Evidence for a common metal ion-dependent transition in the 4-carboxyglutamic acid domains of several vitamin K-dependent proteins.
The murine monoclonal antibody H-11 binds a conserved epitope found at the amino terminal of the vitamin K-dependent blood proteins prothrombin, factors VII and X, and protein C. The sequence of polypeptide recognized by antibody H-11 contains 2 residues of gamma-carboxyglutamic acid, and binding of the antibody is inhibited by divalent metal ions. By using a solid-phase immunoassay with 125I-labeled antibody and immobilized vitamin K-dependent protein, binding of the antibody to the vitamin K-dependent proteins was inhibited by increasing concentrations of calcium, manganese, and magnesium ion. The transition midpoints for antibody binding were in the millimolar concentration range and were different for each metal ion. In general, the transition midpoints were lowest for manganese ion, intermediate for calcium ion, and highest for magnesium ion. Antibody H-11 bound specifically to a synthetic peptide corresponding to residues 1-12 of human prothrombin that was synthesized as the gamma-carboxyglutamic acid-containing derivative. Binding of the antibody to the peptide was not inhibited by calcium ion. These data suggest that inhibition of antibody H-11 binding by divalent metal ions is not due simply to neutralization of negative charge by Ca2+. This transition which is conserved in vitamin K-dependent proteins containing the H-11 antigenic site is likely due to a structural transition of the amino-terminal polypeptide possibly from a random (accessible) to ordered (inaccessible) structure.
Church
W R
WR
Department of Biochemistry, University of Vermont, Burlington 05405.
Boulanger
L L
LL
Messier
T L
TL
Mann
K G
KG
D015055:1-Carboxyglutamic Acid; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002118:Calcium; D002413:Cations, Divalent; D002417:Cattle; D000939:Epitopes / Q000032:analysis*; D005167:Factor VII / Q000276:immunology*; D005170:Factor X / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D011486:Protein C / Q000276:immunology*; D011516:Prothrombin / Q000276:immunology*; D014812:Vitamin K / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002413:Cations, Divalent; D000939:Epitopes; D009842:Oligopeptides; D011486:Protein C; D014812:Vitamin K; D015055:1-Carboxyglutamic Acid; D005167:Factor VII; D011516:Prothrombin; D005170:Factor X; D002118:Calcium