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Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites

Olga I. Stenina^*,1, Valentin Ustinov^*, Irene Krukovets^*, Tina Marinic^*, Eric J. Topol^*, and Edward F. Plow^*

^* Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and
Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, Ohio, USA

¹ Correspondence: Department of Molecular Cardiology, NB50, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH, 44195, USA. E-mail: stenino{at}ccf.org

SPECIFIC AIMS

Recent genetic studies have associated members of the thrombospondin gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in thrombospondin-4 (A387P) and thrombospondin-1 (N700S). Since Ca²⁺ binding is critical for the structure and function of thrombospondin family members, direct evidence for differences in Ca²⁺ binding by the polymorphic forms was sought. The goal of this study was to identify and characterize how the N700S substitution in TSP-1 and the A387P substitution in TSP-4 affect Ca²⁺ binding.

PRINCIPAL FINDINGS

1. Putative Ca²⁺ binding sites altered in TSP-1 and TSP-4 variants
As an initial step in considering the molecular effects of the SNPs on TSP-1 and TSP-4, we analyzed the homologies of the domains containing the SNPs to known structural motifs. In TSP-4, the change of Ala to Pro at the position 387 occurs in an EGF-like domain, a structure present in multiple proteins including cell surface receptors, extracellular matrix proteins, growth factors and coagulation proteins. Based on the comparison of the third EGF-like domain of TSP-4 to other Ca²⁺ binding EGF-like domains, we anticipated that the A387P substitution in TSP-4 might increase Ca²⁺ binding.

The N700S SNP in TSP-1 is located outside EGF-like domains, in the linker between EGF-like domains and type 3 Ca²⁺ binding repeats. The spacing of acidic amino acids in the 692 to 703 segment surrounding the SNP is similar to Ca²⁺ binding motifs in other proteins, e.g., EF-hand sequences in calmodulin and EF-hand-like, hairpin-like turn sequences, GEFDGDL, in the integrin _IIB subunit.

In addition to homology based analyses, the secondary structure of the two segments of TSP-1 (amino acid residues 690-722, corresponding to the linker between type 2 and type 3 domains) and TSP-4 (amino acid residues 386-431, corresponding to the third EGF-like domain) were predicted. Using two programs, SOPM and Protean (DNA Star), differences in secondary structure between the SNP variants were predicted.

Taken together, these comparative and predictive analyses suggest that the N700S SNP in TSP-1 and the P387A SNP in TSP-4 could alter structure and Ca²⁺ binding function.

2. Metal ion binding by synthetic peptides corresponding to TSP-1 and TSP-4 variants
To test the prediction that the sequence containing the SNP in TSP-1 is a Ca²⁺ binding site and that the S700 substitution alters this function, we synthesized the following two peptides: (690)GEDTDLDGWPNENLV and (690)GEDTDLDGWPSENLV. Terbium luminescence spectroscopy was used to quantify the cation binding properties of these peptides. The results of Tb³⁺ luminescence experiments with the two TSP-1 peptides are shown in Fig. 1 A. Both TSP-1 peptides are indeed capable of binding a single metal ion. The Tb³⁺ dissociation constants were: (K_N700) 1.3 x 10^–6 ± 0.3 x 10^–6 M for the N700 TSP-1 variant peptide and (K_S700) 4.6 x 10^–6 ± 0.4 x 10^–6 M for the S700 variant peptide. To assess the relationship between the Tb³⁺ and divalent cation binding sites, the reduction in emission intensity at 545 nm was determined upon the addition of increasing amounts of Ca²⁺, Mg²⁺, or Mn²⁺ to 10 µM TSP-1 variant peptides equilibrated with 10 µM Tb³⁺. All three metal ions bound to the two peptides, and their affinities of binding to S700 variant were consistently lower compared with the N700 variant.

View larger version (18K): [in this window] [in a new window]   Figure 1. Binding of the luminescent Ca2+surrogate terbium to synthetic TSP-1 peptides and fragments. A) TSP-1 peptides. TSP-1 variant synthetic peptides were added to 10 µM Tb3+ and luminescence was measured at 545 nm after 5 min at room temperature. Every point is an average 3 independent measurements. An unrelated peptide of similar size, which does not bind metal ions, was used as a negative control. B)TSP-1 fragments. 1.5 µM N700 (1), S700 (2), and triple mutant fragment (3) (candidate coordinating Ds changed to As) were incubated for 5 min with increasing amounts of Tb3+, then the luminescence intensity was measured. Every point is an average 3 independent measurements.

When similar experiments were performed with synthetic peptides corresponding to A387 or P387 SNP variants in TSP-4, (378)TDIDECRNGACWPN and (378)TDIDECRNGPCWPN, no Tb³⁺ binding was detected. Proper disulfide bonding and ß-hydroxylation of the asparagine are important for Ca²⁺ binding to EGF-like domains. Binding of Ca²⁺ to the third EGF-like domain of TSP-4 may depend on one or both of these features, which would be missing in the synthetic peptides.

