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Helicobacter pylori urease binds to class II MHC molecules on gastric epithelial cells to initiate apoptosis
All8 AGA ABSTRACTS • G0484
STRUCTURAL-FUNCTIONAL RELATIONSHIPS FOR THE 13-SUBUNIT OF THE H,K-ATPASE. CJ. Fan, D.C. Chow and J.G. Forte. Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720. The gastric H,K-ATPase, similar to the ubiquitous Na,K-ATPase, is a heterodimeric membrane protein consisting of two subunits, an ct (HKa) and a ]3 subunit (HK[3). HKa is a multi-membrane-spanning, 114 kDa peptide; HK13is a highly glycosylated, single membranespanning unit with a 34kDa core peptide. We previously showed that HKI~ is involved in proton pumping activity and is highly resistant to pepsinolysis in its native state. Here we use tubulovesicle membranes isolated from parietal cells to further study the native conformation of HKI3 as it may relate to some protective function for the holoenzyme. A monoclonal antibody specific for a site within the first ten, N-terminal, amino acids (a.a.) of HK13 was used to monitor proteolysis of the cytoplasmic domain. Clostripain (Arg specific) cleaves the cytoplasmic domain in the native enzyme. However, the cytoplasmic domain is resistant to lysyl endopeptidase (Lys specific) and chymotrypsin (specific for aromatic a.a, Leu, Met and Ala) until the enzyme is treated with TX-100, Using a biotinylation protection assay, we show that the dose-dependence of exposure of the Lys 8 & Lys 9 corresponds to the response for irreversible inactivation of the enzyme by TX-100, suggesting that Lys 8 & 9 of HK[3 are involved in functional interactions, possibly with l-IKct, in the native state. These results led us to further study the conformation of the extracellular domain of HK13. Using a monoclonal antibody specific for a.a. 226231 of HKI3, we show that the extracellular domain requires full denaturation by SDS to be sensitive to trypsin cleavage. Chymotrypsin cleaves the extracellular domain provided that vesicles are permeabilized by nondenaturing detergents such as saponin or TX-100. These data suggest that charged a.a. within the extracellular domain are sufficiently hidden so that full denaturation must occur before these sites can be exposed for trypsinolysis. Hydrophobic a.a. do not require full denaturation to be exposed and therefore are more readily accessible to digestion with chymotrypsin. These results allow us to formulate a model of the 13-subunit where hydrophobic residues are exposed and charged groups are hidden in the extracellular domain of the native enzyme. Ready interaction of the protein with the aqueous extracellular environment would be provided by the extensive glycosylation of HK13. This model also provides a basis for resistance of the protein to large shifts in [H+] associated with secretion and the ability to remain stable within the harsh luminal environment of the stomach. (Support by NIH grant DK38972; CJF is a Student Research Fellow of the Am. Digestive Health Fndn.) • G0485
HELICOBACTER PYLORI UREASE BINDS TO CLASS II MHC MOLECULES ON GASTRIC EPITHELIAL CELLS TO INITIATE APOPTOSIS. XJ. Fan, H. Gunasena, M. Gonzales, R. Almanza, Z. Cheng, G. Ye, C. Barrera, S.E. Crowe, P.B. Ernst and V.E. Reyes. Departments of Pediatrics, Microbiology & Immunology, Internal Medicine and Sealy Center Molecular Sciences. University of Texas Medical Branch, Galveston, TX. BACKGROUND: Colonization of the gastric epithelium by Helicobacter pylori results in damage to the gastric epithelium that may include peptic ulcer and gastric cancer. We have previously demonstrated that H. pylori binds to class II MHC on gastric epithelial cells and signals apoptosis. However, it is not clear which bacterial proteins bind to class II MHC and trigger apoptosis. Since urease is a major product of H. pylori that has