A polymorphism in thrombospondin-1 associated with familial premature coronary artery disease alters Ca2+ binding.

A single nucleotide polymorphism that results in substitution at residue 700 of a serine (Ser-700) for an asparagine (Asn-700) in thrombospondin-1 is associated with familial premature coronary artery disease. The polymorphism is located in the first of 13 Ca2+ -binding motifs, within a consensus sequence in which Asn-700 likely coordinates Ca2+. Equilibrium dialysis of constructs comprised of the adjoining epidermal growth factor-like module and the Ca2+ -binding region (E3Ca) demonstrated that E3Ca Ser-700 binds significantly less Ca2+ than E3Ca Asn-700 at low [Ca2+]. The hypothesis that this difference is due to loss of a binding site in Ser-700 protein was tested with truncations of E3Ca containing four (Tr4), three (Tr3), two (Tr2), or one (Tr1) N-terminal Ca2+ -binding motifs. The Ser-700 truncation constructs bound 1 fewer Ca2+ than matching Asn-700 constructs and exhibited decreased binding affinities. Intrinsic fluorescence of a tryptophan at residue 698 (Trp-698) in the most N-terminal motif was cooperatively quenched by the addition of Ca2+ to Asn-700 Tr2, Tr3, and Tr4 constructs. In Ser-700 constructs, quenching of Trp-698 was incomplete in the Tr2 and Tr3 constructs and complete only in the Tr4 construct. Ca2+ -induced quenching of Ser-700 constructs required higher [Ca2+] and was slower as shown in stopped-flow experiments than quenching of Asn-700 constructs. Such differences were not found with Tb3+, which quenched the fluorescence of Asn-700 and Ser-700 constructs equivalently. Thus, the Ser-700 polymorphism alters a rapidly filled, high affinity Ca2+ -binding site in the first Ca2+ -binding motif. Slower Ca2+ binding to adjoining motifs partly compensates for the change.

just C-terminal to residue 700 to be an insert (Fig. 1), and we hypothesized that the linker is the first of 13 (rather than 12) Ca 2ϩ -binding motifs. Three mutations in this part of TSP-5 are associated with PSACH or EDM1 (reviewed in Ref. 19), also suggesting that the linker represents a Ca 2ϩ -binding motif. We further hypothesized that the presence of the Ser-700 polymorphism disrupts this site, but cooperative binding of Ca 2ϩ to adjacent Ca 2ϩ -binding motifs also triggers the conformational change reported by Trp-698. To test these hypotheses, we quantified Ca 2ϩ binding to the E3Ca proteins and truncation sets with an asparagine or serine at residue 700. Occupancy of binding sites was related to Ca 2ϩ -induced fluorescence quenching as assessed by equilibrium and stopped-flow fluorescence.

Cloning of Common and Polymorphic Truncation Constructs-To
facilitate baculovirus-mediated protein expression and subsequent purification, we used the pAcGP67.coco transfer vector in which cloning sites are flanked by 5Ј DNA encoding a signal sequence and 3Ј DNA encoding a polyhistidine tag (20). E3Ca Asn-700 and E3Ca Ser-700 were made as described previously (17). Truncated proteins were designed to preserve the disulfide connectivity (10). DNA encoding residues 648 -717 (Tr1), 648 -740 (Tr2), 648 -753 (Tr3), or 648 -776 (Tr4) that contained the third EGF-like module and truncations of the Ca 2ϩbinding repeats were generated by PCR amplification using E3Ca Asn-700 or E3Ca Ser-700 as template. The limits of these proteins are depicted in Fig. 1. The forward primer contained a BamHI site, and the reverse primer contained a PstI site. The PCR products were inserted into BamHI and PstI sites of pAcGP67.coco. Correct sequences of PCRamplified DNAs were verified by DNA sequencing.
