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| The structure consists of 415 amino acides that are separated into an amino terminal [[Gla domain]] (12 Gla residues), which is a characteristic feature of all vitamin K–dependent factors. The Gla region is followed bt two tandem EGF domains (residues 85-127), activation peptide region which is cleaved off upon activation of FIX to FIXa , and a serine protease domain, which contains enzymatic activity.
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| Before protein is secreted into the circulation it undergoes posttranslational modifications, which include gamma-carboxylation, beta-hydroxylation, N and O-linked glycosylation, sulfation, and phosphorylation. Gamma-carboxylation is a vitamin K–dependent process in which the gamma-carboxylation of the glutamic acid residues forms gamma-carboxyglutamyl (Gla) residues in the mature protein and requires reduced vitamin K, oxygen, and carbon dioxide to perform its functions (I will give you an image later). In this modification the negative charge allows the FIX to undergo a conformational change in order to interact with Ca2+ and interact with the anionic phospholipid membrane. In the absence of calcium the Gla residues are exposed to the solvent, however upon its binding the protein exposes its hydrophobic residues which are inserted into the lipid membrane.
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| Hydroxylation occurs in the EGF-1 domain of FIX on aspartic acid residues to form a erythro-β-hydroxy aspartic acid (HYA). The addition of a hydroxyl group (OH) to aspartic acid involves the recognition of a Cys-X-Asp-X-X-X-X-Tyr/Phe-X-Cys-X-Cys consensus sequence (reference 334).
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| Glycosylation involves the addition of a carbohydrate in the activation peptide of FIX. The activation peptide is cleaved off at residues R145-A146 and R180-V181, thus converting the zymogen to an active form.
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| The serine protease domain of FIX accounts for half of its mass and contains a conserved catalytic triad made of Asp, His, and Ser. The binding pockets of these vitamin-K depended proteases recognize a small number of amino acids sequences allowing them to cleave at arginyl residues with high substrate specificity. Serine’s hydroxyl group carries out the nucleophillic attack. While the imidazol ring of hisidine takes up the liberated proton and the carboxylate ion of Asp stabilizes the developing charge.
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| Deficiency of this glycoprotein leads to hemophilia B, a common X linked inherited coagulation disorder that affects 1 in 30, 000 males (reference 458, 464). Females are rarely affected by this disorder unless there is a mutation in the FIX gene (465, 466). The mutations that have been identified in the factor IX gene and lead to proteins reduced activity include; large and small deletions, point mutations, and missense mutations.
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| Gene Structure and Expression:
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| The gene for factor IX is located on the long arm of chromosome X between positions 26.3- and 27.1 and contains eight exons and seven introns.
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| Factor IX (FIX) is the precursor of a serine protease required for blood coagulation. The structural organization of FIX is similar to that of other vitamin-K-dependent plasma proteins, such as Factor VII (FVII) and Factor X (FX) [1]. These proteins consist of an N-terminal -carboxyglutamic acid (Gla) domain, which is necessary for phospholipid binding, two epidermal growth factor (EGF)-like domains, an activation peptide region and the C-terminal serine protease domain with the catalytic centre [1,2]. FIX circulates in plasma as a single-chain zymogen and can be activated by either activated-FVII–tissue-factor complex or activated Factor XI (FXIa) [3,4]. Upon activation the peptide bonds Arg145–Ala146 and Arg180–Val181 are cleaved to yield a two-chain, disulphide-linked molecule, activated FIX (FIXa), and an activation peptide of 35 amino acids. The primary role of FIXa in coagulation is to convert FX into activated FX (FXa) in a process that requires calcium ions, a membrane surface and a protein cofactor, activated Factor VIIIa (FVIIIa; for review see [5]). FIXa alone displays extremely low proteolytic activity towards FX, but when assembled into the FX-activating complex it becomes a potent activator of FX [6,7].
