Binding of Calcium to Anticoagulant Protein S: Role of the Fourth EGF Module
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- 作者：Kristina E. M. Persson ; Johan Stenflo ; Sara Linse ; Yvonne Stenberg ; Roger J. S. Preston ; David A. Lane ; Suely M. Rezende
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：September 5, 2006
- 年：2006
- 卷：45
- 期：35
- 页码：10682 - 10689
- 全文大小：189K
- 年卷期：v.45,no.35(September 5, 2006)
- ISSN：1520-4995
文摘
Protein S is an anticoagulant protein containing a Gla (enclosing 
-carboxyglutamic acids)module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules,and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C(APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities.The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have nowinvestigated this module through studies of recombinant wild-type protein S and a naturally occurringmutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previouslyreported secretion defect. In the wild-type protein, Asn217 is normally 
-hydroxylated. The binding ofcalcium to wild-type protein S is characterized by four high-affinity binding sites with KD values rangingfrom 10-7 to 10-9 M. Three of these binding sites are located in EGF modules. Using surface plasmonresonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected thecalcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S,involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition ofbinding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.
-carboxyglutamic acids)module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules,and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C(APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities.The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have nowinvestigated this module through studies of recombinant wild-type protein S and a naturally occurringmutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previouslyreported secretion defect. In the wild-type protein, Asn217 is normally -hydroxylated. The binding ofcalcium to wild-type protein S is characterized by four high-affinity binding sites with KD values rangingfrom 10-7 to 10-9 M. Three of these binding sites are located in EGF modules. Using surface plasmonresonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected thecalcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S,involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition ofbinding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.