Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells

The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain.     

双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FVⅢ基因

摘要用双载体转运凝血VⅢ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制,但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用,该丈用双载体转B结构域大部缺失型FVⅢ（BDD-FVⅢ）的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为（125＋29）ng／mL,明显高于共转野生型重链和轻链基因细胞的（75＋23）ng／mL;Coatest法显示细胞分泌的凝血活性为（0．784±0．29）U／mL．也明显高于共转野生型重链和轻链基因细胞（0．34＋0．12）U／mL。结果表明,重、轻链间的二硫键形成可提高双载体转FVⅢ基因的功效,为进一步在动物体内转基因提供了实验依据。     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.     

双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FⅧ基因

用双载体转运凝血Ⅷ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制,但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用,该文用双载体转B结构域大部缺失型FⅧ(BDD-FVⅢ)的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为(125±29)ng/mL,明显高于共转野生型重链和轻链基因细胞的(75±23)ng/mL;Coatest法显示细胞分泌的凝血活性为(0.78±0.29)U/mL,也明显高于共转野生型重链和轻链基因细胞(0.34±0.12)U/mL。结果表明,重、轻链间的二硫键形成可提高双载体转FⅧ基因的功效,为进一步在动物体内转基因提供了实验依据。     

Inactivation of factor VIII by activated protein C and protein S

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.     

Expression of blood clotting factor VIII:C gene in capillary endothelial cells.

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.     

Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain.

Human coagulation factor V is an integral component of the prothrombinase complex. Rapid activation of prothrombin is dependent on the interactions of this nonenzymatic cofactor with factor Xa and prothrombin in the presence of calcium ions and a phospholipid or platelet surface. Factor V is similar structurally and functionally to the homologous cofactor, factor VIII, which interacts with factor IXa to accelerate factor X activation in the presence of calcium and phospholipids. Both of these cofactors, when activated, possess homologous heavy and light chains. Binding to anionic phospholipids is mediated by the light chains of these two cofactors. In bovine factor Va, a phosphatidylserine-specific binding site has been localized to the amino-terminal A3 domain of the light chain. In human factor VIII, on the other hand, a region within the carboxyl-terminal C2 domain of the light chain has been shown to interact with anionic phospholipids. We have constructed a series of recombinant deletion mutants lacking domain-size fragments of the light chain of human factor V (rHFV). These mutants are expressed and secreted as single-chain proteins by COS cells. Thrombin and the factor V activator from Russell's viper venom process these deletion mutants as expected. The light chain deletion mutants possess essentially no procoagulant activity, nor are they activated by treatment with factor V activator from Russell's viper venom. Deletion of the second C-type domain results in essentially complete loss of phosphatidylserine-specific binding whereas the presence of the C2 domain alone (rHFV des-A3C1, which lacks the A3 and C1 domains of the light chain) results in significant phosphatidylserine-specific binding. The presence of the A3 domain alone (rHFV des-C1C2) does not mediate binding to immobilized phosphatidylserine. Increasing calcium ion concentrations result in decreased binding of recombinant human factor V and the mutant rHFV des-A3C1 to phosphatidylserine, similar to previous studies with purified plasma factor V and phospholipid vesicles. These results indicate that human factor V, similar to human factor VIII, possesses a phosphatidylserine-specific binding site within the C2 domain of the light chain.     

Reconstitution of human factor VIII from isolated subunits

Human factor VII heterodimers were fractionated into component heavy and light chains using an anti-light chain specific monoclonal antibody immunosorbant. Neither the light chain nor the heavy chain alone possessed activity. Factor VII activity was reconstituted by recombining the subunits in the presence of Mn2+ or Ca2+. Reconstitution of activity also showed ionic strength dependence suggesting the importance of hydrophobic and electrostatic interactions. All factor VIII heavy chains (93 to 210 kDa) recombined with the 83 kDa light chain as judged by retention of all reconstituted heterodimeric forms by the monoclonal immunosorbant. Maximum specific activity (3 units/micrograms) was obtained at a 1:1 molar ratio of light chain:heavy chain. The presence of von Willebrand factor enhanced the rate of factor VIII reconstitution as much as 5-fold. This effect was both ionic strength-dependent and dose-dependent up to a 25-fold weight excess of von Willebrand factor over factor VIII.     

von Willebrand factor mediates protection of factor VIII from activated protein C-catalyzed inactivation.

