title: Factor IX Zutphen: a Cys18-->Arg mutation results in formation of a heterodimer with alpha 1-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.
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000032:analysis; D000510:Alpha-Globulins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005164:Factor IX / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D000510:Alpha-Globulins; D008670:Metals; C016001:alpha-1-microglobulin; C096799:factor IX Zutphen; D015055:1-Carboxyglutamic Acid; D005164:Factor IX; D001120:Arginine; D003545:Cysteine; D002118:Calcium