Expression and Purification of Recombinant Proteins-Recombinant, infectious virus were generated as described previously (20). Passage three of the virus (Ͼ10 8 plaque-forming units/ml) was used to infect High-Five insect cells (Invitrogen) at a multiplicity of infection of five in SF-900 II serum-free medium at 22°C. After 60 -65 h, conditioned medium was harvested and dialyzed into 10 mM MOPS, 0.3 M NaCl, and 2 mM CaCl 2 (pH 7.5). Dialyzed medium was incubated with Ni 2ϩnitrilotriacetic acid resin overnight at 4°C, a column was poured with protein-bound resin, and protein was eluted in buffer containing 300 mM imidazole. Purified protein was dialyzed into 10 mM MOPS, 0.15 M NaCl, and 2 mM CaCl 2 , pH 7.5. Protein was stored in aliquots at Ϫ80°C and thawed in a 25°C water bath prior to use. All proteins were produced in yields ranging from 15 to 30 mg/liter conditioned medium and were soluble at concentrations of Ͼ1 mg/ml. Ca 2ϩ Binding-To determine the number of Ca 2ϩ bound to Asn-700 and Ser-700 proteins, we performed equilibrium dialysis. Proteins were initially treated with 4 mM EDTA to remove Ca 2ϩ and then dialyzed exhaustively at 4°C into buffer containing 5 mM MOPS and 0.1 M NaCl (pH 7.5). Ca 2ϩ -depleted protein (45 l) was then dialyzed at 37°C for 4 h into the same buffer (350 l) containing [Ca 2ϩ ] varying from 10 M to 2 mM plus trace 45 CaCl 2 (Amersham Biosciences) (21,22). Following dialysis, the radioactivity of the protein and buffer samples was determined by scintillation counting, and the protein concentration of the protein samples was determined by BCA protein assay (Pierce Intrinsic UV Fluorescence and Titration with Ca 2ϩ or Tb 3ϩ -Prior to fluorescence assays, recombinant proteins were treated with 4 mM EDTA to remove the Ca 2ϩ and then dialyzed at 4°C into buffer containing 5 mM MOPS and 0.1 M NaCl (pH 7.5), as described above. Dialyzed Asn-700 or Ser-700 proteins were titrated with Ca 2ϩ or Tb 3ϩ at 37°C in an Aminco SLM 8100 or a JY Horiba Fluoromax 3 fluorimeter in 1-cm path length cells. Samples were excited at 295 nm. For samples titrated with Ca 2ϩ , emission spectra from 310 to 400 nm were recorded at each [Ca 2ϩ ] from 0 mM to saturating [Ca 2ϩ ]. For samples titrated with Tb 3ϩ , emission spectra were collected from 310 to 560 nm. The change in fluorescence intensity (⌬F) relative to the Ca 2ϩ -depleted protein from 0 mM to saturation was calculated as where F 0 is the total fluorescence at 0 mM Ca 2ϩ or Tb 3ϩ , and F is the total fluorescence at a given [ Stopped-flow Fluorescence-Stopped-flow was performed using an SX.18MV-R dual-syringe spectrophotometer (Applied Photophysics, Leatherhead, UK). Changes in tryptophan fluorescence over time were determined by exciting proteins at 295 nm and monitoring fluorescence using a 335-nm cut-off filter. All measurements were taken at 37°C in 5 mM MOPS and 0.1 M NaCl, pH 7.5. The samples were incubated in the stopped-flow syringe for 4 min to allow for thermal equilibration, and then equal volumes of protein and Ca 2ϩ were mixed. The reaction mixture contained a final concentration of 5 M protein and various [Ca 2ϩ ]. The dead time of the instrument was ϳ1 ms. Kinetic traces were fitted to a second order exponential, where F is fluorescence at time t, A 1 and A 2 are amplitudes, k 1obs and k 2obs are observed rate constants, and C is the fluorescence at infinite time. Kinetic traces were analyzed using Igor Pro (WaveMetrics Inc., Lake Oswego, OR). Validity of the fit was evaluated by the residuals of the fit and the square value.

