Mutagenesis of a Potential Immunoglobulin-binding Protein-binding Site Enhances Secretion of Coagulation Factor VIII

Swaroop, Manju; Moussalli, Micheline J.; Pipe, Steven W.; Kaufman, Randal J.

doi:10.1074/jbc.272.39.24121

Cited by 100 publications

(92 citation statements)

References 26 publications

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“…Several residues within the A1 and A3 domains of fVIII have been identified that are important for fVIII structure and/or expression. Residues within the amino acid sequence 226 -336 in the A1 domain are important for the interaction of fVIII with the endoplasmic reticulum lumenal protein BiP (GRP78), which has been proposed to limit expression of fVIII (36,37). Within this region there are 13 amino acid differences between human and porcine fVIII.…”

Section: Fig 4 Fviii Expression Levels Versus Steady-state Rna Levelsmentioning

confidence: 99%

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Doering

Healey

Parker

et al. 2004

Journal of Biological Chemistry

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Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency. Factor VIII (fVIII)1 is a plasma protein that functions in proteolytically activated form as a cofactor within the intrinsic pathway of blood coagulation to increase the rate of proteolytic activation of factor X by activated factor IX. fVIII contains a domain structure designated A1-A2-B-ap-A3-C1-C2 that is defined based on internal sequence homology (1, 2). The fVIII A domains share homology with the copper-binding protein ceruloplasmin (3, 4), which has an A1-A2-A3 domain structure in which the three A domains are arranged along a pseudo-3-fold axis of symmetry (5). Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junction into A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits. fVIII circulates in the plasma as an inactive heavy chain/light chain heterodimeric procofactor that is non-covalently bound to von Willebrand factor. Proteolytic activation of fVIII by thrombin results from cleavages at Arg-372 between the A1 and A2 domains, Arg-740 between the A2 and B domains, and Arg-1689 between the ap and A3 domains. During this process, the covalent linkage between the A1 and A2 domains is lost, and the B domain and 41-residue ap are released, producing a heterotrimeric,

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Section: Fig 4 Fviii Expression Levels Versus Steady-state Rna Levelsmentioning

confidence: 99%

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Doering

Healey

Parker

et al. 2004

Journal of Biological Chemistry

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“…As FVIII is expressed at very low levels in vivo, the requirements for FVIII secretion have been characterized in cultured cells that express heterologous FVIII genes. These studies demonstrated that FVIII forms non-disulfide-bonded high molecular weight aggregates that are retained within the ER through interaction with the protein chaperones Ig-binding protein (BiP/ GRP78), calnexin, and calreticulin (8)(9)(10)(11). In addition, FVIII trafficking from ER to the Golgi complex is facilitated through interaction with the lectin LMAN1/MCFD2 complex (12,13).…”

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confidence: 99%

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Malhotra¹,

Miao

Zhang³

et al. 2008

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

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597

560

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Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.factor VIII ͉ oxidative stress ͉ unfolded protein response A lthough endoplasmic reticulum (ER) stress and oxidative stress are closely linked events, the molecular pathways that couple these processes are poorly understood (1). Reactive oxygen species (ROS) originate during oxygen-using cellular metabolic processes, such as oxidative phosphorylation within mitochondria. The ER provides a unique oxidizing environment for protein folding and disulfide bond formation before transit to the Golgi compartment. During disulfide bond formation ROS are formed as a product of electron transport from thiol groups in proteins through protein disulfide isomerase (PDI) and ER oxidoreductase 1 (ERO1) to reduce molecular oxygen to form the oxidant hydrogen peroxide. It has been suggested that oxidation of cysteine residues during disulfide bond formation in the ER may significantly contribute to oxidative stress (2, 3). The unfolded protein response (UPR) is an adaptive signaling pathway designed to prevent the accumulation of misfolded proteins in the ER lumen. Studies also suggest the UPR is designed to minimize the stress of oxidative protein folding (2). The UPR is signaled through the protein kinases inositolrequiring protein 1␣ and PKR-related ER kinase and the activating transcription factor 6␣ (4, 5). Chronic unresolved accumulation of unfolded proteins in the ER leads to apoptosis. To elucidate the relationship between unfolded protein accumulation in the ER lumen, oxidative stress, and apoptosis, we have analyzed the secretion of coagulation factor VIII (FVIII), a large glycoprotein that is deficient in the X chromosome-linked bleeding disorder hemophilia A. As FVIII is prone to misfolding in the ER lumen, FVIII expression provides a unique approach to manipulate the ER stress response that does not rely on pharmacological intervention, where any connection between ER stress and ROS would be difficult to dissect.FVIII is comprised of three domains in the ord...

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“…The molecular mechanism responsible for FVIII inefficient secretion, is mainly caused by the association of the A1 domain in the HC with several protein chaperones, including the immunoglobin-binding protein (BiP) in the lumen of the endoplasmic reticulum (ER) during intracellular processing, and thus the release of FVIII from BiP is dependent on a high concentration of intracellular ATP [15]. In addition, N-linked oligosaccharides in the B-domain of the FVIII heavy chain can also facilitate FVIII secretion by participating in the folding interactions within the ER as well as potentially helping facilitate ER-Golgi transport [16].…”

Section: Discussionmentioning

confidence: 99%

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Zhu

Liu

Jiang

et al. 2012

Sci. China Life Sci.

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Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately (239±56) ng mL 1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately (1.09±0.25) IU mL 1 above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.coagulation factor VIII, dual-vector gene delivery, inter-chain disulfide bonding, heavy chain secretion, coagulation activity Citation: Zhu F X, Liu Z L, Miao J, et al. Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors. Sci China Life Sci, 2012, 55: 521 -526,

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Mutagenesis of a Potential Immunoglobulin-binding Protein-binding Site Enhances Secretion of Coagulation Factor VIII

Cited by 100 publications

References 26 publications

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

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