Summary Background Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665, or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 112, 2761, 2008). Objectives In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on the stability. Methods Studies assessing factor VIII stability involved monitoring decay rates of factor VIII at 55°C or in guanidinium, decay of factor VIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nM) factor VIII concentration. Results and Conclusions Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield factor VIII reagents with improved stability parameters.

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of L-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron-sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron-sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mössbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 Å resolution [H. Sakuraba, H. Tsuge, K. Yoneda, N. Katunuma, and T. Ohshima, (2005) J. Biol. Chem. 280, pp. 26645-26648]. This protein does not contain a CXXCXXC motif, and no Fe/ S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 μmol min -1 mg -1 , significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 μmol min -1 mg -1 ), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated ⌬C2) to characterize the contributions of the C1 domain to function. Binding affinity of the ⌬C2 variant to phospholipid vesicles as measured by energy transfer was reduced ϳ14-fold. However, the activity of ⌬C2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions (ϳ4-fold) were observed in the functional affinity of ⌬C2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability (ϳ3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as ϳ60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function. Factor (F)2 VIII, a plasma protein that is decreased or defective in individuals with hemophilia A, is expressed as both single chain and heterodimer forms. The latter consists of a heavy chain (HC) comprised of A1(a1)A2(a2)B domains and a light chain (LC) comprised of (a3)A3C1C2 domains, with the lowercase a representing short (ϳ30 -40 residues) segments rich in acidic residues (see Ref (1) for review). FVIII is activated by thrombin-or FXa-catalyzed cleavages at the a1A2, a2B, and a3A3 junctions. The resulting product, FVIIIa, is a heterotrimer comprised of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa (FIXa) in the membrane-dependent conversion of zymogen FX to the serine protease, FXa (see Ref.(1) for review).Early structural studies implicated the C2 domain as making the predominant contribution to membrane binding through a combination of electrostatic and hydrophobic interactions (2). In addition, the C2 domain contains apparent secondary binding sites for von Willebrand factor (VWF) (3, 4), thrombin (5), and FIXa (6). The intermediate resolution x-ray crystal structures of FVIII (7, 8) indicate that the C1 and C2 domains are aligned such that both domains may interact with the phospholipid surface. A similar alignment was noted for factor Vai, the activated protein C-inactivated form of the homologous protein, factor Va (9). Recent results comparing isolated FVIII C2 domain with a C1C2 protein showed enhanced affinity of the latter in platelet binding suggesting direct and/or indirect roles for C1 in this interaction (10). It was also reported tha...

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 ϳ3.7-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 ϳ4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ϳ4.6-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. Factor F (FVIII)2 is a plasma protein that is decreased or defective in individuals with hemophilia A. FVIII is expressed as both single chain and heterodimer forms in heterologous cells. The latter consists of a heavy chain composed of A1(a1)A2(a2)B domains and a light chain composed of (a3)A3C1C2 domains, with the lowercase "a" representing short segments rich in acidic residues (see Ref. 1 for review). FVIII is activated following limited proteolysis catalyzed by thrombin or FXa at Arg-372 (a1A2 junction), Arg-740 (a2B junction), and Arg-1689 (a3A3 junction). The resulting product, FVIIIa, is a heterotrimer composed of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for FIXa in the membrane-dependent activation of FX to FXa (see Ref 1 for review).Earlier structural studies identified the C2 domain as making a primary contribution to anionic phospholipid membrane binding through a combination of electrostatic and hydrophobic interactions (2). More recently, the intermediate resolution x-ray structures of FVIII (3, 4) showed that the C1 and C2 domains are aligned such that both domains may interact with the phospholipid membrane surface. In addition, these structures show close contact between A1 (heavy chain) and C2 (light chain) domains. We recently reported that an FVIII variant lacking the C2 domain retained the capacity to bind phospholipid membranes, ...

Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg372 and Arg740 in the FVIII heavy chain and Arg1689 in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg372, which appears to be rate-limiting. Examination of the P3–P3′ residues flanking each P1 site revealed that those sequences at Arg740 and Arg1689 are more optimal for thrombin cleavage than at Arg372, suggesting these sequences may impact reaction rates. Recombinant FVIII variants were prepared with mutations swapping scissile bond flanking sequences in the heavy chain individually and in combination with a second swap or with a P1 point mutation. Rates of generation of A1 and A3-C1-C2 subunits were determined by Western blotting and correlated with rates of cleavage at Arg372 and Arg1689, respectively. Rates of thrombin cleavage at Arg372 were increased ∼10- and ∼3-fold compared with that of wild-type FVIII when it was replaced with P3–P3′ residues flanking Arg740 and Arg1689, respectively, and these values paralleled increased rates of A2 subunit generation and procofactor activation. Positioning of more optimal residues flanking Arg372 abrogated the need for initial cleavage at Arg740 to facilitate this step. These results show marked changes in cleavage rates correlate with the extent of cleavage-optimal residues flanking the scissile bond and modulate the mechanism for procofactor activation.

Summary Background Neutralizing factor (F) VIII antibodies develop in ~30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined following immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Results Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of <12 residues. Conclusions IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of less than 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions.

EssentialsFactor VIIIa (FVIIIa) is unstable due to loss of A2; D666 and Y1792 contribute to its stability. We conducted a study to identify the interactions made at these residues at the A2-A3 interface. We present evidence for stabilizing interactions between D666-S1787 and T657-Y1792 in FVIIIa. A D666C/S1788C variant with a disulfide A2-A3 linkage has a FVIIIa decay rate that is 1% of wild-type.Summary. Background: Factor (F)VIIIa activity and stability depends on the non-covalent association of the A2 subunit with the A1/A3C1C2 dimer, but the interactions that contribute to A2 association are not well defined. Previous work had shown that D666A and Y1792F mutations at the A2-A3 interface resulted in increased FVIIIa decay, suggesting that the residues were involved in bonding interactions important for FVIIIa stability. Objectives: Several potential hydrogen bonding partners of D666 and Y1792 across the A2-A3 interface were selected from the low-resolution FVIII crystal structure, and we used mutagenesis and biochemical analysis to examine the bonding interactions occurring at D666 and Y1792. Methods: Using a series of stability and functional analyses, we analyzed FVIII variants in which D666 and Y1792 were each swapped with the residues of potential bonding partners. Results and conclusions: We present evidence for hydrogen bonds between D666 and S1787 and between Y1792 and T657 that are important for FVIIIa stability. D666S/S1787D and T657Y/Y1792T variants each displayed wild-type (WT)-like FVIIIa stability and performed like WT FVIII in a series of functional analyses, whereas D666S, S1787D, and Y1792T single variants showed increased FVIIIa decay and a T657Y variant had little FVIIIa activity. These results suggest that WT hydrogen bonds are disrupted with the single mutations but maintained in the swap variants. Furthermore, mutation of D666 and S1788 to cysteine resulted in disulfide bond formation across the A2-A3 interface, confirming the close proximity of D666 and S1787, and this covalent attachment of the A2 subunit significantly increased FVIIIa stability.

No abstract