3. Expression and characterization of TSP-1 and TSP-4 fragments
We expressed fragments of TSP-1 and TSP-4 containing 3 EGF-like domains (type 2 repeats) and the linker region between type 2 and type 3 repeats. The fragments corresponded to amino acids 547-722 of TSP-1 (containing either the N700 or S700 substitution) or to amino acids 325-491 in TSP-4 (containing either the A387 or P387 substitution). Increasing amounts of Tb³⁺ were added to 1.5 µM solution of TSP fragments, and the Tb³⁺ luminescence was measured using an excitation wavelength of 285 nm and an emission wavelength 545 nm. The results of these binding experiments are shown in Fig. 1B for TSP-1 fragments and Fig. 2 A for TSP-4 fragments. The N700 TSP-1 fragment bound Tb³⁺ in a manner similar to the synthetic peptide contained within this region. Based upon Scatchard plots of the data, 0.93 ± 0.2 mol of Tb³⁺ were bound per mole of monomeric TSP-1 fragment with a K_d of 1.8 x 10^–6 ± 0.8 x 10^–6 M. We created a mutant TSP-1 fragment, in which Asp at positions 692, 694, and 696 were replaced by Ala (DTDLDG to ATALAG) to disrupt the putative Ca²⁺ binding site. When this mutant fragment was used in Tb³⁺ luminescence experiments, no binding was detected. Differences in Tb³⁺ binding properties of the S700 and N700 TSP-1 fragments were even more dramatic than observed for variant synthetic peptides. Rather than the 3.5-fold difference (K_d=1.3x10^–6 M±0.3x10^–6 for N700 vs. K_d 4.6x10^–6±0.4x10^–6M for S700 in peptides), the fragments displayed a 10-fold difference (K_d 9.8x10^–8±3.2x10^–8 M for N700 vs. K_d 9.1x10^–7±2.2x10^–7 M for S700 in protein fragments).

View larger version (13K): [in this window] [in a new window]   Figure 2. Binding of Tb3+ to full-length TSP-4 and TSP-4 fragments. A) Tb3+ binding by TSP-4 fragments. 1.5 µM A387 (1) or P387 (2) were incubated for 5 min with increasing amounts of Tb3+, then luminescence intensity was measured. Every point is an average 3 independent measurements. B) Binding of Tb3+ to full-length TSP-4. 1.5 µM solutions of purified recombinant TSP-4 were used to measure Tb3+ luminescence in increasing concentrations of Tb3+.

The A387 TSP-4 fragment bound 2 Tb³⁺ ions per monomer [(K_d=2.8x10^–6±0.8x10^–6M]. As predicted, substitution of Ala to Pro in P387 fragment of the third EGF-like repeat resulted in higher affinity of metal ion binding [(K_d 1x10^–6±0.4x10^–6 M (Fig. 2A )]. A Scatchard plot of these data was nonlinear, suggesting a cooperative interaction or multiple sites differing in affinity. Substitution of Ala to Pro in the P387 fragment led to an additional Tb³⁺ binding site. These three sites were of similar affinities [(K_d 1x10^–6±0.4x10^–6 M] as Scatchard plots were linear.

EDTA did not reverse Tb³⁺ binding to P387 TSP-4 fragment. However, Tb³⁺ binding was reversed by BAPTA, a stronger Ca²⁺ chelator.

We focused on potential differences in the Ca²⁺ binding properties of the A387 and P387 variants in full-length TSP-4 using Tb³⁺ luminescence and intrinsic tryptophan fluorescence. The results of experiments in which varying amounts of Tb³⁺ were added to 1.5 µM TSP-4 are presented in Fig. 2B . The full-length TSP-4 variants did not differ in their intrinsic tryptophan fluorescence spectra, with or without added Ca²⁺, suggesting that changes resulting from the substitution are local.

4. The effect of A387P SNP in TSP-4 and N700S SNP in TSP-1 on intrinsic tryptophan fluorescence and calcium sensitivity of TSP fragments
There are 2 Trp in our TSP-1 fragments (W570 and W698) and a single Trp in the TSP-4 fragments (W454). These were excited at 295 nm, and emission spectra in the 310 – 400 nm range were recorded in the absence of Ca²⁺, upon addition of 2 mM Ca²⁺ and then 10 mM Ca², followed by 15 mM EDTA. Alterations in fluorescence intensity were detected in both N700 TSP-1 and P387 TSP-4 fragments but not in S700 TSP-1 or A387 TSP-4, consistent with the lower metal binding affinity predicted from the Tb³⁺ luminescence experiments. Unlike for N700 TSP-1 fragment, in P387 TSP-4, the change in intrinsic fluorescence upon Ca²⁺ addition was not reversed by the addition of EDTA.

CONCLUSIONS AND SIGNIFICANCE

In this study, we provide data to demonstrate that the A387P SNP in TSP-4 and the N700S SNP in TSP-1 affect the Ca²⁺ binding properties of the TSPs. In each TSP, a single Ca²⁺ binding site is directly altered by the SNP substitution. Naturally occurring point mutations in TSP-5 result in the loss of a local Ca²⁺ binding site, and these mutations are associated with dramatic structural and functional changes. The alterations in the Ca ²⁺ binding sites in TSP-1 and TSP-4 induced by the substitutions appear to be more subtle but are yet sufficient to alter properties that are critical for the regulation of cellular dynamics in the blood or vessel wall.

Our data indicate that the SNPs in TSP-1 and TSP-4 have direct effects on Ca²⁺ binding functions. Each substitution directly perturbs a single cation binding site. However, the changes in metal ion binding of the disease-associated SNPs exert the opposite effect on the Ca²⁺ binding properties of TSP-1 and TSP-4 variants, decreasing the former and increasing the latter. Nevertheless, these opposing effects result in the similar pathophysiological consequences, the occurrence of premature cardiovascular disease. Further structural studies are required to determine the global effects on these substitutions on the overall conformations of TSP-1 and TSP-4 and how alterations in Ca²⁺ binding exert their effects on the functions of the two TSP family members.

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic representation of the experimental design.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3712fje;

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