Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.
Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.
Evenäs
P
P
Department of Clinical Chemistry, Wallenberg Laboratory, Lund University, University Hospital Malmö, Sweden.
García De Frutos
P
P
Linse
S
S
Dahlbäck
B
B
D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D017931:DNA Primers / Q000235:genetics; D006023:Glycoproteins; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011485:Protein Binding; D017293:Protein S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000925:Anticoagulants; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D011994:Recombinant Proteins; D015935:Complement C4b