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INTRODUCTION |
Tissue transglutaminase
(TG)1 (EC 2.3.2.13) is a
widely distributed intra- and extracellular
calcium-dependent enzyme, which catalyzes the formation of
high molecular mass complexes of its substrate proteins by creating
isopeptide cross-links from glutamine and lysine residues and releasing
ammonia (1, 2). TG is suggested to be involved in matrix maturation and
stabilize the tissue with cross-links that are resistant to normal
proteolysis (1, 2). TG is closely related to wound healing which
suggests a role for it in tissue remodeling and repair (3, 4).
Immunohistochemical data have also demonstrated the presence of TG in
mineralizing cartilage and bone (5, 6) and the enzyme is thought to
participate in matrix cross-linking before the tissue undergoes
calcification (5, 6). The number of proteins serving as glutaminyl
substrates for TG is restricted indicating the physiological importance
of its functions (1). The roles of TG and the actions of its enzymatic products, meaning high molecular weight proteins, are still unclear.
Osteopontin (OPN), a prominent and potentially multifunctional acidic
phosphoglycoprotein (7, 8), is a substrate of TG (9-11). OPN is a
major product of bone forming cells, osteoblasts, but is not specific
to bone. It is also synthesized in other types of tissues and found in,
e.g. inner ear, brain, kidney (7), and atherosclerotic
plaques (13, 14), and it is also secreted into milk (12) and urine
(15). Its production is also related to immunity, infection, and cancer
(8). Osteoblasts express OPN at an early developmental stage of bone
formation (16, 17). In bone, OPN is deposited into unmineralized matrix
prior to calcification and thereon localized at various tissue
interfaces, e.g. cement lines, lamina limitans, and between
collagen fibrils of fully matured hard tissues (18). Recent knock-out
mice experiments by Liaw et al. (19) indicate that OPN, more
specifically, functions in tissue repair, matrix organization, and
collagen fibrillogenesis.
The role of polymeric OPN, resulting from cross-linking by TG, is
unknown. We have previously demonstrated that osteocalcin inhibits TG
activity in vitro as measured by cross-linking of osteopontin (20). Since recent gene knock-out experiments have demonstrated that osteocalcin is an inhibitor of bone formation (21),
our results suggest that TG activity and the OPN aggregates may be
involved in enhancement of biomineralization or matrix maturation that
precedes it. In this study the collagen binding properties of polymeric
and monomeric OPN were investigated since this feature could be pivotal
for the maturation and organization of the bone matrix as well as for
the mineralization event. The collagen types examined were the fiber
forming collagens, types I and II, III and V, which are synthesized in,
e.g. bone, cartilage, and vascular smooth muscle cells (22),
and type IV, which is a basement membrane collagen (23). We provide
evidence that OPN, as a high molecular weight complex form, exhibits
significantly increased binding ability to all tested collagens. This
may result from an altered conformation of the OPN after polymerization
as observed by circular dichroism measurements. A more stabile
structure and amplified collagen binding property, after treating OPN
with an enzyme that is intimately involved in tissue repair, brings further support to OPN's role as a tissue remodeling protein and gives
an insight into the functions of the polymeric OPN.
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EXPERIMENTAL PROCEDURES |
Materials--
OPN antibody was a gift from Dr. E. Sørensen
(University of Aarhus, Denmark). TG antibody was generously donated by
Dr. D. Aeschlimann (University of Wisconsin). Anti-collagen type I,
anti-rabbit IgG alkaline phosphatase conjugate, guinea pig tissue
transglutaminase (specific activity: 2 units/mg), collagen types I
(from calf skin), II (from bovine tracheal cartilage), III (from human
placenta), IV (from human placenta), V (from human placenta), bovine
serum albumin, 5-bromo-4-chloro-3-indolylphosphate/nitro blue
tetrazolium, and FAST p-nitrophenyl phosphate alkaline
phosphatase substrate were purchased from Sigma. Anti-collagen type V
antibody was purchased from Rockland Immunochemicals (Gilbertsville,
PA). Anti-casein and anti-ovine IgG were from ANAWA Trading SA (Wangen,
Switzerland). 125I-Radionuclide was obtained from NEN Life
Science Products, Inc. (Boston, MA). IODO-BEADS® were from
Pierce, Inc.. The membrane blocking reagent from digoxigenin luminescent detection kit for nucleic acids was from
Boehringer-Mannheim Gmbh (Mannheim, Germany). PD-10 desalting columns
were from Bio-Rad and polyvinylidene difluoride Immobilon P membranes
from Millipore (Bedford, MA). CentriplusTM concentrators
were purchased from Amicon Inc. (Beverly, MA). Nunc-ImmunoTM microtiter plates with MaxiSorp surface were
from Nalge Nunc International (Naperville, IL). Instaview Universal
protein stain was from BDH Laboratory Supplies (Poole, United Kingdom).