Factor VIII, a cofactor of the intrinsic clotting pathway, is proteolytically inactivated by the vitamin K-dependent serine protease, activated protein C in a reaction requiring Ca2+ and a phospholipid surface. Factor VIII was inactivated 15 times faster than factor VIII in complex with either von Willebrand factor (vWf) or the large homodimeric fragment, SPIII (vWf residues 1-1365). Free factor VIII or factor VIII in complex with a smaller fragment, SPIII-T4 (vWf residues 1-272), were inactivated at the same rate, suggesting that this effect was dependent upon the size of factor VIII-vWf complex rather than changes in factor VIII brought about by occupancy of the vWf-binding site. Thrombin cleavage of the factor VIII light chain to remove the vWf-binding site eliminated the protective effects of vWf. In the absence of phospholipid, high levels of the protease inactivated both free and vWf-bound factor VIII at equivalent rates. Using the same conditions, isolated heavy chains and the heavy chains of factor VIII were proteolyzed at similar rates. Taken together, these results suggested that, in the absence of phospholipid, inactivation of factor VIII is independent of factor VIII light chain and further suggest that vWf did not mask susceptible cleavage sites in the cofactor. Solution studies employing fluorescence energy transfer using coumarin-labeled factor VIII (fluorescence donor) and synthetic phospholipid vesicles labeled with octadecyl rhodamine (fluorescence acceptor) indicated saturable binding and equivalent extents of donor fluorescence quenching for factor VIII alone or when complexed with SPIII-T4. However, complexing of factor VIII with either vWf or SPIII eliminated its binding to the phospholipid. Since a phospholipid surface is required for efficient catalysis by the protease, these results suggest that vWf protects factor VIII by inhibiting cofactor-phospholipid interactions.     

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.     

Topography of the human factor VIII-von Willebrand factor complex

Factor VIII circulates in noncovalent complex with von Willebrand factor (vWf). The topography of this complex was evaluated by fluorescence energy transfer using factor VIII subunits modified with N-(1-pyrenyl)maleimide (NPM; fluorescence donor) and vWf-derived fragments modified with 7-diethylamino-3-[4'-maleimidylphenyl]-4-methyl coumarin (CPM; fluorescence acceptor). Results from a previous study indicated an interfactor VIII subunit distance of 20 A separating Cys528 and Cys1858 in the factor VIII heavy and light chains, respectively (Fay, P.J., and Smudzin, T. M. (1989) J. Biol. Chem. 264, 14005-14010). Fluorophore modification of the vWf SPIII homodimer (residues 1-1365) indicated multiple attachment sites at Cys126/135/1360 as determined from sequence analysis of fluorescent tryptic peptides derived from the modified protein. Based upon donor quenching data, an interfluorophore distance of approximately 28 A was calculated separating NPM-factor VIII light chain or factor VIII reconstituted from NPM-light chain plus unmodified heavy chain, from CPM-SPIII. A similar value (29 A) was obtained for NPM-light chain paired with CPM-SPIII-T4 (vWf residues 1-272), suggesting that donor quenching resulted primarily from modified residue(s) Cys126/135 in the acceptor. No energy transfer was observed for the NPM-heavy chain/CPM-SPIII pairing. However, when NPM-heavy chain was reassociated with unmodified light chain prior to reaction with CPM-SPIII or CPM-SPIII-T4, energy transfer was observed with calculated interfluorophore distances of approximately 31 and 34 A, respectively. Levels of acceptor resulting in maximal donor quenching suggested an equimolar stoichiometry of factor VIII (light chain)/vWf fragment in the reconstituted complexes. These results indicate a close spatial arrangement among the A3 domain of factor VIII light chain, the A2 domain of factor VIII heavy chain, and the NH2 terminus region of vWf in the factor VIII-vWf complex.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Formation of intermolecular disulfide bonds on nascent immunoglobulin polypeptides.

