scholarly journals 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

1995 ◽  
Vol 311 (3) ◽  
pp. 753-759 ◽  
Author(s):  
E G C Wojcik ◽  
M van den Berg ◽  
I K van der Linden ◽  
S R Poort ◽  
R Cupers ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Metal Ion ◽  
Human Factor ◽  
Factor Viia ◽  
Cysteine Residue ◽  
Factor Ix ◽  
Disulphide Bond ◽  
Molecular Defect ◽  
Human Factor Ix ◽  
Gla Domain

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.

Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

1996 ◽  
Vol 75 (01) ◽  
pp. 070-075 ◽  
Author(s):  
E G C Wojcik ◽  
P Simioni ◽  
M v d Berg ◽  
A Girolami ◽  
R M Bertina
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Cysteine Residue ◽  
Factor Ix ◽  
Disulphide Bond ◽  
Clotting Factors ◽  
High Molecular Weight Complex ◽  
Low Concentrations ◽  
Gla Domain ◽  
Formation Of Complexes

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

scholarly journals Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 718-721 ◽  
Author(s):  
MN Huang ◽  
CK Kasper ◽  
HR Roberts ◽  
DW Stafford ◽  
KA High
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Cleavage Site ◽  
Human Factor ◽  
Dna Amplification ◽  
Factor Ix ◽  
Molecular Defect ◽  
Human Factor Ix ◽  
Amplification Technique ◽  
Activation Peptide

Abstract A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

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

1998 ◽  
Vol 1382 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Emiel G.C Wojcik ◽  
Wing-Fai Cheung ◽  
Marieke van den Berg ◽  
Irma K van der Linden ◽  
Darrel W Stafford ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Specific Antibodies ◽  
Human Factor Ix ◽  
Gla Domain

scholarly journals Calcium binding to the first EGF-like module of human factor IX in a recombinant fragment containing residues 1-85

FEBS Letters ◽  
1998 ◽  
Vol 421 (2) ◽  
pp. 100-104 ◽  
Author(s):  
Kristina E.M Persson ◽  
Jan Astermark ◽  
Ingemar Björk ◽  
Johan Stenflo
Keyword(s):  
Calcium Binding ◽  
Human Factor ◽  
Factor Ix ◽  
Recombinant Fragment ◽  
Human Factor Ix

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

1990 ◽  
Vol 9 (2) ◽  
pp. 475-480 ◽  
Author(s):  
P.A. Handford ◽  
M. Baron ◽  
M. Mayhew ◽  
A. Willis ◽  
T. Beesley ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Human Factor ◽  
Factor Ix ◽  
Calcium Binding Site ◽  
High Affinity ◽  
Human Factor Ix

scholarly journals 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

1997 ◽  
Vol 323 (3) ◽  
pp. 629-636 ◽  
Author(s):  
Emiel G. C. WOJCIK ◽  
Marieke VAN DEN BERG ◽  
Swibertus R. POORT ◽  
Rogier M. BERTINA
Keyword(s):  
Metal Ion ◽  
Three Dimensional ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Dimensional Structure ◽  
Amino Terminus ◽  
Human Factor Ix ◽  
Carboxyglutamic Acid ◽  
Factor Xia ◽  
Gla Domain

The propeptide of human coagulation factor IX (FIX) directs the γ-carboxylation of the first 12 glutamic acid residues of the mature protein into γ-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mutation of Arg-4 in the propeptide abolishes propeptide cleavage and results in circulating profactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg-4 → Trp), factor IX Bendorf (Arg-4 → Leu) and factor IX Seattle C (Arg-4 → Gln). These variant profactor IX molecules bind normally to anti-FIX:Mg(II) antibodies, which indicates that the mutations do not seriously affect γ-carboxylation. Metal ion titration of the binding of variant profactor IX to conformation-specific antibodies demonstrates that the calcium-induced conformation is destabilized in the variant molecules. Also the binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (> 5 mM) calcium concentration. The three-dimensional structure of the Gla domain of FIX in the presence of calcium indicates that the acylation of the amino-terminus, rather than the presence of the propeptide, was responsible for the destabilization of the calcium-induced conformation. In order to confirm this, the α-amino group of Tyr1 of FIX was acetylated. This chemically modified FIX showed a similar destabilization of the calcium-induced conformation to variant profactor IX. Our data imply that the amino-terminus of FIX plays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the binding to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing the calcium-induced conformation.

Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 718-721
Author(s):  
MN Huang ◽  
CK Kasper ◽  
HR Roberts ◽  
DW Stafford ◽  
KA High
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Cleavage Site ◽  
Human Factor ◽  
Dna Amplification ◽  
Factor Ix ◽  
Molecular Defect ◽  
Human Factor Ix ◽  
Amplification Technique ◽  
Activation Peptide

A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

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