for 1 min to remove this high salt solution. All injections were performed in a random fashion using the RANDOM command in the automated method. The response at equilibruim (Req) was calculated as the average response over the last 10 s of association. Data were analyzed by both linear (Scatchard) and nonlinear regression. Linear transformation (43Limbird L.E. Kenakin T. Cell Surface Receptors: A Short Course on Theory and Methods. Martinus Nijhoff, Boston1986: 51-96Google Scholar) was performed using Excel 97 software, fitting the equation R eq/C = −R eq/K d +R max/K d, whereR eq is the response at equilibrium, Cis the concentration of analyte in solution, K d is the equilibrium dissociation constant, and R maxis the maximal specific binding to the surface. Nonlinear regression was performed using GraphPad PRISM 2.0 fitting the binding isotherm directly (R eq =C·R max/(C +K d). Our previous data, as well as those of other laboratories, show that suPAR stimulates the activity of scuPA at concentrations equal or close to the K d (27Manchanda N. Schwartz B.S. J. Biol. Chem. 1991; 266: 14580-14584Abstract Full Text PDF PubMed Google Scholar, 28Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29Wang J. Mazar A. Quan N. Schneider A. Henkin J. Eur. J. Biochem. 1997; 247: 256-261Crossref PubMed Scopus (16) Google Scholar). In the initial experiments, we examined the effect of increasing the concentrations of the ligands on the activity of the resultant complex. ScuPA (5 nm) was incubated with increasing concentrations of suPAR. As expected, the addition of an equimolar concentration of suPAR stimulated the plasminogen activation activity of scuPA (Fig.1). An additional increase in the concentration of suPAR resulted in a further increase in scuPA activity. The stimulatory effect of suPAR was dose-dependent and saturable. SuPAR alone had no plasminogen activation activity. The presentation of the data as a reciprocal plot (Fig. 1 B) shows that half-maximal stimulation was achieved at a suPAR concentration of 30 nm. To prove that the apparent saturation resulted from a limited scuPA/suPAR interaction and was not the result of other limiting factors, such as the amount of plasminogen, or of the chromogenic plasmin substrate, we added additional amounts of scuPA in the plateau region of the curve (scuPA/suPAR ratio 5/80). Fig.2 shows that by increasing the concentration of scuPA from 5 to 10 nm at a fixed concentration of suPAR (80 nm, which gave a maximal response at 5 nm suPAR), a further increase of scuPA activity was obtained. In addition, Fig. 2 shows that the stimulatory effect of suPAR on scuPA activity could be abolished by the addition of ATF. This observation supports the conclusion that the observed plateau was the result of a limited interaction with suPAR. The inhibitory effect of ATF indicates that the additional interactions depend on the occurrence of primary binding between scuPA and suPAR through the ATF. To further support the role of suPAR in the stimulatory effect, we added polyclonal anti-uPAR antibodies. Fig. 2 shows that the anti-uPAR antibodies had the same inhibitory effect as ATF, whereas irrelevant IgG has no effect on the activity of the complex (not shown). Fig. 3 shows that at a scuPA/suPAR ratio of 1:2, the V max, but not theK m, was greater than with equimolar concentrations. The increase in the V max could also be the result of contamination of suPAR by uPA, but this possibility could be excluded because suPAR alone had no activity (Fig. 1) and ATF inhibited the activity. Another possibility is the presence in suPAR of a protease that cleaves scuPA to tcuPA. To exclude this possibility, we incubated scuPA with suPAR for 24 h. Under these conditions, scuPA continued to migrate as a single band when analyzed by reducing SDS-PAGE as described elsewhere (28Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) (data not shown). These results suggest that suPAR establishes novel interactions with scuPA that are apparent only at higher concentrations of the ligands and that participation of these binding epitopes induces additional conformational changes in the complex, leading to further activation. To support these conclusions, we used a clot lysis assay. Fig.4 A shows that the scuPA-mediated fibrinolysis of human plasma-derived clots was stimulated in a dose-dependent and saturable manner by suPAR concentrations that exceeded those of scuPA and theK d. In previous publications, we showed that suPAR had almost no effect on tcuPA activity when both ligands were present in equimolar concentrations (28Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 41Higazi A.A.-R. Bdeir K. Hiss E. Arad S. Kuo A. Barghouti I. Cines D.B. Blood. 