A1 subunit-mediated regulation of thrombin-activated factor VIII A2 subunit dissociation

Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, Kd, were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant.     

Contribution of A1 subunit residue Q316 in thrombin-activated factor VIII to A2 subunit dissociation

Blood coagulation factor VIII (fVIII) is activated by thrombin to form an A1/A2/A3-C1-C2 heterotrimer, which functions as a cofactor for factor IXa during intrinsic pathway factor X activation. Human thrombin-activated fVIII (fVIIIa) decays rapidly because of first-order dissociation of the A2 subunit, which may function to regulate the coagulation mechanism. The three fVIII A domains each consist of two cupredoxin-like subdomains. Substitution of the COOH-terminal A1 subdomain of porcine fVIIIa, which decays more slowly than human fVIIIa, reduces the dissociation rate constant for fVIIIa decay. Examination of a human fVIII A1-A2-A3 homology model [Pemberton, S., et al. (1997) Blood 89, 2413-2421) revealed a possible interaction between Q316 in the FG helix of the COOH-terminal A1 subdomain and M539 in the FG helix of the NH2-terminal A2 subdomain, which are sites where human and porcine fVIII differ. Decays of purified recombinant human and porcine fVIIIa and the human fVIIIa mutants Q316H, M539L and Q316H/M539L were compared at 23 and 37 degrees C. The decay rates of the Q316H and Q316H/M539L mutants, but not the M539L mutant, were significantly slower than human fVIIIa. These results indicate that the FG helix of the COOH-terminal A1 cupredoxin-like subdomain of fVIII may be under selective pressure by the requirements of hemostatic balance.     

Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog

The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The activations of human and porcine fVIII by thrombin were studied by a two-stage coagulation assay using human citrated plasma as the standard. The peak activation of porcine fVIII was 10-fold greater than human fVIII (1.1 x 10(6) unit/mg versus 1.1 x 10(5) unit/mg). The proteolytic fragmentation of fVIII by thrombin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was not different between human and porcine fVIII, yielding previously identified bands corresponding to fragments A1, A2, A3-C1-C2, and the B domain. Following activation by thrombin, human fVIII was subjected to cation-exchange (Mono S) high performance liquid chromatography at pH 6.0 under conditions that yields stable, heterotrimeric (A1/A2/A3-C1-C2) porcine fVIIIaIIa (Lollar, P., and Parker, C.G. (1990) Biochemistry 28, 666-674). Coagulant activity was recovered in a single peak that was less than 0.5% that of porcine fVIIIaIIa (1.2 x 10(4) unit/mg versus 2.6 x 10(6) unit/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak fraction revealed bands corresponding to the A3-C1-C2 and A1 fragments but only trace levels of the A2 fragment. In contrast, activation of human fVIII by thrombin followed by Mono S HPLC at pH 5.0 produced a peak with 10-fold greater activity (1.2 x 10(5) unit/mg) than at pH 6.0 and which contained significant amounts of the A2 fragment. We conclude that human fVIIIIIa, like porcine fVIIIIIa, is a heterotrimer and propose that its apparent decreased coagulant activity is due to weaker association of the A2 subunit.     

Subunit structure of thrombin-activated porcine factor VIII

Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 with fragment(s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 degrees C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148,000 to 161,000 as the loading concentration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 calculated from SDS-PAGE analysis (148,000) or from the amino acid sequence of human fVIII (159,000). This establishes the major species in the preparation as a fVIIIA1/A2/A3-C1-C2 heterotrimer and additionally indicates either weak self-association of the trimer and/or incomplete association of the individual subunits to form the trimer. Velocity sedimentation of activated fViii revealed a single boundry (S020,w = 7.2 S).(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependent denaturation of thrombin-activated porcine factor VIII

Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.     

