©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Ion Binding to Thrombospondin 1 (*)

(Received for publication, September 14, 1994)

Tina M. Misenheimer Deane F. Mosher (§)

From the Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have quantified the binding of Ca to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with CaCl(2). Ca binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 µM CaCl(2), 90% occupancy at 100 µM CaCl(2), and a Hill coefficient of 2.4 ± 0.2. The average apparent K was 52 ± 5 µM. Maximum binding, assuming M(r) = 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.


INTRODUCTION

Thrombospondin 1 (TSP1) (^1)is a 450-kDa multifunctional glycoprotein composed of three identical 150-kDa subunits connected by disulfide linkages. TSP1 is secreted from the alpha-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.


MATERIALS AND METHODS

Purification of TSP1

TSP1 was purified from indated human platelets obtained from the Badger Red Cross (Madison, WI) by heparin affinity chromatography and gel filtration chromatography(4) . The chromatography resins were equilibrated in 10 mM Tris, 0.15 M NaCl, pH 7.4, buffer (TBS) containing 300 µM CaCl(2) unless otherwise specified.

Ca Binding Assay

The ability of purified TSP1 to bind Ca was measured directly by dialyzing 1 µM TSP1 against TBS containing 0-300 µM CaCl(2) plus 2.25 µCi of CaCl(2) (622 µCi/µmol, Amersham Corp.) at 4 °C. TSP1 (250 µl) was put into dialysis bags, and after dialysis against 9.75 ml buffer, four 40-µl and three 10-µl aliquots were taken for radioactivity and protein determination, respectively. The dialysate buffer was also sampled for radioactivity. Spectrapor dialysis tubing (M(r) cutoff 12,000-14,000; Fisher) was treated prior to use to remove trace metals (15, 16) and washed well with distilled water. All water was glass-distilled and passed through a mixed-bed deionization resin system (Millipore, Bedford, MA). The Ca concentration of TBS, assayed using Fura-2 (Molecular Probes, Eugene, OR) as described by Grynkiewicz et al.(17) , was approximately 140 nM. Protein was determined using the Bio-Rad protein assay with TSP1 as a standard (M(r) = 450,000 and = 0.918 A/mg/ml)(18) . Metal ion competition studies were performed by dialyzing 1 µM TSP1 into TBS containing 0-200 µM metal ion (ZnCl(2), CuCl(2), MnCl(2), MgCl(2), CoCl(2), CdCl(2), or BaCl(2)), 55 µM CaCl(2), and CaCl(2).

Data Analysis

The amount of Ca bound per TSP1 trimer was calculated and plotted as a function of the log of Ca concentration. The initial Ca concentration in these calculations took into account Ca present both inside (due to Ca in the TSP1 preparation) and outside the dialysis bag. The data near the midpoint of the transition in the binding curve were analyzed by Hill plots as described by Limbird(19) . The Hill equation can be written as: log(B/B(max) - B) = nlog(U) - logK where B is the concentration of bound ligand, B(max) is the maximum concentration of bound ligand, U is the concentration of unbound ligand, n is the Hill coefficient, and K is an equilibrium dissociation constant. The B(max) was assumed to be the highest point in the binding curve, while the apparent K(d) was calculated from the x-intercept (x-intercept = log(apparent K(d))).

Trypsin Proteolysis Experiments

Trypsin proteolysis was performed on some samples after dialysis of TSP1 into either TBS containing 0-160 µM CaCl(2) or TBS containing various levels of Ca and other metal ions (Zn, Cu, Mn, Mg, Co, Cd, or Ba). Dialyzed TSP1 (10 µg) was incubated with 1/100 (w/w) tosylphenylalanyl chloromethyl ketone-treated trypsin (Sigma) for 20-24 h at 4 °C. The digestions were stopped by addition of reducing sample buffer followed by SDS-polyacrylamide gel electrophoresis (PAGE). Some gels were analyzed by immunoblotting using the MA-I monoclonal antibody whose epitope maps to amino acids 877-1009 in the carboxyl-terminal end of TSP1(3, 7, 20) . The Coomassie Blue-stained gels were analyzed by densitometric scanning using a GS 300 Transmittance/Reflectance scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA).


RESULTS

Ca binding to TSP1 was quantified via equilibrium dialysis using CaCl(2) (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(2) and 90% occupancy at 100 µM CaCl(2). The apparent K(d) 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(2) plus various levels of CaCl(2) 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(2) (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(2) 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(2) 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(2) instead of 300 µM CaCl(2). After digestion, the protein was dialyzed against TBS containing 55 µM CaCl(2) and CaCl(2) 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(2) 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(2). 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(2) (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(2)) and 55 µM CaCl(2) 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(2) of Ca binding to TSP1. Ca binding was determined via dialysis as described under ``Materials and Methods'' with ZnCl(2) (0-200 µM) and 55 µM CaCl(2) 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




DISCUSSION

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(d) of 52 ± 5 µM. This K(d) 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 beta-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(2). 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 alpha-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 alpha-granules (34) and alpha-granules constitute approximately 8% of the volume of platelets(35) . Therefore, alpha-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 alpha-granules where Zn levels are high, while upon release from the alpha-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.


FOOTNOTES

*
This research was supported by National Institutes of Health grants F32-HL08640 and R01-HL49111. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine, University of Wisconsin-Madison, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1535; Fax: 608-263-4969.

(^1)
The abbreviations used are: TSP, thrombospondin; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor.


ACKNOWLEDGEMENTS

We thank Dr. Jack Lawler for the kind gift of the MA-I antibody.


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