©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CaSuppresses Cell Adhesion to Osteopontin by Attenuating Binding Affinity for Integrin (*)

Dana D. Hu(§)(¶) , John R. Hoyer (1), Jeffrey W. Smith (§)

From the (1) Department of Vascular Biology (VB-1), The Scripps Research Institute, La Jolla, California 92037 and the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Osteopontin (OPN) is an extracellular matrix protein that supports osteoclast adhesion to the bone by binding to integrin . We measured the binding between OPN and integrin with recombinant human OPN and the urinary form of human OPN, uropontin. Recombinant OPN was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and cleaved from glutathione S-transferase with Factor Xa. The mass of this form of OPN (rOP27) is 27,046 Da. rOP27 is truncated at arginine residue 228, 69 amino acids short of the native carboxyl terminus. Uropontin and rOP27 support RGD-dependent cell adhesion and to bind purified integrin with similar affinities. Further study showed that OPN is the only known naturally occurring RGD-containing protein with a much greater affinity for than for the platelet integrin . Most importantly, we find that physiologic levels of Cablock cell adhesion to OPN. Measurement of binding constants between rOPN and purified integrin with surface plasmon resonance showed that the affinity between rOPN and is 26-fold lower in Ca( K= 1.1 10 M) than in Mn( K= 4.3 10 M) and 9-fold lower than in Mg( K= 1.3 10 M). In bone, the resorbing osteoclast generates elevated levels of extracellular Ca, therefore the findings presented here suggest a previously unappreciated mechanism for the modulation of bone resorption by extracellular Ca.


INTRODUCTION

Osteopontin (OPN)() is a highly acidic secreted glycoprotein that was originally identified as a constituent of bone matrix, but recent investigation reveals the presence of OPN in milk (1) , placenta (2) , urine (3) , leukocytes (4) , and some tumor cells (5, 6) . OPN has at least two established biochemical activities. 1) OPN contains an Arg-Gly-Asp (RGD) integrin recognition motif (2, 7, 8) and mediates cell adhesion and cellular signaling by binding to integrin (9, 10, 11, 12) , and 2) OPN can regulate the growth of calcium crystals (3, 13) , presumably by virtue of its high density of aspartic acid residues. OPN has roles in cellular transformation, tissue mineralization, and immune function that have been recently reviewed (14, 15

Bone is the organ system where OPN was first identified and where it almost certainly has an important role in development and homeostasis. OPN is a major noncollagenous protein in bone, and in developing bone it is localized at the interface of cartilage to bone transition (16) . The position of this protein and its highly acidic nature indicate that it could regulate the mineralization of bone. OPN also contains an RGD sequence that can bind to integrin adhesion receptors. Integrins are a large family of heterodimers that mediate cellular adhesion and signaling (17, 18) . The interaction of OPN with integrin has received much interest because the binding of these two proteins is thought to be essential for osteoclast adhesion to bone, an event that initiates bone resorption (19) . In fact, studies done in vitro lend strong support to this hypothesis because RGD peptides and monoclonal antibodies against block bone resorption in culture models (20, 21, 22) . It has also been hypothesized that integrin is a homing receptor that directs osteoclast precursors to OPN in bone matrix (19) . Moreover, it has been suggested that the binding of soluble OPN to on osteoclasts transduces signals that influence osteoclast function during bone resorption (10, 12) . Based on this collective body of data, it is reasonable to hypothesize that inhibitors of the interaction between OPN and integrin could be applied to prevent osteoporosis.

Although several studies have shown that OPN can mediate cell adhesion and signaling through integrin (9, 10, 11, 12, 23) , the binding affinity between these two proteins is not known. Thus, a major objective of this study was to measure the binding affinity between integrin and OPN. Furthermore, both proteins contain multiple divalent ion binding motifs. These motifs are important to the function of because ligands for this integrin can be placed into two categories, those whose binding is potentiated by Caand those whose binding is blocked by Ca. Given that and OPN interact at the bone surface, an environment subject to extremes in divalent ion, it is important to decide which category OPN is in. Thus, another goal of this study was to learn how divalent cations regulate binding between these two proteins. Our characterization was performed with UP, a native urinary form of OPN that is glycosylated and phosphorylated, and with a recombinant fragment of OPN (rOP27) consisting of residues 1-228. Our studies show that UP and rOP27 have comparable affinities for integrin . However, neither protein can bind the homologous platelet integrin . Both forms of OPN display identical abilities to support cell adhesion. In addition, our studies reveal that physiological levels of Casignificantly reduce the affinity of OPN for and block cell adhesion. Consequently, regulation of OPN binding to integrin by extracellular Caappears to provide a new mechanism for the modulation of bone resorption.


