abstract book network symposium 2011 münster · [email protected] cola cordifolia (cav.)...

14th-16th October, 2011 Münster

MCGS First Indo-German International Research Training Group

Presensts

Network Symposium 2011

IRTG |MOLECULAR AND CELLULAR GLYCO-SCIENCES 2


Table of contents

General Information ................................................ 4

Accomodation Conference Venue Poster Session Venue

City

Schedule ................................................................. 5 Invited Groups ......................................................... 8 Abstracts ................................................................. 9


General Information

Accomodation Hotel-Restaurant Überwasserhof e.K. Überwasserstraße 2 48143 Münster. http://www.ueberwasserhof.de/

Conference Venue Main Lecture Hall Katholisch-soziale Akademie FRANZ HITZE HAUS Kardinal-von-Galen-Ring 50 48149 Münster. www.franz-hitze-haus.de

Poster Session Venue Picasso Museum Picassoplatz 1 48143 Münster. www.graphikmuseum.de Muenster City (in English) http://www.muenster.de/en/


Schedule

Friday (14.10.2011)

9:00 - 9:30

Registration Prof. Bruno Moerschbacher (University of Muenster) Co-ordinator MCGS Opening speech Venue : Franz Hitze Haus Kardinal-von-Galen-Ring 50, 48149 Münster

9:30 - 10:45

Prof. Marc Ostermeier (Johns Hopkins University, USA) A protein therapeutic modality founded on molecular regulation. Jay Choi Engineered protein switch as a biosensor.

10:45 - 11:15 COFFEE BREAK

11:15 - 12:30

Prof. Naoto Shibuya (Meiji University, Japan) Glycosignals and their receptors in plant immunity. Dr. Tomonori Shinya Characterization of receptor proteins using affinity cross-linking with biotinylated ligands.

12:30 – 14:30 LUNCH BREAK

14:30 – 15:45

Prof. Berit Smestad Paulsen (Oslo University, Norway) Structural elements in pectins of importance for their effects on the immune system.

15:45 - 16:15 COFFEE BREAK

16:15- 17:15

Dr. Sergey Vakhrushev (Copenhegen University, Denmark) Zinc finger nuclease glycoengineered SimpleCells: a novel approach to decode O-glycoproteome (GalNAc-type).

18:00 - 19:30

Local Excursion Night-watchman city tour (Meeting point Rathhaus)

20:00

Dinner at esCape Hafenweg 46, 48155 Münster, Germany


Saturday (15.10.2011)

9.00 – 10.15

Prof. Lena Kjellen (Uppsala University, Sweden) Regulation of heparan sulfate biosynthesis. Fredrik Noborn and Prof. em. Ulf Lindahl Multiple facets of heparan sulfate involvement in amyloid diseases.

10.15 – 10:45 COFFEE BREAK

10:45– 11:15

Prof. Bruno Moerschbacher Structure-function relationships of chitosans.

11.15 – 12:15

Prof. Dr. Andreas Hensel Polysaccharides from Plants and Lichen: structural features and in vitro applications. Dr. Alexandra Deters Xyloglycans and their interaction with cellular structures.

12:15 – 12:45

Prof. Dr. Francisco M. Goycoolea Bioresponsive biopolymer-based nanomaterials for biomedicine and biotechnology

12:45 – 14.00 LUNCH BREAK

14.00 -14:30

Dr. Michael Mormann Mass spectrometry of biologically active carbohydrates and their interactions with proteins.

14:30- 15:00

Dr. Kay Grobe A new role of heparan sulfate in sonic hedgehog shedding and its functional activation.

15:00 -15:30

PD Dr. Martin Götte Role of the heparan sulfate proteoglycan syndecan-1 as a modulator of breast cancer cell proliferation, motility and invasiveness.

15:30-16:00 COFFEE BREAK

17:00—18:00

Guided tour Picasso Museum Picassoplatz 1, 48143 Münster.

18:15

Poster Session with Dinner and cocktails in Picasso Museum


Sunday (16.10.2011)

9:30 – 13:00

Breakfast and Brunch ‘FUSION breakfast and Brunch’ At Rice and Royals, Frauenstr. 51-52 in 48143 Münster

14:00 – 18:00

Lab visits and Group discussions

18:30

Guided tour ‘Mühlenhof-Freilicht Museum’ Dinner with Music band At Mühlenhof-Freilicht Museum Theo-Breider-Weg 1, Münster.


Invited Groups

Prof. Marc Ostermeier

Department of Chemical and Bimolecular Engineering, USA

http://www.jhu.edu/chembe/faculty-template/MarcOstermeier.html http://www.jhu.edu/chembe/ostermeier/

Prof. Naoto Shibuya

Department of Life Sciences, Meiji University Kanagawa, Japan http://rwdb2.mind.meiji.ac.jp/Profiles/2/0000118/prof_e.html http://www.meiji.ac.jp/cip/english/graduate/science/index.html

Prof. Lena Kjellen

Uppsala University, Department of Medical Biochemistry and Microbiology, Sweden

http://www.imbim.uu.se/medigly/contact.html www.imbim.uu.se/forskning/kjellenresearch.html

Assistant Prof. Sergey Vakhrushev

Department of Cellular and Molecular medicine (ICMM), Denmark http://icmm.ku.dk/Forskergrupper/CCG/people/

Prof. Berit Smestad Paulsen School of Pharmacy, University of Oslo, Norway http://www.mn.uio.no/farmasi/english/research/projects/maliplants/participants/berit.html


Abstracts

Abstracts are listed alphabetically according to the

presenter´s family name


Two novel pectin-like polysaccharides with immunomodulating properties from the bark of the malian medicinal tree cola cordifolia

Ingvild Austarheim1, Terje E. Michaelsen3, Marit Inngjerdingen4, Frode Rise5, Drissa Diallo6, Berit S. Paulsen1

1School of pharmacy, Department of Pharmaceutical Chemistry, Oslo, Norway 2Norwegian Institute of Public Health, Oslo, Norway 3Institute of Immunology, Oslo University Hospital, Norway 4Department of Chemistry, University of Oslo, Norway 5Departement du Medicine Traditionelle, Bamako, Mali

[email protected] Cola cordifolia (Cav.) R.Br. (Sterculiaceae) is a medicinal tree used by the traditional healers in Mali (West-Africa). Among other indications, hot water extracts of the bark of this tree are used to treat wounds. As polysaccharides will readily be extracted with hot water, they will be present in the traditionally prepared remedy; hence our hypothesis is that the polysaccharides may be responsible or partly responsible for the claimed activity and is therefore of interest to elucidate the structure. Structure elucidation: From a 50°C water extract of the stem bark, two fractions were obtained by a combination of different types of anion exchange chromatography. Problems with high viscosity due to divalent ions were overcome by passing the solution through a chelex 100 column. The simplest of the two polysaccharides isolated, CC1P1, has the novel repeating structure [2->)[β-D-Gal(1->3)]α-L-Rha(1->4)α-D-GalA(1-> ]1500-2000 and molecular weight of approximately 500KD. The other, CC1P2, has the same main structure as CC1P1, but differs from CC1P1 by having 4-MeO-GlcA or 2-OMe-Gal in terminal positions as well. The molecular weight of CC1P2 is approximately the same as for CC1P1. Results form GC-MS, NMR (COSY, TOCSY, HSQC, HSQC-NOESY and HMBC), atomic force microscopy (AFM) will be discussed. Biological activity: CC1P1 and CC1P2 have been tested for macrophage activation and complement modulating activity. The simple CC1P1 shows a higher effect in the complement assay compared to the macrophage activation assay. The more complex CC1P2 shows a higher effect in the macrophage assay than in the complement assay.

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Mapping the binding sites of heparan sulfate/heparin on hedgehog

Shyam Bandari, Stefanie Ohlig and Kay Grobe. Westphalian Wilhelms University of Muenster, Institute for Physiological Chemistry and Pathobiochemistry, Waldeyerstrasse 15, D-48149 Muenster, Germany, [email protected] Signaling proteins binding to heparan sulfate (HS) proteoglycans have been known to play a crucial role in signal transduction and regulation of tissue distribution of signaling molecules. The fly morphogen hedgehog (Hh) is one such secreted signaling molecule that is involved in establishing the basis of fly body plan and the molecule remains important during later stages of embryogenesis and metamorphosis. We asked which hedgehog residues are responsible for binding to heparan sulfate/heparin. It has previously been suggested that positively charged residues in highly conserved Cardin-Weintraub (CW) motif of hedgehog proteins are responsible for binding to heparan sulfate/heparin. To test this idea we have mutated positively charged residues arginine 93, 95 and 97 to alanine and analysed mutant protein binding to heparan sulfate and heparin. Interestingly, we found that these residues are not mediating the HS binding. Instead, the most striking feature is that mutations in the CW motif affects the multimer formation, binding of hedgehog to heparan sulfate which is relevant compared to heparin is different. Hedgehog wild type protein multimerizes and monomers bind to HS very efficiently compared to the CW mutated protein in which multimer formation is also disturbed. In both the cases multimers are getting eluted at very low salt concentration. Based on these observations, we suggest a new function of CW- residues in Hh multimerization and propose the presence of CW unrelated HS binding site.

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Towards a site-specific methyltransferase

Brian Chaikind1 and Marc Ostermeier2

1Chemical Biology Interface Program, Johns Hopkins University 2Department of Chemical and Biomolecular Engineering , Johns Hopkins University [email protected] DNA methylation is an important form of epigenetic control, resulting in transcriptional repression. Methylation of CpG sites plays a role in embryonic development, imprinting, and chromosomal inactivation. Additionally, aberrant methylation patterns are associated with numerous disease states, including cancer. The ability to methylate a specific desired site, therefore, would be useful as a tool to study the effects of methylation at a particular promoter and, potentially, as a protein therapeutic. We have designed a methyltransferase whose activity is highly biased towards an eighteen base pair target site. We have accomplished this by splitting the prokaryotic M.HhaI into heterodimeric fragments and fusing each fragment to a separate zinc finger. Because each zinc finger recognizes a specific nine base pair sequence of DNA, the M.HhaI fragments localize and preferentially methylate CpG sites in between these zinc fingers. Optimization of these constructs has lead to minimal off target activity and high levels of targeted methylation.