been detected on the outer surface of the organism, we hypothesized that urease binds to class II MHC on gastric epithelial cells and initiates apoptotic signals. METHODS: The binding of FITC-conjugated urease to the gastric epithelial cell lines KATO III, N87 and AGS was determined by flow cytometry. To further determine the interaction of urease with class II MHC, urease-coated Sepharose beads were incubated with proteins from [35S]methionine metabolically labeled gastric epithelial cells as well as HLA-DRl-transfected (1D12) and untransfected COS-I cells. The bound proteins were eluted and characterized after separation by gel electrophoresis. Apoptosis induction of the cell lines by urease was measured by ELISA and DNA ladder formation. RESULTS: Since gastric epithelial cells are exposed to interferon-T (IFN-'/) during infection with H. pylori, we showed that IFN-T increased the binding of urease to two gastric epithelial cell lines, KATO III and N87, which are class II MHC +, but not to another gastric epithelial cell line, AGS, which is class II MHC-. The binding of urease to KATO III and N87 proteins was blocked by anti-class MHC antibodies. Also, urease bound only to HLA-DRItransfected COS-1 (1D12) cells but not to HLA-DR1- COS-1 cells. To confirm the interaction of urease with class II MHC, 96-well plates were coated with urease, BSA or coating buffer alone. Then, affinity-purified class II MHC from [35S] metabolically labeled gastric epithelial cells were added to each well and the bound proteins were eluted. Gel electrophoresis analysis of the eluted material showed the presence of class II MHC ot and 13chains from the wells which were coated with urease but not from the control wells. To test whether the binding of urease to class II MHC was associated with the induction of apoptosis by urease, KATO III, N87, and AGS cells were exposed to various concentrations of urease and the DNA fragmentation was measured by ELISA. Urease induced apoptosis in KATO III and N87 cells that are class II MHC+, but not in AGS cells that lack class II MHC. Fab antibody fragments recognizing class II MHC blocked the induction of apoptosis by urease in a concentration-dependent manner. Thus, urease was capable of inducing apoptosis of gastric epithelial cells, likely through an interaction with class II MHC molecules on the gastric epithelium. These observations uncover a new role for 1-1.pylori urease in associated diseases.
GASTROENTEROLOGY Vol. 114, No. 4
This research was funded in part by NIH grants (DK51677 and DK50669) and the James W. McLaughlin Fellowship Fund. • G0486
PRESENCE OF NO-SYNTHASE INHIBITOR ADMA IN H. PYLORI INFECTED ANTRAL MUCOSA. Lars F~indriks, Charlotte yon Bothmer*, Hans L~Snroth, Lars Olbe and Anders Pettersson. Departments of Physiology and Pharmacology* and Surgery, Sahlgrenska University Hospital, Gtiteborg University, Sweden. A recent study demonstrates that proteolyzed water extracts of H. pylori contain the potent NO synthase inhibitor asymmetric-dimethyl arginine (ADMA) (1). The possible presence of ADMA in human H. pylori infected antra/mucosa was investigated. Methods: Twelve asymptomatic volunteers underwent endoscopical examination. Six of these were H. pylori negative (mean age: 33, f/m:3/3) and the remaining six were H. pylori positive (mean age: 54, f/m: 3/3), as evidenced by use of culture and the 13C-urea breath test. A modified HPLC method was used to analyse L-argiuine and ADMA (1,2) in mucosal biopsies from the antral part of the stomach. Results: The mucosal L-arginine concentrations were largely similar in H. pylori infected individuals (range: 0,67 - 3,3 lamol/g wet weight, n=6) as compared to uninfected controls (range: 0,26 - 2,4 lamol/g w.w., n=6). Mean concentration of ADMA was markedly higher in the H. pylori infected specimens than in the uninfected ones.