RESULTS
The Ser-700 Polymorphism Subtly Alters the Titration of Ca 2ϩ Binding to E3Ca-Equilibrium dialysis with 45 Ca 2ϩ was performed on E3Ca Asn-700 and polymorphic E3Ca Ser-700, constructs of TSP-1 that contain the third EGF-like repeat together with the complete Ca 2ϩ -binding region. Ser-700 protein reproducibly bound 1-2 fewer Ca 2ϩ at [Ca 2ϩ ] of 60 -200 M ( Fig. 2A). At saturating [Ca 2ϩ ] of Ͼ250 M, the assay was less reproducible, and there was overlap between the two proteins in the estimates of the number of Ca 2ϩ bound. E3Ca Asn-700 FIG. 1. Sequence of E3Ca and truncations. The sequence of the third EGF-like repeat (E3) is in light gray. The sequence of the Ca 2ϩbinding repeats (Ca) is in black. The Ca 2ϩ -binding repeats were initially defined as a linker (Link) and seven T3 repeats (T3 1 -T3 7 ) (18). The disulfide bond pattern (solid lines) is consecutive (10). By x-ray crystallography, it is known that two types of Ca 2ϩ -binding motifs are found in the T3 5 -T3 7 repeats: an N-type motif and a C-type motif (9). Highlighted in bold are residues found to coordinate Ca 2ϩ in a recent crystal structure of this part of the protein (9). The linker contains the polymorphic residue (Asn-700; bold, italicized, and underlined) and a disulfide-linked loop with a glycosylation site (NAT). Trp-698 used as a reporter is also located in the linker sequence (bold, italicized). Constructs were truncated before the cysteine residues at Asn-717 (Tr1), Asp-740 (Tr2), Asn-753 (Tr3), and Asn-776 (Tr4) as shown by vertical lines. Residues 701-714 are boxed and depicted as an inset, thereby allowing the linker to be visualized as a C-type Ca 2ϩ -binding motif. The linker is therefore numbered as motif 1, and five of the original seven T3 repeats (18) that contain double motifs have been subdivided. The resulting 13 motifs are classified as N-or C-type according to convention of Kvansakul et al. (9).
Initial Ca 2ϩ Binding Events Alter Tryptophan Fluorescence-The titration of Ca 2ϩ binding to the E3Ca constructs ( Fig. 2A) was similar to titration of the characteristic far UV circular dichroism spectral changes, which reveals transitions of 150 Ϯ 15 M [Ca 2ϩ ] (n H ϭ 5.1) and 168 Ϯ 7 M (n H ϭ 6.1) for E3Ca Asn-700 and E3Ca Ser-700, respectively (17). In contrast, titration of the fluorescence of a sole tryptophan, Trp-698, reveals transitions at lower [Ca 2ϩ ], 70 Ϯ 2 and 110 Ϯ 6 M for E3Ca Asn-700 and E3Ca Ser-700, respectively (17). To compare the three types of titration curves, the Ca 2ϩ binding data ( Fig.  2A) were fit to sigmoid curves, and the number of Ca 2ϩ bound at various [Ca 2ϩ ] was calculated. With these estimates, we plotted the change in signal of the fluorescence and far UV CD described previously (17) for E3Ca proteins against the number of Ca 2ϩ bound (Fig. 2B). The change in CD signal for both proteins, when plotted as a function of bound Ca 2ϩ , demonstrated an initial steeper slope so that the curve was 75% complete when 15 Ca 2ϩ were bound. The curve reached a plateau as the last 8 -10 Ca 2ϩ were bound. In contrast, the curves for the fluorescence signal of E3Ca Asn-700 and E3Ca Ser-700 were nearly complete when only 3-4 and 5-6 Ca 2ϩ , respectively, bound to the proteins.
These results suggest that the first several Ca 2ϩ that bind alter the environment of Trp-698 and therefore may bind to residues near Trp-698. The results also suggest that the presence of the Ser-700 polymorphism alters initial Ca 2ϩ binding to residues near Trp-698 since more bound Ca 2ϩ was required to change the environment of Trp-698 (Fig. 2B). To test these hypotheses directly, we generated four truncations of wild-type and polymorphic E3Ca that contain the third EGF-like repeat together with increasing numbers of C-terminal Ca 2ϩ -binding motifs (Fig. 1). Constructs were truncated based on the known disulfide connectivity of TSP-2 (10) to prevent the presence of an unpaired cysteine. Therefore, Tr1, Tr3, and Tr4 lacked the C-terminal sequences required for a complete C-type motif (9) (Fig. 1).
The Ser-700 Polymorphism Disrupts Ca 2ϩ Binding to Truncated Proteins-Binding of 45 Ca 2ϩ to Asn-700 constructs by equilibrium dialysis was sigmoidal for Tr4 Asn-700, Tr3 Asn-700, and Tr2 Asn-700 ( Fig. 3, A-C). Removal of motifs from Asn-700 proteins caused a decrease in the maximal number of Ca 2ϩ bound (Table I) (Table I). The calculated Hill coefficients for Tr4 Asn-700, Tr3 Asn-700, and Tr2 Asn-700 indicated positive cooperativity (Table I). Although the Hill coefficients did not significantly differ depending on the truncation size, all were lower than the Hill coefficient for Ca 2ϩ binding to E3Ca Asn-700 (Table I). The titration curve for Tr1 Asn-700 ( Fig. 3D) did not reach a plateau at high [Ca 2ϩ ] (Ͼ800 M), and at this input concentration, Tr1 Asn-700 bound 1.2 Ϯ 0.1 Ca 2ϩ (X Ϯ S.E., n ϭ 4).
