WESTFÄLISCHE WILHELMS-UNIVERSITÄT MÜNSTER Pharmazeutische Biologie und Phytochemie Glycoconjugates from plants as antiadhesive Compounds against Helicobacter pylori Ribes nigrum L. Abelmoschus esculentus (L.) MOENCH Inaugural-Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften im Fachbereich Chemie und Pharmazie der Mathematisch-Naturwissenschaftlichen Fakultät der Westfälischen Wilhelms-Universität Münster vorgelegt von Jutta Messing aus Bocholt -2013-
TABLE OF CONTENTS TABLE OF CONTENTS 1 I. LIST OF ABBREVIATIONS 7 II. SUMMARY 12 1. Introduction 14 1.1. Helicobacter pylori 14 1.1.1. History and discovery of the bacterium 14 1.1.2. Microbiology 15 1.1.3. Transmission 15 1.1.4. Prevalence and epidemiology 16 1.1.5. Colonization 18 1.1.6. Outer membrane proteins (OMPs) 19 1.1.7. The blood group antigen binding adhesin (BabA) 19 1.1.8. The sialic acid binding adhesin (SabA) 22 1.1.9. Pathogenesis 22 1.1.10. Diagnosis, treatment and recurrence 24 1.1.11. Alternative treatments 25 1.2. Ribes nigrum L 26 1.3. Abelmoschus esculentus (L.) MOENCH 28 1.4. Aim of this work 30 2. Results 31 2.1. Isolation of the F2 fraction from Ribis nigri semen 31 2.1.1. Isolation of the raw Polysaccharides 31 2.1.2. Isolation of the F2 fraction from Ribis nigri semen RPS by ion-exchange chromatography (IEC) 32 2.2. Analytical investigations on the carbohydrate part of F2 34 2.2.1. Carbohydrate content of fraction F2 34 2.2.2. Uronic acid content of fraction F2 35 2.2.3. Arabinogalactan protein (AGP) content of fraction F2 36 2.2.4. Molecular weight determination by GPC on Superose 6 39 2.2.5. Molecular weight determination of F2 by HP-SEC 42 2.2.6. SDS-PAGE 47 2.2.7. Determination of monosaccharide composition of F2 by HPAEC-PAD 49 1
2.2.8. Determination of D-/L-configuration of monosaccharides by capillary zone electrophoresis (CZE) 54 2.2.9. Linkage analysis of partially methylated alditol acetates (PMAA) 55 2.3. Analytical investigations on the protein part of F2 62 2.3.1. Protein content offraction F2 62 2.3.2. Determination of the amino acid composition offraction F2 by HPAEC-PAD 63 2.4. Distribution of carbohydrates and proteins in F2 65 2.5. Antiadhesive activity of F2 against H. pylori 68 2.5.1. Preparations before activity testing 68 2.5.1.1. Identification of H. pylori 68 2.5.1.2. Correlation of optical density (OD) and colony forming units (CFU) 70 2.5.1.3. Determination of direct cytotoxicity towards H. pylori 70 2.5.2. Influence of F2 towards the adhesion of H. pylori to human gastric mucosa tissue sections 71 2.5.3. Quantitative in vitro flow cytometric adhesion assay 74 2.5.4. Influence of F2 towards AGS cell viability 78 2.6. interaction and binding properties offraction F2 towards H. pylori 80 2.6.1. Dot blot overlay assay 80 2.6.2. Hemagglutination assay 82 2 6.3- Investigation on binding properties and interaction of F2 and okra extract with BabA and SabA by Radioimmunoassay (RIA) 84 2.6.3.1. Radiolabeling of F2 and okra extract with 12S I 85 2.6.3.2. Binding of F2 and okra extract to different H. pylori strains 88 2.6.3.3. Influence of F2 and okra extract on H. pylori binding to Lewis b 92 2.6.3.4. Influence of F2 and okra extract on H. pylori binding to sialyl-lewis" 96 2.7. Further characterization of F2 99 2.7.1. Hydrolytic degradation experiments 103 2.7.2. Enzymatic degradation experiments 108 2.7.2.1. Treatment of F2 with endo-arabinanase and a-l-arabinofuranosidase 109 2.7.2.2. Treatment of F2 with endo-ß- 1,6-galactanase 112 2.7.2.3. Treatment of F2 with ß-glucuronidase 115 2.7.2.4. Treatment of F2 with ß-xylanase 118 2.8. Activity testing of F2 subfractions and subfractions derived from ß-glucuronidase treatment 12o 2.9. Elucidation of the Potential antiadhesive activity of glucuronic acid 125
2.9.1. Adhesion assay with carbohydrate coupled Polymerie probes 125 2.9.2. Adhesion assay with magnetic nanoparticles 129 3. Discussion 137 3.1. Structural characterization of F2 138 3.2. Antiadhesive activity of F2 and okra extract 141 3.3. The role of glucuronic acid in antiadhesive Compounds 146 4. Materials and Methods 149 4.1. Instruments and consumables 149 4.1.1. Laboratory equipment and instruments 149 4.1.2. Consumable goods 152 4.2. Chemicals and reagents 153 4.2.1. General chemicals and reagents 154 4.2.2. Carbohydrates and amino acids 156 4.2.3. Chemicals and reagents for microbiology, cell culture and molecular biology 157 4.2.4. Buffers, solutions and media 160 4.2.4.1. Periodate treatment of