A polymorphism in thrombospondin-1 associated with familial premature coronary heart disease causes a local change in conformation of the Ca2+-binding repeats.
A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca2+-binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca2+-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 Ca2+. The third epidermal growth factor-like repeat dramatically altered the Ca2+-dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC50) of Ca Asn-700 was 390 microM, and the EC50 of E3Ca Asn-700 was 70 microM. The Ser-700 polymorphism shifted the EC50 to higher Ca2+ concentrations (Ca Ser-700 EC50 of 950 microM and E3Ca Ser-700 EC50 of 110 microM). 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 microM Ca2+, in which both E3Ca Asn-700 and Ser-700 are in the Ca2+-replete conformation at 37 degrees C, the fluorescence of E3Ca Ser-700 reverted to the Ca2+-depleted spectrum at 50 degrees C compared with 65 degrees C for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.
Hannah
Blue-leaf A
BL
Department of Medicine, Medical Sciences Center, University of Wisconsin, Madison, Wisconsin 53706, USA. bahannah@students.wisc.edu
Misenheimer
Tina M
TM
Annis
Douglas S
DS
Mosher
Deane F
DF
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003327:Coronary Disease / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D011110:Polymorphism, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics*; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D011994:Recombinant Proteins; D019700:Thrombospondin 1; D012694:Serine; D004815:Epidermal Growth Factor; D014364:Tryptophan; D002118:Calcium