From the
Osteopontin (OPN) is an extracellular matrix protein that
supports osteoclast adhesion to the bone by binding to integrin
Osteopontin (OPN)
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
Although several studies have shown that OPN can
mediate cell adhesion and signaling through integrin
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.
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
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 A
On-line formulae not verified for accuracy
To measure
k
On-line formulae not verified for accuracy
The pGEXaOP plasmid was used to transform the lon and ampT protease-deficient E. coli strain BL26.
Following induction with
isopropyl-1-thio-
A series of binding studies were performed to derive kinetic
constants, k
This study reveals four important aspects of the binding
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
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
Another
major finding that attests to the functional equivalence of rOP27 and
UP is that neither protein can bind to platelet integrin
The effect of divalent cations
on OPN binding to
The data presented here
could also be important in clinical settings where a major problem is
Mg
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
It is likely that the
effect of Ca
In this study the affinity of OPN for integrin
Although factors that
modulate the affinity of
The
association ( k
. 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 Ca
block 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
.
(
)
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
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.
(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 Ca
and 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 Ca
significantly
reduce the affinity of OPN for
and
block cell adhesion. Consequently, regulation of OPN binding to
integrin
by extracellular
Ca
appears to provide a new mechanism for the
modulation of bone resorption.
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.
Purification of Recombinant Osteopontin
To express
recombinant osteopontin as a fusion protein with GST, BL26
Escherichia coli. (Novagen, FompT
hsdS
(r
m
)
gal dcm lac) were transformed with pGEXaOP. Following
selection of bacterial colonies producing the fusion protein, the
transformants were allowed to grow until A
approached 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
A
returned to base line. The fusion protein was
eluted with 5 mM glutathione (reduced form). GST-OPN was
dialyzed against TBS .
. 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.
by 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
10
cells/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
The ability of rOPN and
UP to bind integrins
and
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
10
cpm/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 ( k
and
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).
, 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 k
is
derived from Equation 3.
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.
-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 IC
for rOP27 and UP
are 30 nM and 27 nM, respectively. This competition
assay was performed under tracer conditions, so that the IC
is 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.
Ca
We previously found that CaInterferes with Cell Adhesion to
OPN
can
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 Ca
on the interaction between OPN and
. As an initial test, cell adhesion
studies were performed in either Ca
,
Mg
, or Mn
and across a
concentration range of rOP27. As shown in Fig. 3 A, the
adhesion of MG63 cells was greater in Mn
than in
Mg
. Ca
failed to support cell
adhesion. Efforts to induce cell adhesion by increasing the
concentration of Ca
or 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 Ca
did 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,
Ca
inhibited 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 Ca
were 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
10
cells/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
Ca
can inhibit cell adhesion to osteopontin, an
experiment was performed in which MG63 cells (1.5
10
cells/ml) were allowed to adhere to rOP27 in adhesion buffer
containing a range of Ca
in 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
To
determine whether the effects of Ca
and rOP27
on 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 Mn
than in buffer containing Ca
(Fig. 4).
Similar results were found with
I-UP, in which the
binding in Ca
was only 5-20% of total binding
in Mn
. Thus, the effect of Ca
on
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 ( k
and 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.
and k
,
for the binding between
and GST-OPN
in different divalent ions. An example of kinetic analysis of binding
of GST-OPN to
in Ca
and Mn
is 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 Mn
than 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 k
can be derived from this
analysis because dissociation of bound
from GST-OPN is evident as a decrease in RU. The value of
k
is derived by plotting
ln(RU
/RU
) as a function of time
(Fig. 5 C). Dissociation of
from GST-OPN is much faster in Ca
than in
Mn
(3.8
10
s
versus 7.8
10
s
). The kinetic parameters of binding in all
three cations are summarized in . The overall
K
between GST-OPN and
is lowest in buffer containing
Mn
( K
= 0.43
nM). Thus binding affinity is greatest in
Mn
. The K
is 26-fold
higher in buffer containing Ca
. The
K
in Mg
falls in
between the values for the other two ions. We conclude that divalent
ions influence both k
and k
for binding of OPN to
and that
Ca
is 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 Ca
on the on and off-rate
of receptor-ligand binding. A, overlaid sensorgrams of
OPN-integrin binding obtained in Ca
and
Mn
are shown. A sample of 25 µl of
at 10 µg/ml in binding buffer
containing either 2 mM Ca
or 0.2 mM
Mn
was 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
. k
in
Mn
(
), and Ca
(
) was
determined as described under ``Experimental Procedures.'' A
typical standard deviation for k
is 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 Mn
or
Mg
are present (Fig. 3 B), we measured
the influence of Ca
on ligand association and
dissociation when Mn
was present in the binding
reaction. Binding between GST-OPN and
was measured across different Ca
concentrations
while the [Mn
] was set at 0.2 mM
(Fig. 6). The inclusion of Ca
decreases the
rate of ligand association ( k
) when Mn
was present. In fact, as the levels of Ca
were
increased, k
reached the value measured in 2
mM Ca
alone (). 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
Ca
in the dissociation phase along with
Mn
did not influence ligand off-rate, even though
ligand dissociation is nearly 5-fold faster in Ca
than 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 Mn
and across a
range of Ca
(values shown in figure). The resulting
sensorgrams are shown. Calculation of on-rates from this sensorgram
showed that as Ca
was increased, the association rate
constant approached that measured in binding reactions where only
Ca
was present (Table I). When Mn
was present, Ca
did not influence ligand
dissociation rate (not shown).
Osteopontin Is an RGD-containing Ligand with Substantial
Affinity Preference for
Integrin
over
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 IC
for
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.
. 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
Ca
on cell adhesion results from a reduction of the
association rate and an increase in the dissociation rate 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.
. 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.
. 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
K
between
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
.
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
Ca
during bone resorption
(35) , and the
dietary intake of Ca
, Mg
and
Mn
are 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
Ca
is 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 Ca
from 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 Ca
by ceasing to resorb bone; therefore, it was suggested that
osteoclasts have a Ca
receptor
(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
``Ca
receptor.''
imbalance, with between 6 and 20% of all patients
showing abnormal magnesium levels
(50) . Moreover, other studies
have indicated that Mg
deprivation can cause improper
bone remodeling
(47, 51) . The regulation of the binding
between
and OPN by physiologic
levels of Ca
and Mg
underscores the
importance of maintaining the proper ion balance in vivo.
Since Ca
fails to support binding of OPN to
and because Mn
is
found in very low concentrations in the body, it is likely that
Mg
is the physiologic ion that supports OPN binding
to
. Therefore, the ratio of
Ca
to Mg
in vivo could
have a major impact on osteoclast activity and bone homeostasis.
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 Ca
can suppress the ligand binding function of
several integrins
(53, 54, 55) . Additionally,
we previously found that Ca
can 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.
on 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 Ca
using circular dichroism.
(
)
Thus, the binding of Ca
to 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 Ca
binding site
(14) . A similar hypothesis has been suggested for
thrombospondin, which also has an RGD sequence within a Ca
binding motif. It was suggested that the binding of
Ca
to this site could interfere with integrin binding
(56) .
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 K
between 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.
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
) 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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.