(Received for publication, September 14, 1994)
From the
We have quantified the binding of Ca to
platelet thrombospondin 1 (TSP1) using equilibrium dialysis with
CaCl
. Ca
binding to TSP1 was
found to be cooperative with 10% occupancy at 15-20 µM CaCl
, 90% occupancy at 100 µM CaCl
, and a Hill coefficient of 2.4 ± 0.2. The
average apparent K
was 52 ± 5
µM. Maximum binding, assuming M
= 450,000 and
= 0.918 (A280/mg/ml), was 35
± 3 Ca
/TSP1. This value is close to the 33
sites (11 per subunit) predicted based on homology of the epidermal
growth factor (1 site) and aspartate-rich (10 sites) regions to known
Ca
binding sequences. Ca
protected
the aspartate-rich region from trypsin proteolysis, but not until
nearly all of the Ca
binding sites were filled. At
lower occupancy of Ca
binding sites, several limited
tryptic digest products were obtained. This finding and the previous
demonstration of extensive thiol-disulfide isomerization within the
aspartate-rich regions suggest that subregions of the aspartate-rich
region are stabilized in different conformers. Zn
,
Cu
, Mn
, Mg
,
Co
, Cd
, and Ba
were tested for their ability to modulate Ca
binding and protease sensitivity of TSP1. Zn
inhibited 40% of the Ca
binding but neither
protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward
trypsin proteolysis. These results provide direct evidence for high
capacity, cooperative and specific binding of Ca
to
conformationally labile aspartate-rich repeats of TSP1.
Thrombospondin 1 (TSP1) ()is a 450-kDa
multifunctional glycoprotein composed of three identical 150-kDa
subunits connected by disulfide linkages. TSP1 is secreted from the
-granules of platelets upon platelet activation and is normally
present only in very low quantities in plasma(1, 2) .
TSP1 is also secreted by a number of cultured cells. Each TSP1 monomer
contains an amino-terminal heparin binding domain, a connecting region
that has the cysteines that form the interchain disulfide linkage,
three type 1 modules also found in properdin, three type 2 (EGF-like)
modules, 13 tandem aspartate-rich type 3 repeats, and a globular
carboxyl-terminal domain. The different domains mediate binding of TSP1
to cells, platelets, and numerous proteins such as collagen,
fibronectin, heparan sulfate proteoglycan, laminin, fibrinogen,
plasminogen, and histidine-rich glycoprotein(1, 2) .
Thirty-six potential Ca binding sequences (12 per
monomer) have been proposed in the type 3 repeat region of TSP1 based
on sequence homology with Ca
binding sites of other
proteins(3, 4) . TSP1 has been shown to interact with
Ca
cooperatively, as ascertained by changes in its
circular dichroism and trypsin digestibility at various Ca
concentrations(5) . The presence of Ca
alters the structure and function of TSP1. Rotary shadowing has
revealed that, in the presence of Ca
, the
carboxyl-terminal portion of TSP1 enlarges while the stalk region
shortens(6, 7, 8, 9) . TSP1 contains
3 equivalents of free thiols per
trimer(10, 11, 12) . The free thiol in each
monomer is distributed among 12 different cysteine residues due to
intramolecular thiol-disulfide isomerization(11) .
Ca
decreases the susceptibility of these thiols to
titration with iodoacetamide and N-ethylmaleimide(11) . Adhesion of cells to TSP1 (4, 13) and inhibition of cathepsin G by TSP1 (14) are Ca
-dependent. These results suggest
that Ca
regulates conformations of the RGD and
candidate protease inhibitory sequences in the aspartate-rich region
and that this regulation has profound effects on TSP1 functions. To
characterize the binding sites for Ca
more completely
we have performed equilibrium dialysis experiments, to quantify the
number and affinity of Ca
binding sites, and studied
specificity of binding. We related Ca
binding to the
trypsin digestibility of TSP1 and found additional evidence to map
Ca
binding to the conformationally labile type 3
repeats.