EXPERIMENTAL PROCEDURES

Construction of Recombinant Osteopontin Expression Vectors

The cDNA encoding full-length human OPN was modified and inserted into a GST-based expression vector to generate recombinant osteopontin. A 510-base pair cDNA encoding base pairs 157-666 of human osteopontin was generated by polymerase chain reaction using the cDNA for OPN as a template. Importantly, this form of OPN contains the 14-amino acid sequence in the amino terminus that is known to be alternatively spliced (2, 8) . The primers for this amplification were 1) 5`-G-GCT-CTA-GAC-ATA-CCA-GTT-AAA-CAG-GCT-GAT-TCT and 2) 5`-GTC-TGT-AGC-ATC-AGG-GTA-CTG-GAT (Integrated DNA Technologies, Inc). The 5` primer contains an XbaI restriction site introduced for subcloning and the cDNA sequence encoding the first 10 amino acids of OPN. The 3` primer is complementary to the cDNA sequence of OPN between base pairs 643 and 666. The polymerase chain reaction product was digested with XbaI, which cuts in the polymerase chain reaction primer, and with NdeI, which cuts internal to the polymerase chain reaction primer. The fragment was gel-purified and used to replace the corresponding DNA segment in the full-length human OPN cDNA. This procedure eliminated the eukaryotic signal sequence and introduced a 5` XbaI site for further subcloning. The insert obtained was cloned into the Bluescript plasmid and the proper cDNA sequence of this construct (pBSrOP) was verified by dideoxy sequencing.

To insert the cDNA for OPN into expression vectors, pBSrOP was excised with XbaI and XhoI and subcloned into the polylinker of pGEX(KG) distal to a polyglycine kinker (24) . The thrombin cleavage site of this vector was replaced with a Factor Xa site by subcloning a SmaI- XhoI fragment from this plasmid into the polylinker of pGEX-5x-1. The resulting construct, pGEXaOP encodes GST linked to the amino terminus of OPN by a polyglycine kinker and Factor Xa cleavage site. All designations for amino acid residues in this report use the isoleucine following the eukaryotic signal sequence as amino acid one.

Purification of Recombinant Osteopontin

To express recombinant osteopontin as a fusion protein with GST, BL26 Escherichia coli. (Novagen, FompT hsdS(rm) gal dcm lac) were transformed with pGEXaOP. Following selection of bacterial colonies producing the fusion protein, the transformants were allowed to grow until Aapproached 1 absorbance unit. Isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 0.5 mM to induce expression of GST-OPN. After a 3-h fermentation at 37 °C, cells were harvested and centrifuged at 5000 rpm at 4 °C. Bacterial pellets were frozen at 70 °C and thawed at room temperature. The bacterial pellet was suspended in TBS (50 mM Tris-HCl, 100 mM NaCl, pH 8.0), incubated on ice for 30 min with 2 mg/ml of lysozyme in the presence of phenylmethylsulfonyl fluoride (2.0 mM), benzamidine (5.0 mM), and EDTA (1 mM). Triton X-100 was added to the lysate to a final concentration of 1%. The suspension was pressed 4 times with a nitrogen bomb at 500 psi and then centrifuged at 12,000 rpm for 45 min. The supernatant containing the fusion protein was batch adsorbed to glutathione-agarose resin (Sigma) by rocking for 1.5 h at ambient temperature, and the resin was then washed with TBS containing protease inhibitors until the Areturned to base line. The fusion protein was eluted with 5 mM glutathione (reduced form). GST-OPN was dialyzed against TBS .

To cleave OPN from GST, the fusion protein was concentrated to 3-5 mg/ml using Aquacide and dialyzed against TBS, pH 8.1, containing 5 mM CaCl. The GST-OPN was incubated with Factor Xa (bovine plasma, Pierce) at an enzyme to protein ratio of 1:200 at room temperature for 2 h. The cleaved product was separated on a DEAE column (10 100 mm, Protein-Pak AP-1, Waters), and recombinant osteopontin (rOPN) was eluted at 300 mM NaCl in a salt gradient of 100-800 mM. rOPN was further purified by a C4 reverse phase column (Vydac) eluting at 40% acetonitrile in a 5-45% gradient. The mass of rOPN was determined by electrospray mass spectroscopy. The amino-terminal amino acid sequence of rOPN was determined by automated Edman degradation and corresponded to the predicted amino terminus based on the position of the Factor Xa cleavage site.

UP was purified under nondenaturing conditions from batches of human urine by monoclonal antibody affinity chromatography as described previously (3) . Importantly, amino acid sequence analysis shows that UP contains the 14 amino acids in the amino terminus of the molecule that can be alternatively spliced (2, 8) . In addition, results from amino acid composition analysis of UP are generally consistent with the predicted composition of the full-length molecule (3) . The concentration of uropontin was determined by enzyme-linked immunosorbent assay titration using standardized batches of UP. The concentration of the UP standards was determined by both Lowry protein determination and by the total content of amino acids as determined by the ninhydrin reaction after alkaline lysis. These two methods gave identical results, whereas protein assays based on Coomassie Blue dye binding greatly underestimated the protein content of samples of UP. UP concentration was also measured by optical density at 280 nm, from which an absorbance of 1.5 was found to correspond to 1 mg/ml of UP. Many studies in this report were done to compare the integrin binding function of UP and rOPN. Therefore, the concentrations of rOPN and UP were standardized by Aby assuming that this protein has an extinction coefficient of 1.5. Protein concentrations were also measured by the bicichonic protein assay (Pierce Chemical Co.), which yielded identical concentrations for both rOP27 and UP.