Altered interactions between the A1 and A2 subunits of factor VIIIa following cleavage of A1 subunit by factor Xa

Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1(336)) was previously shown to possess similar affinity for A2 and retain approximately 60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys(36) that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1(37-336)) showed little if any affinity for A2 (K(d)>2 microm), whereas factor VIIIa reconstituted with A2 plus A1(37-336)/A3-C1-C2 dimer demonstrated significant cofactor activity ( approximately 30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1(37-336) showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K(m) for factor X when A1 was cleaved at Arg(336). These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K(m) for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasma-based assays.     

Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer.

Heterotrimeric factor VIIIa was reconstituted from isolated A2 subunit and A1/A3-C1-C2 dimer of thrombin-activated human factor VIII in a reaction that was sensitive to pH. Maximal levels of reconstituted factor VIIIa at pH 6.0 were as much as 20-fold greater than were values observed at pH 7.5. The presence of factor IXa and phospholipid resulted in a marked increase in factor VIIIa reconstituted at physiologic pH. However, the resultant factor VIIIa was unstable due to slow proteolysis of the A1 subunit. Factor IXa modified by the active site-specific reagent dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DEGR-IXa) increased the level of factor VIIIa reconstituted from subunits to a similar extent as was observed for unmodified factor IXa and yielded stable factor VIIIa. This enhancement was saturated above a 1:1 molar ratio of DEGR-IXa to factor VIIIa subunits and could be blocked by an anti-factor IX antibody, suggesting that the DEGR-IXa-dependent increase in factor VIIIa reconstitution correlated with assembly of the factor X-ase complex. At a saturating amount of DEGR-IXa, the level of factor VIIIa reconstitution at pH 7.5 approached values obtained at pH 6.0. Fluorescence polarization measurements indicated that factor VIIIa altered binding of DEGR-IXa to phospholipid. However, neither the A2 subunit nor the A1/A3-C1-C2 dimer alone produced this effect. This result suggested that both A2 and A1/A3-C1-C2 were necessary for association of the cofactor with factor IXa. These results suggest a model in which assembly of the intrinsic factor X-ase complex stabilizes factor VIIIa through inhibition of subunit dissociation.     

Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by approximately 100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of approximately 1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.     

The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Recently, we showed that isolated A2 subunit enhanced the kcat for factor IXa-catalyzed activation of factor X by approximately 100-fold ( approximately 1 min-1), whereas isolated A1 or A3-C1-C2 subunits showed no effect on this rate (Fay, P. J., and Koshibu, K. J. (1998) J. Biol. Chem. 273, 19049-19054). However, A1 subunit increased the A2-dependent stimulation by approximately 10-fold. The Km for factor X in the presence of A2 subunit was unaffected by A1 subunit, whereas the kcat observed in the presence of saturating A1 and A2 subunits ( approximately 15 min-1) represented 5-10% of the value observed for native factor VIIIa (approximately 200 min-1). An anti-A1 subunit antibody that blocks the association of A2 eliminated the A1-dependent contribution to factor IXa activity. Inclusion of both A1 and A2 subunits resulted in greater increases in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa than that observed for A2 subunit alone and approached values obtained with factor VIIIa. These results indicate that A1 subunit alters the A2 subunit-dependent modulation of the active site of factor IXa to synergistically increase cofactor activity, yielding an overall increase in kcat of over 1000-fold compared with factor IXa alone.     

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs     

Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa.

Factor VIIIa is a heterotrimer of the factor VIII heavy chain-derived A1 and A2 subunits plus the factor VIII light chain-derived A3-C1-C2 subunit. While the A1 and A3-C1-C2 subunits can be isolated as a stable dimer, the A2 subunit is weakly associated with the dimer. In the human protein, the association of A2 with dimer is reversible and governed by a pH-dependent dissociation constant. Using the specific activity of factor VIIIa as an indicator of trimer concentration, the Kd (pH 6.0) was determined to be 28 nM whereas at the more physiologic pH (pH 7.4) this value was approximately 260 nM. Results from pH shift experiments confirmed the reversible binding of A2 to dimer as did the capacity for high levels of exogenous A2 subunit to inhibit the spontaneous decay of factor VIIIa activity. A2 subunit associated with the A1 subunit in the A1/A3-C1-C2 dimer based upon the capacity for free A1 subunit to inhibit the reconstitution of factor VIIIa from A2 subunit and dimer. These results indicate that the primary mechanism for the spontaneous decay of human factor VIIIa is the reversible dissociation of A2 subunit from the A1 subunit of the A1/A3-C1-C2 dimer.     

Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit

Heterodimeric human factor VIII was proteolytically activated by catalytic levels of thrombin to yield the (labile) active cofactor factor VIIIa possessing an initial specific activity of approximately 80 units/microgram. Activation paralleled the generation of fragments A1 and A2 derived from the heavy chain and A3-C1-C2 derived from the light chain. Chromatography of factor VIIIa, on Mono-S buffered at pH 6.0 resulted in separation of the bulk of the A2 fragment from a fraction composed predominantly of A1/A3-C1-C2 dimer plus low levels of A2 fragment. Only the latter fraction contained clotting activity (approximately 20 units/microgram) which was stable and represented a less than 10% yield when compared with the peak activity of unfractionated factor VIIIa. Further depletion of A2 fragment from Mono-S-purified factor VIIIA, achieved using an immobilized monoclonal antibody to the A2 domain, yielded a relatively inactive A1/A3-C1-C2 dimer (less than 0.4 unit/microgram). Factor VIIIa (greater than 40 units/microgram) was reconstituted from the A1/A3-C1-C2 dimer plus the A2 fragment in a reaction that was Me(2+)-independent and inhibited by moderate ionic strength. Reassociation of A2 required the A1 subunit in that the A2 subunit associated weakly if at all to A3-C1-C2 in the absence of A1. These results indicated that human factor VIIIa is a trimer represented by the subunits A1/A2/A3-C1-C2 and that the A2 subunit is required for expression of factor VIIIa activity.     

Cofactor activities of factor VIIIa and A2 subunit following cleavage of A1 subunit at Arg336.

Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K(d) = 1.8 microm). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg(336) by activated protein C or factor IXa is inactivating. A truncated A1 (A1(336)) showed similar affinity for A2 subunit (K(d) = 0.9 microm) and stimulated its cofactor activity to approximately 50% that observed for native A1. However, A1(336) was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1(336). Fluorescence energy transfer demonstrated that, although Fl-A1(336)/A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.     

Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability

Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ～3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ～4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ～4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.     

Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit

Factor VIIIa can be reconstituted from A2 subunit and A1/A3-C1-C2 dimer in a reaction that is facilitated by slightly acidic pH. We recently demonstrated that a truncated A1 (A1(37-336)) possessed markedly reduced affinity for A2 compared with intact A1, but retained 30% of native factor VIIIa activity in the presence of A3-C1-C2. We now identify A1-interactive regions for A2 using A1 fragments derived from a limited tryptic digest. Unfractionated trypsin-cleaved A1 inhibited reconstituted factor VIIIa activity. Two fragments, designated A1(37-121) and A1(221-336), markedly inhibited factor VIIIa reconstitution with either native A1 (K(i)=340 and 194 nM, respectively) or with A1(37-336) (K(i)=69 and 116 nM, respectively) at pH 6.0. A third fragment designated A1(122-206) did not possess inhibitory activity. At pH 7.2, the A1(221-336) partially inhibited reconstitution, whereas the A1(37-121) possessed little if any inhibitory activity. Both fragments inhibited factor VIIIa reconstitution as judged by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1 forms at pH 6.0. Furthermore, covalent cross-linking between A2 and A1(37-121) but not A1(221-336) was observed following reaction with a zero-length cross-linker. These findings demonstrate the presence of an extended, pH-dependent A2-interactive surface within regions 37-121 and 221-336 of A1. This interactive surface appears conformationally labile in the truncated A1 as judged by its apparent stabilization following association with A3-C1-C2.     