Ca binding to TSP1 was quantified via
equilibrium dialysis using
CaCl
(Fig. 1). Preliminary experiments showed that the system
approached equilibrium after 2 h. Four different TSP1 preparations were
dialyzed for 4, 18, or 24 h, and similar results were
obtained. The TSP1 was purified in the presence of 300 µM Ca
unless otherwise noted, and the concentration
of Ca
present in the TSP1 preparation was taken into
account when calculating the Ca
binding. The
following average results ± S.E. were obtained for five
different experiments using these four different TSP1 preparations.
TSP1 was found to bind maximally 35 ± 3 Ca
per
trimeric TSP1 with 10% occupancy at 15-20 µM CaCl
and 90% occupancy at 100 µM CaCl
. The apparent K
was 52
± 5 µM. The average Hill coefficient for
Ca
binding to TSP1 was 2.4 with a range of 1.9 to
3.1, indicating that Ca
binding exhibited positive
cooperativity.
Figure 1:
Ca binding to TSP1.
Ca
binding was determined directly by a 24-h dialysis
at 4 °C as described under ``Materials and Methods.'' The
data from one experiment are presented along with its Hill plot
analysis (inset). Linear regression analysis of the Hill plot
near the transition midpoint resulted in a straight line with a Hill
coefficient of 1.92 (correlation coefficient =
0.99)
Results varied according to Ca concentrations of the TSP at the start of dialysis. TSP1 purified
and stored in the presence of 20 µM Ca
maximally bound only 24 ± 3 Ca
per
trimeric TSP1 (data not shown). Extensive dialysis (24-48 h) into
buffer containing 300 µM Ca
was required
before the TSP1 purified in 20 µM Ca
bound equivalent amounts of Ca
as TSP1 purified
in the presence of 300 µM Ca
.
We
compared direct binding of Ca to trypsin proteolysis
by dialyzing TSP1 samples into TBS containing
CaCl
plus various levels of CaCl
and then dividing the
sample into two parts for quantitation of binding and digestion with
trypsin at 4 °C for 24 h. At Ca
concentrations
less than 60 µM, a 90-kDa tryptic fragment of
TSP1 predominated, while at higher levels of Ca
,
mostly larger fragments were observed (Fig. 2A). The
production of a 30-kDa band derived from the heparin binding
amino-terminal portion of TSP1 was independent of Ca
concentration (6) and therefore used as a reference when
quantitating protein in the Coomassie Blue-stained fragments. These
patterns of Ca
-dependent tryptic digestion are
similar to those reported
elsewhere(5, 6, 7) . Immunoblots of the
trypsin digests with monoclonal antibody MA-I, an antibody whose
epitope maps to amino acids 877-1009 in the carboxyl-terminal
end of the 1152-residue TSP1
polypeptide(3, 7, 20) , revealed that
immunoreactive 61-, 40-, and 36-kDa bands
appeared at low Ca
concentrations, whereas bands of
>100 kDa blotted in digests done at high Ca
concentrations (Fig. 2B). The appearance of the
immunoreactive 61-, 40-, and 36-kDa bands
coincided with the appearance of the 90-kDa fragment mentioned
above. The 90-kDa fragment was not recognized by MA-I. These
results indicate that the sites of increased tryptic susceptibility are
600-300 residues from the carboxyl terminus of TSP1 (residues
552-852) overlapping the aspartate-rich type 3 repeat region
(residues 674-932).
Figure 2:
Trypsin digestion of TSP1. TSP1 samples
were dialyzed into different concentrations of CaCl (as
noted above gel) and then digested with trypsin (1/100, w/w) as
described under ``Materials and Methods.'' The samples were
then analyzed by SDS-PAGE under reducing conditions. A, gels
were stained with Coomassie Blue. Arrows indicate the 90- and
30-kDa bands used to determine the ratios shown in Fig. 3. B, immuno-blotting with MA-I. Arrows point to the
61-, 40-, and 36-kDa immunoreactive bands.
Figure 3:
Ca binding versus trypsin proteolysis of TSP1. Trypsin proteolysis was performed on
TSP1 samples after dialysis in
CaCl
to
determine Ca
binding. The combined data from two
experiments are shown. Densitometric scanning of the Coomassie
Blue-stained SDS-PAGE gel was used to quantify trypsin proteolysis.