Instrumentation--
Chromatography was performed with Pharmacia
LKB GradiFrac chromatography system and Pharmacia LKB FPLC instrument.
HPLC purifications were done with Hewlett-Packard 1050 HPLC system.
Protein sequencing was performed with Applied Biosystem 477 A
sequencer, amino acid analysis with LKB 4151 Alpha Plus amino acid
analyzer, and mass spectrometry with Bruker Biflex MALDI-TOF mass
spectrometer. Circular dichroism was measured with JASCO J-720
spectropolarimeter (Institute of Biotechnology, University of Helsinki, Finland).
Protein Purification--
OPN was isolated from bovine milk by
the method of Sørensen and Petersen (12). OPN was further purified by
reverse phase-HPLC (Vydac C-4 column) using 0.1% trifluoroacetic acid
as buffer A and 70% isopropyl alcohol in 0.1% trifluoroacetic acid as
buffer B in a 60-min linear gradient from 0 to 70% buffer B at a flow rate of 1 ml/min. HPLC fractions containing OPN were pooled and concentrated with a microconcentrator having a molecular mass cut-off
of 10 kDa. The homogeneity of OPN was confirmed by amino acid analysis,
N-terminal sequencing, SDS-PAGE, and MALDI-TOF mass spectrometry.
Preparation and Purification of High Molecular Weight
Osteopontin--
OPN (1 mg) was treated with TG (enzyme:substrate
ratio, 1:2.5 (w/w)) for 24 h at 37 °C. The 2-ml reaction
mixture contained 50 mM Tris-HCl, pH 8.0, 2.5 mM CaCl2, and 1 mM dithiothreitol. After incubation the reaction mixture was subjected to desalting using
PD-10 column and 20 mM Tris-HCl, pH 8.0, as an eluent. The elution was monitored by measuring the absorbance at 280 nm of manually
collected fractions. The protein containing fractions were pooled and
adsorbed into a Mono-Q anion exchanger equilibrated with 20 mM Tris-HCl, pH 8.0 (buffer A). Proteins were eluted with FPLC using a gradient of 1 M NaCl in buffer A (0-45% in
10 min, 45-75% in 30 min, 75-100% in 10 min). Protein peaks were
desalted in PD-10 column, eluted with 0.1 M
NH4HCO3, pH 8.0, aliquoted, and lyophilized.
The preparations were analyzed by 8.5% SDS-PAGE stained by Instaview
Universal protein staining and by Western blotting using specific OPN
and TG antibodies.
Radioiodination of Proteins--
Osteopontin, purified polymeric
osteopontin and collagen type I were radiolabeled with 125I
using IODO-BEADS® following the protocol provided by the
manufacturer. Shortly, 20 µg of each protein was labeled with 2 mCi
of 125I (100 µCi/µg of protein). The labeling was
performed in 0.1 Tris-HCl, pH 6.5, for 30-40 min. Free
125I was removed from the reaction mixture by Sephadex G-25
chromatography using a PD-10 column. Monomeric and polymeric
osteopontin were eluted with 5 mM
NH4HCO3, pH 8.0, and collagen type I with 50 M Tris-HCl, pH 6.5. Radiolabeling was confirmed by running
the labeled proteins in 8.5% SDS-PAGE followed by overnight
autoradiography of the gels.
Cross-linking of Osteopontin and Casein with Increasing Amounts
of Tissue Transglutaminase--
Cross-linking reactions were conducted
in a total volume of 100 µl of 50 mM Tris-HCl, pH 8.0, containing 2.5 mM CaCl2 and 1 mM
dithiothreitol. Reaction mixtures contained 2.5 µg of OPN or casein
and 5, 10, 20, 50, 100, 500, and 1000 ng of the enzyme, yielded
increasing amounts of cross-linked forms of the substrates. The
reactions were carried out for 2 h at 37 °C. TG was omitted from control experiments. For the collagen binding experiments in the
absence of Ca2+, the reactions were terminated with 0.25 M EDTA before applying to ELISA plates. OPN cross-linking
reactions that were analyzed by Western blotting were terminated by
lyophilization. Western blotting was performed as described previously
(20). Casein cross-linking was analyzed by 12% SDS-PAGE, each sample
containing 20 µg of casein.
