Phospholipid vesicles interfere with the binding of antibody fragments to the light chain of factor VIII

2005 ◽  
Vol 93 (05) ◽  
pp. 833-841 ◽  
Author(s):  
Deborah Lewis ◽  
Niels Bovenschen ◽  
Koen Mertens ◽  
Jan Voorberg ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Membrane Binding ◽  
Phospholipid Vesicles ◽  
C2 Domain ◽  
Wild Type ◽  
Single Chain ◽  
Type Protein ◽  
Wild Type Protein ◽  
C1 Domains

SummaryFactor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223, W2313 and V2314 at the tips of β-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factor V form a phospholipid binding site, but residues in the A3 and C1 domains are also thought to contribute to membrane binding. Phage display technology was previously used to isolate factor VIII light chain specific single-chain variable domain fragments (scFv) from patients with factor VIII inhibitors. Phospholipid vesicles inhibited the binding of factor VIII to scFvsWR1 and WR16 (epitope: E2181-M2199) with half saturation values of 23 and 47 μM respectively. The single point mutant F2200A factor VIII light chain bound to WR1 and WR16 with a much lower affinity than wild type protein suggesting that residue F2200 is also included in the epitopes of these scFvs. Binding of factor VIII to C2-specific scFvsWR13 and EL14 (epitope: K2207-M2321) was not inhibited by phospholipid vesicles. Consistent with this, F2200A factor VIII light chain bound to these scFvs with the same affinity as the wild type protein. However, phospholipid vesicles also inhibited the binding of factor VIII to the A3-C1-specific scFvs KM36 (epitope: Q1778-D1840) and KM38 (epitope: S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 μM, respectively, suggesting that the A3 and/or C1 domains may contribute to membrane binding of the cofactor.

Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 558-563 ◽  
Author(s):  
Edward N. van den Brink ◽  
Ellen A. M. Turenhout ◽  
Julian Davies ◽  
Niels Bovenschen ◽  
Karin Fijnvandraat ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Neutralizing Antibodies ◽  
Chain Gene ◽  
Gene Repertoire ◽  
C2 Domain ◽  
Single Chain ◽  
Phage Display Technology ◽  
Light Chain Gene ◽  
Germline Genes

A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by VH domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the VH1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study.

Effect of Membrane Binding Upon Intramolecular Dynamics of the Factor VIII C2 Domain.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3168-3168 ◽  
Author(s):  
Dennis Pantazatos ◽  
Christopher Gessner ◽  
Virgil Woods ◽  
Gary E. Gilbert
Keyword(s):  
Exchange Rate ◽  
Factor Viii ◽  
Membrane Binding ◽  
Phospholipid Vesicles ◽  
C2 Domain ◽  
Amide Hydrogen ◽  
Binding Peptide ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Membrane Interactive

