Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Alpha-1-antitrypsin (α 1 AT) was identified from hemofiltrate library as PT inhibitor. A – C , PT (10 ng/ml = 0.0000962 μM) and 20 μl of a fraction of the hemofiltrate library or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. Cells were left untreated as further control. Then, the cells were lysed and Gαi which has not been ADP-ribosylated during the intoxication with PT was ADP-ribosylated and biotin-labeled via the incubation with PTS1 and biotin-labeled NAD + . Subsequently, the biotin-labeled Gαi was detected via Western Blot, while Hsp90 served as control for equal protein loading. The bar graph ( A ) shows the quantifications of Western Blot signals from one experiment testing the samples as single values or duplicates depending on sample availability, while ( B , C ) show the corresponding Western blot images. The intensity values of the bar graph are given as x-fold of the untreated control (Con), normalized to Hsp90, mean ± SD (n ≥ 1). D , PT (10 ng/ml) and 20 μl of the respective chromatographic subfractions or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. Cells were left untreated as further control. Subsequently, the experiment was performed as described in ( A ). The intensity values of the bar graph are given as x-fold of the untreated control (Con), normalized to Hsp90 staining, mean ± SD (at least n = 1 at most n = 2 from two independent experiments). ( Blue line : chromatogram from the chromatographic fractionation process, gray bars = screening result, green bars : screening results that were considered as hits and or subjected to further chromatographic fractionation). E , sample carbamidomethylation, followed by proteolytic digestion, and mass spectrometry analysis revealed the presence of α 1 AT. The sequence coverage compared to the mature polypeptide was 93.5% with a α 1 AT precursor amino acid sequence range of 25 to 418. F , α 1 AT (Uniprot: P01009-A1AT_HUMAN) was the main component of the active chromatographic subfractions 34_55_56 to 58. Abbreviations are used to identify all chromatographic fractions. From left to right , the number of the active chromatographic fraction used for each next fractionation step is given in chronological order. Therefore, 34_55_56 to 58 is referred to as the chromatographic fractions 56 to 58 derived from fraction 34_55, obtained from fraction 34. Subfractions 34_55_56 to 58 are 56, 57, and 58, obtained from 34_55 ( Fig. 1 C , right side), which showed the highest activity and were subjected together to sequencing. We used this nomenclature to abbreviate the names. The same applies to the other names, for example, 35_48 to 49 indicates fractions 48 and 49 from the fractionation of 35. FCS, fetal calf serum; Gαi, α-subunit of inhibitory G protein.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Solvent, Incubation, Control, Labeling, Western Blot, Staining, Fractionation, Mass Spectrometry, Sequencing, Derivative Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Effect of α 1 AT on ADP-ribosylation status of Gαi in PT-treated CHO-K1 and A549 cells. A , schematic representation of experimental setup for the sequential ADP-ribosylation assay. PT and α 1 AT were either directly added to cells for 4 h or preincubated for 15 min before addition to cells. B – C , PT (10 ng/ml) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. Cells were left untreated as further control. After the incubation, the cells were lysed and Gαi, which had not been ADP-ribosylated during the intoxication with PT, was ADP-ribosylated and biotin-labeled via the incubation with PTS1 and biotin-labeled NAD + . Subsequently, the biotin-labeled Gαi was detected via Western blot, while Hsp90 served as a control for equal protein loading. The bar graph ( B ) shows the quantification of Western blot signals from four independent experiments, while ( C ) shows results of a representative experiment. The intensity values of the bar graph are given as x-fold of the untreated control (Con), normalized to Hsp90, mean ± SEM (n = 8 values from four independent experiments). D and E , PT (10 ng/ml) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were preincubated for 15 min at room temperature and added to CHO-K1 cells in FCS-free medium for 4 h at 37 °C. Cells were left untreated as further control. Subsequently, the experiment was performed as described in ( B and C ). Values are given as mean ± SEM (n = 8 values from four independent experiments). F and G , PT (50 ng/ml = 0.000476 μM) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added directly to A549 cells in FCS-free medium and incubated for 4 h at 37 °C. Cells were left untreated as further control. Subsequently, the experiment was performed as described in ( B and C ). Values are given as mean ± SEM (n = 8 values from four independent experiments). H and I , PT (50 ng/ml) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were preincubated for 15 min at room temperature and added to A549 cells in FCS-free medium for 4 h at 37 °C. Cells were left untreated as further control. Subsequently, the experiment was performed as described in ( B and C ). Values are given as mean ± SEM (n = 8 values from four independent experiments). J and K , PT (10 ng/ml) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-containing medium and incubated for 4 h at 37 °C. Cells were left untreated as further control. Subsequently, the experiment was performed as described in ( B and C ). Values are given as mean ± SEM (n = 6 values from three independent experiments). L and M , different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h ( L ) or 24 h ( M ) at 37 °C. As a positive control for cell death, 20% DMSO was added. After the incubation, the MTS reagent was added to the cells and incubated for 45 min at 37 °C. The absorbance was measured using a plate reader at 490 nm. The intensity values of the bar graph are given as x-fold of the untreated control (Con), mean ± SEM (n = 9 values from three independent experiments). ( B , D , F , H , J , L , and M ) significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only ( B , D , F , H , and J ) or untreated control (Con) ( L and M ) (∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns not significant). α 1 AT, α 1 -antitrypsin; DMSO, dimethylsulfoxide; FCS, fetal calf serum; Gαi, α-subunit of inhibitory G protein; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PT, pertussis toxin; WB, Western blot.