Characterization of a monoclonal antibody B1 that recognizes phosphorylated Ser-158 in the activation peptide region of human coagulation factor IX.
Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as gamma-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K(d) value of 5 nm in the presence of Ca(2+) (EC(50) = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca(2+) dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca(2+). These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca(2+)-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.
Atoda
Hideko
H
Department of Biochemistry, Meiji Pharmaceutical University, Noshio, Kiyose, Tokyo 204-8588, Japan.
Yokota
Emi
E
Morita
Takashi
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D000939:Epitopes; D005164:Factor IX / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas; D008969:Molecular Sequence Data; D010455:Peptides; D010768:Phosphoserine / Q000737:chemistry*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012694:Serine; D012910:Snake Venoms
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D012910:Snake Venoms; D010768:Phosphoserine; D012694:Serine; D005164:Factor IX