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Polymorphisms A387P in thrombospondin‐4 and N700S in thrombospondin‐1 perturb calcium binding sites
Author(s) -
Stenina Olga I.,
Ustinov Valentin,
Krukovets Irene,
Marinic Tina,
Topol Eric J.,
Plow Edward F.
Publication year - 2005
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.05-3712fje
Subject(s) - thrombospondin , amino acid , binding site , recombinant dna , biochemistry , binding domain , chemistry , genetics , gene , biology , enzyme , metalloproteinase
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 Ca 2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca 2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca 2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb 3+ luminescence as an index of Ca 2+ binding. The Tb 3+ binding constants placed the TSP‐1 region affected by N700S polymorphism among other high‐affinity Ca 2+ binding sites. The affinity of Ca 2+ 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 Ca 2+ 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 Ca 2+ binding properties. Since these substitutions exert the opposite effects on Ca 2+ 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.