BSA and preparation of blocking buffer IV 164 4.3. Plant material and test Compounds 165 4.3.1. Plant material 165 4.3.2. Test Compounds 165 4.4. Cell lines 165 4.5. Human stomach tissue sections 165 4.6. Bacterial strains 166 4.7. Isolation of Polysaccharides from Ribis nigri semen 167 4.7.1. Isolation of the raw Polysaccharides from Ribis nigri semen 167 4.7.2. Isolation ofthe F2 fraction from Ribis nigri semen RPS 168 4.8. Preparation of okra extract 168 4.9. Analytical methods 169 4.9.1. Colorimetric methods 169 4.9.1.1. Determination of the total carbohydrate content in F2 according to Monsigny et al. (1988) 169 4.9.1.2. Determination of the total uronic acid content in F2 according to Blumenkrantz and Asboe-Hansen (1973) with modifications according to Kram (1984) 170 4.9.1.3. Determination ofthe total protein content in F2 170 4.9.1.4. Semi-quantitative detection of arabinogalactan proteins according to van Holst et al. (1985) 171
4 TABLE OF CONTENTS 4.9.2. Hydrolysis, reduction, derivatization and partially degradation 171 4.9.2.1. Hydrolysis of Polysaccharides with trifluoroacetic acid (TFA) 171 4.9.2.2. Hydrolysis of Polypeptides with HCl (6 Mol/L) 171 4.9.2.3. Hydrolysis of Polypeptides with NaOH (4.2 Mol/L) 172 4.9.2.4. Derivatization for determination of D-/L-configuration according to Noe and Freissmuth (1995) 172 4.9.2.5. Reduction of glycoproteins according to Taylor et al. (1972) 172 4.9.2.6. Methylation analysis of glycoproteins according to Hakomori (1964) with modification according to Harris (1984) 173 4.9.2.7. Hydrolytic degradation, with additional ethanol precipitation 175 4.9.2.8. Enzymatic degradation 177 4.9.3. Semi-purification of Onozuka RIO cellulase according to Okemoto et al. (2003) 179 4.9.4. Chromatographie methods 180 4.9.4.1. lon-exchange chromatography (IEC) 180 4.9.4.2. Gel-permeation chromatography (GPC) 180 4.9.4.3. High-performance size-exclusion chromatography (HP-SEC) 182 4.9.4.4. High-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) 184 4.9.4.5. Gas chromatography (GC) 187 4.9.5. Electrophoretic methods 188 4.9.5.1. Capillary zone electrophoresis (CZE) for determination of D-/L-configuration of carbohydrates 188 4.9.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) 188 4.9.5.2.1, Detection of AGPs in SDS-PAGE by Yariv reagent staining 189 4.9.5.2.2. Detection of proteins in SDS-PAGE by silver staining i89 4.9.6. Dialysis 190 4.10. Methods for cell culture and microbiology 190 4.10.1. General working procedures 190 4.10.2. Sterilization 190 4.10.3. Working procedures for cell culture 191 4.10.3.1. Defrosting of AGS cells 191 4.10.3.2. Cultivation of AGS cells 191 4.10.3.3. Passaging of AGS cells 191 4.10.3.4. Cryopreservation of AGS cells 191
4.10.3.5. Calculation of cell density 192 4.10.4. Working procedures for microbiology 192 4.10.4.1. Cultivation of H. pylori 192 4.10.4.2. Cryopreservation of H. pylori 193 4.10.5. Correlation of optical density (OD) and colony forming units (CFU) 193 4.10.6. Identification of H. pylori 194 4.10.6.1. Isolation of bacterial DNA 194 4.10.6.2. DNA quantification 194 4.10.6.3. Real-time Polymerase chain reaction (PCR) 194 4.10.6.4. Primers for real-time PCR 195 4.10.7. FITC labeling of H. pylori 197 4.11. Determination of cell viability and cytotoxicity, MTT-assay (Mosmann 1983) 197 4.12. Agar diffusion test 198 4.13. Adhesion assay of H. pylori with human gastric tissue sections 198 4.14. Quantitative In vitro flow cytometric adhesion assay of H. pylori with AGS cells 200 4.15. Adhesion assay of H. pylori with AGS cells determined by fluorescence intensity 202 4.16. Dot blot overlay assay 203 4.17. Hemagglutination assay 205 4.18. H. pylori adhesin interaction studies by radioimmunoassay (RIA) 206 4.18.1. Radiolabeling of glycoproteins with the chloramine T method according to Hunter and Greenwood (1962) 206 4.18.2. Radioimmunoassay (RIA) with H. pylori 207 4.18.2.1. Determination of binding properties of F2 and okra extract towards different H. pylori strains 207 4.18.2.2. Determination of inhibitory effects of F2 and okra extract towards Lewis b binding of H. pylori 207 4.18.2.3. Determination of inhibitory effects of F2 and okra extract towards sialyl-lewis* binding of H. pylori 208 4.19. Adhesion assay of H. pylori to carbohydrate coupled Polymerie probes 208 4.20. Adhesion assay of H. pylori to magnetic nanopartides 209 4.21. Statistics 210 4.22. Software 211 III. REFERENCES 212
PUBUCATIONS 238 CURRICULUM VITAE 239