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Engineered protein switch as a biosensor

Jay H. Choi and Marc Ostermeier Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland [email protected] Engineered protein switches have a number of exemplary properties for sensing applications including a large dynamic range, high specificity for the activating ligand, and a modular architecture that will facilitate fine-tuning of the desired properties. We have previously demonstrated the creation of protein that behave as switches by the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 b-lactamase (BLA). Such recombination can result in hybrid genes that encode allosteric enzymes in the BLA enzyme activity of the hybrid protein is regulated by maltose. Such recombination can also result in genes that confer to E. coli cells maltose-dependent resistance to b-lactam antibiotics even though they do not encode allosteric enzymes. These ‘phenotypic switch’ genes encode fusion proteins whose cellular accumulation is a result of a specific interaction with maltose. However, the fundamental mechanism and protein design rules for these phenotypic switches are not well understood. Our studies have revealed that the linker regions between the MBP and BLA domains have important roles in dictating the behavior of phenotypic and allosteric switches. By exploiting the role of the linker regions, we have developed new types of maltose-activated protein switches that can be regulated by additional input signal such as redox condition or temperature. These double-regulated protein switches demonstrate that protein switches can be applied to develop complex biosensors or synthetic biological systems.

4


MAMP-mediated priming and synergy: Closely related positive crosstalk of signaling cascade for effective mobilization of defense responses

Yoshitake Desaki1*, Ippei Otomo1, Daijiro Kobayashi1, Yusuke Jikumaru2, Yuji Kamiya2, Balakrishnan Venkatesh3, Shinji Tsuyumu3, Hiroshi Takatsuji4, Hanae Kaku1 and Naoto Shibuya1 1Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan 2RIKEN plant science center, Yokohama, Kanagawa 203-0045, Japan 3 Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan 4National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan *Present address: ZMBP, University of Tübingen, Tübingen, Germany [email protected]

In the course of evolution, plants have developed effective defense mechanisms, known as priming and synergy. These mechanisms can more rapidly and extensively mount defense responses to pathogen infection. It has been reported that the priming state is established by the first contact of pathogens and functions to prepare for the next infection. Synergistic response was observed when the plants were simultaneously treated with different kinds of elicitors. Manipulation of these plant systems seems to provide an advantageous approach for the development of disease resistant crops compared to the direct expression of defense genes from the viewpoint of minimizing the side effect of transgenes on plant growth. However, the mechanisms controlling these sophisticated defense systems are largely unknown.

Bacterial LPS is one of the major cell wall components of gram-negative bacteria and acts as a typical MAMP (microbe-associated molecular pattern) elicitor, triggering immune responses in plants, including rice1). LPS is also known to have an ability to induce priming state in plants2). To study the mechanism of LPS-induced priming in more detail, we established a model system using a cultured rice cells and two purified MAMPs, chitin oligosaccharides and LPS. We found that a low concentration of LPS pretreatment clearly primed chitin oligosaccharide-induced ROS generation and gene induction in the rice cells. Priming effect was also observed for chitin-induced JA and JA-Ile biosynthesis, indicating their contribution for the amplification of downstream responses. On the other hand, these phytohormones seemed not to play a role in the establishment of priming state for early responses such as ROS generation as well as the biosynthesis of JA/JA-Ile themselves. Moreover, when we changed the time length of LPS pretreatment, we found even simultaneous treatment of LPS and chitin oligosaccharide resulted in the enhancement of ROS generation and defense gene expression as similar to those observed for the priming by LPS pretreatment. These results suggested that the LPS-mediated priming could be established very rapidly and also the presence of close relationships between priming and synergy. 1) Y. Desaki et al., Plant Cell Physiol., 47, 1530 (2006). 2) M.A. Newman et al., Plant J., 29, 487 (2002)

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Xyloglycans and their interaction with cellular structures

Alexandra Deters Westfalian Wilhelm’s University Muenster, Institute for Pharmaceutical Biology and Phytochemistry, Hittorfstr. 56, 48149 Muenster, Germany, [email protected] Investigations have shown that polysaccharides exert structure and cell type dependent effects on the cell physiology of human skin cells [1, 2]. The well established in vitro cultures of normal human epidermal keratinocytes (NHEK), HaCaT-keratinocytes and normal human dermal fibroblasts (NHDF) are functional for cell physiologic studies and mechanistic studies. The research of this group of IRTG-MCGS is focused on the essential polysaccharide structure necessary for the activity, its cellular target and the differences between keratinocytes and fibroblasts. For investigations an acidic arabinoxylan on Ispaghula seed husk (P1) and the xyloglucan of Tamarind were chosen due to their known structure and the amazing proliferation stimulating activity of P1 on NHEK [3]. The influence of cellular physiology is checked by BrdU proliferation ELISA and by colorimetric as well as luminescence assays for energy metabolism. Mechanistical studies base on gene expression analysis via qRT-PCR or Microarrays, MAPK activity and cellular uptake by fluorescence activated laser scan microscopy. Essential structure is elucidated by enzymatic breakdown followed by structure elucidation and tested on the cellular system. In progress it was observed that P1 also stimulated the proliferation of NHDF but gene expression analysis and microscopic analysis showed a different way of activity. The tamarind xyloglucans showed a similar stimulating activity on skin cells. It has been started to isolate a xylose specific lectin to obtain a tool for detection of xylose containing polysaccharides without FITC-labeling to detect the polysaccharides bound to proteins outside the cells or internalized by endosomes. Results will give an overview about the cellular targets of polysaccharides as well as the signal pathways involved to stimulate the skin cell proliferation. 1. Deters AM., Schröder KR., Hensel A. J Cell Phys, 2005, 202: 717-722 2. Gescher K., Deters AM. Journal of Ethnopharmacology, 2011, dx.doi.org/10.1016/j.jep.2011.05.042 3. Deters, A.M., Schröder, K.R., Smiatek, T., Hensel, A., Planta Medica, 2005, 71 (1): 33-39.

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Smart nanoparticle against aspergillus spp.

J.P. Fuenzalida, Bruno Moerschbacher, F.M. Goycooleaa. Westfälische Wilhelms-Universität Münster Institut für Biologie und Biotechnologie der Pflanzen Schloβgarten 3, 48149 Münster, Germany [email protected] Infections caused by Aspergillus have emerged as an important cause of life-threatening diseases in immunocompromised patients. Aspergillus spp are a class of insidious fungi that can infect different organs such as the brain, liver, and lungs.[1] We are designing nanobiotechological platforms based on polysaccharides and proteins to target specific carbohydrate motifs in the cell wall of Aspergillus, so as to achieve targeted drug delivery to the lung. As a first step towards this end, we are studying the fundamental characteristics of the polyelectrolyte nanocomplex formed between alginate (Mw ~4kD; M/G ~1,42) and chitosan (Mw ~13 kD; DA ~1,4% ) obtained spontaneously by mixing of the two polyelectrolytes. Figure 1 shows the size, polydispersity, and absorbance (λ=520 nm) of the nanocomplexes formed at varying n+/n-(-COO-/-NH3) charge ratios of the two polysaccharides. Notice that the size of the formed nanocomplex attains a maximum value at a value n+/n- well below the full stoichiometry (~0.6). Beyond this point, at greater n+/n- the size of the nanocomplexes decreases under a monotonic trend and attain a fairly constant size of ~ 90 nm. We are currently exploring the effect of varying the Mw of alginate component, the pH and the counter ion concentration on the characteristics of these systems. 1. Gastebois, A., et al.,. Future Microbiology, 2009. 4(5): p. 583-595.

Fig 1: Alginate – Chitosan nanoparticle characterized in size (Z-Ave), Polydispersity index (PDI) and absorbance a 520 nm (Abs)

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Role of the heparan sulfate proteoglycan syndecan-1 as a modulator of breast cancer cell proliferation, motility and invasiveness

Martin Götte and Sherif A. Ibrahim Department of Gynecology and Obstetrics, Münster University Hospital, Domagkstr. 11, D-48149 Münster, Germany Introduction: The heparan sulfate proteoglycan Syndecan-1 (Sdc-1) acts as a coreceptor for growth factors and chemokines, modulating cellular growth and migration during embryonic development, wound repair and malignant transformation. Sdc-1 plays major roles in tumor cell adhesion, -invasion and metastasis, and acts as a co-receptor for the hepatocyte growth factor (HGF) receptor c-Met in multiple myeloma. We have recently demonstrated a coexpression of Sdc-1, c-Met and angiogenic factors in ductal carcinoma in situ of the breast. The present study aimed at an investigation of the role of the functional interplay of Sdc-1 and c-Met in breast cancer progression. Methods: The siRNA-technology was employed to knock down Sdc-1 mRNA expression in the human breast cancer cell lines MDA-MB-231 and MCF-7. Functional consequences were studied by Western blotting for HGF-dependent kinase activation, in cell proliferation assays, in vitro-wound repair cell migration studies, time-lapse video microscopy, and in matrigel invasion chambers. Changes in gene and protein expression were studied by Affymetrix gene array analysis, qPCR and Western blotting. Results: siRNA-mediated targeting of Sdc-1 expression lead to significantly reduced HGF-dependent phosphorylation of c-Met and downstream signalling cascades of the MAPK and Akt pathways. HGF-dependent tumor cell proliferation and migration were significantly decreased. In contrast, in vitro invasiveness and cell motility of Sdc-1 depleted MDA-MB-231 cells were significantly increased in a HGF-independent manner. Affymetrix analysis, qPCR and Western blotting revealed a dysregulation of genes involved in regulating proteolytic activity, cytoskeletal function and E-cadherin expression as a mechanism for the invasion-promoting effect of Sdc-1 depletion. Discussion: Our data demonstrate a co-receptor role for Sdc-1 in HGF/c-Met signalling, which affects cell proliferation and motility. The proinvasive effect of c-Met is overruled by Sdc-1 dependent regulation of protease activity and expression, as well as E-cadherin function. Conclusions: Sdc-1 emerges as a novel target for glycosaminoglycan-based approaches of breast cancer therapy.

