Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the gamma-carboxyglutamic acid-containing region.
Thrombin cleaves protein S at arginine residues 52 and 70 resulting in loss of cofactor activity and reduced Ca2+ ion binding. After thrombin cleavage the NH2-terminal region containing gamma-carboxyglutamic acid (Gla) is linked to the large COOH-terminal fragment by a disulfide bond. Measurements of the rate of disulfide bond reduction by thioredoxin in intact protein S showed that the disulfide bonds are largely inaccessible to thioredoxin in the presence of Ca2+ ions, whereas in the presence of EDTA apparently all of the disulfide bonds are rapidly reduced. Probing the reactivity of the disulfide bonds in thrombin-modified proteins indicated that the thrombin cleavage induces a conformational change in the protein. After thrombin cleavage of protein S, the domain containing gamma-carboxyglutamic acid could be removed by selective reduction with thioredoxin followed by alkylation of the sulfhydryl groups. Ca2+ ion binding was compared in intact protein S, thrombin-modified protein S, and Gla domainless protein S. The intact protein S bound several Ca2+ ions, and the binding was not saturable. Thrombin-modified protein S, whether intact or with the Gla domain removed by selective reduction, bound two to three Ca2+ ions with a KD of 15-20 microM. The Gla domain in thrombin-modified protein S thus does not contribute significantly to the high affinity Ca2+ ion binding. Thrombin cleavage of protein S may be of physiological importance in the regulation of blood coagulation.
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003956:Dialysis; D004220:Disulfides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017293:Protein S; D013879:Thioredoxins / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology
D004220:Disulfides; D005971:Glutamates; D006023:Glycoproteins; D010446:Peptide Fragments; D017293:Protein S; D013879:Thioredoxins; D015055:1-Carboxyglutamic Acid; D004492:Edetic Acid; D013917:Thrombin; D002118:Calcium