In contrast to the subtle differences of Ca 2ϩ binding to E3Ca Asn-700 and E3Ca Ser-700 ( Fig. 2A), greater differences in Ca 2ϩ binding to Ser-700 as compared with Asn-700 truncation constructs were observed ( Fig. 3 and Table I). For all truncated proteins, the presence of the Ser-700 polymorphism caused a decrease in the number of bound Ca 2ϩ as compared with wild type at all [Ca 2ϩ ] tested. The titration curves for Ser-700 truncations were also shifted to higher [Ca 2ϩ ]. For instance, the titration curve for Tr4 Ser-700 was sigmoidal (Fig. 3A) and reached a binding plateau of 4.8 Ϯ 0.5 Ca 2ϩ , lower than that for Tr4 Asn-700. The EC 50 of Ca 2ϩ binding for Tr4 Ser-700 was 420 Ϯ 40 M, higher than that observed for the Tr4 Asn-700. Ca 2ϩ bound to Tr3 Ser-700, but the titration curve did not reach a plateau at high [Ca 2ϩ ] (Fig. 3B). There was little binding of Ca 2ϩ to Tr2 Ser-700, except at [Ca 2ϩ ] greater than 600 M (Fig. 3C), and Tr1 Ser-700 did not bind Ca 2ϩ in the concentration range accessible to study by equilibrium dialysis (Fig. 3D).
Ca 2ϩ -induced Quenching of Tryptophan Fluorescence Depends on the Size of the Truncation and the Presence of the Ser-700 Polymorphism-Since the presence of the Ser-700 polymorphism in truncation constructs caused a decrease in the number of Ca 2ϩ bound, we next investigated whether this altered the ability of the proteins to achieve the Ca 2ϩ -bound conformation as assessed by the fluorescence properties of Trp-698. Asn-700 and Ser-700 truncation constructs were excited at 295 nm to excite Trp-698 specifically, and emission spectra were collected at 310 -400 nm in the presence of various [Ca 2ϩ ]. In the absence of Ca 2ϩ , all truncated constructs emitted at a peak wavelength ( max ) of ϳ350 nm (Fig. 4, A-H). In the presence of 3 mM [Ca 2ϩ ], the tryptophan fluorescence of Tr4 Asn-700, Tr3 Asn-700, and Tr2 Asn-700 was quenched ϳ5-fold (Fig. 4, A, C, and E). Ca 2ϩ also caused a shift in the max of these constructs to 337 nm. The addition of Ca 2ϩ to Tr4 Ser-700 caused ϳ4-fold quenching of fluorescence (Fig. 4B) and a shift in max to 341 nm. The quenching of Tr3 Ser-700 and Tr2 Ser-700 was less, 2.3-and 1.6-fold, respectively, with a smaller shift in max to 346 nm (Fig. 4, D and F). Little fluorescence quenching was seen for Tr1 Asn-700 and Tr1 Ser-700, 1.3-and 1.1-fold, respectively, with no shift in max (Fig. 4, G and H).
The fractional change in total fluorescence for each protein relative to 0 M [Ca 2ϩ ] was calculated at each [Ca 2ϩ ] and then plotted versus M [Ca 2ϩ ] (Fig. 5). With the exception of Tr2 Ser-700 and both Tr1 proteins, the titration curves for each protein were sigmoidal and exhibited positive cooperativity. Similar to E3Ca Asn-700, which titrated at 70 M (17), Tr4 Asn-700 had an EC 50 of 80 Ϯ 1 M (Table I). Further truncation caused the fluorescence of Tr3 Asn-700, Tr2 Asn-700, and Tr1 Asn-700, to titrate at increasing [Ca 2ϩ ] ( Table I). The cooperativity of the fluorescence change for Tr4 Asn-700 (2.4 Ϯ 0.1) was decreased as compared with E3Ca Asn-700 (3.8) (17). Al-though Tr3 Asn-700 exhibited similar cooperativity as Tr4 Asn-700, further truncation to Tr2 and Tr1 Asn-700 resulted in decreased cooperativity (Table I). The presence of the Ser-700 polymorphism caused the fluorescence of the truncated pro-  teins to titrate at higher [Ca 2ϩ ] than the Asn-700 proteins, and this difference was magnified in each successive truncation (Table I). Consistent with this trend, Tr4 Ser-700 titrated at higher [Ca 2ϩ ] (240 Ϯ 1 M) as compared with E3Ca Ser-700 (110 M) (17). Similar to E3Ca proteins, the cooperativity of the fluorescence transition for Tr4 Ser-700 (3.0 Ϯ 0.2) was higher than that for Tr4 Asn-700 (2.4 Ϯ 0.1). However, further truncation resulted in decreased cooperativity for Tr3 Ser-700 and Tr2 Ser-700 as compared with matching Asn-700 constructs ( Table I). The presence of the Ser-700 polymorphism in Tr1 resulted in minimal titration of the tryptophan fluorescence at only high [Ca 2ϩ ] (Fig. 5).