1998; 92: 2075-2083Crossref PubMed Google Scholar). The next question to be addressed was, therefore, whether interactions established at higher concentrations of suPAR affected tcuPA activity. Fig. 4 B shows that tcuPA-mediated fibrinolytic activity on human plasma clots was stimulated by suPAR. Marginal stimulation (10%) was observed at a 1:1 concentration ratio, and a further increase of suPAR concentration induced a dose-dependent and saturable stimulation. Maximal stimulation (more than 5-fold) was obtained at tcuPA/suPAR ratios of 1:6. To exclude the possibility that the stimulation was the result of suPAR activity on contaminating scuPA, tcuPA was analyzed by SDS-PAGE. Overloaded gels did not reveal the presence of scuPA in the preparation (not shown). To determine whether the effect of suPAR was due to binding to tcuPA or to some other mechanism, we examined the effect of ATF on the stimulation by suPAR. Fig. 4 B shows that the effect of suPAR on tcuPA could be completely inhibited by ATF and that suPAR had no effect on the activity of LMWuPA. Anti uPAR antibodies abolished the effect of suPAR on tcuPA (not shown). In addition, ATF inhibited the stimulatory effect of suPAR on scuPA-mediated fibrinolysis, and suPAR had no effect on LMWscuPA activity (Fig. 4 A). It is widely accepted that uPA binds to suPAR at a 1:1 ratio. uPA binds to its receptor with high affinity and a K d of 1 nm. The observation that suPAR exerts a stimulatory effect at concentrations that exceed the 1:1 ratio and the theoretical saturating concentrations suggests the existence of another kind of interaction of suPAR with uPA. This additional interaction proceeds with relatively lower affinity and leads to additional functional modification of the uPA/uPAR complex. This hypothesis is supported by the observation that the activity of the resultant complex is higher and by data from the literature that demonstrate that uPA-induced signal transduction is observed only at concentrations that are much higher than would be predicted from the K d of the bimolecular complex formation (30Koopman J.L. Slomp J. de Bart A.C. Quax P. Verheijen J.H. J. Biol. Chem. 1998; 273: 33267-33272Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 42Li C. Liu J.-N. Gurewich V. J. Biol. Chem. 1995; 270: 30282-30285Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In an attempt to verify the existence of previously unknown interactions between uPA and uPAR, we used several different approaches. First, we used the size exclusion technique. Fig.5 A shows that suPAR exhibited a complex equilibrium when analyzed by SEC-HPLC. SuPAR migrated in two peaks at 4.7 and 8.1 min retention time (RT). In contrast, scuPA migrated as a single peak of 10.9 RT(not shown). Fig. 5 B shows that on SDS-PAGE, scuPA and suPAR injected into the HPLC run as single bands. In an attempt to examine the effect of scuPA on the equilibrium between the different forms of suPAR, we incubated scuPA with suPAR at several molar ratios. Fig. 5 B shows the results of the addition of scuPA at a 1:2 ratio (suPAR excess). Under these conditions, no monomeric scuPA was present, and the second suPAR peak migrated at 8.4 rather than 8.1 min. SDS-PAGE analysis of the protein present in this peak demonstrated that it represents a scuPA-suPAR complex (Fig. 6 B). Fig. 5 C presents the results of the addition of scuPA at a 1:1 ratio. Under these conditions, scuPA shifted both peaks of suPAR to a peak of RT = 8.4 min, which represents the scuPA/suPAR complex (Fig. 6 B). These data suggest that the higher molecular mass form of suPAR is able to interact with scuPA, albeit at a lower affinity. It should be noted that the monomeric scuPA-suPAR complex elutes with aRT (8.4 min) comparable with the 8.1-min peak of suPAR. This fact suggest that the suPAR that eluted with RT = 8.1 min is a dimer. The size exclusion technique shows the status of the isolated ligands and complexes but does not provide information as to the situation under equilibrium conditions. To examine the status of the reactants under equilibrium conditions, we used dynamic light scattering. The dynamic light scattering measures translational diffusion coefficients (D T) of proteins through monitoring the scattered light of protein in solution. Based onD T, the hydrodynamic radius (R h) of the protein can be calculated using the Stokes-Einstein equation: R h =k b T/6πηD T,with Boltzmann's constant (k b), solvent viscosity (η), and temperature Kelvin (T). The molecular weight of the protein is then estimated by fitting the measuredR h to a standard curve derived from 25 globular proteins. Proteins that are either nonspherical in shape or partially unfolded will show apparent molecular weights that are higher than their theoretical molecule weights. Fig. 7 A shows the regularization histogram of scuPA, and illustrates that scuPA is primarily a single species in aqueous solution. Using a monomodal cumulatant analysis, scuPA was found to have an averaged hydrodynamic radius (R h) of 3.77 nm with 16% polydispersity (0.6 nm), corresponding to an apparent molecular mass of 73 kDa, based on the standard size/weight relationship for globular proteins. The matrix-assisted laser desorption ionization (MALDI) mass spectrum of scuPA and SDS-PAGE of scuPA gave a molecular mass of 48,140 Da and ∼47 kDa, respectively (data not shown), and both are close to its theoretical molecular mass, 46,511 Da, derived from protein sequence. The difference of apparent molecular weight from mass when compared with theoretical calculation is believed to be due to the contribution of the carbohydrate present in the protein. In contrast to the monodispersity of scuPA, the regularization histogram of suPAR (Fig. 7 B) reveals some large aggregates with an R h
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