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| The majority of enzymes involved in blood coagulation, including FIXa, belong to the family of trypsin-like serine proteases. A prominent characteristic of these proteases is the presence of a number of surface loops that display high variability in size and amino acid sequence. These loops border the substrate-binding groove and therefore are likely to control the substrate specificity and enzyme activity of each individual enzyme (for review see [8,9]). This notion is exemplified by a recent study from our laboratory in which mutations in one of these surface loops of FIXa, i.e. loop 256–268 [c91–101] (with the chymotrypsin numbering in brackets), enhanced the catalytic activity towards synthetic and natural substrates [10].
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| Furthermore, we have demonstrated that the reactivity of FIXa towards FX and antithrombin is impaired when loop 199–204 [c34–40] is exchanged for the corresponding sequence of related coagulation enzymes [11]. These mutational effects on FIXa activity were abolished in the presence of FVIIIa, suggesting that the surface loops are reoriented upon FVIIIa binding. This cofactor effect may provide an explanation for the fact that the haemophilia B database contains no mutations in loop region 199–204 [12]. Examination of the three-dimensional structure of human FIXa [13] reveals that the opposite edge of the substrate-binding groove is constituted by another surface loop comprising residues 315–322 [c146–154] (Figure 1). It seems conceivable, therefore, that this loop, which has previously been referred to as autolysis loop [14] or Variable Region 4 [15], serves a role similar to that of loop 199–204. One argument against this view, however, is the notion that in loop 315–322 several mutations have been identified that are associated with severe haemophilia B. These mutations occur at positions 316 (Lys Glu), 317 (Gly Arg, Trp or Glu) and 320 (Ala Asp or Val) [12]. In the present study we investigated this distinctive feature of loop 315–322 in more detail by functional analysis of FIXa variants in which Lys316 [c148] was replaced by Glu or Ala. Lys316 was selected for mutagenesis because this amino acid has, in contrast to Gly317 and Ala320, a large basic side chain that is fully exposed to solvent and as such is a candidate for being in direct contact with substrates. The FIX mutants were expressed in mammalian cells, purified by immunoaffinity chromatography and subsequently activated by FXIa. Functional characterization of the activated mutants revealed that Lys316 is indispensable for efficient activation of FX in both the absence and presence of FVIIIa.
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| [http://www.ebi.ac.uk/pdbe-srv/view/entry/1pfx/jmol PORCINE FACTOR IXA] | | [http://www.ebi.ac.uk/pdbe-srv/view/entry/1pfx/jmol PORCINE FACTOR IXA] |
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| <applet load='1lbg' size='450' frame='true' align='right' scene='Factor_IX/Gladomain/1' /> | | <applet load='1lbg' size='450' frame='true' align='right' scene='Factor_IX/Gladomain/1' /> |
| <applet load='1lbg' size='450' frame='true' align='right' scene='Factor_IX/Rfnscene/1' /> | | <applet load='1lbg' size='450' frame='true' align='right' scene='Factor_IX/Rfnscene/1' /> |
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| ******************************************************************************************************************************
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| '''Factor IX''' (plasma thromboplastin component, Christmas factor, or hemophilia B factor) is a single-chain vitamin K-dependent procoagulant glycoprotein. It is synthesized by the liver hepatocyte as a [[pre-prozymogen]] that requires extensive posttranslational modification. The pre-prozymogen contains a pre-pro sequence that is followed by a polypeptide region. The pre-peptide is a hydrophobic signal peptide at its amino terminal that transports the growing polypeptide into the lumen of the Endoplasmic Reticulum. Once inside the ER, this signal peptide is cleaved by signal peptidase. The [[pro-peptide]] contained in the protein induces the docking of the polypeptide to the vitamin K-dependent carboxylase (γ-glutamyl carboxylase), where is modified by γ-carboxylation. The posttranslational modification creates a fully gamma-carboxylated mature zymogen which can now associate with anionic phospholipid surface.