H.pylori negatives
ADMA (nmol/g wet weight)
L-arginine/ADMA
0,25 _+0,5
5910 _+990
16,4 -+ 2,6**
148 -+ 60**
(n = 6)
H. pylori positives (n = 6)
(** p < 0.01, Students t-test, unpaired samples) Conclusion: H. pylori infected antral mucosa have a restricted NOsynthetizing capacity due to the markedly lowered ratio between the true substrate (L-arginine) and the competitive antagonist (ADMA). These results may have implications on H. pylori associated functional disturbances and impaired host defences in the infected gastroduodenal mucosa. References: 1. F~indriks et al. Gastroenterology 1997 113:1570-1575 2. Pettersson etal. Journal of Chromatography 1997 692:257-62 • G0487
PATIENTS WITH FUNCTIONAL DYSPEPSIA RESPONDING TO OMEPRAZOLE HAVE A CHARACTERISTIC REFLUX P A T T E R N . P.G. Farup, ¢. Hovde, R. Torp, S. Wetterhus, Dept. of Medicine, GjCvik County Hospital, Gj~vik, Norway. The effect of acid secretion inhibitors in patients with functional dyspepsia (FD) is equivocal. One previous trial indicates effect in a subset of patients with a peculiar gastroesophageal reflux pattern with numerous short reflux episodes (1). The primary aim of this double blind trial, which was a supplement to a large multicentre trial, was to compare the number of gastroesophageal reflux episodes in responders and non responders to omeprazole. Patients and methods. Twenty-four patients (men/women: 11/13; mean age 49 years) with persistent or recurrent epigastric pain and/or discomfort experienced during the last 3 days prior to randomisation, and with at least one month's history with symptoms for at least 25% of the time, were included. Patients with structural and biochemical explanation for their symptoms, and those with heartburn and/or regurgitation as their main symptom were excluded. An upper endoscopy and a 24 h esophageal pH measurement were performed before randomisation to treatment with omeprazole 10 mg or 20 mg (combined to one group with 14 patients) or placebo (10 patients) for 4 weeks. The patients were asked for sufficient relief of dyspeptic symptoms by a simple question at four weeks. Results: The number of patients with sufficient relief of symptoms in the omeprazole and placebo group was 8/14 (57%) and 2/10 (20%) respectively (p=0.07). The table shows the results of 24 h esophageal pH measurement in patients treated with omeprazole divided into those with and those without sufficient relief of symptoms. The results are given as number or mean (SD), and Mann Whitney U test is used for comparison of the two groups.
Number of patients Fraction of time pH < 4 (%): Total Fraction of time pH < 4 (%): Upright Fraction of time pH < 4 (%): Supine Total number of reflux episodes Number of reflux episodes > 5 min.
Suffmient response 8 6.1 (4.2) 7.0 (5.4) 4.0 (7.4) 57 (17) 3.3 (3.7)
Not surf. response 6 3.6 (2.9) 3.3 (2.2) 3.9 (5.4) 25 (12) 1.8 (2.1)
Statistics
p = 0.30 p = 0.16 p = 0.97 p < 0.003 p = 0.65
In the omeprazole group, the number of patients with sufficient relief of symptoms was 0/5 (0%) in those with <-32 reflux episodes, and 8/9 (89%) in those with > 32 reflux episodes (p < 0~003).
STRUCTURAL-FUNCTIONAL RELATIONSHIPS FOR THE 13-SUBUNIT OF THE H,K-ATPASE. CJ. Fan, D.C. Chow and J.G. Forte. Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720. The gastric H,K-ATPase, similar to the ubiquitous Na,K-ATPase, is a heterodimeric membrane protein consisting of two subunits, an ct (HKa) and a ]3 subunit (HK[3). HKa is a multi-membrane-spanning, 114 kDa peptide; HK13is a highly glycosylated, single membranespanning unit with a 34kDa core peptide. We previously showed that HKI~ is involved in proton pumping activity and is highly resistant to pepsinolysis in its native state. Here we use tubulovesicle membranes isolated from parietal cells to further study the native conformation of HKI3 as it may relate to some protective function for the holoenzyme. A monoclonal antibody specific for a site within the first ten, N-terminal, amino acids (a.a.) of HK13 was used to monitor proteolysis of the cytoplasmic domain. Clostripain (Arg specific) cleaves the cytoplasmic domain in the native enzyme. However, the cytoplasmic domain is resistant to lysyl endopeptidase (Lys specific) and chymotrypsin (specific for aromatic a.a, Leu, Met and Ala) until the enzyme is treated with TX-100, Using a biotinylation protection assay, we show that the dose-dependence of exposure of the Lys 8 & Lys 9 corresponds to the response for irreversible inactivation of the enzyme by TX-100, suggesting that Lys 8 & 9 of HK[3 are involved in functional interactions, possibly with l-IKct, in the