Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma -carboxyglutamyl (Gla) residues.
Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on prothrombin fragment 1, and Ca(2+) strongly inhibited binding to prothrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.
Brown
M A
MA
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden.
Stenberg
L M
LM
Persson
U
U
Stenflo
J
J
D015055:1-Carboxyglutamic Acid / Q000032:analysis* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D019774:Blood Coagulation Factor Inhibitors / Q000276:immunology; D001779:Blood Coagulation Factors / Q000276:immunology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D020916:Conotoxins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012910:Snake Venoms / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014812:Vitamin K / Q000378:metabolism* / Q000502:physiology
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D019774:Blood Coagulation Factor Inhibitors; D001779:Blood Coagulation Factors; D002135:Calcium-Binding Proteins; D020916:Conotoxins; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins; D012910:Snake Venoms; D014812:Vitamin K; D015055:1-Carboxyglutamic Acid