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|
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| The structure consists of 415 amino acides that are separated into an amino terminal [[Gla domain]] (12 Gla residues), which is a characteristic feature of all vitamin K–dependent factors. The Gla region is followed bt two tandem EGF domains (residues 85-127), activation peptide region which is cleaved off upon activation of FIX to FIXa , and a serine protease domain, which contains enzymatic activity.
| |
|
| |
| Before protein is secreted into the circulation it undergoes posttranslational modifications, which include gamma-carboxylation, beta-hydroxylation, N and O-linked glycosylation, sulfation, and phosphorylation. Gamma-carboxylation is a vitamin K–dependent process in which the gamma-carboxylation of the glutamic acid residues forms gamma-carboxyglutamyl (Gla) residues in the mature protein and requires reduced vitamin K, oxygen, and carbon dioxide to perform its functions (I will give you an image later). In this modification the negative charge allows the FIX to undergo a conformational change in order to interact with Ca2+ and interact with the anionic phospholipid membrane. In the absence of calcium the Gla residues are exposed to the solvent, however upon its binding the protein exposes its hydrophobic residues which are inserted into the lipid membrane.
| |
|
| |
| Hydroxylation occurs in the EGF-1 domain of FIX on aspartic acid residues to form a erythro-β-hydroxy aspartic acid (HYA). The addition of a hydroxyl group (OH) to aspartic acid involves the recognition of a Cys-X-Asp-X-X-X-X-Tyr/Phe-X-Cys-X-Cys consensus sequence (reference 334).
| |
|
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| Glycosylation involves the addition of a carbohydrate in the activation peptide of FIX. The activation peptide is cleaved off at residues R145-A146 and R180-V181, thus converting the zymogen to an active form.
| |
|
| |
| The serine protease domain of FIX accounts for half of its mass and contains a conserved catalytic triad made of Asp, His, and Ser. The binding pockets of these vitamin-K depended proteases recognize a small number of amino acids sequences allowing them to cleave at arginyl residues with high substrate specificity. Serine’s hydroxyl group carries out the nucleophillic attack. While the imidazol ring of hisidine takes up the liberated proton and the carboxylate ion of Asp stabilizes the developing charge.
| |
|
| |
| Deficiency of this glycoprotein leads to hemophilia B, a common X linked inherited coagulation disorder that affects 1 in 30, 000 males (reference 458, 464). Females are rarely affected by this disorder unless there is a mutation in the FIX gene (465, 466). The mutations that have been identified in the factor IX gene and lead to proteins reduced activity include; large and small deletions, point mutations, and missense mutations.
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-----This page is still under construction-------
Factor IX (plasma thromboplastin component, Christmas factor, or hemophilia B factor) is a 57-kDa vitamin K-dependent procoagulant glycoprotein. It is synthesized by the liver hepatocyte as a pre-prozymogen that requires extensive posttranslational modification. The pre-prozymogen contains a pre-peptide (hydrophobic signal peptide) at its amino terminal that transports the growing polypeptide into the lumen of the Endoplasmic Reticulum. Once inside the ER, this signal peptide is cleaved by a signal peptidase. A pro-peptide functions as a recognition element for a vitamin K-dependent carboxylase (γ-glutamyl carboxylase) which modifies 12 glutamic acid residues to gamma-carboxyglutamyl (Gla) residues. These residues are required for the association with the anionic phospholipid surface through Ca2+-dependent binding. The Gla domain is followed by two epidermal growth factor domains (EGF-1 and EGF-2). The N-terminus of EGF-1 contains a Ca2+ binding site, while the C-terminus connects to a hydrophobic pocket of EGF-2 and a salt bridge through Lys122 (EGF-1 residue) and Gln74 (EGF-2). EGF-2 connects to the serine protease domain through a linker peptide and is required for a proper orientation and folding of serine proteases. To have a physiologically active factor IX, two cleaves must occur to remove a 35 amino acid region that precedes the catalytic region. The first cleave is at Arg145, generating an inactive FIXα. The second cleavage is at Arg180 results in a catalytically active molecule FIXaβ. This resulting heterodimer is held by a disulfide bridge at Cys132-Cys289. The serine protease domain contains a catalytic triad of His221, Asp269, and Ser365. Upon cleave at Arg180, Val181 can form a salt bridge with Asp364, which is a characteristic of active serine proteases. The active FIXa, can then interact with its cofactor, FVIIIa, to form a membrane-bound Xase complex, which activated FX to FXa.
PORCINE FACTOR IXA