A Calcium-dependent Anti-human Coagulation Factor IX Monoclonal Antibody That Recognizes Phospho-serine (158) of the Activation Peptide Region

Abstract number: P1255

Atoda H, Yokota E, Morita T

One murine monoclonal antibody against human blood coagulation factor IX, B1, recognizes the human coagulation factor IX (hFIX) activation peptide (AP) region in the presence of metal ions, such as Ca²⁺ (EC₅₀= 0.58 mM) or Mg²⁺, but not in the absence of metal ions. We investigated the mechanism of Ca²⁺-dependent binding of B1 and AP.

Methods:  AP derived from human plasma FIX was deglycosylated by treatment with N-glycanase or neuraminidase and endo-a-N-acetylgalactosaminidase. Recombinant AP (rAP) was expressed in E. coli as a fusion protein with thioredoxin using a pET102 directional TOPO expression kit, and the AP region (35 amino acid residues) was cleaved off by trypsin and purified by reverse phase HPLC. The mass of rAP and N- and O-deglycosylated AP were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a Voyager-DE (Applied Biosystem).

Results:  The deglycosylation of AP did not affect the binding of B1 to AP, indicating that carbohydrate moieties have no influence on the formation of the epitope recognized by B1. B1 failed to bind to rAP even at concentrations over 1 µM. The masses of N- and O-deglycosylated AP, 4051.9, and of recombinant AP, 3969.36, were detected by MALDI-TOF MS. Amino acid sequence analysis of N- and O-deglycosylated AP revealed that Ser-13 of AP was masked by some modifications other than glycosylations. The binding activity of B1 to AP was eliminated by acid phosphatase treatment of AP. B1 bound to the synthetic peptide containing phosphoserine at position 13 of AP (AP 5–19) but not to a shorter peptide (AP 7–17).

Conclusion:  These results indicate that the Ser-13 of the activation peptide is phosphorylated and B1 recognizes the epitope including phosphoserine-13 and several hydrophobic amino acids around phosphoserine-13.