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Bioresponsive biopolymer-based nanomaterials for biomedicine and biotechnology

Francisco M. Goycoolea Institut für Biologie und Biotechnologie der Pflanzen Westfälische Wilhelms-Universtät Münster Schlossgarten 3; 48149 Münster, Germany Tel: +49 2518324864 Fax: +49 2518323823 [email protected] The fundamental physicochemical and biological properties of polysaccharides and proteins are being exploited in the rational development of innovative nanobiotechnological bioresponsive ("smart") platforms for use in biomedicine and biotechnology. Several approaches are being pursued in this direction. On the one hand, pH-sensitive nanoparticles have been obtained by ionotropic gelation of chitosan with pentasodium tripolyphosphate (TPP), concomitant complexation with alginate (ALG) and subsequent crosslinking with genipin (GNP), a natural crosslinking agent of chitosan and proteins. These particles have been studied by synchrotron SAXS that has enabled to demonstrate that they possess a core-shell structure in agreement with evidence gathered from TEM images. The overall thickness of the shell was dependent on the pH and seemed to be smaller in acetate buffer (pH 4.5) than in simulated gastric fluid (pH 1.2). The in vitro release of metronidazol after incubation during 1 h was found to be twice as much at pH 4.5 than at 1.2. Insulin-loaded nanoparticles of this type also exhibit a pH-sensitivity and, in principle, perform as expected so as to achieve a high insulin bioavailability by oral delivery. Under another approach, nanoparticles intended for selectively recognize and quench bacterial metabolites involved in quorum sensing (QS) of Gram(-) bacteria, as an strategy to exert a selective targeting and interfere with the pathogenic and virulent responses. To this end, CS-TPP and CS-TPP-GNP nanoparticles are being studied in their capacity to adsorb acyl-homoserin-lactone (AHL) bacterial metabolites well-known to mediate QS processes in a large number of pathogenic bacteria. We are also investigating other approaches, such as the loading of the NPs with anti-quorum sensing compounds which can act as competitors of the AHLs at the interaction with their receptor, blocking the AHL-mediated signalling and QS gene expression. The effect of these NPs has been tested against a modified E. coli reporter used as a biosensor of QS. Ongoing studies in our laboratory are also addressing the mucoadhesive properties of CS and other polysaccharides (e.g. karaya gum) so as to develop novel nanotechnological approaches that can be used in antibiotic-free stomach localized therapy against Helicobacter pylori, for example, by the delivery of molecules known to inhibit the bioadhesion of the pathogen. In parallel, we are developing surface-modified ALG-CS and ALG-protein based nanospheres and nanocapsules with a view to harness specific targeting properties in the treatment of pulmonary aspergillosis infection.

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A new role of heparan sulfate in sonic hedgehog shedding and its functional activation

Kay Grobe Institute for Physiological Chemistry and Pathobiochemistry, Waldeyerstraße 15, University Hospital Münster, Germany, [email protected] Paracrine Sonic Hedgehog (Shh) signaling is a main determinant of embryonic development and contributes to cancer progression and the maintenance of adult stem cells. The understanding of Shh release and activity, however, has been impeded by its unusual biology. Shh is covalently modified by N-and C-terminal lipidations, which in turn affects numerous aspects of Shh localization, release, movement, and activity. These are only poorly understood, but have all been linked genetcally to heparan sulfate (HS) expression. We thus asked why the sequence of lipid modification, firm attachment to the cell surface, multimerization, and multimer release rather than direct discharge is essential for effective Shh secretion and function, and how these processes depend on HS.

In this presentation, we suggest a new model that integrates multiple controversial and poorly understood features of Shh biology. We found that HS-regulated, ADAM-mediated proteolytic shedding of multimeric Shh from the cell surface results in its solubilization in truncated, unlipidated form. In this scenario, Shh N-palmitoylation is the prerequisite for membrane-proximal proteolytic cleavage of an N-terminal peptide during solubilization that otherwise blocks the Shh binding site to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for Shh biological activity. Analysis of the human Shh crystal structure supports this finding, demonstrating that Ptc-binding sites are occupied in trans by N-terminal peptides of adjacent morphogens in the cluster. Moreover, this structure supports a role of HS in Shh multimerization. Based on these findings, we suggest new mechanisms of Shh multimerization and activation/release.

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Investigation of molecular mechanisms of chitosan perception in brachypodium distachyon

Ekaterina Gubaeva, Nour Eddine El Gueddari and Bruno M. Moerschbacher Westfalische-Wilhelms University of Muenster, Institute of Plant Biology and Biotechnology, Hindenburglatz 55, 48143 Muenster, [email protected] Wheat stem rust (Puccinia graminis f. sp. tritici) is historically the most damaging disease of wheat (Triticum aestivum L.). In susceptible wheat plants, the stem rust fungus invades its host through stomata and establishes functional haustoria within host cells as sophisticated organelles allowing the pathogen to participate in the hosts metabolism and complete the fungal life cycle by generating new uredospores. In highly resistant wheat cultivars, infected plant cells react hypersensitively and die within a few hours after penetration. This mechanism of programmed cell death prevents the formation of a functional haustorium and efficiently stops further infection (Menden et al., 2006). Epi-fluorescence microscopy with the fluorescence-labeled lectin wheat germ agglutinin (WGA) revealed that surfaces of infection structures formed on the plant cuticle expose chitin, whereas surfaces formed after invading the host expose chitosan (Gueddari et al., 2002). Conversion of chitin to chitosan during intercellular growth may protect plant pathogenic fungi from being lysed by extracellular plant chitinases and keep them invisible for the plant’s immune system (Gueddari et al., 2002). An ability to recognize chitosan fragments leading to activation of the host immune system with subsequent hypersensitive response is one of the possible reasons for resistance to wheat stem rust. We have shown that partially N-acetylated chitosans and chitooligosacharides are able to elicit an oxidative burst, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, non-wounded wheat leaves (Vander et al., 1998). It was recently reported that perception of chitin oligosacharides contributes to disease resistance to the blast fungus Magnaporthe oryzae in rice and barley (Kishimoto et al., 2010, Tanaka et al., 2010). A plasma membrane glycoprotein CEBiP (chitin elicitor binding protein) and a receptor kinase OsCERK1 (chitin elicitor receptor kinase) were identified as critical components for chitin signaling in rice, as well as the receptor kinase CERK1 in Arabidopsis (Shimizu et al., 2010, Miya et al, 2007). However, unraveling the chitin/chitosan signaling system in wheat is difficult due to its complex genome. Brachypodium distachyon is currently being established as a model species for temperate grasses and cereals, because of many advantages such as short life cycle, small plant size, and small genome. Brachypodium is closely related to the major cereals wheat and barley; mutant screens have been successful in identifying both resistant and susceptible lines (Garvin D.F.). Identification of the components of chitin/chitosan signaling system in Brachypodium distachyon can contribute in better understanding the chitosan-based mechanism of antifungal resistance, including to wheat stem rust. One of the interesting questions in this area and one that we would like to pursue is the possible involvement of the chitin receptor in chitosan perception, using Brachypodium distachyon as a model plant.

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Polysaccharides from Plants and Lichen: structural features and in vitro applications

Andreas Hensel University of Münster, Institute for Pharmaceutical Biology and Phytochemistry Plant-derived polysaccharides compile an extremely heterogeneous group of polymers, differing in sugar composition, linkage types, fine structure and secondary substitution. Major goal of ongoing work is the establishment of a variety of analytical methods for unambiguous and effective structure elucidation of polysaccharides. This can only be done by using a whole bunch of different methods, enabling us to get the polymer fine structure in a puzzle-like manner from monomer composition, linkage analysis, reductive-labeling of uronic acids, NMR and MS experiments, additionally to cleavage of the polymers by specific enzymes to oligosaccharides. Due to the fact that many endogenous processes of human cell physiology are regulated by glycosylated compounds plant-derived oligo- and polysaccharides can be used for specific influencing such processes in order to obtain new compounds for physiological engineering. Skin cell proliferation and differentiation, cell-cell adhesion and functionality of immunological cells can be triggered specifically by exogenous polysaccharides, using these polymers as a suitable tool for development of future medical agents.

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Bio-Activity Matrices for Partially Acetylated Chitosan Oligomers

Thejaswi Kalagara and Bruno M. Moerschbacher Institute of Biochemistry and Biotechnology for Plants, University of Muenster, Muenster 48161, Germany. [email protected]

Chitotriosidase (ChT) is well known to be a human chitinase which belongs to glycosyl hydrolase family 18. ChT is a processive, endo-acting enzyme which is able to cleave chitotriose as the smallest substrate, hence the name chitotriosidase. As part of the innate immune system, ChT is expressed in humans exclusively by professional phagocytes such as macrophages and neutrophiles. Unlike lysozyme, ChT also hydrolyze the partially de-acetylated chitin derivative, chitosan. Chitosan is known to have broad range of biological activities partly due to its better water solubility compared to chitin and also the presence of positive charges at slightly acidic, physiological pH. These properties prompted the development of a number of biomedical applications which however, due to poor reproducibility, have not yet matured to the point of success. It is likely that the physicochemical structure of chitosan influences its biological properties. Here, the main parameters that come into play are its degree of acetylation (DA) and its pattern of acetylation (PA) that determine the fate of chitosan in a biological system. Solubility of chitosan is related to the DA (i.e. the percentage of N-acetylglucosamine units) as deacetylated glucosamine units are protonated in acidic media. We have recently proposed that the biological functionality of chitosan is determined not only by DA but also by its PA (i.e. the arrangement of acetylated and deacetylated units in a polymer chain) which ultimately determines the type of products formed if ChT is present in a target tissue. The main objective of our studies is, therefore, to understand the pattern of hydrolysis of chitosan (of different PA and DA) in vivo by human chitotriosidase and further, the role of chitosan oligomers produced by ChT in influencing various cascades of biological events, e.g. in wound healing. So far, we studied, how the recombinant ChT hydrolyze different chitosan’s (having difference in DA and PA) and further analyzing the oligomers. Presently, we are trying to understand whether different chitosan oligomers (with different degree of polymerization and acetylation) have difference in its activity in human cells. Finally, this may help us to know, the better suitable chitosan’s for biomedical applications. Key words: Chitotriosidase; degree of acetylation; pattern of acetylation

13


Regulation of heparan sulfate biosynthesis

Lena Kjellén Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden, [email protected] Heparan sulfate proteoglycans influence embryonic development as well as adult physiology through interactions with protein ligands. The interactions depend on heparan sulfate structure, which is determined largely during biosynthesis by Golgi enzymes. The biosynthesis enzymes are subject to regulation on both transcriptional, translational and posttranslational levels. In addition to the enzymes, other regulatory proteins and the concentration of the sulfate donor PAPS may also greatly influence the final structure of the heparan sulfate chains produced. Heparan sulfate biosynthesis can be studied in vitro using recombinant enzymes, in cells manipulated to overexpress or to lack components of the biosynthesis machinery, or in model organisms such as mice, zebrafish, Drosophila melanogaster or C. elegans.

