- D. Gilliland (1)
- A. Haeger (2)
- J.P. Knox (3)
- I. Liakos (1)
- Y. Lu (1)
- S.E. Marcus (1)
- J.D. Mikkelsen (1)
- I. Moller (1)
- M. Morra (1)
- M.D. Nagel (1)
- M. Rodrigues-Valverde (1)
- H.A. Schols (3)
- S. Siboni (1)
- P. Ulvskov (1)
- M. Vayssade (1)
- E. Velzenberger (1)
- Y. Verhertbruggen (1)
- R.P. Verhoef (3)
- P. Vigneron (1)
- C.D. Volpe (1)
- A.G.J. Voragen (1)
- W.G.T. Willats (1)
Fingerprinting complex pectins by chromatographic separation combined with ELISA detection
Verhoef, R.P. ; Lu, Y. ; Knox, J.P. ; Voragen, A.G.J. ; Schols, H.A. - \ 2009
Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1808 - 1817.
polysaccharide rhamnogalacturonan-ii - enzymatically-tailored pectins - hairy ramified regions - cell-walls - plant-cell - arabinogalactan-proteins - extracellular polysaccharides - monoclonal-antibodies - structural features - galacturonic acid
Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-a-l-arabinan and (1,4)-ß-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.
Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts
Nagel, M.D. ; Verhoef, R.P. ; Schols, H.A. ; Morra, M. ; Knox, J.P. ; Ceccone, G. ; Volpe, C.D. ; Vigneron, P. ; Bussy, C. ; Gallet, M. ; Velzenberger, E. ; Vayssade, M. ; Cascardo, G. ; Cassinelli, C. ; Haeger, A. ; Gilliland, D. ; Liakos, I. ; Rodrigues-Valverde, M. ; Siboni, S. - \ 2008
Biochimica et Biophysica Acta. General subjects 1780 (2008)7-8. - ISSN 0304-4165 - p. 995 - 1003.
hairy ramified regions - growth-factor receptors - surface-chemistry - fibronectin conformation - arabinogalactan-proteins - monoclonal-antibodies - human keratinocytes - integrin binding - adsorbed fibronectin - rhamnogalacturonan-i
Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR- causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR- show the opposite behaviour to the one observed on MHR-B and --grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.
High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles
Moller, I. ; Marcus, S.E. ; Haeger, A. ; Verhertbruggen, Y. ; Verhoef, R.P. ; Schols, H.A. ; Ulvskov, P. ; Mikkelsen, J.D. ; Knox, J.P. ; Willats, W.G.T. - \ 2008
Glycoconjugate Journal 25 (2008)1. - ISSN 0282-0080 - p. 37 - 48.
oligosaccharide microarrays - arabinogalactan-proteins - glycomics - pectin - polysaccharides - generation - epitope - carrot - homogalacturonan - glycoproteins
Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials. Keywords Carbohydrate microarrays - Plant cell walls - Monoclonal antibodies - Hierarchical clustering