Structural characterization of a polymorphism in thrombospondin-1 associated with familial premature coronary artery disease

A single nucleotide polymorphism in thrombospondin-1 that results in substitution at residue 700 of a serine (S700) for an asparagine (N700) is associated with familial premature coronary artery disease. The polymorphism is in loop 1 of 13 Ca2+ binding loops, within a consensus sequence where N700 likely coordinates Ca2+. We hypothesized that the S700 polymorphism alters the conformation of and Ca2+ binding to the loops. We expressed constructs that contained the Ca2+ binding loops (Ca), the Ca2+ binding loops with the first EGF-like module (E3Ca), and truncations of E3Ca that contained the first one, two, three, or four loops (loop constructs). The intrinsic fluorescence of a tryptophan (W698) residue served as a reporter for Ca2+ binding as it was cooperatively quenched upon addition of Ca2+.The third EGF-like module increased the sensitivity of the Ca 2+-binding loops to Ca2+. A construct containing the EGF-like module with the first two Ca2+-binding loops was the minimal N700 construct required to attain a Ca2+-bound conformation. Ca2+-induced quenching of S700 constructs required higher [Ca 2+] and was slower in stopped-flow experiments than N700 constructs. Equilibrium dialysis demonstrated that S700 constructs bound one less Ca 2+ than matching N700 constructs and exhibited decreased binding affinities. Thus, the S700 polymorphism causes loss of a rapidly filled, high affinity Ca2+-binding site in loop 1. Lower affinity, slower Ca 2+ binding to the adjacent three loops in the polymorphic protein partly compensates for the loss.Structural differences between N700 and S700 TSP-1 are found at [Ca 2+] present in the endoplasmic reticulum (ER). To detect a trafficking defect in the platelet, we measured serum levels of TSP-1 from patients having NN, NS, or SS genotypes. TSP-1 N700 and TSP-1 S700 were also tagged with GFP to monitor trafficking through a fibroblast cell that was either untreated or stressed with increased temperature and decreased ER [Ca2+]. Our data indicate that the S700 polymorphism does not cause a trafficking defect in the platelet or in the fibroblast. Thus future studies are required to determine how structural changes in the Ca2+-binding loops due to the S700 polymorphism result in functional changes that lead to coronary artery disease.