14


NDST1 and NDST2 have divergent impact on mast cell development

Anders Dagälv, Katarina Holmborn, Inger Eriksson, Lena Kjellén & Magnus Åbrink Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden, [email protected] Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, while lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. While MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1+/- and NDST1-/- MCs is higher (≈ 30%) than in control MCs where ≈95% of the 35S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored mast cell proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.

15


Role of 3-O sulfated heparan sulfate in breast cancer pathogenesis

Archana Vijaya Kumar, Ezeddin Salem Gassar, Martin Götte Department of Gynecology and Obstetrics, Münster University Hospital, Domagkstr. 11, D-48149 Münster, Germany. [email protected] ; [email protected]

Cellular function and phenotype are highly influenced by heparan sulphate glycosaminoglycans (HSGAGs). Present at the cell–tissue–organ interface, they have been shown to have crucial regulatory roles in normal physiological processes, such as embroyogenesis, as well as in pathophysiological conditions, like tumour onset and progression. As specific motifs within HS define its ligand binding properties and specificity, defined sulfation patterns in HS may determine its physiological function and its function in malignant disease. During biosynthesis of HS, which occurs in the Golgi apparatus, the polysaccharide backbone is synthesized, which is subjected to various modifications. These modifications include N-deacetylation and N-sulphation of glucosamine, C5-epimerization of GlcA to form IdoA residues, 2-O-sulphation of GlcA or IdoA residues, as well as 6-O-sulphation and 3-O-sulphation of glucosamine residues. Regarding a possible role in the pathogenesis of malignant diseases, heparan sulphate 3 o sulfotransferase 2 (HS3ST2) is an attractive candidate enzyme. A 3-O-sulfated group plays an essential role in binding of antithrombin III by heparin and heparan sulfate, a process potentially modulating metastatic behavior. The HS3ST2 gene is silenced by promoter methylation in a variety of cancers, suggesting a potential role in the pathogenesis process. Increased 3-O-Sulfation of heparan sulfate due to plasmid-based ectopic overexpression of HS3ST2 in human breast cancer cells leads to moderately increased cell proliferation, significantly increased invasiveness in the Matrigel invasion chamber assay. Increased 3-O-Sulfation of heparan sulfate results in increased bFGF-mediated MAPK activation and also in increased general MAPK activation / expression. Increased MAPK activation in HS3ST2-overexpressing cells depends on heparan sulfate, as determined by heparitinase digestion assays. Affymetrix gene array screening revealed upregulation of proteases and downregulation of protease inhibitors in HS3ST2-overexpressing cells. qPCR showed significant upregulation of proteases in HS3ST2-overexpressing cells. Increased proteolytic activity was confirmed by gelatin zymography. Moreover, HS3ST2-overexpression is associated with an upregulation of the cell-cell adhesion molecules E-cadherin and Cadherin 11. Finally, HS3ST2-overexpression is associated with highly increased expression of the transcription factor TCF4, Beta catenin and Lrp 6, which are components of the wnt-signalling pathway. HS3ST2-overexpression is also associated with increased expression of components of Notch pathway. In summary, these results suggests that altered HS structure leads to altered activation of signalling pathways, which in turn results in changes in tumor cell behavior.

16


Nanobiotechnological platforms for stomach mucosal local therapy targeted to helicobacter pylori

Bianca Menchicchi, Andreas Hensel, F. M. Goycooleaa

Westfälische Wilhelms-Universität Münster Institut für Biologie und Biotechnologie der Pflanzen Schloβgarten 3, 48149 Münster, Germany [email protected] The gram-negative bacteria Helicobacter pylori is responsible of chronic gastric infection and inflammation associated with the development of duodenal ulcer and gastric carcinoma due to its persistent colonization of the mucous layer of the stomach and difficult eradication. An important requirement for a promising drug delivery nanosystem acting locally against the adhesion of Helicobacter pylori is the ability of the materials to interact first with the stomach mucous. The mucoadhesion properties of several known and less conventional polysaccharides (e.g. chitosan, karaya gum, mesquite gum, alginate, carboxymethyl cellulose) with well established biocompatibility and biodegradability, documented use in traditional medicine, pharmaceutical and/or food fields, are being currently investigated. To this end, a capillary microviscosimetric method is being used to probe interaction of the polysaccharides with mucin in the dilute regime. Synergistic or antagonistic deviations of the viscosity values (�) from those expected from additivity are being interpreted as the result of heterotypic interaction of the two components. Preliminary results for the mixture of chitosan and mucin at varying composition of the two components notice that at low MUC/CS mass ratios there seems to be a synergistic increase of �rel, while at higher MUC/CS mass ratios a monotonic decrease that attains a minimum at MUC/CS~75/25 is observed. We are currently exploring in further detail the mechanisms involved in this interaction and the potential of this system in the rational design of MUC/CS nanoparticles.

17


Glycoconjugates as antiadhesive compounds against helicobacter pylori outer membrane proteins

J. Messing and A. Hensel University of Münster, Institute of Pharmaceutical Biology and Phytochemistry, Hittorfstr. 56, 48149 Münster, Germany [email protected] Background: Selective blocking of H. pylori adhesins by using defined exogenous oligo- or polysaccharides from plant origin can be a new strategy for preventing the adhesion process and therefore the subsequent infection caused by H. pylori. Especially the use of such antiadhesive compounds after bacterial eradication could help to reduce the risk of reinfection. A polysaccharide from Ribes nigrum L. was isolated and identified to possess sufficient antiadhesive activity [1.] in several in vitro assays. Methods: In situ and in vitro adhesion assays were performed with FITC-labelled H. pylori on human stomach tissue [1., 2.] resp. on AGS-cells [3.]. Dot blot overlay assays with immobilized ligands (Lewisb-HSA, sialyllactose, fetuin, laminin etc.) of the bacterial adhesins on PVDF membranes [4.] were used to evaluate the blocking specificity of the polysaccharide. Agar-diffusion test was used to exclude direct cytotoxicity. Structural elucidation was performed by SEC, HPLC-PAD, GC-MS and methylation analysis. Results: The isolated fraction F2 from blackcurrant seeds showed antiadhesive activity. The polymer with a mean MW of 2000kDa was identified as a type II arabinogalactan protein with high amounts of galactose, arabinose and xylose with a 1,3-linked galactan backbone, and intense branching via position 6 of the galactose residues. Reduction of bacterial adhesion was concentration dependent with 10-15% in the range of 1mg/ml, related to the maximal possible adhesion. Investigation by dot blot assays with immobilized glycoproteins showed specific blocking of the BabA and fibronectin binding adhesin whereas other adhesins were not influenced. The test compound did not show any direct cytotoxicity. Conclusion: This leads to the idea that this polysaccharide contains sugars motives which are suitable blocking substances against BabA. References: 1. Lengsfeld, C., A. Deters, G. Faller, and A. Hensel, Planta Medica, 2004, 70, 620-626 2. Falk, P., K. A. Roth, T. Boren, T. U. Westblom, J. I. Gordon, and St. Normark, Proceedings of the

National Academy of Sciences of the United States of America, 1993, 90, 2035-2039 3. Niehues, M., and A. Hensel, The Journal of Pharmacy and Pharmacology, 2009, 61, 1303-1307 4. Ruhl, S., A. L. Sandberg, M. F. Cole, and J. O. Cisar, Infection and Immunity, 1996, 64, 5421-5424

18


Structure-function relationships of chitosans

Bruno M. Moerschbacher WWU Münster University, Institute of Plant Biology and Biotechnology, Hindenburgplatz 55, 48143 Münster, Germany, [email protected] Chitosans are a family of linear polysaccharides of ß-1,4-linked glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) units. Being derived from the renewable resource chitin, the second most abundant organic molecule on earth, and being bioactive and biocompatible, chitosans are among the most interesting and most promising bioma-terials with many potential applications in biotechnology, cosmetics, food, veterinary, medical, pharmaceutical, or agricultural sciences. The main obstacle in developing marketable products based on chitosans are their often observed lack in reliability in giving specific biological activities, such as rendering plants resistant to disease or leading to scar-free wound healing. We have argued that this is at least in part due to the use of poorly defined chitosan preparations, and a basic lack of understanding structure-function relationships of chitosans. Therefore, we began a thorough investigation of the influence of different structural aspects on the biological activities of well characterised chitosans. We were able to show that both the physico-chemical properties and the biological functionalities of partially acetylated chitosans in aqueous solutions greatly depend on the degree of polymerisation (DP) and the degree of acetylation (DA) of the chitosan used. Based on theoretical speculations concerning the interaction with potential receptors or targets or with potentially metabolizing enzymes in a target tissue, we have then predicted that the biological activities will in addition be influenced by the pattern of acetylation (PA) of the chitosan used. Today’s partially acetylated chitosans are produced using chemical methods, either by homogeneous or heterogeneous deacetylation of fully acetylated chitin or by chemical reacetylation of fully deacetylated chitosan. All of these methods seem to yield chitosans with randomly distributed acetyl-groups (random PA). We now propose to use chitosan modifying enzymes (CME) to generate chitosans with non-random PA, such as processively acting chitin deacetylases to yield chitosans with block-PA, or sequence-specific chitosan hydrolases yielding chitosan oligomers with non-random PA. We are currently running extensive discovery programs for such enzymes, cloning and heterologously expressing their genes, and characterising the recombinant proteins and their products. The first novel CMEs and fingerprinting techniques developed for the analysis of the PA of their products will be described, as well as the results of our initial bioactivity assays on these novel chitosans with non-random PA.

19


Mass spectrometry of biologically active carbohydrates and their interactions with proteins

Lukas Witt1, Mona Schulte1, Gnanesh Kumar B S1,2, Gottfried Pohlentz1, Jasna Peter-Katalinić1, and Michael Mormann1 1University of Muenster, Institute of Medical Physics and Biophysics, Robert-Koch-Str. 31, D-48149 Muenster, Germany; 2University of Hyderabad, Department of Biochemistry, Hyderabad, Andhra Pradesh 500046, India. [email protected] Characterization of molecular structure of complex carbohydrates, i.e. glycoconjugate glycans, oligosaccharides and polysaccharides by mass spectrometry is considered as an essential prerequisite for understanding of their biological function in countless processes. Mass spectrometry (MS) as a method of high sensitivity and accuracy is applied for structural identification of biopolymers. Structural parameters of proteins relevant for their biological function include determination of the peptide chain length, N- and C-termini, their sequence and the sites of disulfide bridges and other posttranslational modifications. We are currently focussing on the selective enrichment of glycopeptides derived from enzymatic digestions by use of zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction based sample preparation methods followed by their thorough investigation by means of quadrupole time-of-flight (Q-TOF) MS and high resolution Fourier-transform ion cyclotron resonance (FT-ICR) MS employing various activation methods such as electron capture dissociation, infrared multiphoton dissociation and low-energy collision-induced dissociation for comprehensive structural characterization. FT-ICR MS is used as a powerful tool for glycoscreening of complex mixtures giving rise to e.g. demonstration of the presence of overlapping nearly isobaric ionic species in glycoconjugate samples obtained from congenital disorder of glycosylation (CDG) patient’s urine or unravelling complex sulfation pattern in glycosaminoglycans. We are currently exploring the potential of ZIC-HILIC-based chromatography hyphenated to ESI-QTOF mass spectrometry for the compositional analysis and structural elucidation of complex heparin oligosaccharide mixtures. Furthermore, we are using mass spectrometric methods for the investigation of the non-covalent interaction of glycoconjugates with carbohydrate-binding proteins such as plant and bacterial lectins.

20


Effects of slight acidic arabinoxylans from plantago ovata seed husk on human skin cells

Wei Nie, Alexandra Deters Westfalian Wilhelm’s University Muenster, Institute for Pharmaceutical Biology and Phytochemistry, Hittorfstr. 56, 48149 Muenster, Germany, [email protected] Plantago ovata (Ispaghula), Forsk, Plantaginaceae is an indigenous product of south Asia. The Plantago ovata seed husk has a high content of water-soluble polysaccharide (P1) which is an arabinosyl(galactosyluronic acid)rhamnosylxylan1. P1 has been shown to increase the proliferation of NHEK2. The effect of Plantago ovata seed husk polysaccharide (P1) is investigated on human skin cells: normal human epidermal keratinocytes (NHEK), human adult low calcium high temperature keratinocytes (HaCaT) and normal human dermal fibroblasts (NHDF). First of all the investigations focus on influence of P1 on cell physiology: 1) BrdU incorporation for quantification of cell proliferation, 2) MTT and WST-1 tests for indication of cell viability and 3) LDH test for determination of necrotic cytotoxicity were chosen. In order to figure out the fundamental mechanism studies of internalization of P1 into the cells and influence of P1 on gene expression of the cells were carried out. P1 showed significant enhance of cellular proliferation and cell viability of keratinocytes and fibroblasts whereas no necrotic cytotoxicity was detected. Activity was shown to be independent to used concentration. Even low amount of 0.01 µg/ml showed a significant effect. Using laser confocal microscope it was observed that P1 was rapidly internalized into the cells after an incubation time of 3h. Gene expression analysis by PIQORTM Skin Microarray revealed that the most influence on the expression of NHEK lies on genes of extra cellular matrix (ECM) and cytokine signaling followed by transcription factors and cell metabolism as well as cytoskeleton. Remarkable regulations on gene expression of NHDF were observed concerning cytokine signaling, cell metabolism, transcription factors as well as protein trafficking. Interestingly gene expression of cytokines is mostly down-regulated. One exception was the CTGF up-regulated in NHDF and involved in ECM production, chemotaxis, proliferation and integrin expression. In conclusion P1 promotes human skin cells proliferation significantly. The data obtained are supported by gene expression analysis and microscopy studies. The focus of further studies lies on the examination of the effective structure of P1 and to itemize the involved signal pathways. 1Sandhu JS., Hudson GJ., Kennedy JF., Carbohydrate Research, 1981, 93: 247-259 2Deters AM., Schröder KR., T. Smiatek T, Hensel A., Planta Medica, 2005, 71: 33-39

21


The role of Uronyl 2-O sulfotransferase in cell proliferation and adhesion

K. Nikolovska, D. G. Seidler Westphalian Wilhelm's University of Münster, Institute for Physiological Chemistry and Pathobiochemistry (Waldeyer Str. 15, 48149 Münster, Germany),[email protected] Chondroitin sulfate (CS) and dermatan sulfate (DS) are proteoglycans present on the cell surfaces and in the extracellular matrix (ECM). They are involved in binding and regulating a number of distinct proteins, including cytokines and growth factors. Furthermore, it is found that CS/DS have strong impact on vital cell characteristics such as cell adhesion, cell proliferation and migration. Structurally, CS/DS are linear, sulphated polysaccharides containing a repeating disaccharide unit. CS disaccharide units consistent of D-glucuronic acid (GlcA) and D-N-acetylgalactosamine (GalNAc) can be modified by sulfate groups mainly at C-4 and/or C-6 of GalNAc and/or C-2 of GlcA. DS is a variant of CS where proportions of GlcA are substituted with iduronyl (IdoA) residues by epimerization during polymerization. In addition, the IdoA of DS is frequently 2-O sulfated. Several sulfotransferases are involved in the creation of the sulfation pattern of the GAG chain. The enzyme of interest in this project is uronyl 2-sulfotransferase (UST). UST is involved in the transfer of sulfate group to the C-2 position of the IdoA residues of DS. However, the exact mechanism of UST action has not yet been revealed. To analyze the role of the enzyme in cell behavior, UST was cloned in pcDNA 3.1 vector and transfected in mammalian CHO K1 cell line. Two high UST expressing and one medium UST expressing clone were selected and analyzed for UST expression by comparison to wild type cell line (CHO K1). Successful transfection was confirmed by mRNA expression, Western blot and immunofluorescence microscopy. Three fold increase of UST expression was detected on mRNA and protein level in the high UST expressing clones. Furthermore, cell surface GAGs were extracted and analyzed for total uronic acid concentration and sulfate content. Higher concentration of uronic acid along with threefold increase of total sulfate concentration was found in the high expressing CHO UST clones compared to CHO K1 cells. FACE analyses after ABC chondroitin lyase digestion of the extracted GAGs (one high expressing CHO-UST clone and CHO K1) revealed two fold increase in 4-O sulfated and 6-O sulfated structures in the high expressing CHO-UST clone. This was confirmed by RT-PCR which showed overexpression of chondroitin 4-sulfotransferase and dermatan 4-sulfotransferase in the same CHO-UST high expressing clone, compared to the CHO K1. Additionally, the proliferation and adhesion characteristics of one CHO UST high expressing clone and CHO K1 cell lines were analyzed. Higher rate of proliferation was found in CHO-UST cells compared to the CHO K1. The adhesion assay showed presence of 52 % adherent cells in the CHO-UST high expressing cells, and 40 % adherent cells in the CHO K1 cell line. It is likely that the differences in the sulfation pattern in the GAGs extracted from the UST transfected cells induce alterations in their adhesion properties and increase their proliferation rate. This effect is caused by the increased presence of ΔDi4S that promotes cell migration and proliferation, as well as ΔDi6S which enhances cell adhesion properties.

22


Heparan sulfate/heparin promotes tranthyretin fibrillization through selective binding to a basic motif in the protein

Fredrik Noborn, Paul O'Callaghan, Erik Hermansson, Xiao Zhang, John B. Ancsin, Ana M. Damas, Ingrid Dacklin, Jenny Presto, Jan Johansson, Maria J. Saraiva, Erik Lundgren, Robert Kisilevsky, Per Westermark & Jin-Ping Li

Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden,

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

23


Multiple facets of heparan sulfate involvement in amyloid diseases

Fredrik Noborn and Ulf Lindahl Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden, [email protected], A common theme in amyloid diseases (Alzheimer’s disease, prion diseases, type II diabetes, AA and TTR amyloidosis) is the deposition of disease-specific peptide aggregates in tissues. Amyloid peptides bind to heparan sulfate (HS), and HS has been found to promote the aggregation process. Findings in our lab are in accord with this notion, but also point to more complex relations. For instance, mice that overexpress the HS-degrading endo-glucuronidase, heparanase, became virtually resistant to inflammation-induced AA amyloidosis. On the other hand, similar mice showed reduced ability to clear cerebral Aß amyloid aggregates, a hallmark of Alzheimer’s disease.

We have recently in particular studied transthyretin (TTR) -related amyloid disease. TTR is a homotetrameric protein that transports thyroxine and retinol. TTR is found as a main constituent of amyloid fibrils in several distinct clinical forms of amyloidosis. Several familial mutations of TTR have been linked to hereditary forms of TTR amyloidosis and considerable effort has been devoted to study of the aggregation of these mutant proteins. However, the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wildtype (WT) protein. Heparan sulfate (HS) has been found as a prominent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis.

In our presentation we aim at providing an overview of the varied roles of HS in some amyloid diseases.

24


A new role of sonic hedgehog shedding in functional activation of the solubilized morphogen

Stefanie Ohlig, Pershang Farshi, Ute Pickhinke, Johannes van den Boom, Stanislav Jakuschev, Daniel Hoffmann, Rita Dreier & Kay Grobe Institute for Physiological Chemistry and Pathobiochemistry, Waldeyerstraße 15, University Hospital Münster, Germany, [email protected] Hedgehog (Hh) signaling plays major roles in embryonic development and the progression of certain cancers. During secretion, all Hhs (fly Hh and vertebrate Indian Hh, Desert Hh and Sonic Hh) undergo dual lipidation at the protein termini, resulting in immobilization on the surface of the producing cell. This property, however, appears inconsistent with the ability of lipid-modified Hhs to signal far from their sites of synthesis. The mechanisms underlying Hh release and gradient formation are unclear and under intense debate Previous work conducted in our lab demonstrated that lipidated Shh gets released from the producing cell via metalloprotease-mediated shedding, and that Heparan Sulfate (HS) modulates this process. However, contrary to this finding seemed the established role of N-terminal palmitoylation for the biological activity of the released morphogen. The work presented here resolves this paradox by showing that ShhNp N-palmitoylation is the prerequisite for proteolytic cleavage of an N-terminal peptide during solubilization. This peptide normally blocks the ShhNp zinc-coordination site that allows ShhNp to bind to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp biological activity.

25


A protein therapeutic modality founded on molecular regulation

Chapman M. Wright1, R. Clay Wright1, James R. Eshleman2, Marc

Ostermeier1 (presenting author) 1Department of Chemical and Biomolecular Engineering, Johns Hopkins

University, Baltimore, Maryland 21218, USA 2Department of Pathology and Oncology, Johns Hopkins University School of

Medicine, Baltimore, Maryland 21231, USA [email protected]

The exquisite specificity of proteins is a key feature driving their application to anti-cancer therapies. The therapeutic potential of another fundamental property of proteins, their ability to be regulated by molecular cues in their environment, is unknown. We propose a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker of choice. We demonstrate this approach by creating a switchable prodrug-activating enzyme that selectively kills human cancer cells that accumulate the cancer marker hypoxia-inducible factor 1a (HIF-1a). HIF-1a accumulates to a high level in many solid tumors including breast, prostate, colorectal, and pancreatic cancers but is virtually undetectable in normal, well-oxygenated tissues. When HIF-1a accumulates in the cytoplasm it interacts with the CH1 domain of p300 and is transported to the nucleus, where it activates the transcription of many genes. We created a switchable enzyme that is a fusion of this CH1 domain and the enzyme cytosine deaminase from yeast, which can convert the non-toxic prodrug 5-fluorocytosine (5FC) to the chemotherapeutic 5-fluorouracil (5FU). This switch couples the production of 5FU to cellular HIF-1a levels. Thus, expression of the switch in RKO colorectal cancer cells and MCF7 breast cancer cells confers susceptibility to 5FC in a HIF-1a dependent manner. Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases.

26


Structural elements in pectins of importance for their effects on the immune system

Berit Smestad Paulsen University of Oslo, School of Pharmacy, P.O.Box 1068 Blindern, 0316 Oslo, Norway [email protected]

In several cultures medicinal plants have an extended use as wound healing agens and they are also used against illnesses that are related to the immune system. Traditionally the water extracts are used as the medical remedy, and based on this it was natural to study the watersoluble polysaccharides that could be present in these water extracts. We have interviewed traditional healers in Mali on what plants they use for woundhealing, and identified the polysaccharides in the water extracts to be the active ingredients. Pectic polysaccharides from different plants, Vernonia kotchyana, Glinus oppositifolius, Opilia celtidifolia, Biophytum petersianum and Cola cordifolia, are all rich in polysaccharides that have an effect in bioassays related to the immune system. We have studied their finer structure as well as determined what parts of the polymers that is important for the bioactivities. Structure - activity relations for the different polysaccharides will be presented.

27


Dual roles of heparan sulfate in sonic hedgehog (shh) release and stabilization from degradation

Ute Pickhinke1, Stefanie Ohlig1, Roger Lawrence2, Rita Dreier1, and Kay Grobe1

1University of Münster, Institute for Physiological Chemistry and Pathobiochemistry, Waldeyerstraße 15, 48149 Münster, Germany 2University of California San Diego, Department of Cellular and Molecular Medicine, La Jolla, California, USA [email protected] All Hedgehog (Hh) morphogens are released from producing cells despite being synthesized as N-and C-terminally lipidated molecules, a modification that normally results in firm tethering of proteins to the cell membrane. Therefore, a mechanism must exist for the release of lipidated morphogen from the plasma membrane to promote long range signaling. In this work, we describe that in addition to proteolytic ShhNp “ectodomain shedding” by ADAM (A disintegrin and metalloprotease) family members 10, 12 and 17, release of ShhNp from the producing Bosc23 cells can also be mediated by members of the matrix metalloprotease family, MMP2 and MMP9. This redundancy in sheddase function, however, raises the question of how ShhNp release is regulated, as the tight control of secretion is a key step in the regulation of Shh signaling activity. We provide evidence that Heparan sulfate proteoglycans co-expressed on Shh secreting cells play this role. Moreover, in vitro morphogen processing indicates an important role of HS sulfation in this process, shedding light on the various roles that HS plays in Hh biology.

28


Lichen derived polysaccharides: exploring uncharacterized pharmaceutically relevant structure

Zalilawati Mat Rashid, Frank Petereit and Andreas Hensel

Institute of Pharmaceutical Biology and Phytochemistry, Westphalian Wilhelm’s University of Münster, Hittorfstraße 56, 48149 Münster, Germany [email protected] Lichens are composite organisms consisting of a symbiotic association of a fungus with a photosynthetic partner, usually either a green alga or cyanobacterium. The overall aim of this study is to determine chemical characteristics and pharmacological effects of lichen-derived polysaccharides. A widespread lichen in middle Europe is Xanthoria parietina for which no phytochemical data or investigations concerning the respective carbohydrate composition, was currently studied. The dried lichen X. parietina (150 g) was first submitted to successive hexane, dichloromethane, ethyl acetate and methanol extractions (each was extracted in triplicate), in order to extract low molecular and lipophilic components, resulting in fractions XH (0.7%), XDC (1.1%), XEA (0.8%) and XM (10.2%) respectively. From fraction XH, an 1,8-dihydroxy-3-methoxy-6-methylanthraquinone was isolated in yields of 0.44 g and characterized by MS and NMR studies. The defatted lichen residues were then extracted consecutively with cold, aqueous (100°C) and alkaline extractions (0.5 M aq. NaOH), three times each which yielded polysaccharide fractions XC (1.5%), XH (2.6%) and XA (10.7%) respectively prior to ethanol precipitation, dialysis and lyophilisation. The three polysaccharide fractions were analyzed concerning their monomer composition by using GC-MS of alditols acetates and found to contain glucose, galactose, mannose and rhamnose as main constituents in respective increasing composition. For further fractionation of XC and XH GPC was performed on Superose CL 6B stationary phases, resulting in molecular weight of the fractions in range of 400 to 600 kDa compared to standands dextrans.

29


Structural investigations of an arabinogalactan protein from JATROPHA CURCAS l. Seeds Maria Sehlbach, Andreas Hensel

Institute of Pharmaceutical Biology and Phytochemistry Hittorfstr. 15, 48147 Münster, Germany, [email protected] Jatropha curcas L. (Euphorbiaceae), native to the American tropics, is widely spread and cultivated in semiarid tropical regions of India, Africa and America. The manifold use of the plant ranges from traditional herbal medicine to the production of biodiesel fuel1. The unfractionated raw polysaccharide (RPS) from J. curcas endosperm, containing an arabinogalactan protein (AGP), have been previously reported as skin active compounds for wound healing by stimulating under in vitro conditions mitochondrial activity of human skin cells (keratinocytes, fibroblasts) and the ATP status of primary keratinocytes2.

In the present studies the crude RPS was purified by heat-depletion of the co-extracted protein, fractionated by ion exchange chromatography and precipitation of the AGP with β-D-glucosyl Yariv reagent. From this isolation procedure a highly purified AGP was obtained (yield 0.1 %, related to the defatted endosperm). HP-SEC analysis indicated the presence of a homodispers polymer with a mean MW of 80 kDa. The protein moiety of Y2 amounted to 5% (m/m), with Ala, Ser, Lys, Hyp and Glu as the most abundant amino acids in about equal proportions. Asp, Gly, Thr, Val, Leu, Ile and Pro occurred in lower amounts.

The sugar components of Y2 are Gal, Ara, GluA and Rha (10:7:1.5:1). As determined after methylation analysis of native and carboxyl-reduced Y2 Gal was present as β-(1→3)-, β-(1→6)- and β-(1→3)-β-(1→6)-linked D-galactopyranosyl (D-

Galp), Ara as α-(1→5)-linked and terminal L-arabinofuranosyl (L-Araf) residues. Rha occurred only as terminal α-L-Rhap, whereas GluA was mainly present as β-(1→4)-linked D-GlupA and in traces as terminal β-D-GlupA. It is likely, that the β-(1→3)-D-

Galp (12%) builds together with the crosslinked β-(1→3)-β-(1→6)-D-Galp (38%) the backbone of the carbohydrate moiety, which side chains are prolonged by β-(1→6)-D-Galp (2%), α-(1→5)-L-Araf (9%) and β-(1→4)-D-GlupA (6%). These units are substituted with terminal α-L-Rhap (5%), α-L-Araf (27%) and β-D-GlupA (1%) residues.

β-D-glucosyl Yariv reagent was synthesized according to Yariv et al3. The qualitative and quantitative analysis of monomers were performed by HPAEC-PAD after suitable hydrolysis. The D- and L-configuration of the monosaccharides were detected after derivatization with S-(–)-1-phenylethylamine and separation of the enantiomers via CZE. The differentiation between the α- and β- anomers was made by 1H and 13C NMR spectroscopy. Linkage investigations were performed by per- and perdeutero-methylation analysis with methyl iodide or deuteromethyl iodide followed by acetylation and analysis of the resulting PMAA by GC-MS. Uronic acids were, previously to methylation, reduced with sodium borodeuteride. The percentage data is given in mass to mass units.

1. Debnath, M. et al., C Pharm Biotechnology, 2008, 9, 288 ff. 2. Zippel, J. et al., Fitoterapia, 2010, 81, 772 ff. 3. Yariv, J. et al., Biochem J, 1962, 85, 383 ff.

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Glycosignals and their receptors in plant immunity

Naoto Shibuya Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan. [email protected]

Plants have the ability to initiate various defense responses upon recognition of so-called microbe (pathogen)-associated molecular patterns (MAMPs/PAMPs), which are common to various microorganisms but not present in plants1). The system has been shown to play a pivotal role to protect plants from the infection by numerous potential pathogens in nature. Interestingly, this MAMP-mediated immunity has many similarities with innate immunity in animals mediated by the recognition of MAMP molecules through the corresponding pattern recognition receptors.

Cell surface glycans such as chitin and b-glucan from fungal cell walls and bacterial lipopolysaccharide (LPS) are representative MAMPs and recognized by a broad range of plant species. Oligogalacturonides generated from pectic polysaccharides of plant cell walls have also been known to act as a danger signal and trigger defense responses in plants. These signals are also known as damage-associated molecular patterns (DAMPs).

We have been working on the molecular machinery of plants involved in the perception and transduction of chitin oligosaccharides, a representative fungal molecular pattern, and identified two types of plasma membrane (glyco)proteins, CEBiP and CERK1, as the components of chitin receptor in plants2,3). Both CEBiP and CERK1 contain characteristic structure, lysine motif (LysM), in their extracellular domains. Knockout/knockdown of these LysM proteins resulted in the suppression of chitin-induced defense responses, showing the essential roles of these molecules in chitin signaling. Infection experiments also indicated the importance of these chitin receptors in the resistance against pathogenic fungi and, interestingly, some bacteria.

Recent studies further showed that these two types of receptor molecules, CEBiP and OsCERK1, form a receptor complex ligand dependently and cooperatively regulate chitin signaling in rice4). On the other hand, it seems that Arabidopsis does not require CEBiP-like molecules for chitin signaling, though a CEBiP homologue in Arabidopsis is biochemically fully functional, i.e., binds chitin oligosaccharides specifically and sensitively. Thus, the chitin receptor systems in these two model plants seem to be significantly different in their structure and signaling mechanism.

On the other hand, some pathogens evolved to overcome this barrier by developing various “effectors” and got the ability to invade plants. A fungal pathogen of tomato plants, Cladosporium fulvum, secretes a protein containing LysM domains when the fungus invades through the intercellular space of host plant. This protein, Ecp6, binds chitin oligosaccharides generated from the cell surface of the fungus itself, thus preventing their perception by plant receptor, CEBiP5). In another case, bacterial effector, AvrPtoB was shown to inhibit chitin signaling by degrading CERK1 receptor kinase through a mechanism mediated by ubiquitination of this molecule6). These results further indicate the importance of chitin perception and signaling as a barrier to prevent the infection of potential pathogens.

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Another interesting feature of plant-microbe interactions mediated by LysM receptors is the communication between symbiotic microbes and their host legumes. It has been well known that the Nod-factors, variously decorated lipochitooligosaccharides, secreted by rhizobial bacteria induce nodulation in their host plants. LysM-containing plasma membrane proteins, such as NFR1and 5 in Lotus japonicus and LYK3 and NFP in Medicago truncatula, have been shown to play essential roles in the perception of Nod-factors. Thus, pairs of structurally related chitin/lipochitin oligosaccharides and LysM receptors can induce almost opposite responses in plants, defense responses to exclude potential pathogens or symbiotic responses to accept rhizobial bacteria. We recently showed that only a limited alteration of amino acid residues in CERK1 defense receptor could switch the downstream responses from defense to symbiotic responses, indicating the presence of close evolutional relationships between these LysM receptors7)

In conclusion, these studies showed that the recognition of chitin oligosaccharides and related molecules by LysM receptors/proteins play important roles in various aspects of plant-microbe interactions.

1) Boller, T. and Felix, G. (2009) Annu Rev Plant Biol, 60, 379; 2) Kaku, H. et al. (2006) Proc. Natl. Acad. Sci. U. S. A., 103, 11086; 3) Miya, A. et al. (2007) Proc. Natl. Acad. Sci. U. S. A., 104, 19613; 4) Shimizu, T. et al. (2010) Plant J., 64, 204; 5) de Jonge, R. et al. (2010) Science, 329, 953; 6) Gimenez-Ibanez, S. et al. (2009) Curr Biol, 19, 423; 7) Nakagawa, T. et al. (2011) Plant J, 65, 169.


Characterization of receptor proteins using affinity cross-linking with biotinylated ligands

Tomonori Shinya , Hanae Kaku and Naoto Shibuya Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan. [email protected] The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the plant chitin receptor, CEBiP (chitin elicitor binding protein) which is a GPI-anchored membrane protein [1, 2]. A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also applied GN8-Bio for kinetic analysis of chitin binding proteins using Biacore and Streptavidin-immobilized sensor chip. This work demonstrates the applicability of this method to the purification, identification and characterization of plant receptor proteins. [1] Kaku et al., Proc Natl Acad Sci U S A. 2006, 103:11086-91. [2]Shinya et al., Plant Cell Physiol., 2010, 51:262-70.

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Chitin recognition machinery for immune responses in Arabidopsis

Tomonori Shinya, Noriko Motoyama, Tomohiko Osada, Asahi Ikeda, Hanae Kaku and Naoto Shibuya Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan. [email protected] Plants and animals recognize microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) for sensing of microbes. Chitin derived from fungal cell wall is a typical MAMP and its perception leads to immune responses. CEBiP (rice) and CERK1/OsCERK1 (Arabidopsis/rice) have been identified as critical components for chitin signaling in these plants [1, 2, 3]. They are a GPI-anchored protein and a receptor-like kinase (RLK), respectively. To understand whether Arabidopsis requires the presence of CEBiP-like molecule(s) for chitin signaling, we characterized CEBiP homologues in Arabidopsis. One of three CEBiP homologues (AtCEBiP) showed a high-affinity binding for chitin oligosaccharides. The binding characteristics of AtCEBiP were very similar to rice CEBiP. However, the knock-out mutant as well as overexpressing plant of AtCEBiP showed chitin-induced ROS generation similar to wild type Arabidopsis. These results indicated that AtCEBiP is biochemically functional as a chitin binding protein but does not significantly contribute to signaling. In other words, only AtCERK1 seems enough for chitin perception and signaling in Arabidopsis. Thus, the machinery required for chitin perception/signaling in Arabidopsis seems to be significantly different from that of rice, which requires both CEBiP and OsCERK1. Analysis of the chitin binding properties of CERK1/OsCERK1 ectodomains suggested that the differences in their chitin binding, whether bind or not, caused the use of different machineries for chitin perception between Arabidopsis and rice. [1] Kaku et al., Proc Natl Acad Sci U S A. 2006, 103:11086-91. [2] Miya et al., Proc Natl Acad Sci U S A. 2007, 104:19613-8. [3] Shimizu et al., Plant J. 2010, 64:204-14.

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Zinc finger nuclease glycoengineered SimpleCells: a novel approach to decode O-glycoproteome (GalNAc-type)

Sergey Y. Vakhrushev, Catharina Steentoft, Henrik Clausen, and Steven B. Levery Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark , [email protected]

Background

A longstanding obstacle for detailed analysis of glycoproteomes has been extensive heterogeneities in glycan structures and attachment sites on proteins Here we used ZFN targeting[1] of cosmc to glycoengineer stable human cell lines with simple O-glycosylation displaying only truncated Tn and STn O-glycans, designated “SimpleCells” to facilitate specific isolation of the O-glycoproteome as well as identification of O-glycosylation sites.

Methods Human cell lines were transfected with either 4µg of compoZr® C1GalT1C1 DNA or 1 vial of mRNA custom produced (Sigma-Aldrich) from the manufacturer using Nucleofection® according to the manufactures protocol (Lonza). The tryptic O-glycopeptides from SimpleCells were enriched by a single lectin capture step (VVA). Chromatography and mass spectrometry were performed on an EASY-nLC II HPLC and a high resolution ESI-IT/FT-MS LTQ-OrbiTtrap XL (Thermo-Scientific). Results Using the SimpleCell approach, we identified a total of 106 glycoproteins in the first three SimpleCell lines. A total of 200 of individual glycopeptide components were characterized, most of them with sufficiently informative MS2 spectra to provide more than 300 well defined, O-GalNAc glycosylation sites. Of the O-glycoproteins identified, the majority of O-GalNAc sites characterized are novel (>70%). Considered with respect to predictive algorithms, our results showed that to great extent the current (3.1) version of NetOGlyc, for example, is particularly inadequate for predicting isolated O-GalNAc sites. Conclusions The ZFN technology provides an exhilarating new dimension to glycoengineering of cells. The SimpleCell strategy introduced here has allowed further expansion of the O-glycoproteome. Finally, the SimpleCell strategy is amenable to other types of protein glycosylation where structural heterogeneity of glycans hampers isolation and characterization of the particular glycoproteome.

References:

Santiago,Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. U. S. A 105, 5809-5814 (2008)

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Chitosan-based physical- and covalent-crosslinked nanoparticles with capacity to interfere with quorum sensing in Gram(-) bacteria

Celina Vila1,2, Carmen Remuñán-López,1 Carlos Bustamante3, Francisco M. Goycoolea1,2 1 Departament of Pharmacy and Pharmaceutical Technology. Universidad de Santiago de Compostela, 15782 Spain; [email protected] 2 Institut für Biologie und Biotechnologie der Pflanzen Westfälische Wilhelms-Universtät Münster. Schlossplatz 3; 48149 Münster, Germany; [email protected] 3 QB3 Institute. University of California Berkeley. 642 Stanley Hall; 94720-3220 Berkeley, CA Chitosan-based nanoparticles intended for selective recognition and capacity to adsorb acyl homoserine lactones (AHLs), bacterial metabolites involved in quorum sensing (QS) of Gram(-) bacteria, are currently being developed as an strategy to design nanobiotechnological approaches to exert a selective targeting and interfere with the pathogenic and virulent responses. The effect of incubating different type of NPs with N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), a well established AHL molecule known to control QS in Vibrio fischeri and other Gram (-) bacteria [1,2], has been tested. To this end, an E. coli reporter strain, used as a biosensor of QS that expresses GFP after the exogenous addition of OHHL has been proven as an effective tool to test the quorum quenching activity of the NP prototypes. In a series of preliminary assays, significant reduction of GFP expression was observed after culture incubation in the presence of NPs, which could be attributed to a chemical binding affinity between OHHL and the NPs. Interestingly, the same effect was observed when supernatants from the incubation reactions of NPs and OHHL were administered to the biosensor cultures. The chemical affinity of the chitosan-based NPs towards various lactones is currently under evaluation by isothermal titration calorimetry (ITC). Besides the design of new nanostructured materials able to recognize and sequester AHLs, we are also investigating other approaches, such as the incorporation in the NPs of anti-quorum sensing molecules which can act as competitors of the AHLs at the interaction with their receptor, blocking the AHL-mediated signalling and QS gene expression. In this sense we have used de E.coli biosensor to screen a series of molecules, such as a brominated furanone, vanillin, polygodial, among others, obtaining very promising results. The results gathered with our NP prototypes and the AHL-like antagonistic molecules using the fluorescent E.coli biosensor so far look very promising. Therefore, our efforts are focused on the use of these prototypes as a platform to design new nanosystems that can exert an efficient microbial control via its ability to inhibit the expression of the genes involved in AHL-based QS signaling pathways. References

1. Eberhard et al. (1981) Biochemistry 20, 2444-2449 2. Bassler and Losick (2006) Cell 125, 237-246

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ZIC-HILIC chromatography and ESI-QToF mass

spectrometry for the compositional analysis and

structural elucidation of heparin oligosaccharides

Lukas Witt1, Stephan Kirsch1, Stefanie Ohlig2, Ute Pickhinke2, Kay Grobe2,

Jasna Peter-Katalinic1 and Michael Mormann1

1University of Muenster, Institute of Medical Physics and Biophysics, Robert-

Koch-Str. 31, 48149 Münster, Germany 2University of Muenster, Institute for Physiological Chemistry and

Pathobiochemistry, Waldeyerstrasse 15, 48149 Münster, Germany

Heparin belongs to the group of glycosaminoglycans (GAGs), a class of complex glycans that form linear chains of a length up to 100 monosaccharide building blocks or more. Heparin oligosaccharides comprise a backbone of 4GlcAβ1-4GlcNα1-subunits which can be N-sulfated/acetylated and O-sulfated at C2 position of the GlcA and at C6 position of the GlcN residue. The sulfation pattern is not consistent throughout the chain: highly sulfated regions alternate with sequences of a lower degree of sulfation. For characterization GAGs are usually enzymatically digested to oligomers prior to mass spectrometric analysis. In this study, nano-scale zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) was coupled to an electrospray quadrupole time-of-flight (ESI-QToF) mass spectrometer. A mixture of 10 disaccharides exhibiting different sulfation patterns served as a model system to develop and optimize the employed LC-MS approach. Additionally, oligomers differing in dp were submitted to LC-MS analysis as well as enzymatically depolymerized commercial and natural Heparin samples. The novel LC-MS based approach presented here enables the separation of heparin-derived di- and oligosaccharides according to their polarity and sulfation pattern. While chromatographic separation gives rise to discrimination of isomeric analyte species, mass spectrometric analysis allows the discrimination of co-eluting species. The selectivity obtained for separation of highly sulfated species of higher dp is a promising approach for profiling and identification of structural motifs inside highly sulfated heparin regions. The utilization of this ZIC-HILIC approach in the analysis of higher oligomers also revealed a separation of isobaric species eluting in double peaks. MS2 experiments were employed in this context to structurally characterize these isomeric species for unambiguous assignment. The method is highlighted as a rapid, efficient, and reliable method to characterize the heparin disaccharide composition as well as sulfated oligomers of different dp.

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Second-generation protein switches for cancer-activated enzyme prodrug therapy

R. Clay Wright1, Chapman M. Wright1, James R. Eshleman2, Marc Ostermeier1

1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA 2Department of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA [email protected]

Prevailing approaches for developing cancer protein therapeutics focus on creating proteins that therapeutically modulate a cancer marker’s function. Such an approach limits the therapeutic mechanism to those that naturally arise from modulation of the cancer marker and precludes the use of cancer markers for which therapeutic modulation is not feasible. Furthermore, many potential protein therapies lack the desired cancer targeting. The ability to link recognition of any cancer marker with activation of any desired therapeutic function would enormously expand the number of possible protein therapeutics.

Here, we describe a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker. We demonstrate this approach by creating a switchable prodrug-activating enzyme that selectively kills human cancer cells that accumulate the cancer marker hypoxia-inducible factor 1α (HIF-1α). HIF-1α is a protein that accumulates to a high level in many solid tumors including breast, prostate, colorectal, and pancreatic cancers but is virtually undetectable in normal, well-oxygenated tissues.

HIF-1a-activated enzyme switches were created by making fusions of the prodrug-converting enzyme yeast cytosine deaminase (yCD) and the CH1 domain of the p300 protein, which binds HIF-1α. We identified one particular engineered switch gene (haps59) that confers to RKO renal clear-cell carcinoma cells a HIF-1α-dependent increase in sensitivity to the prodrug 5-fluorocytosine (5FC). This increased sensitivity arises from an increase in the cellular production of the chemotherapeutic 5-fluorouracil (5FU) from 5FC in the presence of HIF-1α. However, the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. Using random and cassette mutagenesis combined with a two-tiered genetic selection for improved switches, we have identified variants of haps59 that confer increased 5FC sensitivity in the presence of HIF-1α and reduced 5FC sensitivity in the absence of HIF-1α.

Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases. By introducing protein-level regulation into cancer prodrug therapies, our approach skirts the problematic selective transduction requirement currently limiting enzyme-prodrug therapies. In addition, our approach is complementary to both transcriptional and transductional targeting and might be combined with these approaches to afford a double or triple layer of specificity: at the gene delivery level, at the transcription level and at the protein level.

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β-Glucans as inductors for differentiation of human keratinocytes

D. Zacharski, A. Deters and A. Hensel Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Hittorfstr. 56, 48149 Münster, Germany [email protected] Background: The epidermis is an important tissue of the human skin as it provides life-sustaining protection from external noxes from the environment. It is a dynamic renewing structure and principally formed by highly specialized keratinocytes. During a carefully choreographed program of differentiation epidermal keratinocytes undergo complex morphological and biochemical changes (1, 2). As N-Acetyl-D-glucosamines were shown recently to increase the cellular differentiation, as monitored by the differentiation-specific markers cytokeratins K1/K10 and involucrin (3), β-D-glucans are supposed to induce human keratinocyte differentiation. β-D-glucans are structural components of the cell walls of plants, fungi and bacteria with a great variability in structural features, physical properties, molecular mass, tertiary structure, etc. Nothing is known about the structural features required for this differentiation-inducing effect and on the exact mechanism of the glucans against the cells. The aim of the following study was to identify glucans as inductors for cell differentiation, to establish an in vitro test system for distinct structure-activity relations and to identify the molecular targets for interaction of the glucans with the keratinocytes. Methods: Testing was performed on HaCaT keratinocyte cell line and on primary human keratinocytes isolated from human skin. Initial experiments were performed on HaCaT by MTT for assaying influence of test compounds on cell viability. Furthermore preliminary studies were done by quantitative RT-PCR to define optimal culture conditions and assay methods for monitoring cellular differentiation. Results: Increase of mitochondrial activity was observed for cellobiose (dimeric β-1,4 glucose), lichenan and barley glucan (both β-1,3/1,4 glucans). Besides this, a positive effect was found for xyloglucans (β-1,4/1,6) which was depending on the respective fine structures. RT-PCR studies indicated the ionophore A23187 in combination with CaCl2 to be suitable as positive control. Incubation intervals for test compounds were systematically evaluated to be optimal for 6 and 24 h and for 2 or 3 days depending on the appearance of the primary human keratinocytes. Primers for involucrin, cytokeratin K10, transglutaminase 1 and TNF α were selected for following RT-PCR experiments. 1. Eckert et al., J Invest Dermatol, 2004, 123, 13-22 2. Eckert and Rorke, Environ Health Perspect, 1989, 80, 109-116 3. Deters et al., JPP, 2008, 60, 197-204

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Submerged culture conditions for production of extra-cellular polysaccharides with antitumor activity in vitro by Hypsizigus marmoreus JB06

Bingzhao Zhanga, Guozhen Zhu b, Peisheng Yan a,b,

a School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin150001, China, Tel/fax: 86-631-5687230. E-mail address: [email protected] Address: West culture road 2, Weihai, Shandong 264209, China. b School of ocean, Harbin Institute of Technology at Wei Hai, Wei Hai 264209, China [email protected] In order to establish an efficient procedure for cultivation of Hypsizigus marmoreus and production of extra-cellular polysaccharide with higher antitumor activity, uniform design was applied to optimize the seed broth and submerged culture conditions. The optimal seed broth and condition were firstly determined: 1.0 L aqueous medium containing 35g glucose, 110g potato, 17.5g yeast extract, 17.5g beef extract, 1.0g KH2PO4 and 0.5g MgSO4. After one day pre-culture and 9 days culture, the seed culture in best growth stage was obtained, in which the concentration of mycelium pellet and biomass of mycelium reached to 123 pellets per ml and 8.8g/L. Subsequently, the submerged culture broth and conditions were investigated. The results showed that the optimal culture temperature and C/N ratios were 25 � and 3:1 to 6:1. A concentration of glucose 25.8g/L, soya peptone 19.3g/L, KH2PO4 0.5g/L and sweet potato powder 5.3 g/L were found to be a suitable condition for submerged culture for production of polysaccharides with higher antitumor activity in vitro. Keywords: Hypsizigus marmoreus, Polysaccharides, Submerged culture, Antitumor activity, Uniform design

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Advances in studies on polysaccharides from Radix Codonopsis

Yuanfeng Zou a, Berit Smestad Paulsen a, , Xingfu Chen b, Bingzhao Zhang c

aSchool of Pharmacy, University of Oslo, P .O. Box 1068, Blindern, 0316 Oslo, Norway bCollege of Agronomy, Sichuan Agriculture University, Wenjiang, 611130, China cSchool of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin, 150001, China [email protected] Abstract Radix Codonopsis is one of the traditional Chinese herbal medicines (TCM), which was the root of C. pilosula (Franch.) Nannf. and C. pilosula Nannf.var.modesta L.T.Shen and C. tangshen Oliv. It was used in TCM to lower blood pressure and increase white blood cell counts, cure appetite loss, strengthen the immunize system. Radix Codonopsis was utilized primarily as a substitution for ginseng (Panax ginseng). Polysaccharide is one of the most important effective components of Radix Codonopsis. In this review, we summarize the extraction, isolation, purification, structure analysis; quantify determination, and Pharmacological action of polysaccharides from Radix Codonopsis. There are some different types of polysaccharides from Radix Codonopsis, according to the review, we consider that the researches of polysaccharides from Radix Codonopsis should be more and deeper, especially the structure-activity and quality control. Keywords: Radix Codonopsis; Polysaccharide; Extraction; Chemical component

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Looking forward to see you in our city Muenster!!

abstract book network symposium 2011 münster · [email protected] cola cordifolia (cav.)...

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