RESUMEN
The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.
Asunto(s)
Factor VIII/química , Factor VIII/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Calorimetría , Espectroscopía de Resonancia Magnética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Dominios Proteicos , Factor de von Willebrand/genéticaRESUMEN
Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.
Asunto(s)
Secuencia de Aminoácidos , Factor VIII/genética , Factor VIII/aislamiento & purificación , Expresión Génica , Eliminación de Secuencia , Animales , Células CHO , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cricetulus , Diseño de Fármacos , Factor VIII/biosíntesis , Humanos , Modelos Moleculares , Plásmidos/química , Plásmidos/metabolismo , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.
Asunto(s)
Células CHO , Factor VIII/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas Recombinantes/genética , Animales , Cricetinae , Cricetulus , Factor VIII/biosíntesis , Humanos , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Transcriptoma/genéticaRESUMEN
Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.
Asunto(s)
Hormona de Crecimiento Humana/metabolismo , Polisacáridos/metabolismo , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Semivida , Humanos , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Recombinant factor VIIa (rFVIIa, NovoSeven) is currently the only bypassing agent produced by recombinant technology for the treatment of haemophiliacs whose disease is complicated by inhibitory antibodies. In addition, recombinant production of FVIIa has made it widely available for a variety of purposes and accelerated the growth of our knowledge about FVIIa by generating an abundance of clinical and biochemical data. This fascinating molecule has turned out to be a safe haemostatic agent with great potential in the clinic and has inspired the generation of improved variants currently in (pre-)clinical testing. The present review describes the structural and functional aspects of FVIIa, followed by sections dealing with the manufacturing, therapeutic mechanism of action, clinical development and experience with rFVIIa.
Asunto(s)
Factor VIIa/uso terapéutico , Industria Farmacéutica/métodos , Factor VIIa/efectos adversos , Factor VIIa/química , Factor VIIa/farmacocinética , Hemofilia A/complicaciones , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Hemostáticos/efectos adversos , Hemostáticos/farmacocinética , Hemostáticos/uso terapéutico , Humanos , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Relación Estructura-ActividadRESUMEN
Coagulation factor VII (FVII) is a vitamin K-dependent glycoprotein that undergoes extensive post-translational modification prior to secretion. Secretion of FVII proteins from producer cells is a slow process. To identify bottlenecks for the transport of FVII through the secretory pathway of FVII-producing cells, we analysed the processing of intracellular FVII by pulse-chase of FVII producing CHO cells followed by radioimmuno precipitation, SDS-PAGE, and autoradiography. FVII was coprecipitated with GRP78 and vice versa for at least three hours after synthesis of the labelled FVII, suggesting that nascent FVII is retained in the endoplasmic reticulum (ER). Judged from barium citrate precipitation assay, gamma-carboxylation of the pulse-labelled FVII was a slow process requiring several hours and seemed to be the most important bottleneck in the intracellular processing of FVII. Nevertheless, FVII was not released from the cells immediately after gamma-carboxylation. Gamma-carboxylated FVII accumulated in the cells and migrated as a band with reduced mobility compared to uncarboxylated FVII. This shift in migration was caused by N-glycan processing in the Golgi complex. Thus, the release of FVII from producer cells is delayed by at least two bottlenecks. The major bottleneck appears to be gamma-carboxylation, which determines the rate of transport of FVII out of the ER. Another bottleneck retains FVII in the cells after processing of the N-glycans into complex chains. Cells with an intact gamma-carboxylation machinery appear to posses mechanisms that protect nascent FVII from intracellular degradation and keep FVII in the ER until it is gamma-carboxylated.
Asunto(s)
Retículo Endoplásmico/metabolismo , Factor VII/metabolismo , Aparato de Golgi/metabolismo , Transporte de Proteínas/fisiología , Animales , Células CHO , Ácidos Carboxílicos/metabolismo , Cricetinae , Cricetulus , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Polisacáridos/metabolismo , Factores de TiempoRESUMEN
The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.
Asunto(s)
Calcio/metabolismo , Factor VIIa/química , Ácido Aspártico/química , Calcio/química , Cristalografía por Rayos X/métodos , Ácido Glutámico/química , Humanos , Hidrógeno/química , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Electricidad Estática , Tromboplastina/química , Factores de TiempoRESUMEN
Human coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O-glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.
Asunto(s)
Factor VII/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Factor VII/genética , Glicosilación , Humanos , Mutación , Deficiencia de Vitamina K , Warfarina/farmacologíaRESUMEN
N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.
Asunto(s)
Factor VII/química , Factor VII/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células CHO , Línea Celular , Cricetinae , Glicosilación , Humanos , Mutación , Estructura Terciaria de ProteínaRESUMEN
We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U-C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.
Asunto(s)
Hemaglutininas Virales/química , Hemaglutininas Virales/genética , Morbillivirus/crecimiento & desarrollo , Morbillivirus/genética , Adaptación Biológica , Animales , Antígenos CD , Chlorocebus aethiops , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/crecimiento & desarrollo , Glicoproteínas , Inmunoglobulinas , Virus del Sarampión/genética , Virus del Sarampión/crecimiento & desarrollo , Proteína Cofactora de Membrana , Glicoproteínas de Membrana , Datos de Secuencia Molecular , Mutación Missense , Mutación Puntual/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Receptores de Superficie Celular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Células VeroRESUMEN
The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays. The mRNA levels of more than 3000 cellular genes were compared between uninfected PBMCs and cells infected with either the Edmonston MV strain or a wild-type MV isolate. The MV-induced upregulation of individual genes identified by microarray analyses was confirmed by RT-PCR. In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus. The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-kappaB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level. Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated. These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.