Collagen Binding ELISA--
Collagen types, I, II, III, IV, and
V, originally in 10 mM acetic acid, were neutralized by
diluting them in 50 mM
Na2CO3/NaHCO3, pH 9.7, buffer and
then applied to microtiter wells in a 10 µg/ml concentration (1 µg/well). The plates were incubated with proteins overnight at
4 °C. Wells were washed with 0.1% Tween 20 in 50 mM
Tris-buffered saline, pH 8.0, and blocked with 1% BSA in the washing
buffer. From hereon, the procedure was carried out at 30 °C. OPN or
casein cross-linking reaction mixtures, performed in the presence of
increasing amounts of TG (see above), were applied to wells directly
after incubation and allowed to interact with the coated collagens for
15 min. The wells were washed and incubated with rabbit anti-bovine OPN
IgG (anti-casein IgG for casein) diluted in the washing buffer. After
washing, the wells were subjected to a treatment with anti-rabbit IgG
alkaline phosphatase conjugate (for OPN) or anti-ovine IgG alkaline
phosphatase conjugate (for casein). The bound antibody was detected
with FAST p-nitrophenyl phosphate alkaline phosphatase
substrate, following instructions of the manufacturer. The absorbance
values were measured at 405 nm. Negative control experiments included
binding of OPN (or casein) to BSA, binding of TG-treated OPN (or
casein) to BSA (background) and to plastic and experiments without OPN
(or casein), i.e. studying recognition of TG by OPN (or
casein) antibody.
To test the binding of soluble collagen type V to immobilized OPNs, the
wells were coated with 1 µg of monomeric and purified polymeric OPNs
in 50 mM Tris-HCl, pH 8. In a reversed situation collagen
type V immobilization was performed as described above. In both assays
the interacting proteins were diluted in 50 mM Tris-HCl, pH
8, containing 2.5 mM CaCl2. Otherwise the
procedure followed the above mentioned ELISA assay.
Dot Blot Overlay Assay--
Collagen type I was diluted in 50 M Na2CO3/NaHCO3, pH
9.7, buffer at 10 µg/ml concentration and applied onto a
polyvinylidene difluoride membrane using a dot blot filtration
apparatus (1 µg/dot). Protein samples were allowed to adsorb onto the
membrane for 30 min before vacuum filtration. The membrane was blocked
with 1% BSA in 10 mM imidazole buffer, pH 6.8, containing
60 mM KCl and 2.5 mM CaCl2. After
blocking, the membrane was shortly washed with the same 10 mM imidazole/KCl/CaCl2 buffer. Thereafter, the membrane was overlaid with OPN monomer or polymer diluted in the above
mentioned 10 mM buffer at a concentration of 6 µg of
protein/ml; 1 × 106 cpm/ml of
125I-labeled protein was added into the solution as a
tracer. The proteins were allowed to interact for 1 h at room
temperature. The membrane was washed five times with the 10 mM imidazoleKCl/CaCl2 buffer containing 0.5%
Tween 20, before drying and exposing it to x-ray film for 4 h at
70 °C. After autoradiography individual dots were cut off and the
bound proteins were quantitated by counting the bound
125I-radioactivity with a
-counter.
Dot blot overlay assay was also carried out in reversed situation
immobilizing both monomeric and polymeric OPN and incubating them with
soluble 125I-labeled collagen type I. The procedure was
otherwise the same except that monomeric and polymeric OPN were
immobilized in 5 mM NH4HCO3, pH 8, in a concentration of 50 µg/ml (5 µg/dot) and that the collagen
type I concentration in the overlay incubation was 30 µg/ml. The
amount of 125I-labeled collagen tracer was 1 × 106 cpm/ml.
Quantification of protein binding onto polyvinylidene difluoride
membrane was performed separately by dotting proteins with 10,000 cpm
of iodinated protein tracer onto the membrane. Membrane bound
radioactivity reflected the total amount of membrane-bound protein.
Circular Dichroism Spectroscopy--
Monomeric OPN and purified
polymeric OPN were subjected to conformational analysis using circular
dichroism. The CD spectra were measured at room temperature using a
quartz cell with 0.1-mm optical path length. Spectra were measured in
water from 250 to 190 nm at a speed of 20 nm/min. Ten scans were
acquired and averaged for each sample. To obtain a secondary structure
estimation, the CD data were analyzed using program J-700 1.10.02.
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RESULTS |
Cross-linking of Osteopontin and Casein with Increasing Amounts of
Transglutaminase--
To study the collagen binding property of high
molecular weight OPN, we gradually increased the state of OPN
cross-linking by treating it with increasing amounts of TG. In the
reaction series the amounts of TG corresponded to ratios of 1:500,
1:250, 1:125, 1:50, 1:25, 1:5, and 1:2.5 (w/w of TG protein to OPN
protein). Fig. 1A of the
Western blotted reaction series shows that the OPN band above 120 kDa
increases as the amount of cross-linking enzyme increases in the
mixture. The 2-h treatment did not cross-link all available monomeric
OPN since this form was still present in the reaction mixture.
According to the molecular weight markers, the high molecular weight
OPN is a heterogenous mixture of OPN complexes with molecular masses
ranging from 120 to over 250 kDa. However, the migratory properties of
the monomeric OPN in SDS-PAGE analysis are altered and the 33-kDa OPN
monomer migrates like a 60-kDa protein. Therefore, the molecular
weights of the polymers could be less than the Western blots indicate.
Bovine milk itself also appears to contain some high molecular weight
OPN, which copurifies with the monomeric form (Fig. 1A,
lane 1).

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Fig. 1.
Osteopontin and casein cross-linked with
increasing amounts of tissue transglutaminase. A,
bovine OPN (2.5 µg) was incubated with tissue TG for 2 h at
37 °C. Reaction products were resolved on 8.5% SDS-PAGE,
electroblotted onto a polyvinylidene difluoride membrane, and stained
with a polyclonal antibody against bovine OPN. Lane 1, OPN;
lanes 2-8, OPN cross-linked with 5, 10, 20, 50, 100, 500, and 1000 ng of tissue TG, respectively. Molecular weight markers are
indicated on the left margin. B, casein was
cross-linked with TG for 2 h at 37 °C and reaction products
were analyzed by 12% SDS-PAGE. Lane 1, casein; lanes
2-8, OPN cross-linked with 5, 10, 20, 50, 100, 500, and 1000 ng
of tissue TG, respectively. Molecular weight markers are shown on the
left.
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To study the specificity of the effect of polymerization on OPN's
collagen binding, control experiments with casein were performed. Casein was chosen as it is an acidic phosphoprotein and relatively well
characterized TG-substrate having a molecular weight close to that of
OPN (24, 25). Casein cross-linking experiments were analyzed by 12%
SDS-PAGE. Fig. 1B shows that the TG treatment reduced the
amount of the monomeric casein in the reaction mixture and increased
the formation of cross-linked casein.
In Vitro Produced Polymeric Osteopontin--
For further studies
we cross-linked OPN with TG and purified the reaction products by
Mono-Q anion exchange chromatography. The chromatogram is illustrated
in Fig. 2A and the
electrophoretic analysis of the collected peaks, including their
Western analysis with OPN and TG antibodies in Fig. 2B.
Polymers were found in two fractions, peaks B and
C. SDS-PAGE and Western analyses with OPN antibody showed
that peak C contains more higher molecular mass OPN polymers (Fig.
2B, lane 8), is more homogenous, and contains less impurities (Fig. 2B, lane 7) than the
polymer in peak B (Fig. 2B, lane 4). Further
Western analysis showed slight staining of the polymer in peak C with
the TG antibody indicating the presence of TG-TG or TG-OPN cross-linked
complexes in the preparation (Fig. 2B, lane 9).
The polymer preparation from peak C was used in further studies.

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Fig. 2.