native state. These results led us to further study the conformation of the extracellular domain of HK13. Using a monoclonal antibody specific for a.a. 226231 of HKI3, we show that the extracellular domain requires full denaturation by SDS to be sensitive to trypsin cleavage. Chymotrypsin cleaves the extracellular domain provided that vesicles are permeabilized by nondenaturing detergents such as saponin or TX-100. These data suggest that charged a.a. within the extracellular domain are sufficiently hidden so that full denaturation must occur before these sites can be exposed for trypsinolysis. Hydrophobic a.a. do not require full denaturation to be exposed and therefore are more readily accessible to digestion with chymotrypsin. These results allow us to formulate a model of the 13-subunit where hydrophobic residues are exposed and charged groups are hidden in the extracellular domain of the native enzyme. Ready interaction of the protein with the aqueous extracellular environment would be provided by the extensive glycosylation of HK13. This model also provides a basis for resistance of the protein to large shifts in [H+] associated with secretion and the ability to remain stable within the harsh luminal environment of the stomach. (Support by NIH grant DK38972; CJF is a Student Research Fellow of the Am. Digestive Health Fndn.) • G0485
HELICOBACTER PYLORI UREASE BINDS TO CLASS II MHC MOLECULES ON GASTRIC EPITHELIAL CELLS TO INITIATE APOPTOSIS. XJ. Fan, H. Gunasena, M. Gonzales, R. Almanza, Z. Cheng, G. Ye, C. Barrera, S.E. Crowe, P.B. Ernst and V.E. Reyes. Departments of Pediatrics, Microbiology & Immunology, Internal Medicine and Sealy Center Molecular Sciences. University of Texas Medical Branch, Galveston, TX. BACKGROUND: Colonization of the gastric epithelium by Helicobacter pylori results in damage to the gastric epithelium that may include peptic ulcer and gastric cancer. We have previously demonstrated that H. pylori binds to class II MHC on gastric epithelial cells and signals apoptosis. However, it is not clear which bacterial proteins bind to class II MHC and trigger apoptosis. Since urease is a major product of H. pylori that has been detected on the outer surface of the organism, we hypothesized that urease binds to class II MHC on gastric epithelial cells and initiates apoptotic signals. METHODS: The binding of FITC-conjugated urease to the gastric epithelial cell lines KATO III, N87 and AGS was determined by flow cytometry. To further determine the interaction of urease with class II MHC, urease-coated Sepharose beads were incubated with proteins from [35S]methionine metabolically labeled gastric epithelial cells as well as HLA-DRl-transfected (1D12) and untransfected COS-I cells. The bound proteins were eluted and characterized after separation by gel electrophoresis. Apoptosis induction of the cell lines by urease was measured by ELISA and DNA ladder formation. RESULTS: Since gastric epithelial cells are exposed to interferon-T (IFN-'/) during infection with H. pylori, we showed that IFN-T increased the binding of urease to two gastric epithelial cell lines, KATO III and N87, which are class II MHC +, but not to another gastric epithelial cell line, AGS, which is class II MHC-. The binding of urease to KATO III and N87 proteins was blocked by anti-class MHC antibodies. Also, urease bound only to HLA-DRItransfected COS-1 (1D12) cells but not to HLA-DR1- COS-1 cells. To confirm the interaction of urease with class II MHC, 96-well plates were coated with urease, BSA or coating buffer alone. Then, affinity-purified class II MHC from [35S] metabolically labeled gastric epithelial cells were added to each well and the bound proteins were eluted. Gel electrophoresis analysis of the eluted material showed the presence of class II MHC ot and 13chains from the wells which were coated with urease but not from the control wells. To test whether the binding of urease to class II MHC was associated with the induction of apoptosis by urease, KATO III, N87, and AGS cells were exposed to various concentrations of urease and the DNA fragmentation was measured by ELISA. Urease induced apoptosis in KATO III and N87 cells that are class II MHC+, but not in AGS cells that lack class II MHC. Fab antibody fragments recognizing class II MHC blocked the induction of apoptosis by urease in a concentration-dependent manner. Thus, urease was capable of inducing apoptosis of gastric epithelial cells, likely through an interaction with class II MHC molecules on the gastric epithelium. These observations uncover a new role for 1-1.pylori urease in associated diseases.