Cell Adhesion Assays

The ability of rOPN and UP to support cell adhesion was measured with M21 human melanoma cells, which express integrin as the predominant RGD-sensitive integrin (25) . Other studies were performed with MG63 human osteosarcoma cells, which also express (26, 27) . The method for cell adhesion assays has been previously reported (28) . Briefly, cells were harvested from tissue culture flasks with 200 µM EDTA and resuspended in adhesion buffer (Hanks' balanced salt solution, 50 mM Hepes, pH 7.4, containing 1 mg/ml bovine serum albumin) containing 0.5 mM Mn. Cells (1.5 10cells/ml) were added to wells coated with a concentration range of rOP27 or UP. Following a 90-min incubation at 37 °C, nonadherent cells were removed by gentle aspiration and washing. Adherent cells were detected by colorimetric assay for lysosomal acid phosphatase (29) . A standard curve with cells in suspension showed that all absorbance values were directly proportional to cell number.

Binding Studies With Purified and

The ability of rOPN and UP to bind integrins and was measured using purified receptor-ligand binding assays as described previously (28) . UP and rOP27 were radiolabeled with INa using Iodogen (Peirce Chemical Co.). Typical specific activities were 3-7 10cpm/ng of protein. For binding assays, purified integrin was immobilized in a 96-well Titertek microtiter wells at a coating concentration of 50 ng/well. After incubation overnight at 4 °C, nonspecific protein binding sites on the plate were blocked with 20 mg/ml of bovine serum albumin and 1 mM desired metal ion(s) in TBS. To measure binding to immobilized integrins, IrOP27 or I-UP were added to the plate and incubated for 3 h at 37 °C. Free ligand was removed by three rapid washes with TBS. The ligand-integrin complex was solubilized from the wells with 2 N boiling NaOH, and the radioactivity of the solution from each well was determined by counting. Nonspecific binding was determined by competition with either 50 µM GRGDSP or 5 mM EDTA and is subtracted from the total counts to yield counts for specific binding. Nonspecific binding was typically 7-9% of the total radioactivity bound to the immobilized receptor.

Surface Plasmon Resonance Measurements

Surface plasmon resonance (SPR) is a means of assessing receptor-ligand affinities in real time (30, 31) . SPR was performed using the BIAcore instrument from Pharmacia Biotech Inc.. Initial studies showed that OPN binding could not be detected by SPR when the integrin was immobilized on the sensor chip. Neither rOP27 nor UP could be immobilized on the chip presumably because their low isoelectric point caused electrostatic repulsion between the protein and the negatively charged dextrin surface of the sensor chip. However, the use of the fusion protein GST-OPN allowed successful immobilization of the osteopontin moiety. We presume that the fusion protein was coupled to the sensor chip through the GST moiety. It should be noted that cell adhesion and solid phase binding assays showed that GST-OPN has identical activity to rOP27 and that GST alone had no ability to bind integrin (data not shown). GST-OPN were immobilized on the biosensor chip by the methods outlined by Pharmacia. At the end of each measurement, the sensorchip surface was regenerated with 0.5 ML-arginine. GST-OPN on the surface remained active throughout all the measurements. The on- and off-rate constants ( kand k) between and GST-OPN were obtained from BIAcore measurements as follows. To obtain the association rate constant, k, a range of integrin , [I], was passed through the sensor chip coupled with GST-OPN. Response units (RU) were measured as a function of time. Plots of d(RU)/ dt against RU at different receptor concentrations generated a series of lines with different slopes (Equation 1). The latter is proportional to the concentration of receptor. Therefore, by plotting the slopes of these lines as a function of receptor concentration, a new line is obtained with a slope corresponding to k(Equation 2).

  

On-line formulae not verified for accuracy

To measure k, a pulse containing was passed through the sensor chip. The change in response unit was measured and recorded. At the end of the association phase, the flow was changed to buffer lacking integrin, allowing the bound to dissociate from GST-OPN. The off-rate constant kis derived from Equation 3.

  

On-line formulae not verified for accuracy


RESULTS

Expression of Recombinant Human Osteopontin in E. coli

The pGEXaOP vector was constructed to enable expression of OPN in E. coli as a fusion protein with glutathione S-transferase. This strategy enabled the purification of OPN under nondenaturing conditions using a glutathione affinity column. Since a thrombin cleavage site is present near the RGD sequence of OPN, the previous use of thrombin to liberate rOPN from GST yielded two inactive fragments of OPN (32) . Therefore, we engineered a GST-OPN fusion protein containing a factor Xa cleavage site and a ``glycine kinker'' (24) to facilitate proteolytic cleavage between GST and OPN while avoiding cleavage near the RGD motif.

The pGEXaOP plasmid was used to transform the lon and ampT protease-deficient E. coli strain BL26. Following induction with isopropyl-1-thio--D-galactopyranoside, the GST-OPN fusion protein was typically expressed at between 2 and 10 mg/liter. GST-OPN was purified by affinity chromatography on glutathione-Sepharose. Analysis of the fusion protein by SDS-polyacrylamide gel electrophoresis showed it migrated at a position of 85 kDa (Fig. 1 A, lane 2). The rOPN was cleaved from GST using Factor Xa. rOPN was purified from GST by chromatography on DEAE, yielding rOPN that was approximately 85% pure (Fig. 1 A, lane 3). The remaining minor contaminants were removed by chromatography on a C4 reverse-phase column. The final rOPN was greater than 95% pure (Fig. 1 A, lane 4) and yielded a single amino-terminal amino acid sequence corresponding to the residues on the carboxyl-terminal side of the Factor Xa cleavage site in the fusion protein.