Purification of polymeric osteopontin with
Mono-Q anion exchange chromatography. OPN was cross-linked with TG
(1:2.5 (w/w) TG to OPN) at 37 °C overnight. After concentrating and
desalting, the protein mixture was adsorbed into a Mono-Q anion
exchange column in 20 mM Tris-HCl, pH 8. Proteins were
eluted using a FPLC system with a NaCl gradient and the elution was
monitored at 280 nm. Fractions were collected manually, desalted on a
PD-10 column, and lyophilized. A, Mono-Q chromatogram. Three
peaks obtained were designated as A, B, and C.
B, electrophoretic analysis of the protein peaks collected
from the Mono-Q chromatography. Lanes 1, 4, and
7, represent peaks A, B, and C, respectively, resolved by
8.5% SDS-PAGE. Lanes 2, 5, and 8, represent the
same peaks subjected to Western analysis after 8.5% SDS-PAGE
separation and detected with the OPN antibody. Lanes 3, 6, and 9, represent Western blots of the same peaks detected
with the TG antibody.
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Tissue Transglutaminase Treatment Increases the Binding of
Osteopontin to Collagens--
An enzyme-linked immunosorbent assay was
used to compare the collagen binding properties of the monomeric and
polymeric forms of OPN. Different collagens (types I-V) were
immobilized onto microtiter wells and incubated with OPN samples
cross-linked with increasing amounts of TG. To confirm that all samples
contained equal amounts of OPN, no purification steps were performed
for the cross-linked samples, but they were directly applied onto collagens after incubation. Fig.
3A shows that the binding of OPN to different collagens clearly increased as a function of the
amount of TG in the reaction mixture that contains 2.5 mM CaCl2. Collagens also appeared to be saturated with OPN.
Since the OPN antibody did not detect TG (data not shown), the increase in the binding of OPN to collagens is due solely to the presence of the
polymeric forms of OPN in the sample. When Ca2+ was
chelated from the reaction mixtures with EDTA prior to incubation with
the ligand proteins, a drastic decrease in binding was observed. In
this case, the TG-treated OPN showed binding only to collagen type V
(Fig. 3B).

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Fig. 3.
Collagen binding of tissue
transglutaminase-treated osteopontin and its dependence on
calcium. ELISA microtiter wells were coated overnight at 4 °C
with 1 µg of type I, II, III, IV, and V collagen diluted in 0.05 M Na2CO3/NaHCO3, pH
9.7, buffer. After washing and blocking the wells with 1% BSA, the
immobilized collagens were allowed to interact for 15 min with OPN
samples cross-linked with increasing amounts of TG. Reactions were
prepared as described under "Experimental Procedures" and are
illustrated as Western blots in Fig. 1A. Wells were washed
and subjected to a primary OPN antibody incubation for 1 h and
visualized using an anti-rabbit IgG-alkaline phosphatase conjugate and
p-nitrophenyl phosphate as a substrate. A, assays
performed in the presence of 2.5 mM CaCl2.
B, assays performed after chelating the Ca2+
from the reaction mixtures with 10 mM EDTA before applying
them onto the collagens. From the blocking stage on, the ELISA
procedure was conducted at 30 °C. Error bars represent
standard errors of three independent determinations, each including
triplicate assays (n = 9). , collagen type I; ,
collagen type II; ×, collagen type III; , collagen type IV; ,
collagen type V.
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Casein reaction series, used as a comparison and control, behaved in an
opposite way (Fig. 4, A and
B). Casein appeared to bind to collagens both in the
presence and absence of calcium ions, but the binding decreased as the
amount of TG increased in the reaction mixture, suggesting that TG was
competing with casein in collagen binding. This was confirmed by
detection of increasing amounts of collagen-bound TG in the wells (data
not shown). TG was bound to collagens also in experiments with OPN (data not shown), however, as seen in Fig. 3A, TG does not
block the binding of OPN. These results indicate that casein does not possess specific affinity for collagens either as monomeric or polymeric forms and that binding of OPN to collagens before and after
TG treatment seems to be a specific action of this particular protein
and this post-translational modification.

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Fig. 4.
Collagen binding of casein treated with
increasing amounts of tissue transglutaminase. Casein was treated
with TG for 2 h at 37 °C resulting in increasing amounts of
cross-linked forms of casein (shown in Fig. 1B). These
reaction products were applied onto immobilized collagen types I, II,
III, IV, and V. Protein-protein interaction was allowed to take place
for 15 min at 30 °C followed by washing of the plates and detection
of the collagen bound casein by casein antibody and appropriate
secondary antibody IgG-alkaline phosphatase conjugate. Binding was
visualized using p-nitrophenyl phosphate as alkaline
phosphatase substrate. Absorbances were measured at 405 nm.
