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Identification and Purification of Vitamin K-dependent Proteins and Peptides with Monoclonal Antibodies Specific for γ-Carboxyglutamyl (Gla) Residues

2000; Elsevier BV; Volume: 275; Issue: 26 Linguagem: Inglês

10.1074/jbc.m002298200

1083-351X

Mark A. Brown, Leisa M. Stenberg, U Persson, Johan Stenflo,

Blood Coagulation and Thrombosis Mechanisms

Novel monoclonal antibodies that specifically recognize γ-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.