Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 2

The gene organization of factor IX is nearly identical to that of factor X, factor VII and protein C (see Fig. 107-4). After transcription, a factor IX mRNA is generated that is about 2.8 kb. MLID82272386 MLID83065193  6,7 This mRNA includes a 5' untranslated region and a long 3' untranslated region of 1.4 kb. This 3' sequence contains a poly(A) tail nearly 1.4 kb 3' to the stop codon and the typical processing sequence (AATAAA) 16 bp 5' to this polyadenylation site. This leads to a long, noncoding tail in the mRNA. MLID84236100 MLID86000558  9,11

Synthesis of Factor IX

Factor IX is synthesized in the hepatocyte as a precursor protein; it then undergoes a series of intracellular post-translational modifications, leading to the production of mature, fully active factor IX (Fig. 107-3). The coding sequence of the factor IX gene encodes two peptides that are removed prior to secretion of the mature factor IX into the blood: a 28-residue signal peptide that directs the nascent peptide chain to the endoplasmic reticulum and an 18-residue propeptide between the signal peptide and the mature N terminus of factor IX. MLID83065193  7 The signal peptide is cleaved co-translationally by the signal peptidase. The propeptide contains the g-carboxylation recognition site that directs g-carboxylation of the adjacent glutamic acid residues in the g-carboxyglutamic acid domain of mature factor IX. This recognition element is defined by amino acids –18, –17, –16, –14, and –10 toward the N terminus of this region. The vitamin K-dependent carboxylase binds to the g-carboxylation recognition site within the propeptide. MLID89386645 MLID87102866  25,26 The carboxylation reaction, catalyzed by the carboxylase, requires a reduced form of vitamin K (vitamin KH2), molecular oxygen, carbon dioxide, and the factor IX precursor, uncarboxylated profactor IX, containing glutamic acid residues. 27 The carboxylase is an enzyme with a molecular weight of 94,000 that is sufficient to carboxylate a precursor substrate in the presence of the necessary cofactors. MLID91172787 MLID93281629 MLID93391357 MLID92086858  28–31 The carboxylase is a membrane protein, and its catalytic activity resides on the luminal side of the rough endoplasmic reticulum. MLID79226052 MLID80175363  32,33

In addition, Asp 64 undergoes b-hydroxylation. From the cDNA sequences of factor IX, MLID82272386 MLID83065193  6,7 factor X, MLID84222026  34 protein C, MLID85269639 MLID85014826  35,36 protein S, MLID86313649  37 and factor VII, MLID86205965  38 it is apparent that specific aspartic acid residues in precursor forms of these proteins undergo b-hydroxylation post-translationally. Stenflo et al. 39 have suggested that a consensus sequence consisting of Cys-X-Asp/Asn-X-X-X-Phe/Tyr-X-Cys-X-Cys signals this event. Factor IX contains about 0.4 mol of b-hydroxyaspartic acid/mol of factor IX. MLID88033058  40,41 This modified amino acid is formed from aspartic acid by a post-translational process catalyzed by a 2-oxoglutarate-dependent dioxygenase, MLID89098949  42 which has been partially purified from bovine liver. MLID90256774  43 The b-hydroxylase is present within the endoplasmic reticulum.

Bovine and human factor IX are glycoproteins that contain about 17% carbohydrate. Bovine factor IX contains four N-linked glycosylation sites at residues 158, 168, 173, and 261. MLID75036029 MLID80056619 MLID83213285  44–46 These residues correspond to residues 157, 167, 172, and 261 in human factor IX. However, residue 172 is threonine in human factor IX, and residue 261 lacks the appropriate consensus sequence for N-linked glycosylation. Two O-linked glycosylation sites have been identified in the first EGF domain of factor IX. MLID90062160 MLID92388094  47,48 The O-linked sugar at residue 53 is composed of glucose and xylose in equal molar ratios, with the glucose linked to Ser 53. An analogous trisaccharide has been analyzed that is linked to Ser 53 of bovine factor IX and has the structure D-xylose p a 1-3-D-xylose p a 1-3-D-glucose p b I-O-serine-53. MLID90130422  49 The O-linked sugar at residue 61 is composed of equal molar ratios of galactose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid and is linked to Ser 61 through the fucose residue. MLID92388094 MLID93320029  48,50