title: Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.
abstract: Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.
authors.affiliation: Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. stenino@ccf.org
authors.forename: Olga I
authors.initials: OI
authors.lastname: Stenina
authors.forename: Valentin
authors.initials: V
authors.lastname: Ustinov
authors.forename: Irene
authors.initials: I
authors.lastname: Krukovets
authors.forename: Tina
authors.initials: T
authors.lastname: Marinic
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Topol
authors.forename: Edward F
authors.initials: EF
authors.lastname: Plow
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry; D002319:Cardiovascular System / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D014644:Genetic Variation; D006801:Humans; D016023:Integrins / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013725:Terbium / Q000737:chemistry; D019700:Thrombospondin 1 / Q000235:genetics*; D019699:Thrombospondins / Q000235:genetics*; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D000596:Amino Acids; D002147:Calmodulin; D016023:Integrins; D007477:Ions; D010455:Peptides; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D019699:Thrombospondins; C101864:thrombospondin 4; D013725:Terbium; D014364:Tryptophan; D002118:Calcium