Factor IX Zutphen: a Cys18→Arg mutation results in formation of a heterodimer with α1-microglobulin and the inability to form a calcium-induced conformation

Abstract

Factor IX Zutphen is a variant factor IX molecule isolated from the blood of a patient with severe haemophilia B. The molecular defect in factor IX Zutphen is a Cys18-->Arg mutation as a result of a T-->C transition at residue 6427 of the factor IX gene of the patient. The mutation disrupts the disulphide bond in the Gla-domain between Cys18 and Cys23. The remaining free cysteine residue results in the formation of a 95 kDa complex with alpha 1-microglobulin through an intermolecular disulphide bond. The same complex circulates at high levels in plasma of carriers of the mutation. The variant molecule has a calcium-binding defect, which is shown not to be caused by incomplete gamma-carboxylation. Factor IX Zutphen can not bind to phospholipids and can not be activated by factor XIa or by factor VIIa-tissue factor complex. Two sequential metal ion-dependent conformational transitions (factor IX-->factor IX'-->factor IX*) have been proposed for human factor IX [Liebman (1987) J. Biol. Chem. 262, 7605-7612], based upon the metal ion requirements for binding to anti-factor IX:Mg(II) antibodies, which are specific for the factor IX' conformation, and anti-factor IX:Ca(II) antibodies, which are specific for the factor IX* conformation. We used these conformation-specific antibodies, and antibodies raised against a synthetic peptide corresponding to residues 35-50 of human factor IX [anti-factor IX(35-50)] to study the metal ion-induced conformation of factor IX Zutphen. The disruption of the disulphide bond in the Gla-domain, maybe in combination with the complex with alpha 1-microglobulin, destabilized the factor IX' conformation. The formation of the factor IX* conformation was prevented independent of the presence of alpha 1-microglobulin. The disulphide bond in the Gla-domain is therefore essential for the calcium-dependent conformation and function of factor IX.

Modification of the N-terminus of human factor IX by defective propeptide cleavage or acetylation results in a destabilized calcium-induced conformation: effects on phospholipid binding and activation by factor XIa

“…FIX Boxtel was isolated from plasma (200 ml) by barium citrate precipitation, ammonium sulphate precipitation, immunoaffinity chromatography and ion exchange chromatography. The procedures for the barium citrate and ammonium sulphate precipitation have been described previously [24]. The 70 % ammonium sulphate precipitate was dissolved in a small volume of 50 mM triethanolamine (TEA ; pH 7.5), 150 mM NaCl, 10 mM benzamidine and 10 mM MgCl # and dialysed against the same buffer.…”

“…The protein was dialysed against 50 mM TEA (pH 7.5), 150 mM NaCl. Normal FIX was isolated using the same procedure for small quantities (up to 200 µg) or by previously reported procedures [24] for larger quantities (up to 10 mg). Isolated FIX was more than 95 % pure, as judged by SDS\PAGE.…”

“…Isolated FIX was more than 95 % pure, as judged by SDS\PAGE. Molar concentrations of normal, variant or modified FIX were calculated from the antigen concentration determined in an ELISA using non-Ca(II)dependent antibodies and a calibrated standard [24].…”

“…The ability of normal, variant and modified FIX to bind to anti-FIX : Mg(II) antibodies, anti-FIX : Ca(II) antibodies or phospholipids was studied in a sandwich ELISA, using anti-FIX : Mg(II) antibodies, anti-FIX : Ca(II) antibodies or human brain phospholipids [27] respectively to coat microtitre plates. Bound FIX was detected with non-Ca(II)-dependent anti-(factor IX IgG)-HRP conjugate as described [24] and expressed as the A %&! .…”

“…The metal ion dependence of binding of FIX to anti-FIX : Mg(II) antibodies, anti-FIX : Ca(II) antibodies and phospholipids was determined as described previously [24]. In short, immobilized anti-FIX : Mg(II) antibodies, anti-FIX : Ca(II) antibodies or human brain phospholipids were incubated with a fixed concentration of FIX in the presence of the indicated metal ion concentrations.…”