Figure 1: SDS-polyacrylamide gel electrophoresis analysis and mass spectra of recombinant osteopontin. A, recombinant OPN (50 µg) was analyzed on 10% SDS-polyacrylamide gel electrophoresis at various stages of purification. The lanes on the acrylamide gel correspond to the following: lane 1, molecular weight markers; lane 2, GST-OPN purified by glutathione agarose affinity chromatography; lane 3, rOPN purified by DEAE chromatography; lane 4, rOPN purified on a C4 reverse phase column. B, to obtain the exact mass of rOPN, mass spectroscopy was performed using the electrospray method. The results show that rOPN has a mass of 27,047 Da. This form of OPN is designated as rOP27.



Although rOPN migrated at 60 kDa on SDS-polyacrylamide gel electrophoresis after cleavage from GST, mass spectroscopy showed the mass to be 27,046 Da (Fig. 1 B), thus it is designated as rOP27. Amino-terminal sequence analysis showed the amino terminus to be intact, so this form of OPN is truncated on the carboxyl-terminal side of Arg. The inability to generate a full-length form of OPN in E. coli is not entirely unexpected because OPN is known to be highly sensitive to proteolysis (see ``Discussion'').

UP and rOP27 Have Equivalent Adhesive Capacity and Similar Affinity for Integrin

To evaluate the ability of rOP27 to support cell adhesion, its adhesive activity was compared with that of native human uropontin. Uropontin is identical in amino acid sequence and composition to osteopontin but is purified from urine under nondenaturing conditions (3) . A concentration range of rOP27 and UP were coated onto microtiter wells, and the ability of M21 melanoma cells to adhere to each protein was assessed. The major RGD-binding integrin on M21 cells is (25) . The data in Fig. 2 A show that UP and rOP27 support the adhesion of M21 melanoma cells equivalently. We also found that Fab-9, a function-blocking antibody directed to the ligand binding pocket of (33) , abolished adhesion to OPN proving that adhesion is mediated by (data not shown). Additionally, all adhesion to OPN was blocked by synthetic RGD peptides. Identical results were obtained with several of other human cell lines including MG63 osteosarcoma cell, which also express integrin .


Figure 2: Comparison of the adhesive activity and integrin binding ability of rOP27 and UP. A, the ability of rOP27 to support cell adhesion was compared with that of UP using M21 melanoma cells as described under ``Experimental Procedures.'' Each data point was measured in quadruplicate, and the average is shown. This experiment was performed 6 times with identical results. B, a purified ligand-receptor binding assay was performed to compare the integrin binding ability of rOP27 with that of UP. I-rOP27 (0.2 µg/well) was added to microtiter wells coated with purified integrin (50 ng/well). The binding of I-rOP27 was challenged with a concentration range of unlabeled rOP27 () and UP (). Nonspecific binding determined in the presence of 50 mM RGD was deduced from the total binding. The data are an average of triplicate measurements. This experiment was performed five times with identical results.



Results from our analysis with M21 melanoma cells and from previous studies (10, 22) show that integrin is the cell surface receptor for OPN. To obtain the first measure of binding affinity between these two proteins and to assess potential affinity differences between rOP27 and UP for , a competitive binding assay was performed. I-rOP27 was used as radioligand, and unlabeled rOP27 and UP were used to challenge binding to purified integrin . rOP27 and UP competed almost equivalently for binding to (Fig. 2 B). The ICfor rOP27 and UP are 30 nM and 27 nM, respectively. This competition assay was performed under tracer conditions, so that the ICis a reasonable estimate of K(34) . In addition, numerous direct binding studies using this same assay format demonstrated that had an affinity of between 5 and 30 nM for I-rOP27 and of between 2 and 19 nM for I-UP (not shown). Collectively, these results demonstrate that rOP27 and native UP have essentially identical affinities for as well as equivalent adhesive capacities.

CaInterferes with Cell Adhesion to OPN

We previously found that Cacan either support or inhibit ligand binding to . Ligands such as vitronectin and fibronectin bind to purified in Ca, whereas fibrinogen does not (28) . Because the binding of OPN to is likely to be subjected to extremes in [Ca] during bone resorption (35) , we were interested in determining the effect of Caon the interaction between OPN and . As an initial test, cell adhesion studies were performed in either Ca, Mg, or Mnand across a concentration range of rOP27. As shown in Fig. 3 A, the adhesion of MG63 cells was greater in Mnthan in Mg. Cafailed to support cell adhesion. Efforts to induce cell adhesion by increasing the concentration of Caor rOP27 were unsuccessful (data not shown). Identical results were obtained when UP was used as immobilized ligand and when other cell lines, like M21 melanoma cells were studied (data not shown). Since Cadid not support adhesion to OPN, we tested the ability of this ion to interfere with cell adhesion to OPN. Adhesion to rOP27 was measured in Mn(0.2 mM) or Mg(1 mM) as supporting ions and across a range of competing Ca. As shown in Fig. 3 B, Cainhibited cell adhesion to rOP27 in a dose-dependent manner. Adhesion was completely abolished in the presence of 1 mM Ca. Nearly identical data showing suppressing of adhesion to by Cawere obtained when UP or rat osteopontin were used as immobilized ligands (data not shown).