A, assays carried out in the presence of 2.5 mM
CaCl2. B, assays performed after treating the
reaction mixtures with 10 mM EDTA before applying them onto
the collagens. Error bars represent standard errors of two
independent determinations, each including triplicate assays
(n = 6). , collagen type I; , collagen type II;
×, collagen type III; , collagen type IV; , collagen type
V.
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Since the elevated OPN polymer binding to collagens can also partly
depend on the tendency of the OPN aggregates to bind more antibody, we
quantified the collagen binding in a dot blot overlay assay using
purified radioiodinated proteins. We were also interested in finding
out how collagen types I and V would behave when they were in a soluble
form. This interest was directed to collagen types I and V as they
seemed to bind polymeric OPN most efficiently, collagen type V even in
the absence of calcium ions (Fig. 3B). We radioiodinated
monomeric and polymeric OPN as well as collagen type I as illustrated
in Fig. 5. Radioiodination of collagen
type V was not, however, successful (data not shown) and, therefore, we
used an ELISA binding assay to study the binding of soluble collagen V
to pure immobilized polymeric and monomeric OPN.

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Fig. 5.
Electrophoretic analysis and autoradiography
of radioiodinated monomeric and polymeric osteopontins and collagen
type I. 20 µg of purified osteopontin, its polymeric form, and
collagen type I were labeled with 2 mCi of 125I. Proteins
were resolved on 8.5% gel and autoradiographed. Lane 1, 10 µg of OPN; lane 2, 10 µg of polymeric OPN; lane
3, 10 µg of collagen type I. Lanes 4, 5, and 6, each
lane contains 200,000 counts/min of the respective
125I-labeled protein.
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Fig. 6A represents results
from a dot blot overlay assay, showing that the polymeric OPN bound
more efficiently to immobilized collagen type I than the monomeric OPN.
In the reversed situation only a slight difference in binding of
collagen type I to OPNs was observed. Quantification results are
presented in Table I indicating a 5-fold
increase in OPN binding to collagen type I as a polymer than as a
monomer. Similarily, the polymeric OPN was more efficiently bound to
immobilized collagen type V in the ELISA assay (Fig. 6B). In
the reversed situation, however, soluble collagen type V was more
readily bound to the monomeric form of the immobilized OPNs.

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Fig. 6.
Binding of osteopontin and its polymeric form
to collagen types I and V analyzed by dot blot overlay and ELISA
assays. Collagen types I and V, OPN, and the OPN polymer were
immobilized onto polyvinylidene difluoride membranes for 30 min. Each
collagen dot contained 1 µg of protein and OPN and the polymeric OPN
dots 5 µg of protein. The membranes were blocked with 1% BSA in 10 mM imidazole buffer, pH 6.8, containing 60 mM
KCl and 2.5 mM CaCl2. After washing, the
immobilized proteins were overlaid with an excess of soluble labeled
proteins; collagen type I with soluble monomeric and polymeric OPN and
immobilized OPNs with soluble collagen type I. Each solution contained
106 cpm/ml of radioiodinated protein as a tracer.
Incubation was carried out for 1 h. The membranes were
subsequently washed, dried, and autoradiographed. ELISA assay was
performed as described under "Experimental Procedures" and Fig. 3.
A, binding of soluble monomer OPN and the polymeric OPN to
immobilized collagen type I and vice versa analyzed by dot blot overlay
assays. B, the binding of soluble monomeric and polymeric
OPN to immobilized collagen type V and vice versa analyzed by
ELISA.
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Table I
Binding of monomeric and polymeric osteopontin to collagen type I in
soluble and immobilized forms
Binding was determined by a dot blot overlay assay where proteins were
immobilized onto PVDF filters and overlayed with 125I-labeled
soluble proteins (mean ± S.D.).
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Transglutaminase Treatment of Osteopontin Produces Polymers with
Altered Conformation--
The increased collagen binding property of
OPN after TG treatment led us to test the hypothesis that OPN may
undergo a conformational change during polymerization. Conformational
analyses of both monomeric and polymeric forms of OPN were performed
with CD spectroscopy in water to obtain information also at lower
wavelengths, where salts usually interfere with the signal. Circular
dichroism is expressed as millidegrees, since calculation of OPN's
molar concentration was not possible due to the uncertainty of the
exact molecular weight of the polymeric OPN. The spectrum of the
polymeric OPN (Fig. 7) exhibits a clear
shift in absorbance minimum from 201 to 207 nm and a decrease in
ellipticity between 210 and 240 nm as compared with the spectrum of the
monomeric OPN, indicating a more ordered structure for the polymer.
