Ca2+ Binding to the First Epidermal Growth Factor-like Domain of Human Blood Coagulation Factor IX Promotes Enzyme Activity and Factor VIII Light Chain Binding

Hans ter Maat,Olivier D. Christophe,D. Jasper G. Rees,Koen Mertens,Peter J. Lenting

doi:10.1074/jbc.271.41.25332

Hans ter Maat, Olivier D. Christophe + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.41.25332

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Abstract

Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp64 by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km approximately 1, 2, and 4 x 10(3) M-1 s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent ( approximately700- and 200-fold, respectively), indicating that Asp64 replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.

Highlights

(EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2؉
The Ca2ϩ dependence of wt-factor IXa␤ (FIXa) was indistinguishable from that of pd-FIXa (Fig. 1A), which suggests that these proteins are similar in Ca2ϩ binding to sites involved in substrate hydrolysis
Effect of Synthetic FVIII Peptides on factor X (FX) Activation—Because both FVIII heavy and light chain regions are involved in complex assembly with FIXa, we addressed the possibility that binding of Factor IX (FIX) to one or both of these regions is affected by the Asp64 substitutions

Summary

EXPERIMENTAL PROCEDURES

Materials—Protein A-Sepharose CL4B and CNBr-Sepharose CL4B were from Pharmacia Biotech AB (Uppsala, Sweden). Synthetic Peptides—Peptide Lys1804–Lys1818 represents the FIXa binding site on the corresponding FVIII light chain region (i.e. KNFVKPNETKTYFWK) and was prepared as described previously [11]. Purification of FIX—In order to purify recombinant FIX, the concentrated medium was subjected to immunoaffinity chromatography employing the anti-FIX antibody CLB-FIX 14 (5 mg/ml CNBr-Sepharose). In preliminary experiments recombinant FIX proteins proved indistinguishable from pd-FIX with regard to binding to barium citrate and to the Ca2ϩdependent anti-FIX antibody CLB-FIX 2 (not shown) These data are in agreement with previous reports that established normal ␥-carboxylation in the FIX expression system employed [23, 29, 30]. Recombinant and pd-FIX were converted into FIXa employing FXIa as described [12]. Binding Assays—Binding of normal or mutant FIXa to the immobilized FVIII light chain and calculation of binding parameters were performed as described elsewhere [10]

RESULTS

ϪFVIIIa ϩFVIIIa

DISCUSSION