Generic Techniques
Prof. Dr. Andreas Hensel
Methods for Plant Polysaccharide Analysis
- Gel permeation chromatography
- Ion exchange chromatography
- Affinity chromatography
- GC-FID
- GC-MS
- HPAEC-PAD
- 13C, 1H NMR
- Capillary zone electrophoresis
- ESI-MS
- MALDI-MS
- Deglycosylation of O-/N-glycoproteins
Results of a typical MCGS project, finished 2013
An non-GPI-anchored AGP cluster (Y2) was isolated from the seeds of Jatropha curcas L. (Euphorbiaceae) with 4.8% polypeptides (mainly Ala, Ser, Gly, Hyp, Glu) and a carbohydrate moiety composed of Gal, Ara, GluA, Rha, Man and GlcN. Beside the typical structural features of arabinogalactans, typical N-glycan linker of the complex type (GlcNAc4Man3Gal2Fuc1Xyl1) were identified. O-glycosylation occurred likely via Hyp and to a lesser extent to Thr and Ser. N-glycans from the complex type, carrying at the innermost GlcNAc at position O-3 one terminal α-Fuc-residue, were also present.
MS analysis of the tryptic digest assigned peptides of three major protein groups: fasciclin-like arabinogalactans, xylogen-like proteins and LysM domain-containing proteins. They were impossible to separate further and it is indicated that various homologous protein forms co-exist. Histological investigation of J. curcas seeds revealed the presence of AGPs in the vessels of cotyledons and in the procambium ring of the embryo.
Example of an ungoing MCGS project
Isolation and structural analysis of lichen-derived polysaccharides and influence on immunocompetent human cells
Details on Methodology can be found in these references:
- Sehlbach M, König S, Mormann M, Sendker JD, Hensel A (in press) The arabinogalactan protein cluster isolated from Jatropha curcas L seed embryo contains fasciclin, xylogen and LysM proteins. Carbohydrate Polymers.
- König S, Herrmann A, Hensel A (2013) Assignment of unknown proteins based on mass spectrometric peptide fragmentation. Biomacromolecular Mass Spectrometry. 3:2, 61-75.
- Hermann A, König S, Lechtenberg M, Sehlbach M, Vakrushev S, Peter-Katalinic J, Hensel A (2012) Proteoglycans from Boswellia serrata Roxb. and B. carteri Birdw. and identification of a proteolytic plant basic secretory protein. Glycobiology. 22,1424-1439.
- J. Zippel, A. Deters, D. Pappai, A. Hensel (2009) A high molecular arabinogalactan from Ribes nigrum L.: influence on cell physiology of human skin fibroblasts and keratinocytes and internalization into cells via endosomal transport. Carbohydrate Research 344, 1001-8.
- Deters A, Lengsfeld C, Hensel A (2005) Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocyes in vitro. Journal of Ethnopharmacology,22, 100-107.
- Deters A, Schröder KR, Hensel A (2005) Kiwi fruit (Actinida chinnisis L.) polysaccharides exert stimulating effects on cell proliferation via enhanced growth factors receptors, energy production and collagen synthesis of human keratinocytes, fibroblasts and skin equivalents, Journal of Cell Physiology, 202, 717-722.
Antiadhesive glycoconjugates against Helicobacter pylori
Prof. Dr. Andreas Hensel
Previous work has shown that acidic peptides, glucuronic acid containing arabinogalactans and pectin-like rhamnogalacturonans are able to inhibit bacterial adhesion of H. pylori to stomach tissue under in vitro and ex situ conditions.
For details see:
- Niehues A, Euler M, Georgi G, Mank M, Stahl B, Hensel A (2010) Peptides from Pisum sativum L. enzymatic protein digest with anti-adhesive activity against Helicobacter pylori: structure-activity and inhibitory activity against BabA, SabA, HpaA and a fibronectin-binding adhesion. Molecular Nutrition and Food Research. 54, 1–11.
- Wittschier N, Lengsfeld C, Vorthems S, Stratmann U, Verspohl E Hensel A (2007) Large molecules as antiadhesive compounds against pathogens. Journal of Pharmacy and Pharmacology, 59, 777-786.
- Lengsfeld C, Titgemeyer F, Faller G, Hensel A (2004) Glycosylated compounds from Okra inhibit adhesion of Helicobacter pylori to human gastric mucosa. Journal of Agricultural and Food Chemistry, 52, 1495-1503.
- Lengsfeld C, Deters A, Hensel A (2004) High molecular weight polysaccharides from black currant seed inhibit adhesion of Helicobacter pylori to human gastric mucosa; Planta Medica, 70, 620-626.
These antiadhesive effects are due to interaction of the glycoconjugates with bacterial adhesins, mainly with BabA and SabA. Ongoing projects aim to establish distinct structure-activity relation, and investigate the respective protein-carbohydrate interaction.
It seems interesting that inhibition of a single bacterial adhesion will lead only to about 20 to 30 % reduction in bacterial inhibition, while use of mixtures of different carbohydrates, influencing different adhesins will result in strong diminished inhibition rates. For that a recent, just finished project used a standardized, rhamnogalacturonan and glycoprotein-containing extract (Okra FE) from immature okra fruits with was used for quantitative in vitro adhesion assay with FITC-labled H. pylori J99, AGS cells and FACS evaluation.
Pinpointing of bacterial adhesins affected by FE was performed by dot-blot overlay assay with immobilized Lewisb, sialyl-Lewisa, H-1, laminin, fibronectin. 125I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron-nanoparticles with different coatings were used to investigate the influence of charge-dependency of interaction with H. pylori surface. Okra FE dose-dependently (0.2 to 2 mg/mL) inhibits H. pylori binding to AGS cells up to 70 % without any cytotoxic effects. FE inhibits the adhesive binding of membrane proteins BabA, SabA and HpA to its specific ligands. Radiolabeled compounds from FE showed unspecific binding against different strains of H. pylori, also to BabA/SabA deficient mutants, which indicates interaction with a still unknown membrane structure in vicinity to the adhesins. The binding depends on the charge of the inhibitors. Okra FE does not lead to a subsequent feed back regulation or increased expression of adhesins or virulence factors. Data indicate unspecific interaction of high molecular compounds from okra fruits to the surface of H. pylori, leading to strong antiadhesive effects.
Updated 26.06.2013
