Identification of residues in the Gla-domain of human factor IX involved in the binding to conformation specific antibodies

摘要

The binding of Ca~(2+) induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(II) antibodies recognize a conformational epitope (FIX') that can be induced by several metal ions such as Ca~(2+), Mg~(2+), Mn~(2+) and Ba~(2+), while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX) that can be only induced by Ca~(2+) and Sr~(2+) ions (Liebman et al., J. Biol. Chem., vol. 262 (2987) pp. 7605-7612). The latter conformation is essential for the function of factor IX. In this study we tried to identify residues in the Gla-domain of factor IX which are involved in binding to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For this we substituted residues in recombinant human factor IX for those of factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodies. Re-introduction of factor IX specific residues increased the binding to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicating that the structural integrity of the Gla-domain was not seriously affected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg(II) antibodies, while residues 1-11 are important for binding to anti-factor IX:Ca(II) antibodies.