Figure 3: Ca blocks cell adhesion to OPN. A, the ability of different divalent ions to support adhesion to OPN was tested with human MG63 osteosarcoma cells. Cells (1.5 10cells/ml) were resuspended in adhesion buffer containing either 2 mM Ca(), Mg(), or 0.5 mM Mn() and were allowed to adhere to wells coated with rOP27 at 37 °C for 1.5 h. Adherent cells were quantified by using a colorimetric assay for acid phosphatase (29). Each data point is an average of quadruplicate data points. This experiment was performed four times with identical results. B, to ascertain whether Cacan inhibit cell adhesion to osteopontin, an experiment was performed in which MG63 cells (1.5 10cells/ml) were allowed to adhere to rOP27 in adhesion buffer containing a range of Cain addition to either Mg(1.0 mM) or Mn(0.5 mM). The data are expressed as a percentage of control adhesion in the absence of Ca.



Measurement of Binding Affinity Between Purified and rOP27

To determine whether the effects of Caon cell adhesion to OPN were a result of an effect on binding to integrin , we measured the binding of OPN to purified integrin. Purified receptor binding assays were performed between I-rOP27 and . Substantially more I-rOP27 was bound in Mnthan in buffer containing Ca(Fig. 4). Similar results were found with I-UP, in which the binding in Cawas only 5-20% of total binding in Mn. Thus, the effect of Caon cell adhesion is at least partially a direct effect on OPN binding to integrin .


Figure 4: Ca supports lower binding affinity between rOP27 and purified integrin . The regulation of rOP27 binding to integrin by cations was further studied using a purified ligand-receptor binding assay. Purified integrin was coated on microtiter wells at a concentration of 50 ng/well at 4 °C for 18 h. A concentration range of I-rOP27 in binding buffer containing either 2 mM Ca() or 0.2 mM Mn() was added to the wells. Following incubation for 3 h at 37 °C, free ligand was removed by washing, and bound ligand was determined by counting. Nonspecific binding, determined by including 25 mM EDTA in identical binding assays, was subtracted from total binding. Each data point is an average of triplicate measurements. The data presented are representative of five experiments.



The binding affinity between and OPN are the product of two parameters, ligand association rate and dissociation rate. To determine at which step divalent ions exert their effect, we measured the association and dissociation rate constants ( kand k) between OPN and using SPR. These studies were performed by immobilizing purified GST-OPN on the sensor chip as described under ``Experimental Procedures'' and then passing purified over the chip in the fluid phase. The refractive index of the sensor chip surface changes as a result of integrin binding and is reported as RU (30, 31) . Control studies proved that the binding of an unrelated protein, bovine serum albumin, was negligible and that the binding between GST-OPN and was was blocked by the peptide with sequence GRGDSP but not by a peptide with the sequence SDGPRG (data not shown). Additional experiments showed that integrin does not bind to GST alone.

A series of binding studies were performed to derive kinetic constants, kand k, for the binding between and GST-OPN in different divalent ions. An example of kinetic analysis of binding of GST-OPN to in Caand Mnis shown in Fig. 5. The overlaid sensorgrams in Fig. 5 A were generated by passing purified (5 µg/ml) through the chip containing immobilized GST-OPN. It is evident that ligand association is more efficient in Mnthan in Ca. The association rate constants were derived from the sensorgrams by plotting ( d(RU)/ dt)/RU versus the concentration of fluid-phase (Fig. 5 B). The dissociation rate constants were acquired by saturating the GST-OPN with and then measuring the dissociation of the integrin. The kinetic constant kcan be derived from this analysis because dissociation of bound from GST-OPN is evident as a decrease in RU. The value of kis derived by plotting ln(RU/RU) as a function of time (Fig. 5 C). Dissociation of from GST-OPN is much faster in Cathan in Mn(3.8 10sversus 7.8 10s). The kinetic parameters of binding in all three cations are summarized in . The overall Kbetween GST-OPN and is lowest in buffer containing Mn( K= 0.43 nM). Thus binding affinity is greatest in Mn. The Kis 26-fold higher in buffer containing Ca. The Kin Mgfalls in between the values for the other two ions. We conclude that divalent ions influence both kand kfor binding of OPN to and that Cais least effective in supporting the binding of OPN to .