Trypsin proteolysis is expressed as the ratio of the 90-kDa protein
band to the 30-kDa protein band (Fig. 2); therefore, high ratios
indicate high levels of proteolysis. Analysis of the data using Hill
plots resulted in Hill coefficients of 2.6 and 1.4 for Ca
binding and tryptic digestion,
respectively
Analysis of the relative amount of protein
in the 90- and 30-kDa Coomassie Blue-stained
fragments via densitometric scanning yielded a sigmoidal curve when
plotted as a function of the log of Ca concentration (Fig. 3). A comparison of the Ca
binding curve
to the tryptic digest curve revealed that the aspartate-rich region of
TSP1 was not protected completely from trypsin proteolysis unless
>80 µM Ca
was present. At these
concentrations, >80% of the Ca
binding sites were
filled. Inspection of the MA-I immunoblots (Fig. 2B),
however, demonstrated that the 61-, 40-, and
36-kDa bands were maximal in digests carried out in 70
µM Ca
and lost only when Ca
was <20 µM.
The >100-kDa
proteolytic fragments of TSP1 that immunoblot with MA-I should contain
the full array of aspartate-rich repeats(3) , while the smaller
fragments result from cleavages within the repeats. Therefore, to
compare the Ca binding properties of the fragments,
TSP1 was digested with trypsin (1/100, w/w) in the presence of 300 or
20 µM CaCl
for 24 h at 4 °C. The TSP1 in
20 µM Ca
was obtained by purification of
TSP1 in the presence of 20 µM CaCl
instead of
300 µM CaCl
. After digestion, the protein was
dialyzed against TBS containing 55 µM CaCl
and
CaCl
to quantify Ca
binding.
Tryptic digests of Ca
-replete TSP1 were found to bind
nearly equal amounts of Ca
as intact TSP1, while
digests of TSP1 in 20 µM Ca
bound very
little Ca
(Table 1). This result indicates that
the Ca
binding region remains intact functionally
after trypsin proteolysis in 300 µM, but not in 20
µM, CaCl
and provides evidence that the
majority of Ca
binding sites are located in the
region of increased tryptic susceptibility.
Several metal ions were
examined for their ability to compete with Ca binding (Fig. 4). The competition experiments were performed by
dialyzing TSP1 into TBS containing 200 µM metal ion, 55
µM Ca
, and
CaCl
. A Ca
concentration of
55 µM was used because this concentration of
Ca
is at the transition midpoint of the
Ca
binding curve, and small changes in Ca
binding due to the presence of other metal ions should be
apparent. Both Cu
and Zn
partially
inhibited Ca
binding to TSP1, while
Mn
, Mg
, Co
,
Cd
, and Ba
did not inhibit or
enhance Ca
binding. A dose-response study of
Zn
inhibition of Ca
binding to TSP1
revealed that maximum inhibition (40%) was achieved at 50 µM ZnCl
(Fig. 5). After dialysis of TSP1 against
TBS containing 55 µM Ca
and 0-200
µM Zn
or 20 µM Zn
and 55-200 µM Ca
, 10 µg of TSP1 were trypsinized with 0.1
µg of trypsin for 24 h at 4 °C. No difference in proteolysis
patterns was observed (data not shown). Therefore, Zn
did not protect TSP1 against proteolysis as does
Ca
, nor did it labilize TSP1 toward proteolysis.
Dialysis of TSP1 against TBS containing 20 µM Ca
plus 280 µM metal ion
(Zn
, Cu
, Mn
,
Mg
, Co
, Cd
,
Ba
, or Ca
) followed by
trypsinization also failed to show any change in proteolysis patterns, i.e. the presence of metal ions other than Ca
did not protect TSP1 from trypsinization (data not shown).
Figure 4:
Metal ion competition for Ca binding to TSP1. Ca
binding was determined via
dialysis as described under ``Materials and Methods'' with
200 µM metal ion (XCl
) and 55 µM CaCl
present in the dialysis buffer. Control levels of
Ca
binding to TSP1 in the absence of other metal ions
were normalized to 100% for each experiment (CON); the average
level of control binding was 19 ± 4 Ca
/TSP1.
