Journal: PLOS Pathogens

Article Title: Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion

doi: 10.1371/journal.ppat.1011232

Figure Lengend Snippet: A. Induced-fit docking pose of GlcCer with HRTV Gc protein. HRTV Gc ectodomain (PDB: 5YOW) is represented as ribbons and colored by domains (red, domain I; yellow, domain II; blue, domain III). The fusion loops at the tip of domain II are displayed in orange. The bound head group of GlcCer (framed) is drawn as sticks and colored according to atom type (carbon, green; nitrogen, blue; oxygen, red). B. Interaction of GlcCer with HRTV Gc protein in the lipid-head-group binding pocket. The hydrogen bonds between GlcCer and Gc protein are represented as dashed lines and colored in magenta. C. GlcCer binding groove. The HRTV Gc lipid-head-group binding pocket is shown in surface representation and colored according to electrostatic potential. D. 2D representation of docking interaction of GlcCer with binding site residues of HRTV Gc. The hydrogen bonds are shown in magenta lines. E. HRTV Gc protein directly binds to DOPC, GalCer, and GlcCer. Serially diluted DOPC, GalCer, and GlcCer were spotted onto the PVDF membrane. Protein-lipid overlay assay was performed with an anti-6xHis antibody. F. SPR assay of HRTV Gc binding kinetics to the indicated liposomes at neutral pH. All experiments were performed as duplicates, and the calculated fit is shown as a black line. Equilibrium constants (K D ) are determined by measuring the association rate constant (K a ) and dissociation rate constant (K d ). G. Binding kinetics of HRTV Gc to the indicated liposomes at acidic pH. Sensorgrams measured in replicates by bio-layer interferometry (BLI) and fitting (black) of the interaction of Gc to the indicated liposomes.

Article Snippet: NB-DNJ (Caymanchem, 21065), UGT8 Inhibitor 19 (Caymanchem, 32723), GalCer (Avanti polar lipids, 860521), GlcCer (Avanti polar lipids, 860539), LacCer (Matreya, 1507), OptiPrep density gradient medium (Sigma-Aldrich, D1556), DiOC18 (Thermofisher, D275), R18 (Thermofisher, O246), NBD-GlcCer (Matreya, 1622–001), NBD-GalCer (Matreya, 1633–001), DOPC (Avanti polar lipids, 850375), cholesterol (Avanti polar lipids, 700000), pHrodo green dextran (Invitrogen, P35368) were purchased from the indicated manufacturers.

Techniques: Binding Assay, Membrane, Protein-lipid Overlay Assay (PLOA), SPR Assay, Liposomes

Journal: Communications biology

Article Title: Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

doi: 10.1038/s42003-019-0304-y

Figure Lengend Snippet: Fig. 1 The actions of βγ-CAT were inhibited by AGSLs (gangliosides and sulfatides) via direct interactions. a THP-1 cells were treated with gradient concentrations of neuraminidase and then incubated with βγ-CAT. The IL-1β concentration in the supernatant was then measured by ELISA. *P < 0.05 and **P < 0.01 versus control by using unpaired two-tailed Student’s t-test (n = 3). b THP-1 cells were treated with different types of glycosidase and then incubated with βγ-CAT. The IL-1β concentrations in the supernatant was measured by ELISA. NS, not significant versus control by using unpaired two-tailed Student’s t-test (n = 3). c FITC-labeled βγ-CAT was incubated with different concentrations of gangliosides. The membrane binding of βγ-CAT to THP-1 cells was determined by flow cytometry. The untreated cells were used as a negative control. d βγ-CAT was incubated with different concentrations of gangliosides and added to THP-1 cells. The oligomerization of βγ-CAT was detected by western blotting. The immunoblots are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 7. e FITC-labeled βγ-CAT was incubated with different concentrations of sulfatides and then mixed with THP-1 cells. The membrane binding of βγ-CAT was determined by flow cytometry, the untreated cells were used as a negative control. f βγ-CAT was incubated with different concentrations of sulfatides, then mixed with THP-1 cells. The oligomerization of βγ-CAT was detected by western blotting. The immunoblots are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 7. g βγ-CAT was incubated with different concentrations of lipids and then added to LPS-primed THP-1 cells. The IL-1β concentrations in the supernatant were measured by ELISA. *P < 0.05, **P < 0.01 and ***P < 0.001 represent gangliosides and sulfatides versus sphingomyelin by two-way ANOVA with Bonferroni correction (n = 3). h, i The direct interaction between βγ-CAT and gangliosides (h) or sulfatides (i) was determined using a BLI assay. The BLI interaction curves are representative of three independent experiments. Bars represent the mean ± SD from three independent experiments per condition in a, b and g

Article Snippet: Sulfatides were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test, Labeling, Membrane, Binding Assay, Cytometry, Negative Control, Western Blot

