Helicobacter pylori γ-Glutamyl Transpeptidase Is a Pathogenic Factor in the Development of Peptic Ulcer Disease Min Gong, Samantha Shi Min Ling, Sook.

Helicobacter pylori γ-Glutamyl Transpeptidase Is a Pathogenic Factor in the Development of Peptic Ulcer Disease Min Gong, Samantha Shi Min Ling, Sook Yin Lui, Khay Guan Yeoh, Bow Ho Gastroenterology Volume 139, Issue 2, Pages (August 2010) DOI: /j.gastro Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 GGT activity of H pylori strains isolated from patients with different disease status. GGT activity of 98 H pylori isolates obtained from patients with NUD (n = 44), DU (n = 29), and GU (n =25). Values of GGT activity represent means ± SDs of triplicate measurements and are representative of 3 experiments. *P < .001 for comparison of NUD vs DU or GU, **P = .043 for comparison of DU vs GU. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Purification of native H pylori GGT. M, low-molecular-weight protein marker; 1, cell sonicate before purification; protein fraction eluted from 2, first ion exchange chromatography (IEX); 3, gel filtration; 4, second IEX. Circles indicate the large and small subunit fragments of GGT protein of 38 and 22 kDa, respectively. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 H pylori GGT induces H2O2 generation in AGS or primary human gastric epithelial cells. (A and B) H2O2 generation in AGS cells (5 × 104) with various treatments was examined by a fluorescence-based analysis. Data are means ± SDs of the H2O2 generated from AGS in each sample from 3 independent experiments. (C) H2O2 generation in primary gastric cells (5 × 104) with various treatments. NC, uninfected cells. Cells were treated with SBC, 10 mmol/L serine-borate complex; DFO, 50 μmol/L DFO; GSH, 0.1 mmol/L GSH, and 1 mmol/L glycyl-glycine; Fe3+, 20 μmol/L Fe3+; GGT, HP-nGGT (1.5 mU); BSA, 10 ng bovine serum albumin. The significant difference was compared with H pylori WT-treated cells (*P < .05), untreated cells (**P < .05), GGT-treated cells (***P < .01), and GGT + GSH–treated cells (****P < .01). Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 H pylori GGT induces NF-κB activation. (A) H pylori GGT–mediated I-κB degradation and NF-κB activation in which AGS cells (2 × 107) were treated with H pylori WT, various mutants, or HP-nGGT (500 mU). (B) AGS cells transfected with pNF-κB-MetLuc2-Reporters were cocultured with H pylori WT, various mutants, or HP-nGGT. Values represent the means ± SDs of luciferase activity relative to uninfected control from triplicate measurements and are representative of 3 experiments. *P < .05 for comparison of WT vs Δggt, **P < .05 for comparisons of HP-nGGT in the presence of various inhibitors vs HP-nGGT. (C) Preincubating AGS cells with inhibitors, NAC (10 mmol/L), ST638 (30 μmol/L), and MG132 (30 μmol/L) for 30 minutes blocked the H pylori GGT–mediated I-κB degradation and NF-κB activation. β-Actin was used as loading control. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 H pylori GGT induces IL-8 production. Enzyme-linked immunoabsorbent assay of (A) IL-8 generation from AGS cells (5 × 105) after treatments with H pylori WT, various mutants, or HP-nGGT (15 mU) over a period of 24 hours. (B) IL-8 generation from primary gastric epithelial cells after treatment with H pylori WT, Δggt, and HP-nGGT for 4 hours. (C) Effects of inhibitors on H pylori GGT induced IL-8 production where AGS cells (5 × 105) were pretreated with or without the inhibitors NAC (10 mmol/L), ST638 (30 μmol/L), and MG132 (30 μmol/L) for 30 minutes. After which, the mixtures were cocultured with HP-nGGT for another 6 hours. (D) IL-8 generation from primary macrophages after treatment with H pylori WT, various mutants, or HP-nGGT for 4 hours. *P < .05 of HP-nGGT–treated cells vs negative control. **P < .05 preincubation of various inhibitors in HP-nGGT–treated cells vs HP-nGGT–treated cells alone. ***P < .05 for comparison of cells infected with WT vs Δggt. NC, uninfected cells culture served as negative control. (E) Real-time PCR analysis detecting IL-8 mRNA in AGS cells treated with H pylori WT, various mutants, HP-nGGT, or any one of the inhibitors. Values from the enzyme-linked immunoabsorbent assay test and real-time PCR represent the means ± SDs of triplicate measurements and are representative of 3 experiments. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 H pylori GGT induces oxidative DNA damage in AGS. (A) AGS cells with oxidized DNA (8-OH-dG) as analyzed by flow cytometry. (B) DNA damage in AGS cells as examined with the comet assay. *P < .05 for comparison of WT vs Δggt; **P < .05 for comparisons of HP-nGGT in the presence of NAC vs HP-nGGT. Data are representative of 3 separate experiments. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1A
Gastroenterology , DOI: ( /j.gastro ) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1B

Supplementary Figure 1C

Supplementary Figure 1D

Supplementary Figure 1E

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Supplementary Figure 1G

Helicobacter pylori γ-Glutamyl Transpeptidase Is a Pathogenic Factor in the Development of Peptic Ulcer Disease Min Gong, Samantha Shi Min Ling, Sook.

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Helicobacter pylori γ-Glutamyl Transpeptidase Is a Pathogenic Factor in the Development of Peptic Ulcer Disease Min Gong, Samantha Shi Min Ling, Sook.

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