GASTROENTEROLOGY Vol. 114, No. 4
This research was funded in part by NIH grants (DK51677 and DK50669) and the James W. McLaughlin Fellowship Fund. • G0486
PRESENCE OF NO-SYNTHASE INHIBITOR ADMA IN H. PYLORI INFECTED ANTRAL MUCOSA. Lars F~indriks, Charlotte yon Bothmer*, Hans L~Snroth, Lars Olbe and Anders Pettersson. Departments of Physiology and Pharmacology* and Surgery, Sahlgrenska University Hospital, Gtiteborg University, Sweden. A recent study demonstrates that proteolyzed water extracts of H. pylori contain the potent NO synthase inhibitor asymmetric-dimethyl arginine (ADMA) (1). The possible presence of ADMA in human H. pylori infected antra/mucosa was investigated. Methods: Twelve asymptomatic volunteers underwent endoscopical examination. Six of these were H. pylori negative (mean age: 33, f/m:3/3) and the remaining six were H. pylori positive (mean age: 54, f/m: 3/3), as evidenced by use of culture and the 13C-urea breath test. A modified HPLC method was used to analyse L-argiuine and ADMA (1,2) in mucosal biopsies from the antral part of the stomach. Results: The mucosal L-arginine concentrations were largely similar in H. pylori infected individuals (range: 0,67 - 3,3 lamol/g wet weight, n=6) as compared to uninfected controls (range: 0,26 - 2,4 lamol/g w.w., n=6). Mean concentration of ADMA was markedly higher in the H. pylori infected specimens than in the uninfected ones.
H.pylori negatives
ADMA (nmol/g wet weight)
L-arginine/ADMA
0,25 _+0,5
5910 _+990
16,4 -+ 2,6**
148 -+ 60**
(n = 6)
H. pylori positives (n = 6)
(** p < 0.01, Students t-test, unpaired samples) Conclusion: H. pylori infected antral mucosa have a restricted NOsynthetizing capacity due to the markedly lowered ratio between the true substrate (L-arginine) and the competitive antagonist (ADMA). These results may have implications on H. pylori associated functional disturbances and impaired host defences in the infected gastroduodenal mucosa. References: 1. F~indriks et al. Gastroenterology 1997 113:1570-1575 2. Pettersson etal. Journal of Chromatography 1997 692:257-62 • G0487
PATIENTS WITH FUNCTIONAL DYSPEPSIA RESPONDING TO OMEPRAZOLE HAVE A CHARACTERISTIC REFLUX P A T T E R N . P.G. Farup, ¢. Hovde, R. Torp, S. Wetterhus, Dept. of Medicine, GjCvik County Hospital, Gj~vik, Norway. The effect of acid secretion inhibitors in patients with functional dyspepsia (FD) is equivocal. One previous trial indicates effect in a subset of patients with a peculiar gastroesophageal reflux pattern with numerous short reflux episodes (1). The primary aim of this double blind trial, which was a supplement to a large multicentre trial, was to compare the number of gastroesophageal reflux episodes in responders and non responders to omeprazole. Patients and methods. Twenty-four patients (men/women: 11/13; mean age 49 years) with persistent or recurrent epigastric pain and/or discomfort experienced during the last 3 days prior to randomisation, and with at least one month's history with symptoms for at least 25% of the time, were included. Patients with structural and biochemical explanation for their symptoms, and those with heartburn and/or regurgitation as their main symptom were excluded. An upper endoscopy and a 24 h esophageal pH measurement were performed before randomisation to treatment with omeprazole 10 mg or 20 mg (combined to one group with 14 patients) or placebo (10 patients) for 4 weeks. The patients were asked for sufficient relief of dyspeptic symptoms by a simple question at four weeks. Results: The number of patients with sufficient relief of symptoms in the omeprazole and placebo group was 8/14 (57%) and 2/10 (20%) respectively (p=0.07). The table shows the results of 24 h esophageal pH measurement in patients treated with omeprazole divided into those with and those without sufficient relief of symptoms. The results are given as number or mean (SD), and Mann Whitney U test is used for comparison of the two groups.
Number of patients Fraction of time pH < 4 (%): Total Fraction of time pH < 4 (%): Upright Fraction of time pH < 4 (%): Supine Total number of reflux episodes Number of reflux episodes > 5 min.
Suffmient response 8 6.1 (4.2) 7.0 (5.4) 4.0 (7.4) 57 (17) 3.3 (3.7)
Not surf. response 6 3.6 (2.9) 3.3 (2.2) 3.9 (5.4) 25 (12) 1.8 (2.1)
Statistics
p = 0.30 p = 0.16 p = 0.97 p < 0.003 p = 0.65
In the omeprazole group, the number of patients with sufficient relief of symptoms was 0/5 (0%) in those with <-32 reflux episodes, and 8/9 (89%) in those with > 32 reflux episodes (p < 0~003).