Abstract Abstract 3168 Poster Board III-109 Factor VIII functions as a membrane-bound cofactor in the Factor Xase complex. Binding to a phospholipid membrane enhances activity of the factor VIIIa-factor IXa complex approx. 100,000-fold. While membrane binding increases the net affinities of factor VIIIa for factor IXa and factor X, the major effect of membrane binding is upon the catalytic activity of the assembled complex. The mechanism through which activity is enhanced remains largely unknown. The C2 domain of factor VIII contains the major membrane-binding function. The x-ray crystal structures of the C2 domain and, subsequently, of intact factor VIII have enabled hypotheses about the mechanism of membrane binding and the enhancement of activity. Identified functional motifs of the C2 domain include two pairs of hydrophobic, membrane-interactive amino acids at the tips of “spikes,” on the lower end of the C2 domain and a putative factor IXa-binding peptide on an upper surface. Hydrogen/Deuterium Exchange Mass Spectrometry (DXMS) has proven to be an effective method for characterizing protein conformational change induced by ligand interaction. Amide backbone hydrogens of a protein readily exchange with those in the solvent, contingent upon physical contact of the solvent hydrogen with the amide hydrogen. Therefore the exchange rate in a particular region of the protein is dependent on amide hydrogen solvent accessibility. DXMS is performed by isotopic labeling of a protein at various time points with deuterium oxide (D2O) followed by quenching the exchange and proteolysis to produce overlapping peptides spanning the length of the sequence. The degree of labeling is assessed using liquid chromatography coupled with mass spectrometry analysis. Using this analysis a stability map of the protein can be determined that represents changes in global and local structural dynamics in bound and unbound conditions. We have utilized DXMS to characterize the dynamics of the factor VIII C2 domain (fVIII-C2) in solution and when bound to phospholipid vesicles. fVIII-C2 was produced in E. coli and purified by immobilized metal affinity chromatography and ion exchange chromatography. Preliminary studies indicated that digestion on serial pepsin and V8 protease columns produced peptides spanning the entire fVIII-C2 structure, with overlapping peptides spanning the membrane-interactive “spikes” and the putative factor IXa-interactive peptide. Exchange was rapid on the membrane-interactive spikes in the absence of phospholipid vesicles, confirming flexibility of these structures. Upon binding of fVIII-C2 to phospholipid vesicles, the exchange rate decreased particularly in spike 3 (from 73% to 49% at 10s labeling, upon addition of phospholipid vesicles). The extent of the region with slowed exchange implied increased structural rigidity in addition to any solvent protection that occurs upon immersion of the hydrophobic side chains into the membrane. A marked decrease in exchange rate was also observed in the putative FIXa binding peptide (from 85% to 21% at 10s labeling, upon addition of phospholipid vesicles), while the exchange rates of most of the peptides from the core beta-barrel of fVIII-C2 were not significantly affected. These observations indicate that membrane engagement alters the mobility of membrane interactive spikes and suggest that membrane binding has an allosteric effect on the putative factor IXa binding site. Membrane-induced allosteric changes in the factor VIII C2 domain may contribute to the major enhancement of factor VIIIa-factor IXa activity that occurs upon membrane binding. Disclosures No relevant conflicts of interest to declare.

Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

2021 ◽  
Author(s):  
Yuto Nakajima ◽  
Hiroaki Minami ◽  
Keiji Nogami
Keyword(s):  
Surface Plasmon Resonance ◽  
Factor Viii ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
C2 Domain ◽  
Wild Type ◽  
Cross Link ◽  
Domain Specific ◽  
Cofactor Activity ◽  
Acidic Region

AbstractFactor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.

scholarly journals Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128954 ◽  
Author(s):  
Saara Laulumaa ◽  
Tuomo Nieminen ◽  
Mari Lehtimäki ◽  
Shweta Aggarwal ◽  
Mikael Simons ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neutron Scattering ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Peripheral Membrane Protein

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

2021 ◽  
Author(s):  
Jie Lan ◽  
Chunhui Sun ◽  
Xinping Liang ◽  
Ruixin Ma ◽  
Yuhua Ji ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Transcriptional Activation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Thyroid Dysgenesis ◽  
Type Protein ◽  
Wild Type Protein

Abstract Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.

scholarly journals Membrane-binding properties of the Factor VIII C2 domain

2011 ◽  
Vol 435 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Valerie A. Novakovic ◽  
David B. Cullinan ◽  
Hironao Wakabayashi ◽  
Philip J. Fay ◽  
James D. Baleja ◽  
...  
Keyword(s):  
Factor Viii ◽  
Structural Integrity ◽  
Membrane Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Motif ◽  
C2 Domain ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia

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.

scholarly journals Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-Crystallin on Chaperone Function and Anti-Apoptotic Activity

2021 ◽  
Vol 22 (19) ◽  
pp. 10771
Author(s):  
Sundararajan Mahalingam ◽  
Srabani Karmakar ◽  
Puttur Santhoshkumar ◽  
Krishna K. Sharma
Keyword(s):  
Deletion Mutant ◽  
Mutant Protein ◽  
Wild Type ◽  
Chaperone Function ◽  
Type Protein ◽  
Wild Type Protein ◽  
Cytotoxicity Studies ◽  
Αb Crystallin ◽  
Apoptotic Activity ◽  
Subunit Organization

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

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