Secondary structure estimation based on the spectra (Table
II) suggested that the random coil
structure had decreased in the polymeric OPN from 43.4 to 30.6%. OPN
has been observed to undergo structural alterations at higher
concentrations (17.9 mg/ml) by Gorski et al. (26). In our
experiments the concentrations corresponded to about one-third of that
and, yet, a change in the CD spectrum was clearly observed and the
spectrum was different from the CD spectrum of Gorski et al.
(26). In conclusion, the CD data and the secondary structure estimations suggest that cross-linking of OPN by TG produces covalent polymers, which have a more organized structure than OPN has as a
monomer.

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Fig. 7.
Circular dichroism analysis of monomeric and
polymeric osteopontin. Far UV CD spectra (250-190 nm) of OPN and
the purified OPN polymer from the TG treatment were recorded at room
temperature in water using the following concentrations: OPN, 0.5 mg/ml; OPN polymer, 6.15 mg/ml. The measured rotations are expressed as
millidegrees.
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Table II
Secondary structure predictions of monomeric and polymeric osteopontin
by circular dichroism
OPN was cross-linked with TG and the reaction products were separated
by Mono-Q anion exchange chromatography. Protein peak C containing pure
high molecular mass products was subjected to CD analysis. Monomeric
OPN was also analyzed. Secondary structure predictions were calculated
from the spectra using program J-700 1.10.02.
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DISCUSSION |
We have previously reported that the TG-catalyzed cross-linking of
OPN is inhibited in vitro by osteocalcin (20). In light of
the finding that osteocalcin apparently functions as a mineralization inhibitor in the mouse model (21), the TG-mediated protein aggregation event might have an advantageous effect on mineralization or matrix maturation that precedes it. The results of this study demonstrated that OPN aggregates exhibited a property of increased binding to
collagen as compared with the monomeric form. Another acidic phosphorylated TG-substrate, casein, did not posses this property, indicating the specificity of collagen binding of TG-treated OPN. TG
treatment appeared to introduce a 5-fold amount of OPN onto collagens,
but predominantly when OPN and its polymer were in solution. This might
indicate that a specific conformation, achieved in solution, might be
required for binding. This elevated collagen binding property of
polymeric OPN can result from: 1) its increased affinity for and
association with collagen fibrils, therefore resulting in more rapid
coating of collagen during the incubation; 2) or the polymer may have
more binding sites on collagen resulting in a more efficient coating.
Both could be explained by a conformational change observed in the CD
experiments. Alteration in the OPN conformation of the monomer unit
and/or several OPN molecules packed together could expose or create
motives relevant to its interactive properties with collagen. Most
interestingly, in comparison with other types of collagens, collagen
type V appeared to have a very distinctive and different behavior. It
seemed to bind the polymeric OPN even in the absence of calcium, but
only when the polymer was in solution. Soluble collagen type V also
seemed to have the greatest affinity for immobilized monomeric OPN.
This might not only reflect a special function of collagen type V in
extracellular matrix maturation (or bone formation) sequence, but also
demonstrates the difference between the two forms of OPN.
OPN's in vitro behavior with TG clearly shows that OPN is a
substrate of this enzyme. OPN functioning also as an in vivo
substrate is supported by several studies. The transglutaminase
reactive acceptor glutamines are well conserved in all known OPN
sequences, indicating the significance of the motif to its functions
(27). OPN polymers have been found in different physiological sources such as bone (10), secreted by smooth muscle cells (28), and in milk as
shown by our study. The observation that TG has been found to be active
in bone in areas undergoing mineralization (5, 6) where also OPN has
been localized in high concentrations (29), gives us a reason to
believe that TG and OPN are able to interact in these areas. Indeed,
Sørensen et al. (10) have shown with Western blotting that
EDTA extracts of bovine bone contain high-molecular mass OPN complexes.
Functional or basic biochemical studies of OPN aggregates have not been
performed earlier and the functions of these complexes have only been speculative.