Each bar represents the mean ± S.E. of four
experiments
Figure 5:
Inhibition by ZnCl of
Ca
binding to TSP1. Ca
binding was
determined via dialysis as described under ``Materials and
Methods'' with ZnCl
(0-200 µM) and
55 µM CaCl
present in the dialysis buffer.
Control levels of Ca
binding to TSP1 in the absence
of Zn
were normalized to 100% for each experiment,
with an average level of binding at 17 ± 2
Ca
/TSP1. Each point represents the mean ± S.E.
of four experiments
We have quantified the ability of purified platelet TSP1 to
bind Ca. The data indicate that Ca
binding is cooperative with a Hill coefficient of 2.4 ±
0.2, maximal binding of 35 ± 3 Ca
per trimeric
TSP1 and an apparent K
of 52 ± 5
µM. This K
value is similar to the
transition midpoints of 45 and 50 µM reported previously
for the Ca
dependence of tryptic digests of TSP1 (5) and for the Ca
dependence of a monoclonal
antibody binding to TSP1(9) . It should be stressed that our
results are true for platelet TSP1 that had been purified and kept in
300 µM Ca
up until the time of
experimentation. TSP1 purified in 20 µM Ca
bound less Ca
, presumably due to a
conformational change.
Potential Ca binding
sequences in TSP1 can be identified by examination of the protein
sequence for homology to the consensus sequence for EF-hands in other
Ca
binding proteins(1, 3) . Proteins
with EF-hands bind Ca
within a helix-loop-helix
conformation(21, 22) . Ca
is
coordinated by 6 residues whose vertices approximate an octahedron
(positions are designated as X, Y, Z,
-X, -Y, and -Z). Five of
these residues usually have an oxygen-containing side chain (X, Y, Z, -X, and
-Z), while the coordinating oxygen at position
-Y comes from the main chain. The amino acid at position
-Z usually is a bidentate ligand so that seven oxygens
are involved in Ca
coordination. The type 3 repeats
are usually drawn as a series of seven or eight tandem
repeats(1, 3, 23) , but within the type 3
repeat region of TSP1, 13 sequences have features of the EF-hand
consensus sequence (Fig. 6). All have the amino acid aspartic
acid or asparagine, with aspartate predominating, at positions X, Y, Z, and -X, but only 10
of the 13 have aspartate at position -Z. The other three
sequences have amino acids with non-oxygen containing side chains at
position -Z, so these repeats may bind Ca
less well, or not at all.
Figure 6:
Ca binding sequence
homologies in the type 3 repeats of TSP1. The amino acid sequence of
the type 3 repeats of TSP1 (residues 674-932 counting from the
beginning of the mature protein) are aligned with the addition of gaps
(. . .) to optimize sequence homologies. The underlined cysteines were shown to exist as free thiols by Speziale and
Detwiler(11
EGF-like modules also can bind
Ca (e.g. see Selander-Sunnerhage et
al.(24) ). The structures of the
Ca
-replete and Ca
-depleted
amino-terminal EGF-like domain of factor X have been resolved using
nmr. Binding appeared to be to residues in the consensus sequence for
Ca
binding EGF-like modules amino-terminal to the
first cysteine residue of the domain, (D/N)-(I/V)-(D/N)-(E/D)-C, and
the consensus sequence for the Asp/Asn
-hydroxylase,
C-X-(D/N)-X-X-X-X-(Y/F)-X-C(24) .
The second EGF-like module (amino acids 570-627) of TSP1 conforms
to these sequence rules and has the potential to bind
Ca
.
Between the EGF-like module and the type 3
repeats, TSP1 contains a total of 14 potential Ca binding sequences. Because three of the sequences in the type 3
repeats are lacking an amino acid with an oxygen-containing side chain
at position -Z of an EF-hand, only 11 sites (33 per
trimer) can be considered good candidates for binding
Ca
. Our data support this hypothesis. We observed a
maximum of 35 ± 3 Ca
bound/TSP1. The type 3
repeats are well conserved among TSP1, TSP2(25, 26) ,
TSP3(27) , TSP4(28) , and cartilage oligomeric matrix
protein (29) , and the proposed Ca
binding
amino acids (D/N) are nearly always
conserved(30, 31) . The other TSPs, therefore, almost
certainly bind Ca
like TSP1. TSP3 and TSP4 have an (Y/T)-(I/V)-P-P-G sequence inserted in the 11th type 3 repeat,
however, and it will be interesting to learn the influence of this
sequence insertion on Ca
binding characteristics of
these two proteins.