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Solvent, Incubation, Control, Labeling, Western Blot, Positive Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Effect of the serpin proteins α 1 AT and antithrombin on intoxication of CHO-K1 cells with PT. A – C , PT (10 ng/ml) and α 1 AT (100 μM), antithrombin (AT) (100 μM), and fondaparinux (Fon) (1 g/l) or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells in FCS-free medium and incubated for 4 h at 37 °C. Cells were left untreated as further control (Con) or incubated with AT and Fon only. After the incubation, the cells were lysed and Gαi which has not been ADP-ribosylated during the intoxication with PT was ADP-ribosylated and biotin-labeled via the incubation with PTS1 and biotin-labeled NAD + . Subsequently, the biotin-labeled Gαi was detected via Western Blot, while Hsp90 or Ponceau-S staining served as control for equal protein loading. The bar graphs ( A and B ) show the quantifications of Western blot signals from six independent experiments, while ( C ) shows blots of a representative experiment. The intensity values of the bar graph are given as x-fold of the untreated control (Con), normalized to Hsp90 or Ponceau-S staining, mean ± SEM (at least n = 7 values from six independent experiments). A and B , significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to controls treated with PT only ( A ) or untreated controls (Con) ( B ) (∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns not significant). α 1 AT, α 1 -antitrypsin; Gαi, α-subunit of inhibitory G protein; PT, pertussis toxin.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Solvent, Incubation, Control, Labeling, Western Blot, Staining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Effect of α 1 AT on enzyme activity of PTS1 in vitro . A , schematic representation of experimental setup for the enzyme activity assay. PTS1 and α 1 AT were either directly added to CHO-K1 cell lysate with biotin-NAD + and incubated for 40 min or preincubated for 15 min before addition to cell lysate and biotin-NAD + . B – D , PTS1 (84 nM) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) (Con) were added directly to CHO-K1 cell lysate and biotin-NAD + and incubated for 40 min at room temperature. Cell lysate was left untreated with biotin-NAD + as further control (lysate). Gαi, which was ADP-ribosylated and biotin-labeled via the incubation with PTS1 and biotin-labeled NAD + , was detected via Western blot, while Hsp90 or Ponceau-S staining served as control for equal protein loading. The bar graph ( B ) shows the quantifications of Western blot signals from six independent experiments, while ( C and D ) show blots of representative experiments. The intensity values of the bar graph are given as x-fold of the control (Con), normalized to Hsp90 or Ponceau-S staining, mean ± SEM (at least n = 6 values from six independent experiments). E – G , PTS1 (84 nM) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) (Con) were preincubated for 15 min at room temperature before addition to CHO-K1 cell lysate and biotin-NAD + . Cell lysate was left untreated with biotin-NAD + as further control (lysate). Subsequently, the experiment was performed as described in ( B – D ). The bar graph ( E ) shows the quantifications of Western blot signals from seven independent experiments, while ( F and G ) show blots of representative experiments. The intensity values of the bar graph are given as x-fold of the control (Con), normalized to Hsp90 or Ponceau-S staining, mean ± SEM (at least n = 6 values from seven independent experiments). B and E , significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to controls treated with PT only (∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns not significant). α 1 AT, α 1 -antitrypsin; Gαi, α-subunit of inhibitory G protein; PT, pertussis toxin; WB, Western Blot.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Activity Assay, In Vitro, Enzyme Activity Assay, TNKS1 Histone Ribosylation Assay, Incubation, Solvent, Control, Labeling, Western Blot, Staining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Effect of α 1 AT on binding of PT in CHO-K1 cells. A , PT (1 μg/ml = 0.00952 μM) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells and incubated for 40 min at 4 °C to enable PT binding but not internalization. Cells were left untreated as control. Subsequently, the cells were washed, fixed, permeabilized, and quenching was performed. Blocking was performed and the cells were incubated with primary antibodies for PTS1 ( green ) and α 1 AT ( red ). Primary antibodies were detected via fluorescently labeled secondary antibodies and nuclei were stained using Hoechst ( blue ). Representative images are shown from three independent experiments (n = 3), ( B ) whereas the PTS1 signal is given in the bar graph as x-fold of the PT-treated control (PT), normalized to the Hoechst signal, mean ± SEM (n = 44–45 values from three independent experiments). Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns not significant). α 1 AT, α 1 -antitrypsin; PT, pertussis toxin.