Figure 5: Determining the association and dissociation rate constants between OPN and with SPR. The kinetics of GST-OP binding to was measured with SPR in order to dissect effects of Caon the on and off-rate of receptor-ligand binding. A, overlaid sensorgrams of OPN-integrin binding obtained in Caand Mnare shown. A sample of 25 µl of at 10 µg/ml in binding buffer containing either 2 mM Caor 0.2 mM Mnwas injected on to the sensor chip occupied with GST-OPN. The association was observed at real time over a period of 5 min. B, to measure association rate constant, a series of SPR measurements was performed using a concentration range of . kin Mn(), and Ca() was determined as described under ``Experimental Procedures.'' A typical standard deviation for kis less than 10%. C, the dissociation rate constants were measured by generating a dissociation profile. Following binding of (40 µl of 20 µg/ml), the dissociation rate constants ( k) were determined from the slope of a plot of ln((RU)/(RU)) versus time of dissociation. The standard deviation of the measurements was less than 5%.



Because Cainhibits cell adhesion to OPN when Mnor Mgare present (Fig. 3 B), we measured the influence of Caon ligand association and dissociation when Mnwas present in the binding reaction. Binding between GST-OPN and was measured across different Caconcentrations while the [Mn] was set at 0.2 mM (Fig. 6). The inclusion of Cadecreases the rate of ligand association ( k) when Mnwas present. In fact, as the levels of Cawere increased, kreached the value measured in 2 mM Caalone (). This finding is consistent with our prior studies suggesting the presence of a class of divalent cation binding sites on integrin that regulate ligand association (28, 52) . Interestingly, the inclusion of Cain the dissociation phase along with Mndid not influence ligand off-rate, even though ligand dissociation is nearly 5-fold faster in Cathan in Mn(). This observation suggests that integrins may have distinct classes of divalent ion binding sites that separately regulate ligand association and dissociation. This hypothesis is currently being investigated.


Figure 6: Ca reduces the rate of association between OPN and when Mn is present as a supporting ion. The binding of to GST-OPN immobilized on a sensor chip was measured in 0.2 mM Mnand across a range of Ca(values shown in figure). The resulting sensorgrams are shown. Calculation of on-rates from this sensorgram showed that as Cawas increased, the association rate constant approached that measured in binding reactions where only Cawas present (Table I). When Mnwas present, Cadid not influence ligand dissociation rate (not shown).



Osteopontin Is an RGD-containing Ligand with Substantial Affinity Preference for over

Integrin is closely related to the platelet fibrinogen receptor as the two receptors contain the same subunit and subunits that are 36% identical. These integrins share many of the same ligands (36) . Some ligands have substantial affinity preference for over . However, the only ligand with affinity preference for is the snake venom cerastin, which showed only a 20-fold affinity preference for (37) . Thus, we sought to determine whether OPN has an affinity preference for over platelet integrin . We were unable to detect any binding between I-rOP27 and purified in any of the divalent ions tested (Fig. 7 A). Native UP also failed to bind purified and did not interact with on the surface of activated platelets (not shown). To extend this analysis, the ability of rOP27 to interfere with I-fibrinogen binding to was compared with that of unlabeled fibrinogen. This allowed us to test the inhibitory activity of much higher concentrations of rOP27. As expected, unlabeled fibrinogen blocked the binding of I-fibrinogen to purified . The ICfor this inhibition was near 1 nM. However, 400-fold greater levels of rOP27 had little effect on I-fibrinogen binding to . Fibrinogen binding was inhibited only by less than 10% even at 400 nM of rOP27. Therefore, OPN has little, if any, ability to bind to and is not a physiologic ligand for this integrin. OPN is the first RGD-containing protein that displays such a degree of affinity preference for over .


Figure 7: rOP27 does not bind platelet integrin . A, the affinity of rOP27 for platelet integrin was examined with a purified ligand receptor binding assay. Purified integrin was immobilized in microtiter wells (50 ng/well) and was incubated with a range of concentration of IrOP27 in either 2 mM Ca() or 0.2 mM Mn() for 3 h at 37 °C. No measurable binding was observed in either cation. B, the specificity of rOP27 was further characterized by challenging the binding of I-fibrinogen ( I- Fg; 0.1 nM) to immobilized (50 ng/well) with either unlabeled rOP27 () or fibrinogen (). Nonspecific binding was measured by adding 50 µM RGD peptide in the reaction mixture and was subtracted from the raw data. The result is an average of triplicate measurements. The data are expressed as the percentage of control binding.




DISCUSSION

This study reveals four important aspects of the binding between OPN and integrin . First, we show that the peptide backbone of OPN corresponding to residues 1-228 is sufficient for cell adhesion and for maximal binding affinity for integrin . Second, neither rOP27 nor UP bind to the platelet integrin with measurable affinity, so OPN is not a physiologic ligand for this integrin. Third, and most significant, cell adhesion to OPN is not supported by Ca. In fact, physiologic levels of this ion inhibit cell adhesion to OPN. Finally, we show that the inhibitory effect of Caon cell adhesion results from a reduction of the association rate and an increase in the dissociation rate between OPN and integrin .

The RGD motif within OPN is presumed to provide the structural basis for both cell adhesion and cell signaling. However, the protein backbone of OPN can be modified in several ways, including alternative splicing, differential glycosylations, phosphorylations, and proteolytic processing. Each of these modifications has been hypothesized to regulate integrin binding (7, 14, 15) . However, because of the heterogeneity of OPN purified from tissues, and because of the lack of quantitative information on binding affinity between OPN and integrin , the contribution of modifications of OPN to its functional activity have not been ascertained. Thus, a primary goal of this study was to compare the integrin binding affinity of recombinant OPN with native UP.

