title: Functional properties of the sex-hormone-binding globulin (SHBG)-like domain of the anticoagulant protein S.
abstract: Protein S (PS) possesses a sex-hormone-binding globulin (SHBG)-like domain in place of the serine-protease domain found in other vitamin K-dependent plasma proteins. This SHBG-like domain is able to bind a complement fraction, C4b-binding protein (C4b-BP). To establish whether the PS SHBG-like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG-like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b-BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG-like region is an independent folding unit. We also obtained the first direct evidence that the SHBG-like domain alone is sufficient to support the interaction with C4b-BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b-BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG-like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG-like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.
authors.affiliation: Unité INSERM 428, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris V, Paris, France.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Saposnik
authors.forename: Delphine
authors.initials: D
authors.lastname: Borgel
authors.forename: Martine
authors.initials: M
authors.lastname: Aiach
authors.forename: Sophie
authors.initials: S
authors.lastname: Gandrille
mesh_terms: D000925:Anticoagulants / Q000378:metabolism*; D001466:Barium Sulfate / Q000378:metabolism; D001777:Blood Coagulation; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003169:Complement Inactivator Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D017293:Protein S / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D012738:Sex Hormone-Binding Globulin / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000925:Anticoagulants; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D007074:Immunoglobulin G; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D012738:Sex Hormone-Binding Globulin; D001466:Barium Sulfate; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002118:Calcium