A Polymorphism in Thrombospondin-1 Associated with Familial Premature Coronary Heart Disease Causes a Local Change in Conformation of the Ca2+-binding Repeats

Abstract

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca 2؉ -binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca 2؉ -binding repeats alone (Ca) or with the third epidermal growth factor-like module (E3Ca), without (Asn-700) or with (Ser-700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (Trp-698) adjacent to the polymorphic residue was quenched cooperatively by adding Ca 2؉ . The third epidermal growth factor-like repeat dramatically altered the Ca 2؉ -dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC 50 ) of Ca Asn-700 was 390 M, and the EC 50 of E3Ca Asn-700 was 70 M. The Ser-700 polymorphism shifted the EC 50 to higher Ca 2؉ concentrations (Ca Ser-700 EC 50 of 950 M and E3Ca Ser-700 EC 50 of 110 M). This destabilizing effect is due to local conformational changes, as the Ser-700 polymorphism did not influence the secondary structure of E3Ca or Ca as assessed by far UV circular dichroism. At 200 M Ca 2؉ , in which both E3Ca Asn-700 and Ser-700 are in the Ca 2؉ -replete conformation at 37°C, the fluorescence of E3Ca Ser-700 reverted to the Ca 2؉ -depleted spectrum at 50°C compared with 65°C for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca 2؉ and thermal denaturation.Cardiovascular disease is a leading cause of death in Western societies with over 50% of the cases due to coronary heart disease (CHD) 1 (1). Some patients who develop CHD prematurely (before age 45 in men and before age 50 in women) have a family history of the disease, suggesting genetic bases for premature CHD. A recent case control study (2) identified a single nucleotide polymorphism in thrombospondin-1 (TSP-1) that was strongly associated with familial premature CHD in patients homozygous for the single nucleotide polymorphism. The single nucleotide polymorphism results in the substitution of a serine for an asparagine at residue 700 of TSP-1. TSP-1 is a 450-kDa trimeric extracellular matrix glycoprotein that previously has been observed in atherosclerotic plaques and intimal hyperplasia (reviewed in Ref.3). During arterial injury or upon stimulation with growth factors in vitro, TSP-1 expression in smooth muscle cells is increased (4 -7), and TSP-1 and platelet-derived growth factors synergistically enhance smooth muscle cell migration (8). Patients having two Ser-700 alleles also had 2-fold lower levels of plasma TSP-1 than control patients (2).A TSP-1 monomer contains an N-terminal module, an oligomerization sequence, a procollagen module, three properdin (type 1) modules, three EGF-like (type 2) modules, a number of Ca 2ϩ -binding (type 3) repeats, and a long C-terminal sequence (Fig. 1A). The Ca 2ϩ -binding and C-terminal sequences are unique to TSPs and are highly conserved. For instance, the alignment of human TSP-1 and Drosophila TSP de...

A Polymorphism in Thrombospondin-1 Associated with Familial Premature Coronary Artery Disease Alters Ca2+ Binding

“…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.…”

“…E3Ca Asn-700 and E3Ca Ser-700 were made as described previously (17). Truncated proteins were designed to preserve the disulfide connectivity (10).…”

Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization

“…46 SNPs have also been reported for PEDF and TSP-1 genes, and have been implicated in increased risk for ocular and coronary disease. [47][48][49] Some SNPs have translated into functional variations in which individuals homozygous for a missense mutation in TSP-1 were found to have lower plasma TSP-1 levels compared to control patients. 48 Alternatively, the differential expression of pro-and antiangiogenic genes may be due to differential expression of upstream genes that influence angiogenesis including HIF-1 alpha and inflammatory mediators.…”

Thrombospondins: from structure to therapeutics

“…The Asn700Ser THBS-1 polymorphism is located in repeat 1C of the calcium-binding wire (Figs. 2F and 3) and has been associated with subtle changes in the kinetics of binding of calcium to repeat 1C and adjacent repeats [41,42].…”

“…3). The Ser700 polymorphism is associated with subtle changes in the kinetics of binding of calcium to repeat 1C and adjacent repeats [41,42]. In THBS-5, there are three known diseaseassociated mutations in the wire at the aspartates at position 10 (Asp310Val in repeat 2N, and Asp479His and Asp479Tyr in repeat 11C) (Fig.…”