Toward this end, we expressed a nonmodified form of OPN in E. coli. During our efforts to generate recombinant OPN, we encountered severe truncations of the molecule when the protein was expressed in K12 strains of E. coli. These truncations within rOPN were only identified because of the use of mass spectroscopy. Without this application, extremely misleading data were obtained. For example, rOPN expressed in K12 E. coli migrated at 34 kDa on SDS-polyacrylamide gel electrophoresis and supported cell adhesion. However, mass spectroscopy proved the mass of this amino-terminal fragment to be only 18.8 kDa (data not shown). Consequently, without mass spectral analysis, the 18.8-kDa fragment could have been confused with full-length OPN. Based on these observations it is conceivable that many of the forms of OPN reported in the literature to migrate at different positions on SDS gels are not the full-length protein. We attempted to measure the mass of native UP by spectroscopy, but this protein was too heterogeneous to obtain good measurements, probably because of differential glycosylations and phosphorylations.

Because of the extreme sensitivity of rOPN to proteolysis, we were unable to obtain a full-length form of the molecule using GST-based expression systems. The largest form of OPN we have been able to express and isolate from BL-26 E. coli is rOP27, which extends to arginine 228. The difficulty in expressing a full-length form of OPN is not entirely unexpected, as OPN is notoriously sensitive to proteolysis during purification and even during biosynthesis. For example, OPN is cleaved into two large fragments during its purification from breast milk (1) . This cleavage pattern is thought to be similar to the thrombin cleavage of OPN that has been observed in vivo (1) . Peptide fragments of OPN have been identified in porcine bone (38) , and OPN is cleaved in its carboxyl-terminal domain during biosynthesis in Madin-Darby canine kidney cells (39) . Collectively, these observations suggest that proteolysis of the carboxyl-terminal domain of OPN is a common event that occurs in vivo.

Although rOP27 is truncated in the carboxyl terminus, this recombinant fragment is functionally identical to native uropontin in terms of supporting cell adhesion and the ability to interact with purified . Both rOP27 and UP also display the same requirement for divalent cations for integrin binding, and the same specificity for over . Therefore, we conclude that rOP27 recapitulates all of the integrin-mediated adhesive capacity of UP and that cleavage of OPN in the carboxyl terminus would not necessarily eliminate integrin binding function. One report has indicated that a carboxyl-terminal peptide of OPN that lacks the RGD motif can support cell adhesion by binding to integrin (40) . Our data do not exclude this possibility, but we never observed a difference in the affinity of the truncated rOP27 and full-length UP. As in previous studies, which examined the adhesive capacity of OPN-fusion proteins (32) , our findings show that glycosylation and phosphorylation of OPN are not necessary for integrin binding activity.

Another major finding that attests to the functional equivalence of rOP27 and UP is that neither protein can bind to platelet integrin . Although several snake venom proteins, antibodies, and peptide mimetics have been either designed or identified with preference for (41, 42, 43) , no ligand has demonstrated more than a 20-fold preference for integrin (37) . With purified receptor and radioligand binding assays, our measurements of Kbetween and OPN ranged from 5 to 30 nM at 37 °C. However, even 400 nM rOP27 did not compete substantially for fibrinogen binding to . Thus, OPN appears to be the only RGD-containing protein with a substantial affinity preference for over .

The effect of divalent cations on OPN binding to is especially pertinent because both OPN and contain multiple divalent ion binding motifs (44, 45) . Moreover, the micro environment where these proteins interact, the bone surface, is subject to extreme levels of Caduring bone resorption (35) , and the dietary intake of Ca, Mgand Mnare all known to influence the health of bone (46, 47) . Therefore, we sought to quantify the effects of divalent cations on the binding affinity between OPN and . Surprisingly, we find that Cais a negative regulator of cell adhesion to OPN. This ion is not sufficient for cell adhesion to OPN in vitro, and in fact, can block cell adhesion when supporting ions are present. This finding may explain an important aspect of osteoclast function. Osteoclasts liberate Cafrom mineralized bone during the resorption cycle and increase the free [Ca] beneath the osteoclast to as high as 40 mM(35) . The data in this report indicate that such a rise in [Ca] would preclude the formation of any additional contacts between on the osteoclast and OPN in bone. It has been previously shown that osteoclasts respond to increases in extracellular Caby ceasing to resorb bone; therefore, it was suggested that osteoclasts have a Careceptor (48, 49) . Our findings indicate that the bond between OPN and is sensitive to elevated Ca. Consequently, it is possible that in addition to being the osteoclast receptor for OPN, integrin is also the osteoclast ``Careceptor.''

The data presented here could also be important in clinical settings where a major problem is Mgimbalance, with between 6 and 20% of all patients showing abnormal magnesium levels (50) . Moreover, other studies have indicated that Mgdeprivation can cause improper bone remodeling (47, 51) . The regulation of the binding between and OPN by physiologic levels of Caand Mgunderscores the importance of maintaining the proper ion balance in vivo. Since Cafails to support binding of OPN to and because Mnis found in very low concentrations in the body, it is likely that Mgis the physiologic ion that supports OPN binding to . Therefore, the ratio of Cato Mgin vivo could have a major impact on osteoclast activity and bone homeostasis.

