Journal: Nature Communications

Article Title: Histone functions as a cell-surface receptor for AGEs

doi: 10.1038/s41467-022-30626-8

Figure Lengend Snippet: a Binding of DHA-modified Bt-PA to histone H2B. Recombinant histone H2B immobilized on ELISA plate was incubated with either Bt-PA or DHA-Bt-PA. The binding was detected using streptavidin-HRP. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). b HPLC analysis of DHA-modified Bt-PA capable of binding to H2B. The reaction mixture of Bt-PA and DHA was separated by reverse-phase HPLC, and each fraction was measured for the binding to H2B (middle ) and anti-AGEs antibodies (bottom). The reactions were monitored with absorbance at 200–600 nm (top). The dashed square indicates the region with the binding activity to H2B and anti-AGEs antibody. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). c Binding of modified Bt-PAs to histone H2B. Bt-PA was incubated with DHA, glucose (Glc), and glucose metabolites, such as glycolaldehyde (GA), glyoxal (GO), methylglyoxal (MG), glyceraldehyde (GCA), and 3-deoxyglucosone (3-DG), and the binding to histone H2B was evaluated by solid-phase binding assay. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). d Surface plasmon resonance measurements. The interaction of H2B and either BSA or AGEs (10 µg/ml) was monitored. e Effect of NaCl on binding of AGEs to H2B. H2B immobilized on ELISA plate was incubated with Bt-AGEs (5 μg/ml) under the indicated concentrations of NaCl. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Dunnett’s test (two-sided), relative to AGEs treatment under 0.15 M NaCl. f Binding of acylated proteins to the recombinant histone H2B. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Dunnett’s test (two-sided), relative to the no-treatment control. g Involvement of H2B N -terminal tail region and its electrostatic property in the binding to AGEs. MBP-fusion H2B full length (FL), N -terminal (N-term. (1-35)), N -terminal deletion mutant (ΔN35), acetylated Nterm. (1-35), or N -terminal scrambled fragments (scrambled 1, 2, and 3) were subjected to the solid phase binding assay. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant ( p > 0.05).

Article Snippet: J774A.1 cells, cultured on glass-based dishes, were preincubated with anti CD16/CD32 mAb (93, Biolegend) to block the FcγRII/III receptors and incubated with anti-histone H2B mAb (5HH2-2A8, Millipore, 1:200 dilution) and either 100 μg/ml Bt-BSA or Bt-AGEs at 4 °C for 15 min in HBSS-BSA.

Techniques: Binding Assay, Modification, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Activity Assay, SPR Assay, Control, Mutagenesis

Journal: Nature Communications

Article Title: Histone functions as a cell-surface receptor for AGEs

doi: 10.1038/s41467-022-30626-8

Figure Lengend Snippet: a Binding of AGEs to mouse peritoneal cells. Left histogram, peritoneal cells were incubated alone (black line) or with Bt-BSA (blue line) or Bt-AGEs (red line), stained with streptavidin-APC, and analyzed by flow cytometry. Right two panels, Stained cells with CD11b and F4/80 were analyzed without or with gating on APC (+). b Cell-type specificity for AGEs binding. Peritoneal cells were incubated alone (NT) or with Bt-BSA (BSA) or Bt-AGEs (AGEs) and stained with streptavidin-BV421. Macrophages, Monocytes, Dendritic cells, Neutrophils, Eosinophils were gated and the bindings of Bt-BSA or Bt-AGEs were expressed as median fluorescent intensity (MFI) of BV421. Data are mean ± S.D. ( n = 4, biologically independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant ( p > 0.05). c , d Binding of AGEs to J774A.1 ( c ) and RAW264.7 ( d ) cells. The cells were incubated alone (black line) or with either Bt-BSA (blue line) or Bt-AGEs (red line) and analyzed by flow cytometry. e Colocalization of H2B with AGEs. J774A.1 cells were treated with either Bt-BSA or Bt-AGEs and stained with anti-H2B antibody and streptavidin-alexa568. Scale bar, 10 µm. Representative image of three independent experiments. f Pull-down assay for the detection of H2B as a cell-surface AGEs-binding protein. J774A.1 cells were incubated with Bt-BSA or Bt-AGEs and then incubated with DTSSP. The detergent-resistant membrane fractions were subjected to pull-down, and the resulting precipitates were detected by western blotting using an anti-H2B antibody. Representative image of two independent experiments. g Inhibition of AGEs binding by anti-histones or anti-AGEs receptors antibodies. Cells were preincubated with antibody against histone H1, H2A, H2B, H3, H4, RAGE, AGER1, or CD36 prior to the treatment with Bt-AGEs. Relative AGEs bindings were determined by the MFI of APC. Data are mean ± S.D. ( n = 4 for histone antibodies and n = 3 for known AGEs receptors antibodies, biologically independent experiments). Dunnett’s test (two-sided), relative to the control. h Effect of H2B overexpression on AGEs binding. J774A.1 cells were transfected with a bicistronic construct encoding H2B followed by IRES driving the translation of GFP (upper left panel) and the binding of Bt-AGEs was analyzed ( n = 3, biologically independent experiments). Paired t test (two-sided).

Article Snippet: J774A.1 cells, cultured on glass-based dishes, were preincubated with anti CD16/CD32 mAb (93, Biolegend) to block the FcγRII/III receptors and incubated with anti-histone H2B mAb (5HH2-2A8, Millipore, 1:200 dilution) and either 100 μg/ml Bt-BSA or Bt-AGEs at 4 °C for 15 min in HBSS-BSA.

