Characterization of recombinant epidermal growth factor (EGF)‐like modules from vitamin‐K‐dependent protein S expressed in Spodoptera cells

Abstract

Epidermal growth factor (EGF)-like modules in protein S, a physiological anticoagulant protein that functions as a cofactor to activated protein C, have been expressed in Spodoptera cells using baculovirus. EGF modules 1-3, 1-4, 2-3 and 2-4 were produced on a preparative scale. The isolated modules were more than 95% homogenous, as judged by sequence determination. 45 Ca 2ϩ -ligand blotting experiments indicated that recombinant proteins that contained the fourth EGF module, i.e. EGF 1-4 and 2-4, bound Ca 2ϩ with high affinity. The 45 Ca 2ϩ -ligand blotting results, together with results of competitive binding experiments using monoclonal antibodies as structural probes, indicated that the recombinant proteins had been folded to a native conformation. EGF modules 1-3 and 1-4 inhibited the interaction between activated protein C and protein S, whereas modules 2-3 and 2-4 had no effect on this interaction. It is thus apparent that EGF module 1 is crucial for the interaction between protein S and activated protein C. Moreover, EGF modules 1-4 were approximately 10-fold more effective in inhibiting the interaction than modules 1-3, suggesting a very weak interaction between module 4 and activated protein C or that this module is important to keep module 1 in a conformation that is optimal for interaction with activated protein C.Keywords : protein S; calcium binding; epidermal-growth-factor module.Protein S is a plasma glycoprotein that is a cofactor to acti-module contains one β-hydroxyaspartic acid residue whereas modules 2-4 each contains one β-hydroxyasparagine residue. vated protein C, a regulator of blood coagulation. In the presence of protein S activated protein C degrades the activated forms of Hydroxylation appears to be partial. Moreover, modules 2, 3 and 4 have the typical structural motif that is required for Ca 2ϩ bindthe procoagulant cofactors V and VIII by limited proteolysis, which is necessary to dampen the blood coagulation cascade. ing [6Ϫ8]. The Ca 2ϩ affinity of isolated EGF modules is low (i.e. K d values of 0.6Ϫ8 mM have been reported) [9]. In the The physiological significance of protein C and protein S is evident from the fact that mutations of either protein S or protein intact proteins, however, the Ca 2ϩ affinity appears to be much higher due to effects of the neighboring modules [10Ϫ12]. C, that lead to reduced functional levels of the proteins, increase the risk for venous thrombosis [1]. Protein S is composed of The EGF-module-containing region of protein S appears to be crucial for the interaction with activated protein C [13,14]. multiple modules. An N-terminal module containing γ-carboxyglutamic acid (Gla) is followed by a thrombin-sensitive module Dahlbäck and colleagues have found the interaction between and four tandem modules that are homologous to the epidermal protein S and activated protein C to be inhibited by Fab fraggrowth factor (EGF) precursor. The C-terminal part of protein S ments of monoclonal antibodies only if the epitope is in the first is homologous to a steroid...

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex

“…38 Inhibition studies using in vitroexpressed PS EGF-like domains have supported the role of EGF1 in APC binding. 39 Chemically synthesized EGF1 can also induce a translocation of the APC active site relative to the phospholipid surface similarly to intact PS. 40 Furthermore, several naturally occurring missense mutations affecting EGF1 have been described in association with qualitative PS deficiency.…”

“…45 This suggestion is supported by the association of qualitative PS deficiency with naturally occurring mutations in EGF2 (Lys155Glu 46 and Cys134Phe 47 ). EGF4 of PS has also been suggested to be important to keep EGF1 in optimal alignment for interaction with APC, 39 although a recombinant EGF4 alone has failed to exhibit APC cofactor activity in a clotting assay. 48 The contribution of EGF4 to function is supported by the association of classic qualitative PS deficiency with the splice site mutation Ϫ2 G Ͼ A intron g, which causes deletion of the first 2 residues of EGF4 (Ile-Asp203-204).…”

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

“…[13][14][15][16][17][18][19][20][21] Given that the deleted or replaced domains (TSR and Gla) were adjacent, it is possible that the EGF1 domain might have been nonfunctional in the PS/FII chimeras, resulting in a loss of APC cofactor activity. However, this possibility was ruled out by the equal recognition of wild-type rPS and the PS/FII chimeras by a Ca 2ϩ -and conformationdependent mAb (HPS54) directed against the PS EGF1 domain, which has been shown to inhibit APC cofactor activity 14 (Table 3).…”

“…12 Although the molecular basis of the APC cofactor functions of PS are poorly understood, many efforts have been made to determine the importance of each PS domain in APC cofactor activity. Studies based on epitope mapping with monoclonal antibodies, 13,14 site-directed mutagenesis, [15][16][17] in vitro expression or chemical synthesis of isolated PS domains, [18][19][20] and functional characterization of thrombin-or FXa-cleaved PS 21 support the involvement of the TSR and EGF1 domains in APC binding. Recently, the second EGF-like (EGF2) domain was also shown to play an important though elusive role in optimal expression of APC cofactor activity.…”

Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain

“…Protein S has also been reported to remove factor Xa (FXa) protection of FVa in the prothrombinase complex (21). Several protein S domains have been shown to contribute to APC cofactor activity, including the thrombin-sensitive region, EGF1, EGF2, and the Gla domain (21)(22)(23)(24)(25)(26)(27)(28). A direct role for the APC Gla domain in mediating protein S cofactor activity was suggested by a study in which a recombinant protein C chimera was generated and the protein C Gla domain (residues 1-45) was replaced by that of prothrombin (18).…”