Structural Analysis of N-Linked Sugar Chains of Human Blood Clotting Factor IX

Makino, Yasushi; Omichi, Kaoru; Kuraya, Nahoki; Ogawa, Hideyuki; Nishimura, Hitoshi; Iwanaga, Sadaaki; Hase, Sumihiro

doi:10.1093/oxfordjournals.jbchem.a022738

Cited by 28 publications

(27 citation statements)

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“…Factor IX is a coagulation factor used to treat hemophilia B, a congenital disease caused by a factor IX deficiency (22,23). The protein contains two N-linked glycans, of which approximately 65% are tetraanntennary and 20% are triantennary glycans (24), with a mixture of α2,3/6-linked terminal sialic acid residues. Finally, bovine serum fetuin, although not a therapeutic protein, was chosen on the basis of its well-studied three N-glycan sites, of which approximately 80% are triantennary with a mixture of α2,3/6-linked terminal sialic acid residues (25,26).…”

Section: Resultsmentioning

confidence: 99%

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

Lindhout

Iqbal

Willis

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

108

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The posttranslational modification of therapeutic proteins with terminal sialic acids is one means of improving their circulating half-life, thereby improving their efficiency. We have developed a two-step in vitro enzymatic modification of glycoproteins, which has previously only been achieved by chemical means [Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Int J Pharm 300:125-130). This two-step procedure uses the Campylobacter jejuni Cst-II α2,8-sialyltransferase to provide a primer on N-linked glycans, followed by polysialylation using the Neisseria meningitidis α2,8-polysialyltransferase. Here, we have demonstrated the ability of this system to modify three glycoproteins with varying N-linked glycan compositions: the human therapeutic proteins alpha-1-antitrypsin (A1AT) and factor IX, as well as bovine fetuin. The chain length of the polysialic acid addition was optimized by controlling reaction conditions. After demonstrating the ability of this system to modify a variety of proteins, the effect of polysialylation on the activity and serum half-life of A1AT was examined. The polysialylation of A1AT did not adversely affect its in vitro inhibition activity against human neutrophil elastase. The polysialylation of A1AT resulted in a significantly improved pharmacokinetic profile when the modified proteins were injected into CD-1 mice. Together, these results suggest that polysialylated A1AT may be useful for improved augmentation therapy for patients with a deficiency in this protein and that this modification may be applied to other therapeutic proteins.glycosyltransferase | glycosylation

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Section: Resultsmentioning

confidence: 99%

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

Lindhout

Iqbal

Willis

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

108

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“…Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene.…”

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confidence: 99%

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Østergaard¹,

Bjelke²,

Hansen

et al. 2011

Blood

128

133

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Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K m for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemo- IntroductionFactor IX (FIX) is a vitamin K-dependent glycoprotein and an essential protease of the hemostatic system. The domain organization of FIX is shared with factors VII, X, and protein C and comprises an N-terminal domain rich in ␥-carboxyglutamic acid (Gla), 2 epidermal growth factor-like repeats and a C-terminal trypsin-like protease domain. 1 Together they form a 55-kDa single-chain protease precursor circulating in plasma at a concentration of approximately 90nM (5 g/mL), defined as 1 IU/mL. FIX is converted to the 2-chain activated form by the tissue factor (TF)-factor VIIa (FVIIa) complex or factor XIa (FXIa). Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene. The prevalence of HB is approximately 1 in 25 000 males, and it has been estimated that approximately 84 000 people are affected worldwide. 5 The mainstay in HB treatment is substitution therapy by infusion of plasma-derived or recombinant FIX (rFIX). The therapeutic goal is to prevent bleeding episodes and to provide safe and efficacious treatment of bleedings when they occur. Because of the relatively short half-life of FIX (18-24 hours [6][7][8] ), the recommended prophylaxis regimen consists of 2 to 3 weekly infusions of 40-100 IU/kg 9 FIX to maintain trough levels above 1% and thus shifting patients from a severe to a milder phenotype. When adhered to, prophylaxis in patients without severe joint disorder is efficacious with a frequency of only 0-2 breakthrough bleeds per year in the majority of patients. 8,10 However, the need for multiple weekly infusions present challen...

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“…S4). FIX AP has two N-linked and two O-linked oligosaccharides (4,18). Upon deglycosylation of N-linked sugar with N-glycosidase F, two Asn residues changed to Asp residues, with corresponding increase in mass of 2.0 Da.…”

Section: Discussionmentioning

confidence: 99%

“…Human coagulation FIX undergoes various post-translational modifications including ␥-carboxylation of twelve Glu residues in the Gla domain, attachment of N-linked and O-linked oligosaccharides at the EGF domain and AP region, and ␤-hydroxylation of an Asp residue in the first EGF domain (4). Other post-translational modifications, such as sulfation of a Tyr residue and phosphorylation of Ser residue(s), also have been suggested (5) but precise data on these have not been reported to date.…”

Section: Blood Coagulation Factor IX (Fix)mentioning

confidence: 99%

Characterization of a Monoclonal Antibody B1 That Recognizes Phosphorylated Ser-158 in the Activation Peptide Region of Human Coagulation Factor IX

Atoda

Yokota

Morita

2006

Journal of Biological Chemistry

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Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as ␥-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K d value of 5 nM in the presence of Ca 2؉ (EC 50 ‫؍‬ 0.58 mM). B1 bound to the isolated AP of FIX and retained the Ca 2؉ dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca 2؉ . These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca 2؉ -bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158. Blood coagulation factor IX (FIX)2 is present in an inactive precursor form that consists of a ␥-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, an activation peptide (AP) region, and a serine protease domain (1) (Fig. 1). FIX is activated physiologically by factor XIa or factor VIIa-tissue factor complex to generate FIXa␤ via FIX␣, an inactive intermediate, whereas RVV-X, a protease from Russell's viper venom, can activate FIX to FIXa␤ via FIXa␣, an active intermediate (2). In both cases, AP is removed by peptide bond cleavage at two sites of FIX to generate the final form of activated FIX, FIXa␤, and an AP. Increased plasma FIX level is one of the risk factors of thrombosis (3), and along with the concentration of AP in plasma is a useful index for the activation of the blood coagulation system.Human coagulation FIX undergoes various post-translational modifications including ␥-carboxylation of twelve Glu residues in the Gla domain, attachment of N-linked and O-linked oligosaccharides at the EGF domain and AP region, and ␤-hydroxylation of an Asp residue in the first EGF domain (4). Other post-translational modifications, such as sulfation of a Tyr residue and phosphorylation of Ser residue(s), also have been suggested (5) but precise data on these have not been reported to date. These post-translational modifications take place in the liver and may play important roles for specific activity, secretion, recovery, half-life in the circulation, and ...

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Structural Analysis of N-Linked Sugar Chains of Human Blood Clotting Factor IX

Cited by 28 publications

References 0 publications

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Characterization of a Monoclonal Antibody B1 That Recognizes Phosphorylated Ser-158 in the Activation Peptide Region of Human Coagulation Factor IX

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