Journal: The Journal of biological chemistry

Article Title: Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d.

doi: 10.1074/jbc.275.5.3713

Figure Lengend Snippet: FIG. 2. A composite of SDS-PAGE of pooled peak Stx2d cyto- toxicity-enhancing activity fractions from various steps in the isolation scheme of the Stx2d-activating factor and a lane from a gelatin gel on which the isolated activator was tested for proteolytic activity. Samples from the various steps were concen- trated by precipitation with trichloroacetic acid where appropriate and subjected to electrophoresis on continuous 10% acrylamide gels. All of the lanes were stained with silver stain except the gelatin gel, which was stained with colloidal Coomassie Blue. Samples included: mouse crude small intestinal mucus (Crude Mucus, 300 mg); mucus proteins resuspended in IEC buffer following ammonium sulfate precipitation ((NH4)2SO4 Precipitated, 300 mg); eluant from a Q Sepharose ion ex- change column (IEC Eluant, 27.5 mg); eluant from a Phenyl Sepharose HP hydrophobic interaction column (HIC Eluant, 2.5 mg); Rotofor frac- tion with Stx2d cytotoxicity-enhancing activity (Rotofor Fraction, 0.5 mg; note that the presence of ampholytes in this sample interferes with protein concentration assays, so the protein concentration in this sam- ple was estimated based on densitometric comparisons); and HIC elu- ant subjected to electrophoresis on a 0.2% gelatin gel and then pro- cessed as detailed under “Experimental Procedures” (Gelatin Gel, 2.5 mg concentrated by microspin column). The numbers on the left indicate the positions of molecular mass markers in kilodaltons. The arrow on the right indicates the position of the isolated Stx2d-activating factor that was sequenced.

Article Snippet: The desalted sample was separated by isoelectric focusing in a mini Rotofor cell (Bio-Rad) using 2% BioLyte® ampholytes, pH range 4/6 (Bio-Rad) at a constant power of 10 W. Harvested Rotofor fractions were tested for Stx2d cytotoxicity-enhancing activity, and the peak fractions were pooled.

Techniques: SDS Page, Activity Assay, Isolation, Electrophoresis, Staining, Silver Staining, Protein Concentration

Journal: The Journal of biological chemistry

Article Title: Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d.

doi: 10.1074/jbc.275.5.3713

Figure Lengend Snippet: FIG. 3. Alignment of peptide sequences obtained from mass spectrometry-based sequencing of the 32-kDa protein with the translated amino acid sequence of a mouse gene predicted to be homologous to the human elastase IIIB gene (22). Alignment of peptide sequences 3, 4, and 5 with the predicted amino acid sequence of Mus musculus clone 1001269 (gb:AA771563) (Mouse) and the amino acid sequence of human elastase IIIB precursor (gb:M18692) (Human). Sequences shown are the mature elastase IIIB proteins. X in peptide 4 indicates that the corresponding amino acid was not determined. The lowercase letters in the mouse sequence represent amino acids homol- ogous to the human elastase IIIB that are reported to be in a different reading frame than the rest of the protein (i.e. frameshifts caused by sequencing errors). The asterisk indicates the predicted stop codon in the main open reading frame of the mouse gene. FIG. 4. Stx2d cytotoxicity is specifically enhanced by elastase (A) in a dose-dependent manner (B). A, the elastase-specific inhib- itor elastatinal prevents activation of Stx2d by mouse crude small intestinal mucus and isolated activator (mouse elastase). Crude mucus (50 mg), hydrophobic interaction chromatography eluant (200 ng), or pooled peak activating activity fractions from Rotofor isoelectric focus- ing (30 ng, estimated by densitometry) were preincubated for 30 min in the presence or absence of elastatinal (1 mg, 30 ng, or 30 ng, respec- tively) at 37 °C. Stx2d (60 ng) was then added to each sample, and the samples were incubated an additional 1 h at 37 °C. 10-fold serial dilu- tions of each sample on Vero cells were used to determine the CD50/mg protein. Samples include: Stx2d incubated with mouse crude small intestinal mucus (“Mucus”), in the presence (1) or absence (2) of elasta- tinal; Stx2d incubated with cytotoxicity-enhancing activity-containing fractions eluted from a Phenyl Sepharose HP column (HIC Eluant), in the presence (1) or absence (2) of elastatinal; and Stx2d incubated with pooled peak cytotoxicity-enhancing activity-containing, fractions from isoelectric focusing (Rotofor Peak) in the presence (1) or absence (2) of elastatinal. The data represent geometric means of CD50/mg protein from at least three experiments. The error bars indicate one standard error of the mean. Note that the cytotoxicity of untreated Stx2d was similar to the cytotoxicity of elastatinal-treated samples, and elastati- nal alone does not affect the cytotoxicity of Stx2d (data not shown). B, purified Stx2d (60 ng) was incubated with increasing concentrations of isolated mouse elastase (as indicated on the figure) for 1 h at 37 °C. 10-fold serial dilutions of each mixture was transferred to Vero cells to determine the CD50/sample. Fold activation was calculated as the CD50 for each elastase-treated sample divided by the CD50 for buffer-treated Stx2d control. Each datum square represents the mean of three exper- iments. The error bars indicate one standard error of the means. Squares without error bars indicate errors too small to depict.