The Tr1 Constructs Contain an intact Tb 3ϩ -binding Site-
The results indicate that Asn-700 constructs with two or more Ca 2ϩ -binding motifs bind Ca 2ϩ with high affinity, resulting in quenching and a blue shift in the fluorescence of Trp-698. However, the 5-fold change in intrinsic fluorescence upon the addition of Ca 2ϩ was not present in the Tr1 constructs. Although the equilibrium dialysis data indicated that Tr1 Asn-700 contains binding sites for Ca 2ϩ , the affinity was too weak to characterize binding rigorously. To characterize metal-binding site(s) in the Tr1 protein more definitively, we performed Tb 3ϩ binding studies on Tr1 Asn-700 and Tr1 Ser-700. The lanthanide Tb 3ϩ ( ex ϭ 488, max ϭ 543) emits phosphorescence when excited due to resonant energy transfer from nearby tryptophans (23). Thus, Tr1 proteins were excited at 295 nm to excite Trp-698, and emission spectra were collected at 310 -560 nm in the presence of various [Tb 3ϩ ]. Upon the addition of 30 M Tb 3ϩ , tryptophan fluorescence of Tr1 Asn-700 and Tr1 Ser-700 (310 -400 nm) was similarly quenched (Fig. 6, A and B). In contrast to the Ca 2ϩ -induced quenching observed with the E3Ca proteins and the longer Asn-700 truncations, there was no blue shift in the max . The addition of Tb 3ϩ also resulted in an emission peak at 543 nm (Fig. 6, A and B) that increased in amplitude with increasing [Tb 3ϩ ]. Titration of Trp-698 fluorescence with Tb 3ϩ resulted in EC 50 values of 12.0 Ϯ 3.0 and 12.9 Ϯ 3.5 M for Tr1 Asn-700 and Tr1 Ser-700, respectively. The transition was not cooperative as the Hill coefficients for Tr1 Asn-700 and Tr1 Ser-700 were 1.3 Ϯ 0.1 and 1.1 Ϯ 0.1, respectively. Analysis of the peak at 543 nm revealed similar EC 50 values for Tr1 Asn-700 and Tr1 Ser-700 (data not shown). Thus, these results indicate that Tr1 proteins bind Tb 3ϩ , resulting in quenching of Trp-698 fluorescence due to energy transfer to the bound metal, and that the binding is insensitive to the Ser-700 polymorphism.
The Ser-700 Polymorphism Alters the Rate of Ca 2ϩ Binding-Since the residues around Trp-698 contain a metal ionbinding site that regulates the number and average affinities of bound Ca 2ϩ , we hypothesized that N700S polymorphism may alter the rate of Ca 2ϩ binding to E3Ca and truncated constructs. Therefore, we analyzed Ca 2ϩ -induced quenching of E3Ca Asn-700, E3Ca Ser-700, Tr4 Asn-700, Tr4 Ser-700, Tr3 Asn-700, and Tr2 Asn-700 by stopped-flow fluorescence. These constructs exhibited at least 4-fold quenching of tryptophan fluorescence upon the addition of Ca 2ϩ (Fig. 3) (17), providing a robust signal to monitor as a function of time. Kinetic traces were obtained at high [Ca 2ϩ ] as compared with protein concentration, conditions for pseudo-first order kinetics. Fig. 7 shows representative traces from Tr4 Asn-700 and Tr4 Ser-700.