Based on the results of this study, we suggest that the TG-mediated
cross-linking of OPN may be directed to enhance the "glue-like" adhesion properties required in the processes that need collagen binding, e.g. in the adherence of different tissue
interfacial structures (new and old bone), collagen fibrillogenesis,
and wound healing. The enhanced adhesive property would be highly
important for a protein postulated to function as a "mortar between
bricks" (18). Although OPN is predominantly localized between
collagen fibrils in fully matured hard tissue (29), the reports on its affinity for collagens have shown evidence of no or only moderate attachment (7, 30). The results of our study suggest that OPN has a
significant affinity for collagens, but predominantly as a polymeric
form. TG has been recently characterized as a biological glue for
cartilage-cartilage interfaces (31). Therefore, OPN as a TG substrate
could be an essential component of the "glue" and the
polymerization indeed a prerequisite for its functioning as an adhesive protein.
Since isopeptide bonds produced by TG are resistant to normal
proteolysis (1, 2), the polymeric OPN may substantially contribute to
the overall integrity and strength of the extracellular matrix where it
is present. This kind of strenghtening might be required, for example,
in wound healing. In bone, it is also possible that the binding of OPN
to collagen could be further stabilized by additional cross-linking by
TG since covalent collagen-phosphoprotein complexes have been found
in vivo in bone (32) and several collagens have been
identified as substrates of TG (2, 33). Osteonectin (6) and fibronectin
(11) are also TG substrates indicating the presence of non-collagenous
protein-protein cross-links in the extracellular matrix.
In addition to the plausable adhesive and matrix-stabilizing (or matrix
organizing) properties of the polymeric OPN/TG activity, a
collagen-bound acidic cross-link network could also provide suitable
bedding for mineral growth in bone (34-36). Calcification of the
matrix follows matrix deposition and maturation in the bone formation
sequence, distinguishing it from other types of extracellular matrices.
It has been shown that collagen per se is not able to
calcify (37). To accumulate and crystallize calcium and phosphate into
hydroxyapatite, collagens seem to require other charged molecules on
their surface (37). Bone phosphoproteins have been postulated to
function as such molecules (34). Indeed, phosphoproteins have been
observed to initiate in vitro mineral formation when bound
to collagen (37) and to inhibit crystal growth in vitro when
in solution (38-40). OPN, as a phosphoprotein, could function as an
initiator when bound to collagen as a polymeric form or provide a
protein scaffold together with collagen for other bone matrix
macromolecules, such as bone sialoprotein, to bind and initiate
calcification. The calcium binding properties of OPN were not
specifically altered after polymerization (data not shown). However,
OPN is known as a high capacity calcium binder (30).
A protein aggregation event may have an important role in bone
formation, maturation, and calcification in general. Gorski et
al. (41, 42) have reported that bone acidic glycoprotein-75 (BAG-75) undergoes a spontaneous Ca2+-induced
polymerization, which increases its collagen binding activity. The
aggregated forms of BAG-75 are resistant to reducing and denaturing
conditions indicating their covalent nature, which suggests that
intramolecular cross-links could be present. High molecular weight
complexes of BAG-75 were detected in extracts of mineralizing calvarial
explant cultures (41). More interestingly, newly synthesized BAG-75
from these cultures was present entirely in large macromolecular
complexes, whereas non-mineralizing ROS 17/2.8 cultures produced only
monomeric BAG-75 (41). Other kinds of enzymatic modulation have also
recently been reported to have an effect on bone matrix protein
interaction property. Sasaki et al. (43) demonstrated,
e.g. that cleavage of osteonectin by metalloproteinases
results in a 7-20-fold increase in its binding to collagens.
Those results together with ours suggest that biological functions of
bone matrix proteins may largely depend on their post-translational modifications, conformational alterations, and surrounding ion concentrations, and that especially a covalent aggregation event may
have a pivotal role for tissue maturation and development. In light of
our findings, we suggest that TG activity could result in an altered
function of OPN resulting from altered conformation accompanied with
amplified collagen binding property. Instead of monomeric OPN, the
complex form of OPN might be the OPN that is involved in collagen
fibrillogenesis, matrix maturation, and possibly mineralization.
Importantly, the function of OPN could therefore be regulated by TG
expression and tissue distribution during different stages of tissue
remodeling or bone formation.