1Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, Korea.
2National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA.
Hemophilia A is caused by heterogeneous mutations in
Recombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptide interacted with coagulation factor IX, which is known to bind the A2 domain of activated FVIII.
Recombinant, domain-specific polypeptides are useful tools to study the domain-specific functions of FVIII during the coagulation process, and they may be used for production of domain-specific antibodies.
Hemophilia A (HA) is a common congenital bleeding disorder resulting from a qualitative or quantitative deficiency in coagulation factor VIII (FVIII) . Recently, remarkable progress has been made in understanding the molecular basis of HA. FVIII is a multi-domain protein. It is composed of six domains that are arranged as follows: A1-A2-B-A3-C1-C2 (from N to C terminus) . The FVIII protein is secreted as a heterodimer consisting of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains); these bind to each other in circulating blood via Ca2+ and Cu2+ . FVIIIa (the active form of FVIII) functions primarily as a cofactor for coagulation factor IX (FIX) . FVIII also interacts with diverse proteins such as von Willebrand factor (vWF), thrombin, calnexin, and calreticulin . vWF binding occurs when FVIII is secreted into the bloodstream . The FVIII-vWF complex has a longer half-life and is more stable than FVIII alone; this interaction is important for effective hemostasis . During FVIII maturation, calnexin and calreticulin promote FVIII folding; these interactions are important for FVIII protein quality control in the endoplasmic reticulum and Golgi apparatus . In the case of a
The pET-28a(+) vector was purchased from Novagen (Madison, WI, USA), and the pGEX-4T-2 vector was obtained from Amersham Biosciences (Uppsala, Sweden).
Each FVIII domain was cloned from Hep3B hepatocyte complimentary deoxyribonucleic acid (cDNA). Purification of Hep3B cDNA was performed using an RNeasy Plus Mini Kit (Qiagen; Valencia, CA, USA).
Bacteria harboring fusion plasmids were seeded in 10 mL 2× YT medium containing 100 µg/mL ampicillin (for the GST-tagging vector) or kanamycin (for the 6× His-tagging vector). Cells were cultured at 37℃ until an optical density (at 600 nm; OD600) of 0.4 was achieved. To induce FVIII protein expression, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture medium at a final concentration of 0.5 mM. The cells were incubated for 4 hours (hr) at 30℃. Then, cells were harvested by centrifugation at 3,000× g for 20 min at 4℃. Cells were then resuspended in lysis buffer (137 mM NaCl, 8.0 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, 2.7 mM KCl, 1 mg/mL lysozyme, 5 mM DTT, 0.03% SDS, and 1% Triton X-100, pH 7.4), and the mixture was sonicated for six cycles of 10 sec each at 5 sec intervals. The lysates were centrifuged at 16,000× g for 5 min at 4℃. Lastly, the extract was applied to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblot analysis. Expression conditions were optimized in terms of the IPTG concentration, induction temperature, and incubation duration to obtain a high recombinant protein yield.
For GST-tagged protein purification, agarose-immobilized glutathione was added to the lysates and incubated, with shaking, for 1 hr at room temperature. To remove unbound proteins, mixtures were centrifuged at 1,250× g for 1 min at 4℃, and the supernatant was discarded. The pellets were resuspended in a wash buffer (137 mM NaCl, 8.0 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, and 2.7 mM KCl, pH 7.4) and then loaded onto the Poly-Prep chromatography column. The column was washed twice with 5 mL of wash buffer, and the retained GST-FVIII protein was eluted with a buffer consisting of 137 mM NaCl, 8.0 mM Na2HPO4·7H2O, 1.4 mM KH2PO4 (pH 8.0), 2.7 mM KCl, and 100 mM reduced glutathione (GSH). For purification of His-tagged polypeptides, cells were lysed in lysis buffer (50 mM sodium phosphate containing 300 mM NaCl, pH 7.0) using ultrasonication followed by denaturation in lysis buffer containing 8 M urea. Cells were then centrifuged at 3,000×
To assess the binding activity of the recombinant polypeptides, GST-tagged polypeptides corresponding to FVIII domains were incubated overnight with either Benefix (recombinant FIX from Pfizer) or albumin-free standard human plasma at 4℃. Next, 100 µL 50% glutathione-agarose bead slurry was added to the mixture and incubated on a rotor for 6 hr at 4℃. The beads were washed three times with 1 mL ice-cold GST lysis buffer. After centrifugation at maximum speed for 1 min, the supernatant was discarded. The beads or the eluted proteins were analyzed using SDS-PAGE and immunoblotting.
SDS-PAGE was performed using 10% acrylamide gels. Gels containing separated proteins were stained with Coomassie Brilliant Blue solution. For western blot analysis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using the Trans-blot semidry system (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 4% skim milk solution and incubated with appropriate primary antibodies. Immunoreactive proteins were detected using a secondary antibody conjugated with horseradish peroxidase, and they were visualized using the enhanced chemiluminescence method.
Human FVIII is normally expressed as a large glycoprotein containing the A1-A2-B-A3-C1-C2 domains. After activation by thrombin, FVIIIa transiently circulates as a heterotrimer consisting of A1, A2, and A3-C1-C2, and the B domain is released . To prepare domain-specific DNA constructs from human
For expression of domain-specific recombinant polypeptides, the pGEX4T-2 or pET-28a (+) vectors were introduced into the
His-tagged, domain-specific polypeptides were expressed from the pET-28a(+) vector. Expression was induced using 1 mM IPTG. The mock control consisting of the empty pET-28a(+) vector was the negative control (
For recombinant polypeptide purification, bacteria expressing GST-tagged or His-tagged proteins were cultured until the OD600 reached 0.5, and then they were incubated with 1 mM IPTG. After optimization of GST-tagged domain-specific FVIII polypeptide expression conditions, protein fragments were purified using GST affinity chromatography.
For GST-tagged recombinant polypeptide validation, a GST pull-down assay was performed as described above. During blood clotting, coagulation factor IXa (FIXa) is known to bind FVIII via the A2 domain . Therefore, we tested whether each domain bound to recombinant FIX or platelet-derived FIX in standard human plasma. Results of the GST pull-down assay were shown by western blot analysis using the anti-FIX antibody. GST-tagged FVIII domain polypeptides were identified using the anti-GST antibody. All GST-tagged FVIII domain polypeptides were detected at the expected sizes among the pull-down products along with recombinant FIX (Fig. 3A and 3B). In contrast, recombinant FIX was only detected in complex with the FVIII-A2 domain polypeptide (indicated as rFIX in Fig 3A). The pull-down assay using standard human plasma also showed FIX in complex with the FVIII-A2 domain polypeptide alone (indicated as FIX in Fig. 3B). These results clearly showed that the synthetic polypeptides corresponding to FVIII domains have binding characteristics identical to neutral FVIII in human blood.
Owing to the recent work of several research groups, remarkable progress was made in understanding the molecular basis of HA. The method for profiling
In previous studies, cDNA generated from human leukocyte RNA was used to clone
The FVIII domains were produced in
The main function of FVIIIa is to act as a protein cofactor during blood coagulation. The FVIII protein interacts with diverse proteins in human plasma. Because previous studies focused primarily on the coagulation process, studies regarding the specific roles of each FVIII domain are insufficient. In this study, we report an important step toward understanding the protein-protein interactions of FVIII during blood coagulation.
Cloning of each
Expression of recombinant FVIII polypeptides tagged with GST
Interaction of recombinant FVIII domain polypeptides with FIX. To characterize recombinant FVIII domain polypeptides, a pull-down assay was performed with FIX. GST-tagged polypeptides were purified and incubated with either recombinant FIX