Backbone resonance assignments of the C2 domain of coagulation factor VIII.
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ABSTRACT:
SUBMITTER: Nuzzio KM
PROVIDER: S-EPMC8663028 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Backbone (1)H, (13)C and (15)N resonance assignments are presented for the extracellular domain of tissue factor. Tissue factor is the integral membrane protein that initiates blood coagulation through the formation an enzymatic complex with the plasma serine protease, factor VIIa.
| S-EPMC2947601 | biostudies-literature
Project description:Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.
| S-EPMC8223065 | biostudies-literature
Project description:Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa-Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.
| S-EPMC3670696 | biostudies-literature
Project description:The development of anti-factor VIII antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A, leading to significant increases in morbidity and treatment cost. Using a panel of mAbs against different epitopes on FVIII, we have recently shown that epitope specificity, inhibitor kinetics and time to maximum inhibition are more important than inhibitor titer in predicting responses to FVIII and the combination of FVIII and recombinant FVIIa. In particular, a subset of high-titer inhibitors responded to high-dose FVIII, which would not be predicted on the basis of their inhibitor titer alone. Thus, the ability to quickly map the epitope spectrum of patient plasma with a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients.To map the epitopes of anti-FVIII mAbs, three of which are classic inhibitors and one of which is a non-classic inhibitor, by the use of hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS).The binding epitopes of four mAbs targeting the FVIII C2 domain were mapped with HDX-MS.The epitopes determined with HDX-MS are consistent with those obtained earlier through structural characterization and antibody competition assays. In addition, classic and non-classic inhibitor epitopes could be distinguished by the use of a limited subset of C2 domain-derived peptic fragments.Our results demonstrate the effectiveness and robustness of the HDX-MS method for epitope mapping, and suggest a potential role of rapid mapping of FVIII inhibitor epitopes in facilitating individualized treatment of inhibitor patients.
| S-EPMC3947443 | biostudies-literature
Project description:BackgroundHemophilia A is caused by heterogeneous mutations in F8. Coagulation factor VIII (FVIII), the product of F8, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIII is known to interact with diverse proteins, and this characteristic may be important for hemostasis. However, little is known about domain-specific functions or their specific binding partners.MethodsTo determine F8 domain-specific functions during blood coagulation, the FVIII domains A1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinant polypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged, over-expressed in bacteria, and purified by specific affinity chromatography.ResultsRecombinant 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.ConclusionRecombinant, 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.
| S-EPMC4486152 | biostudies-literature
Project description:Autophagy is a catabolic cellular process that targets cytosolic material, including mitochondria, to the vacuole or lysosomes for degradation. The selective degradation of mitochondria by autophagy is termed mitophagy. Dysfunctional mitophagy, which leads to the accumulation of damaged mitochondria, has been implicated in Parkinson's disease, cancer, cardiac disease and metabolic disease. In Saccharomyces cerevisiae, mitophagy is initiated by the autophagy receptor Atg32, an outer mitochondrial membrane protein. A lack of structural information for Atg32 has hindered our understanding of the molecular mechanisms of mitophagy initiation. To gain new structural insight into Atg32, we have identified the location of a structured domain within the cytosolic region of Atg32 and completed the backbone and side chain resonance assignments for this domain.
| S-EPMC5693731 | biostudies-literature
Project description:BackgroundCoagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies.ObjectivesTo structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin.MethodsThe structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics.ResultsHere, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics.ConclusionsThese improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.
| S-EPMC6940532 | biostudies-literature
Project description: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 DeltaC2) to characterize the contributions of the C1 domain to function. Binding affinity of the DeltaC2 variant to phospholipid vesicles as measured by energy transfer was reduced approximately 14-fold. However, the activity of DeltaC2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions ( approximately 4-fold) were observed in the functional affinity of DeltaC2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability ( approximately 3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as approximately 60-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.
| S-EPMC2919080 | biostudies-literature
Project description:Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 domains. The structure permits unambiguous modeling of the relative orientations of the 5 domains of r-fVIII. Comparison of the structures of fVIII, fV, and ceruloplasmin indicates that the location of bound metal ions and of glycosylation, both of which are critical for domain stabilization and association, overlap at some positions but have diverged at others.
| S-EPMC2214755 | biostudies-literature
Project description:The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.
| S-EPMC5286935 | biostudies-literature