title: Binding of calcium to anticoagulant protein S: role of the fourth EGF module.
abstract: Protein S is an anticoagulant protein containing a Gla (enclosing gamma-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 now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, 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 the calcium-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 of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.
authors.affiliation: Department of Laboratory Medicine, Clinical Chemistry, Lund University, University Hospital, Malmö, 20502 Malmö, Sweden. Kristina.Persson@med.lu.se
authors.forename: Kristina E M
authors.initials: KE
authors.lastname: Persson
authors.forename: Johan
authors.initials: J
authors.lastname: Stenflo
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Stenberg
authors.forename: Roger J S
authors.initials: RJ
authors.lastname: Preston
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.forename: Suely M
authors.initials: SM
authors.lastname: Rezende
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000925:Anticoagulants / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017293:Protein S / Q000737:chemistry* / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D017293:Protein S; D004815:Epidermal Growth Factor; D002118:Calcium