The carboxylation efficiency of the vitamin K-dependent clotting factors: Studies with factor IX
ABSTRACT Haemophilia B is characterized by a deficiency of the gamma-carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach (Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K-dependent proteins in vitro, to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT-FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is gamma-carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.
- SourceAvailable from: Shohreh Khorshidi
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- "Propeptides behave differently, when their affinities towards c -carboxylase is considered. The affinity of propeptides for the c-carboxylase varies over 2 logs, with the lowest affinity for prothrombin that can explain a high efficient ccarboxylation of the human prothrombin (hProt) (Stanley et al. 1999; Blostein et al. 2008). A primary comparison of the nucleotide and amino acid sequences of prothrombin of porcine, canine, cat and human, demonstrated respective similarities of 86.2 and 83 %, between those of human and porcine. "
ABSTRACT: To study the functions of pre-pro leader peptides of the human and porcine prothrombins on the human FIX (hFIX) expression. In silico analysis predicted higher secretion efficiencies for the prothrombins-derived signal peptides, in comparison with the native hFIX signal peptide. Replacements of the hFIX pre-pro sequence with those of the two prothrombins, led to increased levels of transcription of the chimeric transgenes, as compared to the native clone. This was in consistent with the lower minimum free energies, calculated for the recombinant transcripts, based on their secondary structures. Evaluation of secretion efficiency revealed that the highest and lowest FIX secretions belong to signal peptides derived from porcine' prothrombin and hFIX, respectively. Coagulation activities of the FIX expressed from chimeric variants could be increased up to tenfold, relative to the native clone. The feasibility of a leader-peptide replacement for the improvement of both transcription and post-transcriptional processes is described that can be relevant for production the vitamin-K dependent proteins.Biotechnology Letters 06/2015; DOI:10.1007/s10529-015-1868-3 · 1.74 Impact Factor
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- "Color images available online at www.liebertpub.com/hum reductase, and paired basic amino acid cleaving enzyme (PACE)/furin (Blostein et al., 2008; Napolitano et al., 2010; Tie et al., 2011). Our results showed that the clotting activity of de novo-synthesized hFIX was modulated by menaquinone (vitamin K 2 ) in a dose-dependent manner. "
ABSTRACT: Hemophilia is an X-linked bleeding disorder, and hemophilia patients are deficient in a biologically active coagulation factor. This study was designed to combine the efficiency of lentiviral vector transduction techniques with murine adipose tissue-derived stem/stromal cells (mADSCs) as a new method to produce secreted hFIX and treat hemophilia B. mADSCs were transduced with simian immunodeficiency virus (SIV)-hFIX lentiviral vector at multiplicities of infection (MOIs) from 1 to 60, and the most effective dose was at MOI 10, as determined by hFIX production. The hFIX protein secretion persisted over the 28-day experimental period. Cell sheets composed of lentiviral vector-transduced mADSCs were engineered to further enhance the utility of these cells for future therapeutic applications in transplantation modalities. These experiments demonstrated that genetically transduced ADSCs may become a valuable cell source for establishing cell-based gene therapies for plasma protein deficiencies, such as hemophilia.Human gene therapy 01/2013; DOI:10.1089/hum.2012.162 · 3.62 Impact Factor
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ABSTRACT: Ex vivo gene therapy requires a suitable bioreactor for production and delivery of the gene products into a target tissue, and keratinocyte is suitable model in this regard because of its potential for systemic release of proteins. To establish a keratinocyte-specific expression system, a mammalian-based expression plasmid equipped with a 2,240-bp fragment from the human keratin 14 (k14) gene enhancer/promoter region was constructed and used for the insertion of the human coagulation factor IX (hFIX)-cDNA downstream the K14-derived regulatory elements. The human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free media and transfected with the recombinant plasmid. The K14-promoter-driven expression of recombinant hFIX (rhFIX) was evaluated by performing coagulation test as well as enzyme-linked immunosorbent assay on the cultured media collected from the transfected cells at various stages. The rhFIX corresponding transcript and protein were confirmed by performing reverse transcription PCR as well as immunoblotting experiments, respectively. Based on the coagulation activities obtained from the conditioned media of nine isolated clones, the hFIX expression levels vary from 5% to 39% of normal human plasma. Expression levels of the hFIX obtained in this study are comparable to those reported for viral systems. The obtained data supported the potential of keratinocyte for the expression and secretion of biologically active rhFIX and underscore the importance of the examined cis sequences for enhancing gene expression in a mammalian expression system. Besides, it has provided means for further bioengineering strategies to improve the expression efficiency of the hFIX in keratinocytes and other mammalian host cells.Applied biochemistry and biotechnology 11/2010; 162(6):1599-611. DOI:10.1007/s12010-010-8941-0 · 1.74 Impact Factor