Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme

2011 ◽  
Vol 28 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Jafar Vatandoost ◽  
Alireza Zomorodipour ◽  
Majid Sadeghizadeh ◽  
Roghayeh Aliyari ◽  
Mettine H. A. Bos ◽  
...  
Keyword(s):  
Vitamin K ◽  
Factor Ix ◽  
Biologically Active ◽  
Clotting Factor ◽  
S2 Cells ◽  
Drosophila S2 Cells ◽  
Dependent Protein ◽  
Human Clotting Factor

scholarly journals siRNA silencing of calumenin enhances functional factor IX production

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3757-3760 ◽  
Author(s):  
Nadeem Wajih ◽  
Susan M. Hutson ◽  
Reidar Wallin
Keyword(s):  
Vitamin K ◽  
Expression System ◽  
Factor Ix ◽  
High Yield ◽  
Clotting Factor ◽  
Functional Forms ◽  
Dependent Protein ◽  
Sirna Silencing ◽  
Human Clotting Factor ◽  
Functional Factor

AbstractTo improve production of functional fully γ-carboxylated recombinant human clotting factor IX (r-hFIX), cell lines stably overexpressing r-hFIX have been engineered to also overexpress proteins of the γ-carboxylation system. Here we demonstrate that siRNA silencing of calumenin, an inhibitor of the γ-carboxylation system, enhances production of functional r-hFIX produced by engineered BHK21 cells. The production yield of functional r-hFIX was 80% in engineered cells where calumenin had been silenced 78%. We propose that this high-yield expression system can easily be adapted to overproduce functional forms of all members of the vitamin K–dependent protein family.

Expression of biologically active human clotting factor IX (hF IX) in the mammary gland of transgenic mice

1998 ◽  
Vol 43 (15) ◽  
pp. 1294-1298 ◽  
Author(s):  
Ying Huang ◽  
Kezhong Zhang ◽  
Wenying Huang ◽  
Daru Lu ◽  
Ying Huang ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Transgenic Mice ◽  
Factor Ix ◽  
Biologically Active ◽  
Clotting Factor ◽  
Human Clotting Factor

scholarly journals Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

2001 ◽  
Vol 276 (50) ◽  
pp. 46878-46886 ◽  
Author(s):  
B. Nirmala Pudota ◽  
Eric L. Hommema ◽  
Kevin W. Hallgren ◽  
Beth A. McNally ◽  
Susan Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Vitamin K ◽  
Complex Analysis ◽  
Factor Ix ◽  
Contact Site ◽  
Carboxylase Activity ◽  
Catalytic Center ◽  
Dependent Protein ◽  
Different Order ◽  
Active Site Conformation

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to γ-carboxylated Glu residues (Glas) in VKD proteins, which is required for their activity. VKD precursors are targeted to the carboxylase by their carboxylase recognition site, which in most cases is a propeptide. We have identified a second tethering site for carboxylase and VKD proteins that is required for carboxylase activity, called the vitamin K-dependent protein site of interaction (VKS). Several VKD proteins specifically bound an immobilized peptide comprising amino acids 343–355 of the human carboxylase (CVYKRSRGKSGQK) but not a scrambled peptide containing the same residues in a different order. Association with the 343–355 peptide was independent of propeptide binding, because the VKD proteins lacked the propeptide and because the 343–355 peptide did not disrupt association of a propeptide factor IX-carboxylase complex. Analysis with peptides that overlapped amino acids 343–355 indicated that the 343–345 CVY residues were necessary but not sufficient for prothrombin binding. Ionic interactions were also suggested because peptide-VKD protein binding could be disrupted by changes in ionic strength or pH. Mutagenesis of Cys343to Ser and Tyr345to Phe resulted in 7–11-fold decreases in vitamin K epoxidation and peptide (EEL) substrate and carboxylase carboxylation, and kinetic analysis showed 5–6-fold increases inKmvalues for the Glu substrate. These results suggest that Cys343and Tyr345are near the catalytic center and affect the active site conformation required for correct positioning of the Glu substrate. The 343–355 VKS peptide had a higher affinity for carboxylated prothrombin (Kd= 5 μm) than uncarboxylated prothrombin (Kd= 60 μm), and the basic VKS region may also facilitate exiting of the Gla product from the catalytic center by ionic attraction. Tethering of VKD proteins to the carboxylase via the propeptide-binding site and the VKS region has important implications for the mechanism of VKD protein carboxylation, and a model is proposed for how the carboxylase VKS region may be required for efficient and processive VKD protein carboxylation.

EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII

1987 ◽  
Author(s):  
K L Berkner ◽  
S J Busby ◽  
J Gambee ◽  
A Kumar
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Factor Ix ◽  
Biologically Active ◽  
Factor Vii ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Mammalian Expression ◽  
Plasma Factor ◽  
Gla Domain

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.

scholarly journals Association of the Inactive Circulating Matrix Gla Protein with Vitamin K Intake, Calcification, Mortality, and Cardiovascular Disease: A Review

2019 ◽  
Vol 20 (3) ◽  
pp. 628 ◽  
Author(s):  
Stefanos Roumeliotis ◽  
Evangelia Dounousi ◽  
Theodoros Eleftheriadis ◽  
Vassilios Liakopoulos
Keyword(s):  
Cardiovascular Disease ◽  
Renal Function ◽  
Vitamin K ◽  
Serum Levels ◽  
Matrix Gla Protein ◽  
Biologically Active ◽  
Vitamin K Deficiency ◽  
K Deficiency ◽  
Dependent Protein ◽  
Existing Data

Matrix Gla Protein (MGP), a small Gla vitamin K-dependent protein, is the most powerful natural occurring inhibitor of calcification in the human body. To become biologically active, MGP must undergo vitamin K-dependent carboxylation and phosphorylation. Vitamin K deficiency leads to the inactive uncarboxylated, dephosphorylated form of MGP (dpucMGP). We aimed to review the existing data on the association between circulating dpucMGP and vascular calcification, renal function, mortality, and cardiovascular disease in distinct populations. Moreover, the association between vitamin K supplementation and serum levels of dpucMGP was also reviewed.

Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

2001 ◽  
Vol 46 (18) ◽  
pp. 1534-1538
Author(s):  
Hongwei Wang ◽  
Chenbo Ye ◽  
Li Chen ◽  
Xuefeng Wang ◽  
Xinfang Qiu ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
B Mice ◽  
Human Clotting Factor ◽  
Efficient Transfer

VKORc1 Is Under-Expressed in Skeletal Muscle of Humans, Dogs and Mice: Potential Implications for Ectopic Coagulation Factor Expression in Pre-Clinical and Therapeutic Applications

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1477-1477
Author(s):  
Courtney T Connolly ◽  
Armida Faella ◽  
Timothy C. Nichols ◽  
Katherine A. High ◽  
Valder R. Arruda ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Vitamin K ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Biologically Active ◽  
Transcript Levels ◽  
Vitamin K Cycle

Abstract Post-translational modifications of coagulation factors in the liver are essential for function. The vitamin K dependent coagulation proteins (VKCPs) require vitamin K to undergo gamma carboxylation of the glutamic residues in their Gla domain by gamma-glutamyl carboxylase [GGCX]. The vitamin K is then recycled by the action of epoxide reductase [VKORc1] and/or quinone reductase [NQO1]. The hemostatic importance of the vitamin K “cycle” is evidenced by patients who may suffer bleeding complications when anticoagulated with warfarin, which targets the vitamin K cycle. Moreover, the ability of a variety of VKCPs to secrete a biologically active product depends on the removal of their propeptide by the action of the intracellular endoprotease furin [FURIN gene]. Previous in vitro work on recombinant coagulation Factor IX, which is used for hemophilia B treatment, has connected these two processing steps by showing that endogenous VKORc1 as well as FURIN can be limiting factors in high-yield expression systems. In vivo, skeletal muscle (in contrast to liver) has been utilized to express low levels of coagulation Factor IX in the first hemophilia B gene therapy clinical trial. However, our experiments in mice demonstrated that the specific activity of muscle-synthesized Factor IX via gene transfer decreased at the high levels of FIX expression by a limited muscle area (Schuettrumpf J. et al., Blood 2005). These results suggest that in vitro and in vivo expression of biologically-active VKCPs outside the liver may be limited by the host cell post-translational modification machinery. Here, we performed a systematic study to determine the expression profiles of the vitamin K cycle and furin endoprotease genes in human liver and muscle, compared to the mouse. We also established these profiles in two hemophilic dogs, given the extensive use of this animal model in gene-based hemophilia therapies. RNA from liver and skeletal muscle was used as a template for reverse transcription and the subsequent relative quantification of the GGCX, VKORc1, NQO1, and FURIN genes by qPCR in each tissue using a housekeeping reporter gene. For this, a variety of housekeeping genes were investigated in all three species to identify ones with similar transcript levels in both liver and muscle tissue. We identified the housekeeping genes HPRT1, beta actin, and 18s rRNA as equivalently expressed in the liver and skeletal muscle of human, mouse, and dog, respectively. The relative mRNA transcript quantification of the vitamin K cycle genes in humans showed that the transcript levels of GGCX were similar in liver and muscle. In contrast, both VKORc1 and NQO1 were under-expressed in muscle vs. liver (69.5 ± 4.9% and 67.8 ± 12.5%, respectively, P<0.01). In the mouse, VKORc1 transcript levels in the muscle were reduced to 73.8 ± 9.9% vs. liver (P<0.05), while GGCX and NQO1 exhibited similar transcript levels in both tissues. In the dog, we observed a dramatic reduction in VKORc1 and GGCX transcript levels in the muscle vs. liver (11.8 ± 4.2% and 29.5 ± 15.8%, respectively, P<0.01). Surprisingly, NQO1 transcript levels were 253.8 ± 156.7% higher in muscle than liver (P<0.05). Lastly, in all three species tested, transcript levels for FURIN were similar in both muscle and liver. Our results indicate that VKORc1, a key enzyme in the vitamin K cycle, is consistently under-expressed in the skeletal muscle of humans as well as in mice and hemophilic dogs. In contrast, FURIN transcripts are similarly abundant in the liver and muscle of all three species tested. These suggest that the vitamin K cycle but not propeptide processing by furin can be a limiting factor in the secretion of biologically active muscle-expressed VKCPs. As a result, our observations provide (1) a plausible explanation for the inverse relationship between specific activity and Factor IX expression levels in mice following Factor IX gene transfer, and (2) further support for the mouse and dog as useful models for therapies that depend on the muscle-derived expression of VKCPs. Disclosures No relevant conflicts of interest to declare.

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