Journal: Communications biology

Article Title: Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

doi: 10.1038/s42003-019-0304-y

Figure Lengend Snippet: Fig. 2 The BmALP1 and BmTFF3 subunits of βγ-CAT bound to gangliosides and sulfatides, respectively. a Schematic graph of βγ-CAT, BmALP1 subunit and BmTFF3 subunit. The BmALP1 subunit and BmTFF3 subunit of βγ-CAT have apparent molecular weights of 38 kDa and 18 kDa, respectively. b Interactions between MBP-αN and MBP-αC with gangliosides was detected by BLI assay. c Interactions between MBP-αN and MBP-αC with sulfatides was detected by BLI assay. d The binding kinetic curves between MBP-αC and gangliosides were determined by BLI assay. e βγ-CAT was incubated with different concentrations of anti-BmTFF3 polyclonal antibody or control IgG and added to LPS-primed THP-1 cells. The IL-1β concentration in the supernatant was measured by ELISA. Bars represent the mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. control IgG by using unpaired two- tailed Student’s t test (n = 3). f FITC-labeled βγ-CAT was incubated with an anti-BmTFF3 polyclonal antibody or control rabbit IgG and added to THP-1 cells, the binding of βγ-CAT with THP-1 cells was evaluated by flow cytometry. Untreated THP-1 cells are indicated as normal, and THP-1 cells treated with FITC-labeled βγ-CAT are indicated as the control. g βγ-CAT was incubated with an anti-BmTFF3 polyclonal antibody or control rabbit IgG and then added to THP-1 cells, the oligomerization of βγ-CAT was detected by western blotting. The immunoblots are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 8. h The binding of BmTFF3 with different types of sphingolipids was determined by protein-lipid overlay assay. The blots are representative of three independent experiments, the original images of blots are shown in Supplementary Figure 8. i The binding kinetic curves between BmTFF3 and sulfatides was determined by BLI assay. The BLI interaction curves in b–d and i are representative of three independent experiments

Article Snippet: Sulfatides were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA).

Techniques: Binding Assay, Incubation, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Labeling, Cytometry, Western Blot, Protein-lipid Overlay Assay (PLOA)

Journal: Communications biology

Article Title: Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

doi: 10.1038/s42003-019-0304-y

Figure Lengend Snippet: Fig. 4 AGSLs of the cell membrane were essential for the actions of βγ-CAT. a THP-1 cells were treated with PPMP and then incubated with FITC-labeled βγ-CAT. The binding between βγ-CAT and THP-1 cells was detected by flow cytometry. b The oligomerization of βγ-CAT was determined by western blotting. The immunoblots are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 12. c The IL-1β release induced by βγ-CAT was measured by ELISA. *P < 0.05 vs. control by using unpaired two-tailed Student’s t test (n = 3). d–f THP-1 cells in which ganglioside expression was knocked down or gangliosides re-addition, then incubated with βγ-CAT. The binding between βγ-CAT and THP-1 cells was detected by flow cytometry (d), the oligomerization of βγ-CAT was detected by western blotting (e), and the IL-1β concentration was measured by ELISA (f). *P < 0.05 vs. the respective control by using unpaired two-tailed Student’s t test (n = 3). The immunoblots (e) are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 13. g–i THP-1 cells in which sulfatide expression was knocked down or sulfatides re-addition, then incubated with βγ-CAT. The binding between βγ-CAT and THP-1 cells was detected by flow cytometry (g), the oligomerization of βγ-CAT was detected by western blotting (h), and the IL-1β concentration was measured by ELISA (i). *P < 0.05 vs. the respective control by using unpaired two-tailed Student’s t test (n = 3). The immunoblots (h) are representative of three independent experiments, the original images of immunoblots are shown in Supplementary Figure 13. In flow cytometry (a, d, g), the untreated THP-1 cells are indicated as normal, and THP-1 cells treated with FITC-labeled βγ-CAT are indicated as control. Bars represent the mean ± SD from three independent experiments per condition in c, f, i

Article Snippet: Sulfatides were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA).

Techniques: Membrane, Incubation, Labeling, Binding Assay, Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test, Expressing, Concentration Assay

Journal: Communications biology

Article Title: Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

doi: 10.1038/s42003-019-0304-y

Figure Lengend Snippet: Fig. 6 The proposed action model and pathway of βγ-CAT. As a vertebrate-secreted ALP and TFF protein complex, βγ-CAT exerts its functions via an assembly process. The actions of assembled βγ-CAT can be divided into four steps. Step 1. Membrane binding. Once the assembled βγ-CAT encounters target cells, βγ-CAT is subsequently anchored to lipid raft microdomains on the plasma membrane. Therefore, the BmALP1 subunit of βγ-CAT binds to the extracellular glycan headgroup of gangliosides, and the BmTFF3 subunit of βγ-CAT binds to the extracellular glycan headgroup of sulfatides. Step 2. After βγ-CAT binding to the membrane, the BmTFF3 subunit detaches from the membrane, and the BmALP1 subunit of βγ-CAT is endocytosed and enters the cell. Step 3. The endocytic βγ-CAT then oligomerizes and forms pores in the membranes of endolysosomes. Step 4. The formed pores induce a flux of substances, such as ions, ultimately regulating the properties of endolysosomes. As we observed previously, changes in the properties of endolysosomes can trigger various effects, such as the expulsion of pathogen-containing vesicles16 and increased lysosome destabilization, leading to inflammasome- dependent IL-1β release15 and tissue repair10

Article Snippet: Sulfatides were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA).

Techniques: Membrane, Binding Assay, Clinical Proteomics, Glycoproteomics