Identification of residues in the Gla-domain of human factor IX involved in the binding to conformation specific antibodies.
The binding of Ca2+ 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 Ca2+, Mg2+, Mn2+ and Ba2+, while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX*) that can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) 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.
Wojcik
E G
EG
Haemostasis and Thrombosis Research Centre, University Hospital Leiden, The Netherlands. ewojcik@hematology.azl.nl
Cheung
W F
WF
van den Berg
M
M
van der Linden
I K
IK
Stafford
D W
DW
Bertina
R M
RM
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry*; D001666:Binding Sites, Antibody; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry*; D005164:Factor IX / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection
D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins; D005164:Factor IX