نتائج البحث

An internal thioester in a pathogen surface protein mediates covalent host binding

• Authors : Walden, Miriam; Edwards, John M; Dziewulska, Aleksandra M ...

• Source: eLife ; volume 4 ; ISSN 2050-084X

تفاصيل العنوان

Figure 3—figure supplement 1. Effect of acetylation of Lys side chains on fibrinogen (Fg):TED adduct formation. ; The hash symbol indicates protein pre-incubated with sulfo-NHS acetate. Red asterisks highlight the positions of adduct bands.

تفاصيل العنوان

Figure 3. SDS-PAGE analysis of TED:fibrinogen (Fg) adduct formation. ; (A) Stable adducts between fibrinogen and TEDs revealed by SDS-PAGE. Red asterisks, adduct bands. (B) Cys-to-Ala mutants of TEDs do not result in adduct formation after incubation with Fg. (C) Schematic representation of the intermolecular isopeptide bond formed between a Lys residue of fibrinogen Aα and the Gln residue of the TED.

تفاصيل العنوان

Figure 4—figure supplement 1. TED:fibrinogen (Fg) adduct formation in plasma pull down assays. ; An overloaded gel of the plasma pull-down better shows adduct formation between fibrinogen and TEDs, and its dependence on the thioester. P = plasma depleted of the majority of albumin by blue sepharose chromatography. Red asterisks, adduct bands, ‘a’ and ‘d’ denote human serum albumin and Fg γ dimer, respectively.

تفاصيل العنوان

Figure 4. TED:fibrinogen (Fg) adduct formation in plasma pull down assays. ; (A) Schematic representation of the isopeptide domain (green) complementation used in the pull-down assays. Pink triangle, tobacco etch virus protease cleavage site. (B) SfbI and FbaB-TEDs pull down fibrinogen from blood plasma, forming covalent adducts (red asterisks). ‘a’ denotes bands corresponding to human serum albumin. Bands below 31 kDa are breakdown products of tagged TEDs.

تفاصيل العنوان

Figure 5—figure supplement 1. Acetylation of Lys residues in the fibrinogen Aα subunit in absence and presence of SfbI-A40-TED. ; Sequence coverage by peptide mass fingerprinting is indicated by colors mapped onto the sequence of human fibrinogen subunit Aα. Grey, signal sequence (not present in mature fibrinogen Aα); red, trypsin coverage; blue and green, additional coverage obtained by endoproteinase GluC and chymotrypsin digestion, respectively; black, no coverage. All Lys residues are highlighted in boldface. Underlined K indicates that the residue was observed in its acetylated form; non-underlined K were observed only in the non-acetylated form.

تفاصيل العنوان

Figure 5—figure supplement 2. Structure of fibrinogen with important sites labeled. ; The positions of Lys100 (magenta) and RGDF platelet-binding motif (cyan) are highlighted on the crystal structure of Fg (PDB:3GHG). α, β and γ chains are colored blue, green and red, respectively.

تفاصيل العنوان

Figure 5. Mass spectrometric identification of the SfbI-TED and FbaB-TED target residue in fibrinogen (Fg). ; (A) Precursor ion masses identified in tryptic-digest nanoLC-MSE of excised adduct gel bands for different samples. (B) Several charged states are observed in nanoLC-MSE of cross-linked precursor peptides obtained by tryptic digestion of adducts formed by TEDs with fibrinogen or in plasma pull-downs. (C) Fragmentation nanoLC-MSE spectra of the cross-linked precursor obtained for SfbI-A40-TED in a plasma pull-down. The low and high mass range spectrum is shown on the left and right, respectively. Fragmentations observed in nanoLC-MSE are indicated in the schematic drawings above the spectra by hooks, y-series on top of sequences, b-series below sequences. Numbers correspond to positions in the cross-linked fragments; red, SfbI-A40-TED; blue, fibrinogen Aα.

تفاصيل العنوان

Figure 6. The weak integrity of the BBB in the fetal dentate. ; (A) E17.5 embryos and P1 pups were perfused with biotin-conjugated 70 KDa dextran and sections of the DG were stained for biotin. Arrows indicate the dextran outside blood vessels. (B) Gli1-CreERt2 pups (TM at E17.5) were used to stain hedgehog-responding cells (GFP+) and blood vessels (PECAM). Hedgehog-responding Gli1+ dentate progenitors have endfeet wrapping blood vessels forming neurovascular units. Magnified images on the right panels show representative GFP + cells. Yellow dashed lines mark the meningeal blood vessels of the DG. (C) P5 Gli1-GFP pups were perfused with biotin-conjugated cadaverine to reveal the area of the DG with leaky blood vessels. GFP + hedgehog-responding cells are surrounded by the dentate granule neurons uptaking the dyes (arrows). Dashed lines mark the meningeal blood vessels of the DG. (D) P5 CD1 pups were perfused with biotin-conjugated 70 KDa dextran and sections were stained for biotin. (D′) Biotin signals from the section of (D) were used to measure the permeability of blood vessels in the hippocampus (CA1), and the DG (DG). The area (100 μm2) surrounding blood vessels was selected to count the transcytosed dextran dyes (n = 6). (E) Staining for fibrinogen (Fib), a marker for leaky blood vessels, was conducted at E17.5 and P1 in the DG. Arrows indicate the leaky blood vessels. (F) CD41, a marker for platelets, was used to stain the dermal platelets (including megakaryocytes) and circulating platelets in sagittal sections at P1. The boxed area is presented as a high-power image on the right. Student t-test was used to address the statistical significance. ***, p < 0.001. Scale bars: A, D, F = 200 μm, B, C, E = 100 μm.

تفاصيل العنوان

Figure 7. Thioester-dependent binding of SfbI-A40-TED to A549 cells. ; (A) Schematic representation of the covalent cell labeling experiment. After incubation with cells, isopep-tag (iPT) labeled TED is detected by GFP fused to sIPD. (B) SfbI-A40-TED and SfbI-A40-TED:Cys109Ala binding to A549 cells before and after incubation with interleukin-6 (IL6) and dexamethasone (dex). No TED, sIPD-GFP controls. Cell nuclei appear in blue. Scale bars, 200 μm. (C) Western blot of whole-cell extracts of induced and non-induced A549 cells, probed with an anti-fibrinogen α antibody. The same membrane was re-probed with an anti-β-actin antibody (shown in separate panel). MW, molecular weight markers; TED, cells incubated with SfbI-A40-TED-iPT; TED C-A, cells incubated with SfbI-A40-TED:Cys109Ala-iPT.

تفاصيل العنوان