The mechanism of divalent ion regulation of integrin function is complex. In other recent studies, we found that ligands can displace two divalent ions from the platelet integrin and that divalent ion and RGD peptide can compete for binding to the same peptide domain on the integrin subunit (52) . Based on these data, we proposed a ``displacement hypothesis'' suggesting that competition between divalent cations and ligand for the same site on integrin could be an important regulatory event in integrin function. One of the important predictions of this hypothesis is that divalent ion binding could be favored over ligand binding and that divalent ions could interfere with cell adhesion. Evidence has already been presented that physiologic levels of Cacan suppress the ligand binding function of several integrins (53, 54, 55) . Additionally, we previously found that Cacan block fibrinogen binding to integrin (28) . Here, we show that OPN binding to is similarly attenuated by Ca. Thus, it appears that a common mechanism of preventing cell adhesion involves divalent cation suppression of ligand binding to integrins.

It is likely that the effect of Caon binding of OPN to is a result of ion binding to integrin. However, OPN is also a Ca-binding protein (44) , and we have detected conformational changes in OPN induced by Causing circular dichroism.() Thus, the binding of Cato sites on OPN may change protein conformation so that the RGD sequence is not exposed. It was recently hypothesized that the aspartic acid within the RGD motif in OPN could be a part of a Cabinding site (14) . A similar hypothesis has been suggested for thrombospondin, which also has an RGD sequence within a Cabinding motif. It was suggested that the binding of Cato this site could interfere with integrin binding (56) .

In this study the affinity of OPN for integrin was measured with radioligand binding assays and SPR. It is important to emphasize that both methods corroborate the effect of divalent cations on whole cell adhesion, and also show the same pattern of cation dependence with respect to ligand affinity (Figs. 4 and 6 A). Additionally, both approaches have been validated by demonstrating that RGD containing peptides and EDTA interfere with ligand binding. However, it is important to note that substantial affinity differences are observed between the two assays. The Kbetween OPN and is 0.43 nM by SPR, but it is between 5 and 30 nM in radioligand binding assays. In fact, similarly disparate K's have been reported in the literature for the interaction of OPN with on cells. Effective concentrations of OPN range across 6 orders of magnitude, from 1 pM(57) to near nanomolar (12, 58) and even to several micromolar (10) . In each case, data were presented that the effects of OPN on cells are integrin-mediated, but no explanation has been put forth for these affinity differences. Collectively they suggest that the affinity state of is highly sensitive to its environment. In fact, recent study has revealed that the affinity state of many integrins can be modulated by a variety of factors (59) . It is likely that the two assays used here display in different conformations and thus different affinity states. Similarly, it is also reasonable to speculate that prior studies of the interaction of OPN with cells reflects binding of OPN to that is in different affinity states.

Although factors that modulate the affinity of await identification, our data suggest that receptor clustering could be one potential reason for the observed differences in affinity. Because we could not detect the binding of soluble OPN to covalently immobilized on sensor chips, the SPR assay had to be configured so that the integrin was in the solution phase, which contains no detergent. Electron microscopy has previously established that purified integrins assemble ``tail to tail'' in the absence of detergent (60, 61) . Therefore, it is likely that in SPR the affinity measurement is really made between OPN and clusters of the integrin. Consequently, our affinity measurements by SPR are probably more appropriately be termed avidity. Nevertheless, these measurements could be physiologically relevant because it is well recognized that integrins exist in multivalent clusters on cells. Integrins assemble into multivalent structures termed focal contacts in response to ligand. Moreover, we have recently found that integrin is clustered into large aggregates on cells in suspension, even in the absence of ligand.() Based on these observations, there is ample reason to suspect that multivalent interactions are an important physiologic aspect of OPN binding to integrin . The affinity differences we observe between SPR measurements and radio-ligand binding assays and the reported differences in the effective concentrations of OPN suggest that cellular events that modulate the affinity of integrin may regulate the biological function of OPN.

  
Table: Affinity constants between GST-OPN and integrin in different divalent cations

The association ( k) and dissociation ( k) rate constants between GST-OPN and were determined with surface plasmon resonance as described under ``Experimental Procedures'' and in Fig. 6. All SPR measurements were obtained at 25 °C. The levels of each divalent ion used for these measurements are Mn, 0.2 mM; Mg, 2 mM; Ca, 2 mM. The cation concentrations have been shown to be optimal for ligand binding to integrin (28).



FOOTNOTES

*
This study was supported in part by National Institutes of Health Grants CA 56483 and AR 42750 (to J. W. S.) and DK 33501 (to J. R. H.). This is manuscript 8972-VB from the Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported initially by a postdoctoral grant from Monsanto/Searle and subsequently by a postdoctoral grant from the California affiliate of the American Heart Association.

The abbreviations used are: OPN, osteopontin; rOPN, recombinant osteopontin; UP, uropontin; GST, glutathione S-transferase; TBS, Tris-buffered saline; SPR, surface plasmon resonance; RU, response unit(s).

J. Hoyer, unpublished observations.

I. Stuiver and J. Smith, unpublished observations.


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