Roles of phytanoyl-CoA alpha-hydroxylase in mediating the expression of human coagulation factor VIII

The coagulation factor VIII (FVIII) is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Previous transfection studies demonstrated that factor VIII was 10-100-fold less efficiently expressed than the homologous coagulation factor, factor V. To investigate the regulatory mechanisms of FVIII synthesis and secretion, we used the yeast two-hybrid system as an approach to search for proteins that associated with FVIII. The A2 domain (337-740 amino acids) of factor VIII (FVIII-A2) was used as a bait and phytanoyl-CoA alpha-hydroxylase (PAHX) was identified as a binding protein of FVIII-A2. PAHX had potential to interact with the residues 373-508 within the A2 domain, but not with A1 and A3 (the homologous domains of A2). The interaction between the A2 domain and PAHX was independent of the type 2 peroxisomal targeting signal (PTS2) of PAHX. Overexpression of PAHX in FVIII-produced cells decreased the expression of FVIII by about 70%. The elevated expression of von Willebrand factor had no effect on the suppression of FVIII secretion by PAHX. Expression of the green fluorescent PAHX fusion protein in SMMC-7721 cells affected the intracellular trafficking of FVIII-A2. These results suggested that the interaction between PAHX and FVIII-A2 was in part responsible for the low-level expression of factor VIII.     

Production of Recombinant Human Factor VIII in Different Cell Lines and the Effect of Human XBP1 Co-Expression

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.     

Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion

Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.     

Stable and high-level production of recombinant Factor IX in human hepatic cell line

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.     

Storage of factor VIII variants with impaired von Willebrand factor binding in Weibel-Palade bodies in endothelial cells

Background

Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood.

Methodology/Principal Findings

We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH.

Conclusions

Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo.     

Long-term expression of von Willebrand Factor by a VSV-G pseudotyped lentivirus enhances the functional activity of secreted B-Domain-deleted Coagulation Factor VIII

von Willebrand factor (vWF) is a multimeric glycoprotein which functions within the coagulation system. It colocalizes with factor VIII (FVIII) by non-covalent interaction and alters its intracellular trafficking. vWF is also instrumental in maintaining the stability of secreted FVIII. The principal objective of this study was to generate a lentivirus-based vWF expression vector for gene therapy of hemophilia A. We inserted a vWF of 8.8 Kb into a lentiviral vector thereby producing VSV-G-pseudotyped vEx52. However, its titer was quite low, presumably because the length of vWF gene exceeds the size limit of the lentiviral vector. In order to overcome the low-titer, we concentrated the vEx52 and thus increased the efficiency of transduction approximately 6-fold with 1/100th of the volume. However, as concentration requires an additional laborious step, we attempted to enhance the transduction efficiency by deleting exons 24-46 and 29-46 in pRex52 to construct pRex23 and pRex28, and in pvEx52, yielding pvEx23 and pvEx28, respectively. The transfected pRex52 had a profound effect on the activity of secreted FVIII, and this activity declined as domains of vWF were deleted. However, when the domain-deleted vWF-lentiviruses were transduced into K562 cells, the vEx28 increased the activity of the secreted FVIII compared to what was observed with vEx52. This result is probably due to higher efficiencies of transduction and expression while retaining the essential domains required for proper interaction with FVIII.     

The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein C<Subscript>A267T</Subscript> mutant in CHO-K1 cells trough the GRASP55 pathway

Some inherited coagulation factor deficiencies are caused by intracellular retention or degradation of misfolded proteins, and chemical chaperones have been shown to reverse protein misfolding. The purpose of the present study was to investigate whether chemical chaperones may improve secretion of the protein C_A267T (PC_A267T) mutant in a cellular model. Using stably transfected Chinese hamster ovary cells (CHO-K1) expressing PC_A267T we demonstrate that sodium 4-phenylbutyrate (PBA) increased the secretion of PC_A267T by approximately 4-fold in comparison with untreated cells, and that this secretion seemed to follow an unconventional pathway via the Golgi reassembly stacking protein (GRASP55).     

Factor VIII hydrolysis mediated by anti-factor VIII autoantibodies in acquired hemophilia

Acquired hemophilia is a rare hemorrhagic disorder caused by the spontaneous appearance of inhibitory autoantibodies directed against endogenous coagulation factor VIII (FVIII). Inhibitory Abs also arise in patients with congenital hemophilia A as alloantibodies directed to therapeutic FVIII. Both autoimmune and alloimmune inhibitors neutralize FVIII by steric hindrance. We have described FVIII-hydrolyzing IgG in 50% of inhibitor-positive patients with severe hemophilia A that inactivate therapeutic FVIII. In this study, we investigated the presence of autoimmune FVIII-hydrolyzing IgG in patients with acquired hemophilia. Pooled IgG from healthy donors demonstrated moderate FVIII-hydrolyzing activity (56 +/- 26 micromol/min/mol). Purified IgG from 21 of 45 patients with acquired hemophilia demonstrated FVIII hydrolysis rates (mean 219 +/- 94 micromol/min/mol) significantly greater than that of control IgG. Three of four patients followed over the course of the disease had rates of FVIII hydrolysis that co-evolved with inhibitory titers in plasma, suggesting that IgG-mediated FVIII hydrolysis participates, in part, in FVIII inactivation. The present work extends the scope of the diseases associated with FVIII proteolysis and points toward the importance of FVIII as a key target substrate for hydrolytic immunoglobulins. Our data suggest that elevated levels of FVIII-hydrolyzing IgG in acquired hemophilia result from the exacerbation of a physiological catalytic immune response.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Chemical chaperone 4-phenylbutyric acid alleviates the aggregation of human familial pulmonary fibrosis-related mutant SP-A2 protein in part through effects on GRP78

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.     

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII

Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.     

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.     

A和C结构域糖基化位点对凝血八因子的分泌及活性的影响

在血液来源和血液制品安全隐患制约下,基因工程重组凝血八因子已开始替代天然八因子用以治疗血友病等病症,但目前高效表达重组八因子的方法在我国仍有技术瓶颈.该项目利用定点突变技术,通过PCR、质粒抽提、转染以及产物检测这四个步骤来研究重组凝血八因子在A和C结构域添加糖基化位点对八因子表达及活性的影响.结果表明,在重组八因子A结构域Bip结合位点外端增加糖基化位点能够促进八因子的细胞外分泌量,而在Bip结合位点内的突变则不但大大减少了八因子的胞外分泌也使八因子活性几乎全部丧失.与A结构域不同,在八因子C结构域增加糖基化位点的结果表明,无论在蛋白C结合区之内或之外增加糖基化位点不但不能大幅提高八因子的胞外分泌,也使八因子活性基本丧失.这些结果说明只有在A结构域外缘的非Bip结合区进行糖基化改造,才有可能提高八因子分泌并保持其活性.     

Expression of functional recombinant human factor IX in milk of mice

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk β-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.