Enhanced biosynthesis of coagulation factor VIII through diminished engagement of the unfolded protein response

Human and porcine coagulation factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast, a significant proportion of porcine fVIII was localized to the Golgi indicating efficient transit through the secretory pathway. Overexpression of BiP, an integral UPR protein, reduced the secretion of human fVIII by 50%, but had no effect on porcine fVIII biosynthesis. In contrast, expression of BiP shRNA increased human fVIII expression levels. The current data support the model of differential engagement of UPR by human and porcine fVIII as a non-traditional mechanism for regulation of gene product biosynthesis.     

Bovine brain calmodulin-dependent protein phosphatase. Regulation of subunit A activity by calmodulin and subunit B

Calmodulin-dependent protein phosphatase isolated from bovine brain consists of a catalytic subunit A (Mr = 60,000) and a regulatory subunit B (Mr = 19,000) present in equal molar ratios. The two subunits were dissociated by gel filtration in 6 M urea and reconstituted to investigate the role of calmodulin and subunit B in regulating the phosphatase activity of subunit A. The activity of subunit A was stimulated 2-fold by calmodulin, 13-fold by subunit B, and 21-fold by both, indicating that the effects of both were synergistic. Maximum stimulation by calmodulin was observed at a calmodulin to subunit A molar ratio of 2:1 in the presence or absence of subunit B, whereas that by subunit B was observed at a B to A molar ratio of 3:1 in the presence or absence of calmodulin. Calmodulin and subunit B increased the Vmax of subunit A 2- and 5-fold, respectively, but had little effect on the Km for casein. The specific activity of the phosphatase reconstituted from subunits A and B reached 86% that of the native enzyme, whereas that of the holoenzyme reached 90%. Subunit B, even though similar to calmodulin in many respects, did not stimulate the activity of native phosphatase, suggesting that it cannot substitute for calmodulin. Limited trypsinization of subunit A increased its catalytic activity to the level observed with calmodulin; and this activity was further stimulated by subunit B but not by calmodulin. These results indicate that subunit A of phosphatase contains one catalytic domain and two distinct regulatory domains, one for calmodulin, and another for subunit B, that these two proteins do not substitute for one another and that they stimulate subunit A synergistically.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein

Purified porcine atrial muscarinic receptor (mAcChR) was reconstituted with purified porcine atrial inhibitory guanine nucleotide binding protein (Gi) in a lipid mixture consisting of phosphatidylcholine, phosphatidylserine, and cholesterol (1:1:0.1 w/w). 5'-Guanylyl imidodiphosphate (0.1 mM) had no effect on the binding of the muscarinic antagonist L-quinuclidinyl benzilate but converted high-affinity carbachol binding sites (Kd equal to 1 microM) in the reconstituted preparation to the low-affinity state (Kd equal to about 100 microM). Steady-state kinetic measurements of GTPase activity showed that the turnover number was increased from 0.19 min-1 in the presence of the muscarinic antagonist L-hyoscyamine to 2.11 min-1 for the agonist carbachol. The affinity of Gi for GDP was reduced by about 50-fold upon interaction with the carbachol-mAcChR complex, and the observed rate constant for GDP dissociation was increased by 38-fold from 0.12 to 4.5 min-1. Thus, the increase in steady-state GTPase activity observed for muscarinic agonists is largely, if not exclusively, due to the increase in GDP dissociation from Gi--probably the rate-limiting step in the steady-state mechanism. Carbachol-stimulated GTPase was sensitive to ADP-ribosylation of the reconstituted Gi by pertussis toxin, but the high-affinity agonist binding was uncoupled only when the reconstituted preparation was treated with pertussis toxin in the presence of GTP and the agonist acetylcholine. These results suggest that association with the mAcChR protects Gi from ADP-ribosylation by pertussis toxin.