Article Snippet: The desalted sample was separated by isoelectric focusing in a mini Rotofor cell (Bio-Rad) using 2% BioLyte® ampholytes, pH range 4/6 (Bio-Rad) at a constant power of 10 W. Harvested Rotofor fractions were tested for Stx2d cytotoxicity-enhancing activity, and the peak fractions were pooled.

Techniques: Mass Spectrometry, Sequencing, Inhibition, Activation Assay, Isolation, Hydrophobic Interaction Chromatography, Activity Assay, Incubation, Purification, Control

Journal: The Journal of biological chemistry

Article Title: Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d.

doi: 10.1074/jbc.275.5.3713

Figure Lengend Snippet: FIG. 6. Crude mucus from mouse small intestine and isolated mouse elastase alter the mobility of the A2 peptide in activated samples of Stx2d on SDS-PAGE. Stx2d (5.6 mg) was incubated for 2 h at 37 °C with trypsin (1.25 mg in all samples) plus buffer (Control), plus mucus (Mucus, 200 mg), or plus Rotofor peak fractions that contained mouse elastase (Activator, 3 mg, as estimated by densitometry). This incubation followed a preincubation at 37 °C for 30 min in the absence or presence of elastatinal (1Elastatinal, 40 mg). The samples were concentrated by microspin concentration and resolved on a 4–12% NuPAGE Bis-Tris gel (Novex) and then electrotransferred to nitrocel- lulose. The Western immunoblot was developed with affinity-purified anti-Stx2d A2 antiserum incubated overnight at 1:1000 followed by a conjugated secondary antibody and chemoluminesence-based detection. The arrows at the left indicate the mobility of the activated (bottom arrow) versus the nonactivated (top arrow) Stx2d A2 peptides. Note that elastatinal alone did not alter the mobility of the Stx2d A2 subunit (data not shown). The cytotoxicity-enhancing activity for each sample was determined by testing a portion of each sample on Vero cells. The fold activation was determined by dividing the CD50 of each sample by the CD50 for the buffer-treated Stx2d. Fold activation results less than or equal to 1 were considered 0-fold activation.

Article Snippet: The desalted sample was separated by isoelectric focusing in a mini Rotofor cell (Bio-Rad) using 2% BioLyte® ampholytes, pH range 4/6 (Bio-Rad) at a constant power of 10 W. Harvested Rotofor fractions were tested for Stx2d cytotoxicity-enhancing activity, and the peak fractions were pooled.

Techniques: Isolation, SDS Page, Incubation, Control, Concentration Assay, Western Blot, Affinity Purification, Activity Assay, Activation Assay