All traces were biphasic over concentrations between 125 M and 4 mM. Upon the addition of Ca 2ϩ , there was an initial rapid decrease in fluorescence followed by a slower decrease in fluorescence. For Asn-700 constructs, ϳ80% of the total change in amplitude was due to the first phase at concentrations between 0.125 and 1 mM [Ca 2ϩ ]. This increased to ϳ90% at concentrations greater than 1 mM. In addition, for Asn-700 constructs at higher [Ca 2ϩ ], part of the first phase was not detected as it occurred too quickly to be followed by our instrument. For E3Ca Ser-700 and Tr4 Ser-700, the first phase represented less than 70% of the total change in amplitude at [Ca 2ϩ ] less than or equal to 1 mM; this increased to ϳ80% at concentrations greater than 1 mM (data not shown).
The rate of the first phase (k 1obs ) for Asn-700 proteins increased linearly with increasing [Ca 2ϩ ] (Fig. 8A) and was dependent on the level of truncation. The k 1obs at each [Ca 2ϩ ] for E3Ca Asn-700 was slightly higher than k 1obs for Tr4 Asn-700. The k 1obs of Tr3 Asn-700 was similar to that of Tr4 Asn-700, but both were higher than k 1obs of Tr2 Asn-700 for each [Ca 2ϩ ] (Fig.  8A). The presence of the Ser-700 polymorphism in E3Ca Ser-700 and Tr4 Ser-700 caused a decrease in k 1obs such that the k 1obs of both proteins were lower than the k 1obs of Asn-700 Tr4, Tr3, or Tr2 proteins (Fig. 7B). For Ser-700 proteins, k 1obs also increased with increasing [Ca 2ϩ ], but the dependence of k 1obs with [Ca 2ϩ ] was not linear, and the curves of E3Ca Ser-700 and Tr4 Ser-700 were shifted to higher [Ca 2ϩ ] as compared with Asn-700 proteins (Fig. 7B).
There were small differences between the rates of the slow phase (k 2obs ) for wild-type constructs (Fig. 8C). The k 2obs for Asn-700 proteins increased mildly between ϳ1 and 8 s Ϫ1 as a function of [Ca 2ϩ ], values that are ϳ20-fold lower than the k 1obs at similar [Ca 2ϩ ]. The k 2obs was also dependent on the level of truncation, with Tr2 Asn-700 and Tr3 Asn-700 exhibiting slower rates than E3Ca Ser-700 and E3Ca Asn-700 at Ca 2ϩ between 1 and 4 mM (Fig. 7C). The rate constant of the slow phase (k 2obs ) did not significantly differ between Asn-700 and Ser-700 proteins at each [Ca 2ϩ ] (Fig. 8D). DISCUSSION As shown in Fig. 1, two novel but related Ca 2ϩ -binding motifs occur in TSP-1 (9). In both N-and C-type motifs, aspartate residues at positions 1, 3, and 5 coordinate the first Ca 2ϩ (9). The residue at position 7, which is variable among the motifs, provides a main chain carbonyl that contributes to coordinating the first Ca 2ϩ . At position 9, an aspartate or asparagine bridges a water molecule to the first Ca 2ϩ . The 6-fold coordination of the first Ca 2ϩ is completed by an aspartate at position 12 (9). The second Ca 2ϩ is coordinated by aspartates at positions 3, 5, and 12 that also participate in coordination of the first Ca 2ϩ . In C-type motifs, the second Ca 2ϩ is additionally coordinated by residues C-terminal to the 12-residue sequence (9). Equilibrium dialysis of E3Ca indicated that 23-25 Ca 2ϩ ions bind to the Ca 2ϩ -binding repeats. This is higher than what has been observed for TSP-1, TSP-5, and TSP-5 constructs (13)(14)(15)(16)21) but is in agreement with the values predicted by the crystal structure (9) and what has been determined for TSP-2 constructs by atomic absorption spectroscopy (22). Trp-698, which is quenched upon the addition of Ca 2ϩ , is at position 7 of a putative motif (Fig. 1) and would be expected to coordinate Ca 2ϩ via its main chain carbonyl. The N700S polymorphism resides at position 9, where the side chain would be expected to coordinate Ca 2ϩ through water (9). However, these residues have been considered to be part of a linker region (9,18) between the third EGF-like repeat and the Ca 2ϩ -binding repeats. To test the hypothesis that the linker binds Ca 2ϩ , we generated truncations based on the disulfide bond pattern that included the linker or the linker and up to three adjacent Ca 2ϩ -binding motifs. The shortest Asn-700 construct, Tr1, which contains the EGF-like module and residues 692-717, was found to bind both Ca 2ϩ and Tb 3ϩ as assayed by equilibrium dialysis and luminescence, respectively. Tb 3ϩ bound to Tr1 Asn-700 and Tr1 Ser-700 with much higher affinity than Ca 2ϩ binding to either construct, similar to what has been observed with other proteins such as calreticulin (24). Thus, Tr1 contains a strong Tb 3ϩ -binding site, and in the case of the Asn-700 protein, a weaker Ca 2ϩ -binding site.