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Binding Assay, Solvent, Incubation, Control, Blocking Assay, Labeling, Staining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Effect of α 1 AT on detectable PTS1 signal in CHO-K1 and A549 cells. A – B , PT (100 ng/ml = 0.000952 μM) and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added directly to CHO-K1 cells and incubated for 4 h at 37 °C. Cells were left untreated as control (Con). Subsequently, the cells were washed, fixed, permeabilized, and quenching was performed. After blocking, cells were incubated with primary antibodies against PTS1 ( green ) and α 1 AT ( red ). Primary antibodies were detected via fluorescently labeled secondary antibodies, and nuclei were stained using Hoechst ( blue ). Representative images are shown from three independent experiments (n = 3), B , PTS1 signal is given in the bar graph as x-fold of the PT-treated control (PT), normalized to the Hoechst signal, mean ± SEM (n = 45 values from three independent experiments). C , PT (100 ng/ml) and α 1 AT or the respective amount of solvent (H 2 O) were added directly to A549 cells and incubated for 4 h at 37 °C. Subsequently, the experiment was performed as described in ( A ). Representative images are shown from three independent experiments (n = 3), D , PTS1 signal is given in the bar graph as x-fold of the PT-treated control (PT), normalized to the Hoechst signal, mean ± SEM (n = 43–45 values from three independent experiments). B and D , significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns not significant). α 1 AT, α 1 -antitrypsin; PT, pertussis toxin.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Solvent, Incubation, Control, Blocking Assay, Labeling, Staining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Effect of different preincubation schemes of α 1 AT on PT-intoxication and binding of PT to cells. A – B , for the competition study based on the sequential ADP-ribosylation of Gαi six different treatment options using PT (10 ng/ml) and α 1 AT (100 μM) or the respective amount of solvent (H 2 O) were performed in parallel. First and second, α 1 AT or solvent control were preincubated on CHO-K1 cells for 15 min at 37 °C and were either present in the medium during intoxication with PT or washed away prior to PT-intoxication. Third and fourth, PT was preincubated on cells for 15 min at 37 °C and was either present in the medium while α 1 AT or solvent control were added or washed away before adding α 1 AT or water. Fifth, α 1 AT, water (solvent control), and PT were preincubated for 15 min at room temperature before addition to CHO-K1 cells. Sixth, α 1 AT, solvent control, and PT were added directly to CHO-K1 cells. Cells were left untreated as further control (Con). The wash steps were performed using FCS-free medium. After the CHO-K1 cells were treated, the cells were incubated for further 4 h at 37 °C. Then, the cells were lysed and Gαi which has not been ADP-ribosylated during the intoxication with PT was ADP-ribosylated and biotin-labeled via the incubation with PTS1 and biotin-labeled NAD + . Subsequently, the biotin labeled Gαi was detected via Western Blot, while Hsp90 served as control for equal protein loading. The bar graph ( A ) shows the quantification of Western blot signals from three independent experiments, while ( B ) shows blots of a representative experiment. The intensity values of the bar graph are given as x-fold of the untreated control (Con), normalized to Hsp90, mean ± SEM (n = 3 values from three independent experiments). C , The 488-labeled PT (500 ng/ml) was preincubated for 15 min at 4 °C on CHO-K1 cells to enable binding of PT to cells but no internalization. Subsequently, cells were washed by centrifugation, and different concentrations of α 1 AT or the respective amount of solvent (H 2 O) were added to the cells and incubated for further 15 min at 4 °C. Cells were left untreated as control (Con). After that, cells were washed by centrifugation and 488-labeled PT bound to cell surfaces was measured using flow cytometry. Values of median are given as x-fold of the untreated control (Con), mean ± SEM (n = 9 values from three independent experiments). A and C , significance was tested using one-way ANOVA followed by Šídák’s multiple comparison test ( A ) or Dunnett’s multiple comparison test ( C ) and refers to samples treated with PT only ( A , C ) (∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns not significant). α 1 AT, α 1 -antitrypsin; FCS, fetal calf serum; Gαi, α-subunit of inhibitory G protein; PT, pertussis toxin.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: Binding Assay, Solvent, Control, Incubation, Labeling, Western Blot, Centrifugation, Flow Cytometry, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Alpha-1 antitrypsin inhibits pertussis toxin

doi: 10.1016/j.jbc.2024.107950

Figure Lengend Snippet: Interaction of α 1 AT with PT and PTS1 in vitro. A , decreasing concentrations of PTS1 (antibody control), Gαi, and α 1 AT were vacuum aspirated onto a nitrocellulose membrane using the Dot blot system. PBS was aspirated as a control. Subsequently, the membrane was stained with Ponceau-S ( right ), blocked, and cut for incubation with the overlay samples, PT (200 ng/ml = 0.001904 μM), PTS1 (200 ng/ml = 0.0007616 μM), and PBS-T as control. The amount of spotted PTS1 is below the detection limit of Ponceau-S staining. Bound PTS1 was detected using an antibody against PTS1 ( left ). A representative blot is shown of at least three independent experiments (n = 3). α 1 AT, α 1 -antitrypsin; Gαi, α-subunit of inhibitory G protein; PT, pertussis toxin.

Article Snippet: This was followed by washing steps in PBS-T and incubation with the primary antibody for PTS1 (anti-PTS1, clone 63.1G9, sc-57639, Santa Cruz) and peroxidase-coupled anti-mouse antibody for the detection of bound PTS1.

Techniques: In Vitro, Control, Membrane, Dot Blot, Staining, Incubation