High-Resolution NMR Studies of Human Tissue Factor

Nuzzio, Kristin M.; Watt, Eric D.; Boettcher, John M.; Gajsiewicz, Joshua M.; Morrissey, James H.; Rienstra, Chad M.

doi:10.1371/journal.pone.0163206

Cited by 3 publications

(2 citation statements)

References 30 publications

(36 reference statements)

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“…Innovin (Dade Behring, Mississauga, ON) was the source of liposome‐reconstituted purified recombinant human TF. Soluble TF (sTF‐His)39 was expressed in Escherichia coli (strain BL21‐DE3) with a His 6 tag and 5‐amino acid spacer replacing the transmembrane and cytoplasmic domains, and was purified by nickel affinity chromatography. gC was similarly solubilized (gCΔ457t) by substituting the membrane‐spanning domain with His 5 in a baculovirus expression system and was purified using rabbit anti‐gCΔ457t polyclonal antibody (R118) as previously reported 40,41.…”

Section: Methodsmentioning

confidence: 99%

Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced tenase complex oriented on membranes

Lin

Sutherland

Rosell

et al. 2020

Journal of Thrombosis and Haemostasis

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Background The cell membrane‐derived initiators of coagulation, tissue factor (TF) and anionic phospholipid (aPL), are constitutive on the herpes simplex virus type 1 (HSV1) surface, bypassing physiological regulation. TF and aPL accelerate proteolytic activation of factor (F) X to FXa by FVIIa to induce clot formation and cell signaling. Thus, infection in vivo is enhanced by virus surface TF. HSV1‐encoded glycoprotein C (gC) is implicated in this tenase activity by providing viral FX binding sites and increasing FVIIa function in solution. Objective To examine the biochemical influences of gC on FVIIa‐dependent FX activation. Methods Immunogold electron microscopy (IEM), kinetic chromogenic assays and microscale thermophoresis were used to dissect tenase biochemistry. Recombinant TF and gC were solubilized (s) by substituting the transmembrane domain with poly‐histidine, which could be orientated on synthetic unilamellar vesicles containing Ni‐chelating lipid (Ni‐aPL). These constructs were compared to purified HSV1 TF±/gC ± variants. Results IEM confirmed that gC, TF, and aPL are simultaneously expressed on a single HSV1 particle where the contribution of gC to tenase activity required the availability of viral TF. Unlike viral tenase activity, the cofactor effects of sTF and sgC on FVIIa was additive when bound to Ni‐aPL. FVIIa was found to bind to sgC and this was enhanced by FX. Orientation of sgC on a lipid membrane was critical for FVIIa‐dependent FX activation. Conclusions The assembly of gC with FVIIa/FX parallels that of TF and may involve other constituents on the HSV1 envelope with implications in virus infection and pathology.

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Section: Methodsmentioning

confidence: 99%

Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced tenase complex oriented on membranes

Lin

Sutherland

Rosell

et al. 2020

Journal of Thrombosis and Haemostasis

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“…8 Tissue factor (TF), or clotting factors III, also known as cytozyme or thromboplastin, is a single transmembrane glycoprotein containing 263 amino acid residues and holds structural homology with type II cytokine receptors. 9 The complex of TF-FVIIa gradually forms when TF is exposed to blood clotting factor VII/VIIa. Subsequently, TF-FVIIa activates coagulation factors X (FX) and IX (FIX) to initiate the coagulation cascade.…”

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confidence: 99%

Ten Representative Saponins on Tissue Factor Expression in Human Monocytes: Structure–Activity Relationships and Molecular Docking

Zeng

Jiang

et al. 2020

Natural Product Communications

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Saponins have significant bioactivities in treating cardiovascular disease. Whereas there is a lack of in-depth knowledge about how saponins prevent cardiovascular disease. Tissue factor (TF) is the major initiator of the coagulation cascade and plays an important role in hemostasis and thrombosis. However structure–activity relationships (SARs) of saponins inhibiting TF activity have not been discussed in detail at present. To further clarify the relationships between saponins and TF, in this study, 10 representative saponins were selected to study the inhibitory effect on TF procoagulant activity of monocytes by an improved chromogenic substrate method, and the possible SARs were preliminarily analyzed. Furthermore, molecular docking analysis suggested that 4 representative saponins had a good affinity with TF/FVIIa. In addition, a representative saponin, ruscogenin, decreased both messenger ribonucleic acid and protein levels of TF in human monocytes partly due to its downregulation of nuclear factor kappa-light-chain-enhancer of activated B cells and c-Jun N-terminal kinase pathways. In conclusion, this study provides further explanation for the cardiovascular protection of saponins, and the analysis of SARs between inhibiting TF activity and saponins will be helpful to explore the therapeutic TF inhibitors.

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Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy

2017

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Selective isotope labeling is central in NMR experiments and often allows to push the limits on the systems investigated. It has the advantage to supply additional resolution by diminishing the number of signals in the spectra. This is particularly interesting when dealing with the large protein systems which are currently becoming accessible to solid-state NMR studies. Isotope labeled proteins for NMR experiments are most often expressed in E. coli systems, where bacteria are grown in minimal media supplemented with NHCl and C-glucose as sole source of nitrogen and carbon. For amino acids selective labeling or unlabeling, specific amino acids are supplemented in the minimal medium. The aim is that they will be incorporated in the protein by the bacteria. However, E. coli amino-acid anabolism and catabolism tend to interconnect different pathways, remnant of a subway system. These connections lead to inter conversion between amino acids, called scrambling. A thorough understanding of the involved pathways is thus important to obtain the desired labeling schemes, as not all combinations of amino acids are adapted. We present here a detailed overview of amino-acid metabolism in this context. Each amino-acid pathway is described in order to define accessible combinations forC or N specific labeling or unlabeling. Using as example the ABC transporter BmrA, a membrane protein of 600 residues, we demonstrate how these strategies can be applied. Indeed, even though there is no size limit in solid-state NMR, large (membrane) proteins are still a challenge due to heavy signal overlap. To initiate resonance assignment in these large systems, we describe how selectively labeled samples can be obtained with the addition of labeled or unlabeled amino acids in the medium. The reduced spectral overlap enabled us to identify typical spectral fingerprints and to initiate sequential assignment using the more sensitive 2D DARR experiments with long mixing time showing inter-residue correlations.

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High-Resolution NMR Studies of Human Tissue Factor

Cited by 3 publications

References 30 publications

Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced tenase complex oriented on membranes

Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced tenase complex oriented on membranes

Ten Representative Saponins on Tissue Factor Expression in Human Monocytes: Structure–Activity Relationships and Molecular Docking

Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy

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