Crystal Structure of the Calcium-stabilized Human Factor IX Gla Domain Bound to a Conformation-specific Anti-factor IX Antibody

Huang, Mingdong; Furie, Barbara C.; Furie, Bruce

doi:10.1074/jbc.m314011200

Cited by 67 publications

(71 citation statements)

References 50 publications

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“…27 Vitamin K 1 (10 mg/mL) for cell culture was from Abbott Laboratories. A vitamin K internal standard for VKOR activity assay, 2-methyl-3(3,7,11,15,19-pentamethyl-2-eicosenyl)-1,4-naphthalenedione, also known as vitamin K 1 (25) , was from GLSynthesis. Protein C cDNA clone was from Open Biosystems.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction was terminated by the addition of 500 L of isopropanol. The reaction mixture was extracted with 500 L of n-hexane containing 2.52M vitamin K 1 (25) as an internal standard. The upper organic phase containing the vitamins was transferred to a 2-mL brown vial and dried with nitrogen.…”

Section: Vkor Activity Assaymentioning

confidence: 99%

“…This replacement allowed us to use a monoclonal antibody (mAb) specific for the carboxylated gla domain of FIX for quantitative detection purposes. 24,25 We stably expressed this chimeric reporter protein, FIXgla-PC, in HEK293 cells, which are commonly used for the biosynthesis of vitamin K-dependent proteins, and in AV12 cells, which are less efficient in the carboxylation of vitamin Kdependent proteins. 26 Studies have indicated that recombinant protein C produced by HEK293 cells is fully carboxylated and even more active than plasma-derived protein C. Under similar conditions, protein C produced by AV12 cells is partially carboxylated, with 20% anticoagulant activity relative to plasma protein C. 26 This result indicates that HEK293 and AV12 cells might have different vitamin K pathways for making vitamin K-dependent proteins.…”

Section: Introductionmentioning

confidence: 99%

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Functional study of the vitamin K cycle in mammalian cells

Tie

Jin

Straight

et al. 2011

Blood

152

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We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH 2 ) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH 2 , and (4) the primary function of VKOR is the reduction of KO to vitamin K. (Blood. 2011;117(10):2967-2974) IntroductionVitamin K hydroquinone (KH 2 ) is a cofactor for ␥-glutamyl carboxylase (GGCX), which catalyzes the posttranslational carboxylation of specific glutamic acid residues to ␥-carboxyglutamic acid (gla) in a variety of vitamin K-dependent proteins. 1 ␥-Glutamyl carboxylation is essential for the biologic functions of vitamin K-dependent proteins involved in blood coagulation, bone metabolism, signal transduction, and cell proliferation. Concomitant with ␥-glutamyl carboxylation, KH 2 is oxidized to vitamin K 2,3-epoxide (KO). KO must then be converted back to vitamin K (the quinone form) and then to KH 2 by separate 2 electron reductions to support the carboxylation reaction. The cyclic production of KO and the conversion back to KH 2 constitutes the vitamin K cycle (Figure 1). The only enzymes unequivocally identified as part of the cycle are GGCX and vitamin K epoxide reductase (VKOR). 2 Sherman et al first proposed that the reduction of KO to vitamin K is carried out by a sulfhydryl-dependent epoxide reductase that is sensitive to warfarin inhibition. 3 This enzyme is probably VKOR, the only enzyme thus far shown to reduce KO to vitamin K. On the other hand, some reports suggest that the reduction of vitamin K to KH 2 can be accomplished by at least 2 microsomal enzymes called vitamin K reductases. 4 However, other studies 5,6 suggest that one enzyme serves as both the epoxide reductase and the vitamin K reductase, catalyzing both the reduction of KO to vitamin K and that of vitamin K to KH 2 .Wallin proposed that there are 2 enzymes that reduce vitamin K in support of vitamin K-dependent carboxylation. 7 One enzyme is inhibited by anticoagulant drugs such as warfarin, 8 while the other is an NADH-dependent reductase that is resistant to inhibition by warfarin. 4,[9][10][11] Consistent with the latte...

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Section: Methodsmentioning

confidence: 99%

Section: Vkor Activity Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Functional study of the vitamin K cycle in mammalian cells

Tie

Jin

Straight

et al. 2011

Blood

152

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“…Hemizygosity for Asn2Ile in FIX was associated with FIX functional activity of < 1% that of wild type FIX, despite normal plasma FIX antigen. Within the FIX Gla domain, the Asn2 side chain formed interactions with Gla-7 and Gla-25 which are important for Ca 2+ binding (Huang et al, 2004). Asn2 is therefore likely to have a wider role in maintaining conformational stability of the FIX Gla domain during x-loop formation that is likely to be similar in PC.…”

Section: Discussionmentioning

confidence: 99%

The protein C ω‐loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity

et al. 2009

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Summary We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u/dl, amidolytic activity 40 u/dl, anticoagulant activity 9 u/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co‐segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)‐mediated inhibition of plasma thrombin generation from an individual with PC‐Asn2Ile was lower (endogenous thrombin potential (ETP) 56 ± 1% that of ETP determined without sTM) than control plasma (ETP 15 ± 2%) indicating reduced PC anticoagulant activity. Recombinant APC‐Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)‐dependent anticoagulant activity of recombinant APC‐Asn2Ile and binding of recombinant APC‐Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild‐type APC. Asn2 lies within the ω‐loop of the PC/APC Gla domain and this region is critical for calcium‐induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally‐occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

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“…Based on the finding that glacontryphan-M expresses a single, saturable calcium-binding site (K D 0.63 mM) (11), the final structure calculations included six distance restraints between the C␤, the C␥, the C␦1 and C1 carbons of Gla 2 and Gla 4 , to model a bound calcium ion between Gla 2 and Gla 4 . These distance restraints were based on the measured distances between paired Gla residues (Gla 25 and Gla 30 ) involved in the coordination of a single calcium ion in known structures of the calcium-bound human factor IX Gla domain, FIX (1-47) (33,39). Inclusion of these restraints did not significantly perturb the protein backbone conformation or violate any of the NOE-derived distance constraints involving residues in the N terminus.…”

Section: Methodsmentioning

confidence: 99%