Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 4

g-Carboxyglutamic Acid Domain

The NH2 terminal g-carboxyglutamic acid domain of human factor IX includes 12 g-carboxyglutamic acid residues located at positions 7, 8, 15, 17, 20, 21, 26, 27, 30, 33, 36, and 40. MLID77026369 MLID76210956  66,67 The g-carboxyglutamic acid domain defines some of the critical calcium-binding sites of the protein and is required for the interaction of factor IX with membrane surfaces in the presence of calcium ions. MLID84185715  68 Naturally occurring point mutations of glutamic acid residues destined to be g-carboxylated include Glu 7, Glu 8, Glu 17, Glu 21, Glu 27, Glu 30, and Glu 33; patients with these mutations have moderate-to-severe hemophilia, emphasizing the functional importance of these residues. MLID93324412  69 A small loop, formed by cysteine at residues 18 and 23, is common to all of the vitamin K-dependent blood clotting proteins. Point mutations at Cys 18 or Cys 23 result in severe hemophilia.

Epidermal Growth Factor-Like Domains

The g-carboxyglutamic acid domain is linked to two adjacent EGF domains by a short domain rich in aromatic amino acids. The EGF domains, each characterized by a segment of 53 amino acids and a pattern of disulfide-bonded cysteine residues, have distinct functions. The structure of the first EGF domain has been determined by two-dimensional nuclear magnetic resonance spectroscopy. MLID91308127  70 The first EGF domain may function as a spacer, since factor IX, in which the first EGF domain substitutes for the corresponding EGF domain of factor X, has full functional activity. MLID90094389  71,72 However, Astermark et al., MLID92147679  73 using inhibitory peptides, have suggested that the first EGF domain may participate in the interaction between factor X and factor IXa in the tenase complex. Factor IX Alabama, which results from a point mutation in the first EGF domain (Asp 47 B Gly), 74 may be defective in its ability to bind to cell surfaces. 75 However, because the g-carboxyglutamic acid domain is intact, this mutant binds normally to phospholipid vesicles. MLID86131633  76 More recent evidence suggests that the activation of factor X by factor IXa Alabama is not enhanced by factor VIIIa. MLID90285142  77 Naturally occurring mutations at Gln 50, Cys 51, Cys 56, and Gly 60 lead to a severe hemophilia phenotype, but the basis for this defective factor IX function is not known. The first EGF domain contains an unusual amino acid, b-hydroxyaspartic acid, at residue 64. MLID83308813  78 The function of b-hydroxyaspartic acid is unknown. Mutant factor IX in which Asp 64 has been mutated to lysine, valine, or glycine does not have functional activity. MLID88328994  79 A naturally occurring mutant, factor IX London-6 (Asp 64 B Gly), results in mild hemophilia B. MLID89305505  80 However, wild-type recombinant factor IX has been expressed in the presence of dipyridyl, o-phenanthroline, or pyridine 2,4-dicarboxylate, which are inhibitors of 2-ketoglutarate-dependent dioxygenases. MLID89214061  81 The factor IX expressed in this system is fully g-carboxylated but contains <0.1 mol of b-hydroxylated aspartic acid/mol of factor IX, compared with 0.5 mol in the factor IX expressed in the absence of inhibitors. The factor IX that is not b-hydroxylated demonstrates full functional activity when evaluated in a coagulant assay and is indistinguishable from plasma-derived factor IX in an endothelial cell-binding assay.

The second EGF domain is required for the interaction between factor IXa and factor VIIIa in the tenase complex. MLID90094389  71,72 It would appear that this second EGF domain contains contact residues that interact with factor VIIIa, but not directly with the substrate factor X. Some point mutations within this domain have only a moderate effect on function. However, moderate-to-severe hemophilia is associated with mutations of Asn 92, Gly 93, Arg 94, Gly 114, Cys 124, Asn 92, Cys 95, Cys 99, Cys 111, Asn 120, and Ala 127. MLID93324412  69