The observations that Trp-698 in Tr1 Asn-700 is not fully quenched even at high [Ca 2ϩ ] of Ͼ3 mM and Tr1 Asn-700 does not bind Ca 2ϩ with high affinity indicate that either the Ca 2ϩbinding motif is incomplete or additional motifs of TSP-1 are required for high affinity Ca 2ϩ binding to Tr1. Inspection of the linker sequence of TSP-1 reveals that it can be modeled as a C-type motif if residues 701-714 are considered to be an insert (Fig. 1). Our group is in the process of determining the crystal structure of a Ca 2ϩ -replete portion of TSP-2 that contains the EGF-like modules, the Ca 2ϩ -binding repeats, and the lectinlike module. The linker region in this TSP-2 construct binds 2 Ca 2ϩ with the coordination predicted for a C-type motif, and the insert is accommodated as a loop. 2 TSP-1 and TSP-2 have 90% sequence identity and identical spacing in the Ca 2ϩ -binding repeats. Therefore, it is likely that the linker of TSP-1 also functions as a C-type motif and binds 2 Ca 2ϩ cooperatively in conjunction with adjacent motifs. Tr1 includes only the Nterminal half of the motif plus the insert (Fig. 1) and therefore is an incomplete C-type motif. The addition of residues 719 -728 and the adjacent N-type Ca 2ϩ -binding motif to the linker (Tr2 Asn-700) restored cooperative Ca 2ϩ -induced quenching of Trp-698 fluorescence and high affinity binding of approximately 3 Ca 2ϩ when measured by equilibrium dialysis. These results indicate that the N-terminal 2 Ca 2ϩ -binding motifs participate in a linked set of relatively high affinity sites that, when occupied with Ca 2ϩ , quenches Trp-698 in the first motif. The increasing Hill coefficients upon the addition of more Ca 2ϩbinding motifs, for both quenching of Trp-698 and binding of Ca 2ϩ , indicate additional cooperativity among binding sites in N-terminal motifs.
The presence of the Ser-700 polymorphism caused loss of a Ca 2ϩ -binding site as Tr1 Ser-700 did not bind Ca 2ϩ in the range of [Ca 2ϩ ] tested, and Tr2 Ser-700, Tr3 Ser-700, Tr4 Ser-700, and E3Ca Ser-700 bound on average 1 fewer Ca 2ϩ than Asn-700 constructs. The Ser-700 polymorphism in the most N-terminal Ca 2ϩ -binding motif also influenced high affinity Ca 2ϩ binding as evidenced by the shift of the Ca 2ϩ titration curves of Ser-700 proteins to higher [Ca 2ϩ ] as compared with Asn-700 counterparts. In addition, complete quenching of Trp-698 fluorescence in E3Ca Ser-700 required 5-6 Ca 2ϩ rather than the 3-4 Ca 2ϩ required for E3Ca Asn-700. The N700S polymorphism localizes to position 9, where an aspartate or asparagine coordinates Ca 2ϩ through a bridging water molecule (9). The presence of a serine rather than an asparagine at position 9 likely disrupts this water bridge, resulting in loss of a high affinity Ca 2ϩ -binding site. However, the results indicate that binding of Ca 2ϩ to adjacent motifs allows the region around Trp-698 to adopt the Ca 2ϩ -replete conformation.
In equilibrium fluorescence studies, Trp-698 quenching in Tr4 Asn-700 titrated similarly to E3Ca Asn-700, and by stopped-flow fluorescence, the k obs1 of Tr4 Asn-700 was also similar to k obs1 for E3Ca Asn-700 at [Ca 2ϩ ] less than 4 mM. In the presence of the Ser-700 polymorphism, residues present in the largest truncation, Tr4, and high [Ca 2ϩ ] were necessary to induce a Ca 2ϩ -bound conformation that results in quenching of Trp-698 fluorescence. However, Tr4 Ser-700 had a k obs1 that was much lower than even Tr2 Asn-700, and even the presence of all the Ca 2ϩ -binding motifs in E3Ca Ser-700 was not sufficient to restore k obs1 to that observed for Asn-700 protein containing only the first two motifs. Thus, residues in the first Ca 2ϩ -binding motif regulate the rate of formation of the Ca 2ϩbound conformation, as monitored by Trp-698. The Ser-700 polymorphism slows the rate of the first conformational change and decreases the affinity of Ca 2ϩ -binding under equilibrium conditions.