One of the remarkable properties of TSP1 is the
different sensitivity to trypsin proteolysis at high Ca levels versus low Ca
levels(5, 6) . Rotary shadowing has revealed
that in the presence of Ca
the carboxyl-terminal
portion of TSP1 enlarges while the stalk region
shortens(6, 8, 9) . Ca
binding apparently masks the protease sensitive sites by changing
the conformation of TSP1. We found that the aspartate-rich region of
TSP1 is not protected completely from trypsin proteolysis until >80%
of the Ca
binding sites are filled ( Fig. 2and Fig. 3). TSP1 has 3 mol of free thiol per mol of TSP1
trimer(11) . Twelve different thiols in the
Ca
-sensitive carboxyl terminus, including 10 in the
aspartate-rich region (Fig. 6), were labeled. This indicates
that each cysteine was free in only a fraction of the TSP1 population
and that there must be a series of conformations stabilized by
different arrangements of disulfides. The different conformations
likely account for the size heterogeneity of MA-I staining
carboxyl-terminal fragments when TSP1 in intermediate Ca
concentrations was digested with trypsin.
The Ca binding sites in TSP1 are relatively specific for Ca
since of the seven metal ions examined at concentrations up to
200 µM, only Zn
and Cu
partially inhibited Ca
binding to TSP1 ( Fig. 4and Fig. 5). Dixit et al. (9) measured binding of
I-TSP1 to immobilized
monoclonal antibodies that were sensitive to different conformations of
TSP1, and found that TSP1 in 5 mM EDTA bound to the
antibodies, while only a small fraction of the TSP1 in 5 mM Ca
bound to the antibodies. Dialysis of
Ca
-replete TSP into 5 mM Mg
resulted in TSP1 that bound to the antibodies as well as
EDTA-treated TSP1. We, however, found no evidence of Mg
binding to TSP1 even though we looked for both a decrease and an
increase in Ca
binding. Although inclusion of
Cu
resulted in less Ca
binding to
TSP1, Cu
may not actually compete with Ca
for binding to TSP1 but instead catalyze the oxidation of the
free thiol groups in TSP1 as described for guinea pig transglutaminase
by Boothe and Folk(32) .
Zn, in contrast
to Cu
, may be competing for Ca
binding sites. Forty percent inhibition of Ca
binding by Zn
was the maximum observed, and it
was achieved with 50 µM ZnCl
. Unlike
Ca
, however, neither Zn
or any of
the other metal ions examined protects TSP1 from trypsin proteolysis,
or labilizes TSP1 toward proteolysis. This finding indicates that
Zn
is not able to change the conformation of TSP1 in
a manner similar to that of Ca
binding even though it
competes with Ca
binding. We speculate that
Zn
binding may nevertheless be functionally
important. Zn
may bind to sites that are not critical
for proteolysis. The concentration of zinc in plasma is about 15
µM(33, 34) . The amount of zinc in
platelet
-granules, however, may be as high as 2.5 mM,
because zinc levels in platelets are 30-60-fold higher than
plasma levels, 40% of the platelet zinc is located in the
-granules (34) and
-granules constitute approximately
8% of the volume of platelets(35) . Therefore,
-granule
levels of zinc are more than sufficient to inhibit Ca
binding to TSP1. As described above, the Ca
binding repeat region of TSP1, upon release from platelets,
exists in a large number of conformers as ascertained by the 12
different positions of the free sulfhydryl group(11) . Since
Zn
partially inhibits Ca
binding to
TSP1, TSP1 may be present in one set of conformers in the
-granules where Zn
levels are high, while upon
release from the
-granules, the TSP1 may change to a different set
of conformers due to the low level of Zn
in plasma.
Adoption of these conformations may be catalyzed by a protein disulfide
isomerase also released from platelets(36) . Further, TSP1
constitutively secreted by cells through compartments that are not rich
in Zn
may adopt a different set of conformations than
TSP1 released from activated platelets.