1. Volume 132, Issue 5, Pages 1820-1833 (May 2007)
Inhibition of T-Cell Proliferation by Helicobacter pylori γ-Glutamyl Transpeptidase 
Christian Schmees, Christian Prinz, Tilman Treptau, Roland Rad, Ludger Hengst, Petra Voland, Stefan Bauer, Lena Brenner, Roland M. Schmid, Markus Gerhard 
Gastroenterology 
Volume 132, Issue 5, Pages (May 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Fractions from H pylori supernatant inhibiting T-cell proliferation show enzymatic GGT activity. (A) List of secreted proteins from H pylori with a molecular weight between 30 and 66 kilodaltons according to Kim et al and Bumann et al.9,10 (B) Proteins in eluted fractions from size-exclusion chromatography were separated by SDS-PAGE and silver stained. Only fractions b–f inhibited proliferation of human T cells, whereas all other fractions did not. Protein bands corresponding to the inhibitory profile of the fractions are marked by arrows. (C) Enzymatic GGT activity of gel filtration fractions was determined by a spectrophotometric assay as described in Materials and Methods. HP, H pylori.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
GGT-deficient H pylori mutants fail to inhibit lymphocyte proliferation. Cell proliferation of (A) stimulated PBMCs and (C) isolated primary human T lymphocytes in the presence or absence of indicated H pylori supernatant was determined by 3H-thymidine incorporation assay. GGT phenotype of constructed knockout strains was confirmed by enzyme activity assay and immunoblotting using a polyclonal antibody raised against the large GGT subunit (B). For immunoblotting, 30 μg protein of H pylori supernatant was used. Immunoblotting with anti-VacA antibody served as a loading control (see inset). Data represent mean ± SD of 3 independent experiments. *P < .001 as determined by Student t test was considered significant. HP, H pylori; WT, wild-type.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Recombinant HPGGT from E coli shows an inhibitory effect. (A) Silver staining and (B) immunoblotting of purified recombinant HPGGT fractions with anti-GGT antibody directed against its large subunit show processing of GGT. ***, Pro-form; **, large subunit, *, small subunit. (C) Enzymatic activity of recombinant HPGGT (recGGT) expressed in E coli as determined by release of p-nitroanilide from l-γ-glutamyl-p-nitroanilide. (D) Dose-response curve showing inhibition of proliferation of human PBMCs treated with recombinant HPGGT (recGGT 1–3, 3 independent experiments) or purified, acid-activated VacA (VacA 1–2, 2 independent experiments). (E) Recombinant HPGGT showed catalytic activity at pH 2–10. Data represent mean ± SD of 3 independent experiments. (F) Sera from H pylori–positive (lanes 1–9) and –negative (lanes 10–14) patients were tested for the presence of antibodies directed against HPGGT by immunoblotting as described in Materials and Methods. Rabbit anti-GGT antibody (αGGT) was used as a positive control. ***, Pro-form; **, large subunit of HPGGT protein. FT, flow through.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Inhibitory effect of HPGGT depends on its enzymatic activity. Purified GGT from equine kidney displayed (A) catalytic GGT activity but (B) lacked a proliferation-inhibiting effect toward lymphocytes. Site-directed mutagenesis of recombinant HPGGT at Ser 451/452 (S451/452A) (A) abolished GGT enzyme activity and (B) the inhibitory effect. Preincubation of H pylori wild-type supernatant with acivicin (50μmol/L) for 2 hours at 37°C abrogated (C) GGT activity and (D) inhibition of PBMC proliferation. Data represent mean ± SD of 3 independent experiments. *P < .05 as determined by Student t test was considered significant.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Influence of HPGGT on cytokine secretion and apoptosis. Production of cytokines (A) interleukin-2 and (B) interferon gamma by PBMCs was measured after 24 hours by enzyme-linked immunosorbent assay as described in Materials and Methods. Data represent mean ± SD of 3 independent experiments. P values as determined by Student t test are indicated. (C) Jurkat T cells were treated for 24 hours as indicated (gray curves) and stained with Annexin V-FITC and PI. The rate of apoptotic Jurkat T cells was determined by FACS analysis acquiring 10,000 events. The anti-cancer drug staurosporine (blank curve), used as a positive control, strongly induced apoptosis at a concentration of 1 μmol/L. HP, H pylori; WT, wild-type.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Effect of HPGGT on cell cycle progression and signal transduction. (A) Cell cycle analysis of Jurkat T cells treated with or without indicated HPSN for 24 hours. Percentage of cells in G1 phase (lower left), early and late S phase (upper left and right), and G2 phase (lower right) is depicted (y-axis, bromodeoxyuridine-FITC; x-axis, PI). Cellular levels of cell cycle regulatory proteins were determined in the same cells by immunoblotting. (B) A total of 107 PBMCs were incubated with different concentrations of H pylori wild-type (HP WT) and H pylori ΔGGT supernatant (HP ΔGGT) or recombinant HPGGT for 24 and 48 hours and subsequently lysed. A total of 35 μg of total protein was separated by SDS-PAGE and analyzed by immunoblotting. Levels of indicated proteins were determined using the corresponding antibodies. Data were reproduced 2 times with similar results. HPSN, H pylori supernatant.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions