Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling.
Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.
Giri
T K
TK
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Villoutreix
B O
BO
Wallqvist
A
A
Dahlbäck
B
B
de Frutos
P G
PG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D017293:Protein S / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013045:Species Specificity; D013917:Thrombin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D008563:Membrane Lipids; D010743:Phospholipids; D017293:Protein S; D011993:Recombinant Fusion Proteins; D013917:Thrombin