Techniques: Binding Assay, Incubation, Staining, Flow Cytometry, Pull Down Assay, Membrane, Western Blot, Inhibition, Control, Over Expression, Transfection, Construct

Journal: Nature Communications

Article Title: Histone functions as a cell-surface receptor for AGEs

doi: 10.1038/s41467-022-30626-8

Figure Lengend Snippet: a Effect of AGEs on Plg binding to histone H2B in vitro. Recombinant H2B coated on ELISA plate was preincubated with either BSA or AGEs (1, 10, and 100 µg/ml), followed by treatment with Bt-Plg (1 µg/ml). Plg binding to H2B was detected with streptavidin-HRP. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Dunnett’s test (two-sided), relative to the treatment with Plg alone. b Surface plasmon resonance measurements. The interactions of Plg (10 µg/ml) with H2B immobilized on a Biacore sensor chip NTA, following the addition of either BSA or AGEs (10 or 100 µg/ml) were monitored. c Schematic representation of full length and deletion mutants of H2B (upper panel) and effects of AGEs on Plg binding to deletion mutants of H2B (lower panel). H2B mutants coated on ELISA plate were preincubated with either BSA or AGEs (100 µg/ml), followed by treatment with Bt-Plg (1 µg/ml). Plg binding to H2B mutants was detected with streptavidin-HRP. The absorbance value in the absence of competitor was defined as 100% Plg binding. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Student’s t test (two-sided), n.s. not significant ( p > 0.05). d Effect of BSA or AGEs on Plg binding to J774A.1 e Effect of BSA or AGEs on Plg binding to peritoneal macrophage. Cells were preincubated with either BSA, AGEs, or tranexamic acid (a Plg inhibitor). After further incubation with Bt-Plg, Plg binding to cells was analyzed by FACS with streptavidin-APC. Plg binding (% of control) was calculated as described in the Materials and Methods section. Data are mean ± S.D. ( n = 3 and 6, respectively, biologically independent experiments). Student’s t test (two-sided). f , g Effect of AGEs on plasmin formation in the presence of recombinant H2B ( f ) or J774A.1 cells ( g ). Plasmin was measured by the absorbance at 405 nm using plasmin specific substrate S-2251. The rate of plasmin generation was calculated as described in the Materials and Methods section. Data are mean ± S.D. ( n = 3 and 5, respectively, biologically independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant ( p > 0.05).

Article Snippet: J774A.1 cells, cultured on glass-based dishes, were preincubated with anti CD16/CD32 mAb (93, Biolegend) to block the FcγRII/III receptors and incubated with anti-histone H2B mAb (5HH2-2A8, Millipore, 1:200 dilution) and either 100 μg/ml Bt-BSA or Bt-AGEs at 4 °C for 15 min in HBSS-BSA.

Techniques: Binding Assay, In Vitro, Recombinant, Enzyme-linked Immunosorbent Assay, SPR Assay, Incubation, Control

Journal: Nature Communications

Article Title: Histone functions as a cell-surface receptor for AGEs

doi: 10.1038/s41467-022-30626-8

Figure Lengend Snippet: a In vitro infiltration assay. J774A.1 cells were incubated alone or with BSA or AGEs in the presence of Plg. The infiltration activity was assessed by the number of cells across the Matrigel (mean ± S.D., n = 3, biologically independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant ( p > 0.05). b Effect of H2B or RAGE on AGEs-dependent suppression of infiltration. Cells were preincubated with anti-H2B or anti-RAGE antibody and the infiltration activity was assessed (mean ± S.D., n = 3, biologically independent experiments). Tukey–Kramer tests (two-sided), n.s., not significant ( p > 0.05). c Effect of BSA or AGEs on the peritoneal infiltration. BSA or AGEs was given daily by intravenous administration and the number of peritoneal cells was determined 0, 24, 48, and 72 h after TG injection (mean ± S.D., n = 3 for BSA-0 h, AGEs-0 h, and BSA-24 h, n = 4 for BSA-48 h and AGEs-48 h, n = 5 for AGEs-24 h and BSA-72 h, n = 6 for AGEs-72 h, biologically independent experiments). Student’s t test (two-sided). d Inhibition of Plg activation abrogate the effect of AGEs on macrophage infiltration. Mice were administrated with TXA, and the number of peritoneal macrophages was determined 72 h after TG injection (mean ± S.D., n = 5 for AGEs+TXA group and n = 6 for other groups, biologically independent experiments). Student’s t test (two-sided), n.s. not significant ( p > 0.05). e Effect of AGEs on MMP-9 activation. MMP-9 in peritoneal lavage was purified and detected with MMP-9 antibody (upper panel), and act/proMMP-9 ratio were quantified (lower panel; mean ± S.D., n = 4, biologically independent experiments). Dunnett’s test (two-sided), relative to the control. f Expression levels of proinflammatory and anti-inflammatory macrophage markers in peritoneal macrophage. Data are relative expression to BSA-treated mice (mean ± S.D., n = 7, biologically independent experiments). Student’s t test (two-sided). g Effect of AGEs on sepsis model. Mice were treated with saline ( n = 9, black line), BSA ( n = 6, gray line), or AGEs ( n = 7, brown line) 4 h after the CLP procedure. There were significant differences in the treatment with saline vs. AGEs and with BSA vs. AGEs, but not with saline vs. BSA ( p = 0.0004, 0.0099, and 0.4154, respectively, log-rank test).

Article Snippet: J774A.1 cells, cultured on glass-based dishes, were preincubated with anti CD16/CD32 mAb (93, Biolegend) to block the FcγRII/III receptors and incubated with anti-histone H2B mAb (5HH2-2A8, Millipore, 1:200 dilution) and either 100 μg/ml Bt-BSA or Bt-AGEs at 4 °C for 15 min in HBSS-BSA.

Techniques: In Vitro, Incubation, Activity Assay, Injection, Inhibition, Activation Assay, Purification, Control, Expressing, Saline