The initial step of intermolecular covalent assembly of immunoglobulins molecules involves formation of heavy chain-light chain or heavy chain-heavy chain disulfide bonds. Using QAE-Sephadex chromatography to isolate microsomal nascent polypeptides, we have shown that this initial step of intermolecular covalent assembly occurs, to a substantial extent, on nascent heavy chains, as well as on completed heavy chains as previously demonstrated by others. In MPC 11 mouse myeloma cells, completed light chains are assembled covalently to nascent heavy chains, whereas in MOPC 21 mouse myeloma cells, completed heavy chains are assembled covalently to nascent heavy chains. These results are consisted with the heavy-light half-molecule being the major initial intermediate in the assembly of MPC 11 IgG2b and heavy-heavy dimer being the major initial intermediate formed in assembly of MOPC 21 IgG1. The nascent MPC 11 heavy chain must be at least 38,000 daltons in size before assembly with the light chain occurs, even though the heavy chain cysteine involved in this disulfide bond is 131 residues (approximately 15,000 daltons) from the NH2 terminus. In addition, pulse-chase labeling studies of MPC 11 cells have shown that the assembly of completed light chains with the nascent heavy chain must occur within a few minutes of the synthesis of the light chain even though a large excess of unassembled MPC 11 light chains remain inside the cell for an average time of 2 h before being secreted.     

BiP表达下调改善双载体转断裂FVIII基因的功效

双载体转凝血Ⅷ因子（FⅧ）基因可作为一种转基因策略克服腺相关病毒(AAV)载体容量限制,但重链分泌的低效性影响转基因功效. 为提高重链分泌,本文用RNA干扰技术下调内质网内蛋白伴侣分子免疫球蛋白重链结合蛋白(BiP)的表达,观察对HEK293细胞双载体共转FⅧ基因分泌重链和生物活性的影响. 结果显示,RNA干扰可明显下调BiP表达,但不影响细胞生长; ELISA检测BiP下调细胞单独转重链基因时的重链分泌量为98±38 ng/mL,与轻链共转基因时显著升高到157±32 ng/mL,明显高于对照细胞单独转重链基因和共转重链和轻链基因的重链分泌量(分别为29±8 ng/mL和79±19 ng/mL);Cotest法检测显示,BiP下调细胞共转重链和轻链基因细胞分泌的凝血生物活性为0.73±0.23 IU/mL,明显高于对照细胞共转重链和轻链基因(0.39±0.07 IU/mL). 结果表明, BiP表达下调通过促进重链分泌,可提高双载体共转FⅧ基因的功效,为进一步动物体内双AAV载体转FⅧ基因的甲型血友病基因治疗研究提供了实验依据.     

Murine plasma cells secreting more than one class of immunoglobulin. VI. Secretion of completely assembled IgG2b and IgA molecules with segregated heavy chains and free light chains by spontaneous myeloma SAMM 368 in culture.

The antigenic and molecular characteristics of the two immunoglobulins secreted by a single cell line of plasmacytoma SAMM 368 were analyzed by polyacrylamide gel electrophoresis of biosynthesized proteins. Adapted to continuous in vitro cultivation, this BALB/c plasmacytoma secretes at least 98% of its heavy chains as components of fully assembled and isotypically uniform IgG2b and IgA molecules. The IgA is secreted as monomers, dimers, and multimers with chemical properties typical of BALB/c myeloma IgA including disulfide bonded J chain and noncovalently bonded light chains. The noncovalently bonded light chains are monomers rather than dimers. Free light chains are also secreted. The ability to segregate heavy chains is attributed either to chemical, enzymatic, or compartmental regulatory factors operating within these plasma cells.     

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.     

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.     

Protein <Emphasis Type="Italic">trans</Emphasis>-splicing based dual-vector delivery of the coagulation factor VIII gene

A dual-vector system was explored for the delivery of the coagulation factor VIII gene, using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally. A pair of eukaryotic expression vectors, expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII), was constructed. With transient co-transfection of the two vectors into 293 and COS-7 cells, the culture supernatants contained (137±23) and (109±22) ng mL⁻¹ spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL⁻¹ for 293 and COS-7 cells, respectively. The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes. The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting. The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.