Our results demonstrating rapid, high affinity Ca 2ϩ binding to the N-terminal motifs of TSP-1 can be compared with studies of the Ca 2ϩ -binding region of TSP-5 (cartilage oligomeric matrix protein). Similar to Trp-698 in E3Ca (17), Trp-344 at position 7 in motif 4 of TSP-5 is cooperatively quenched in the presence of Ca 2ϩ (13). The intrinsic fluorescence of Trp-344 of TSP-5 has been analyzed in the presence a mutation, D361Y, at position 1 in the adjacent motif 5. Ca 2ϩ titration of Trp-344 fluorescence in a TSP-5 fragment reveals that the D361Y mutation caused a transition at 5-fold higher Ca 2ϩ (ϳ1 mM) as compared with the wild-type TSP-5 fragment (ϳ0.2 mM) (13). In addition, the D361Y mutation shifted the EC 50 of Ca 2ϩ binding by equilibrium dialysis to higher Ca 2ϩ . In contrast, the D469⌬ mutation at position 12 in motif 10 did not alter the titration curves of the fluorescence or Ca 2ϩ binding (13), suggesting that mutations in N-terminal motifs more greatly influence high affinity Ca 2ϩ binding. The importance of the Nterminal motifs in high affinity binding is further demonstrated by a TSP-5 truncation construct that contains only the last 6 Ca 2ϩ -binding motifs. This truncated protein binds less Ca 2ϩ with decreased affinity as compared with a construct containing the entire Ca 2ϩ -binding region (16).
The Ca 2ϩ titration differences between Asn-700 and Ser-700 protein occur in the range of 100 -800 M [Ca 2ϩ ] that is found in the endoplasmic reticulum (reviewed in Ref. 25). However, differences in the rate of Ca 2ϩ binding were observed at all [Ca 2ϩ ], including high diffusible [Ca 2ϩ ] similar to those found in the extracellular fluid (26). Thus, the Ser-700 polymorphism may potentially exert effects both during trafficking of TSP-1 to the cell surface and after secretion of TSP-1 into the extracellular space. The trafficking hypothesis is suggested due to possible analogies between the polymorphism in TSP-1 and mutations in TSP-5 that cause TSP-5 to accumulate in the endoplasmic reticulum of chondrocytes in patients with PSACH or EDM1 (27,28). Patients with mutant TSP-5 have decreased levels of plasma TSP-5 (29), and similarly, patients homozygous for the allele encoding the Ser-700 polymorphism also have decreased levels of plasma TSP-1 (1), suggesting a related problem with secretion. The Ser-700 polymorphism that causes loss of a Ca 2ϩ -binding site and slows the rate of Ca 2ϩ -induced protein folding could influence TSP-1 maturation and transit through the endoplasmic reticulum. The Ser-700 polymorphism alters the conformation of full-length TSP-1, inasmuch as polymorphic TSP-1 is more sensitive to unfolding by urea and guanidine as assessed by intrinsic fluorescence and circular dichroism (30). It will therefore be key to learn whether TSP-1 is accumulated in the endoplasmic reticulum in lesions of patients with familial premature coronary artery disease.
In the extracellular space, the Ser-700 polymorphism may alter interactions of the C-terminal region of TSP-1 with hemostatic proteins such as thrombin (31) and von Willebrand factor (32,33), which bind covalently to TSP-1 via disulfide bonding at the C terminus. Recently, the presence of the Ser-700 polymorphism in TSP-1 has been shown to increase binding of TSP-1 to fibrinogen on the platelet surface, resulting in increased platelet aggregation (30). Increased platelet aggregation is one mechanism by which the Ser-700 polymorphism may cause disease by exerting its effects extracellularly. Further studies are required to learn how loss of a high affinity Ca 2ϩ -binding site associated with slowed kinetics alters the function of TSP-1, leading to familial premature coronary artery disease. Such studies may also uncover beneficial effects of the polymorphism that account for the frequency of ϳ10% for the Ser-700 allele in Caucasian populations (1,34).