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Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus.

ABSTRACT: The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val¹¹⁵, ASP¹¹⁶, Val¹¹⁷, and Lys¹²²) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein-toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

SUBMITTER: Qiao K

PROVIDER: S-EPMC8065724 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease.

Project description:Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.

| S-EPMC7028320 | biostudies-literature

Critical von Willebrand factor A1 domain residues influence type VI collagen binding.

Project description:BACKGROUND:von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. OBJECTIVES:We report here on several mutations that exclusively alter binding to type VI collagen. PATIENTS/METHODS:Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. RESULTS:Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. CONCLUSIONS:VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.

| S-EPMC3809952 | biostudies-literature

Increase of von Willebrand factor with aging in type 1 von Willebrand disease: fact or fiction?

Project description: Not available

| S-EPMC5664402 | biostudies-literature

A common mechanism by which type 2A von Willebrand disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand factor A2 domain FRET construct.

Project description:Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.

| S-EPMC5706690 | biostudies-literature

Molecular coevolution of coagulation factor VIII and von Willebrand factor.

Project description:Ancestral sequence reconstruction provides a unique platform for investigating the molecular evolution of single gene products and recently has shown success in engineering advanced biological therapeutics. To date, the coevolution of proteins within complexes and protein-protein interactions is mostly investigated in silico via proteomics and/or within single-celled systems. Herein, ancestral sequence reconstruction is used to investigate the molecular evolution of 2 proteins linked not only by stabilizing association in circulation but also by their independent roles within the primary and secondary hemostatic systems of mammals. Using sequence analysis and biochemical characterization of recombinant ancestral von Willebrand factor (VWF) and coagulation factor VIII (FVIII), we investigated the evolution of the essential macromolecular FVIII/VWF complex. Our data support the hypothesis that these coagulation proteins coevolved throughout mammalian diversification, maintaining strong binding affinities while modulating independent and distinct hemostatic activities in diverse lineages.

| S-EPMC7876868 | biostudies-literature

Von Willebrand Factor Type A domain of hCLCA1 is sufficient for U-937 macrophage activation.

Project description:The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1β mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.

| S-EPMC6444176 | biostudies-literature

Monitoring of von Willebrand factor inhibitors in patients with type 3 von Willebrand disease using a quantitative assay.

Project description:BackgroundAntibodies inhibiting von Willebrand factor (VWF) develop in a subset of patients with type 3 von Willebrand disease (VWD3) and may be detected by their inhibition of ristocetin cofactor activity (VWF:RCo). Some also inhibit factor VIII activity (VIII:C).AimTo describe monitoring of ten VWD3 patients for VWF inhibitors using a quantitative assay.MethodsVWF inhibitor was measured by comparing VWF:RCo activity of a mix of patient and pooled normal plasma (PNP) with a mix of buffer and PNP, using agglutination of fixed normal platelets in microtiter plates or lyophilized platelets in an aggregometer. VIII:C inhibitor was measured by Bethesda assay. Preanalytical heat treatment of patient plasma was used during treatment episodes.ResultsFour of 10 patients monitored developed VWF inhibitors, two detected during bleeding episodes refractory to treatment and two on routine screening. Data from the first five patients were used to establish an arbitrary unit, VWU, defined as the amount of inhibitor per millilitre of patient plasma inactivating 25% of the activity of 1 mL of PNP. In three of four patients, both VWF:RCo and VIIII:C were inhibited at some time points, although VIII:C inhibition sometimes disappeared. In one patient, no VIII:C inhibition was seen. Two patients remained inhibitor positive more than 15 years after inhibitor detection, one became negative following immune tolerance induction, and one was deceased.ConclusionsVWF inhibitors can be quantitatively monitored in VWD3 patients. Preanalytical heat treatment may be required for their detection post infusion.

| S-EPMC8667261 | biostudies-literature

Destabilization of the von Willebrand factor A2 domain under oxidizing conditions investigated by molecular dynamics simulations.

Project description:The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib ? (GpIb?). This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIb?.

| S-EPMC6141083 | biostudies-literature

Functional characterization of a 13-bp deletion (c.-1522_-1510del13) in the promoter of the von Willebrand factor gene in type 1 von Willebrand disease.

Project description:We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5' of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.

| S-EPMC2981484 | biostudies-literature

Calcium stabilizes the von Willebrand factor A2 domain by promoting refolding.

Project description:Von Willebrand factor (VWF) is a large, multimeric plasma glycoprotein that critically mediates hemostasis at sites of vascular injury. Very large VWF multimers have the greatest thrombogenic activity, which is attenuated by cleavage in the A2 domain by the metalloproteinase ADAMTS13. ADAMTS13 proteolysis requires mechanical force to expose the scissile bond and is regulated by a calcium-binding site within A2. In this study, we characterized the interaction between VWF A2 and calcium by examining the effect of calcium on VWF A2 stability and mechanical unfolding and refolding. Isothermal calorimetry yielded a calcium binding K(d) = 3.8 ± 1.0 μM and reversible thermal denaturation showed that 5 mM calcium stabilized the unfolding transition from 56.7 ± 0.1 to 69.1 ± 0.1 °C. Using optical tweezers to apply tensile force to single domains, we found that calcium did not affect VWF A2 unfolding, but rather enhanced refolding kinetics fivefold, resulting in a 0.9 kcal/mol stabilization in the folding activation energy in the presence of calcium. Taken together, our data demonstrate that VWF binds calcium at physiologic calcium concentrations and that calcium stabilizes VWF A2 by accelerating refolding.

| S-EPMC3309790 | biostudies-literature

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