3^rd Glycobiology World Congress June 26-28, 2017 London, UK

Biography:

Robert Engel is Professor of Chemistry and Biochemistry at Queens College of the City University of New York. He earned his PhD in chemistry in 1966 from The Pennsylvania State University in Chemistry. He has also served in several administrative roles at CUNY, in addition to his teaching and research. His current research efforts are particularly directed toward the regulation of pathogens without the use of antibiotics.

Abstract:

With the increasing development of resistance by bacterial species to the action of classical antibiotics, there has developed a necessity for new approaches toward the control of bacterial regulation and treatment. Efforts of our laboratories have resulted in the generation of a series of surface modifications that result in the destruction of bacterial species of both Gram + and Gram – types rapidly and without the development of resistance to the action. These modified surfaces involve the incorporation of cationic lipids either covalently bound to the surface or embedded within the surface such that the cationic lipid portion protrudes and is able to interact with the bacteria. Our current efforts have been directed toward the development of material that could be used as treatments for bacterial infection, and not simply the prevention of transmission of such pathogens. Glycosides, in several forms, have been demonstrated to be of particular value in the construction of such antibacterial agents. We herein report on several of these glycoside scaffoldings and their ability to hold cationic lipids useful for the treatment of bacterial diseases.

Biography:

Clifford Lingwood completed his PhD at the University of London in1974, and postdoctoral studies at the Universities of Washington and Toronto. He has been a full professor at the University of Toronto since 1997 and is a senior scientist within the Molecular Medicine program of the Research Institute at the Hospital for Sick Children, Toronto Dr Lingwood’s research program is concerned with the biochemistry, chemistry, metabolism and function of glycosphingolipids with a view to the therapy of diseases in which they are involved. He has published more than 200 papers in reputed journals.

Abstract:

ABC cassette transporters differentially control GSL synthesis. The precursor for the synthesis of most complex glycosphingolipids (GSLs) is glucosylceramide (GlcCer) made on the outer Golgi membrane. The mechanism by which GlcCer is flipped into the Golgi has remained a mystery for>40yeras. Based on the use of MDR1(ABCB1) inhibitors, we proposed a role for this multidrug resistance protein. We have now used a newly synthesized photolabile GlcCer crosslinker to identify GlcCer binding proteins in microsomes, using proteomics/mass spectrometry. Three ATP binding ABC cassette transporters were thus identified, ABCB10, ABCB4 and ABCA3. Using SiRNA knockdown, these candidate GlcCer flippases were reduced in DU145 cells. The loss of ABC cassette proteins had a differential effect on selective GSLs. Some were increased e.g. lactosyl ceramide, while others reduced. In each case of ABC cassette knockdown, GalCer was increased. Moreover, MDR1 siRNA knockdown reduced overall GSL content by>50% according to cell line. These studies provide new insights into the complex regulation of GSL biosynthesis by precursor supply, a new basis for the link between cancer/multidrug resistance and GSL biosynthesis, suggest a new link between GlcCer and GalCer-based GSLs and indicate that multiple ABC transporters can transport GlcCer into the Golgi to generate LacCer pools used for the synthesis of different GSLs. Different LacCer pools could provide a partial basis for the biosynthesis of different GSLs within different Golgi regions (figure).

References

1. Mahfoud R, Manis A, Binnington B, Ackerley C, Lingwood CA (2010) A major fraction of glycosphingolipids in model and cellular cholesterol containing membranes are undetectable by their binding proteins. J Biol Chem 285: 36049-36059.

2. Kamani M, Mylvaganam M, Tian R, Binnington B, Lingwood C (2011) Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism. J Biol Chem 286: 21413-21426.

3. Lingwood D, Binnington B, Róg T, Vattulainen I, Grzybek M, et al. (2011) Cholesterol modulates glycolipid conformation and receptor activity. Nature Chem Biol 7: 260-262.

4. Kamani MA, Provencal P, Boutin M, Pacienza N, Fan X, et al. (2016) Glycosphingolipid storage in Fabry mice extends beyond globotriaosylceramide and is affected by ABCB1 depletion. Future Sci OA 2: FSO147.

5. Lingwood CA (2011) Glycosphingolipid Functions. Cold Spring Harb Perspect Biol Biology of Lipids: 149-174.

Biography:

For the past 35 years, Dr. Szewczuk is Full Professor of Immunology and Medicine, Queen’s University, Kingston, Ontario Canada.  He received his B.Sc. (Hon) in Chemistry (U. of Guelph), M.Sc. in Biochemistry (Guelph), Ph.D. in Immunochemistry (U. of Windsor) and post-doctoral training with Gregory W. Siskind, M.D. in cellular immunology at Cornell University Medical College, NYC. Dr. Szewczuk’s recent research has focused on the role of glycosylation in receptor activation with a particular focus of TOLL-like, nerve growth factor Trk,  EGFR  and insulin receptors. He has discovered a novel receptor-signaling platform and its targeted translation in multistage tumorigenesis and engineered drug delivery systems.

Abstract:

Overactivity of the renin-angiotensin system (RAS) involving angiotensin II (ANG II) produced from this system can induce insulin resistance. It has also been linked to the pathophysiology of type 2 diabetes mellitus, hypertension and cancer. ANG II binds to angiotensin receptors, AT1 and AT2, which belong to a class of G protein-coupled receptors (GPCR). The molecular mechanism of ANG II induced insulin resistance and other pathologies is unknown. We have reported a novel neuromedin B (NMBR) GPCR-signaling platform controlling mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) cross-talk in the activation of the insulin receptor (IR) through the modification of the IR glycosylation in human IR-expressing hepatoma (HTC) cells. This novel organizational signaling platform linked to glycosylated receptor tyrosine kinases (RTK) (e.g., EGFR, TrkA, insulin) and TOLL-like (TLR) receptors is identified to regulate receptor activation process, all of which are known to play major roles in pathologies. This signaling paradigm proposes that ligand binding to its receptor on the cell surface induces a conformational change of the receptor to initiate GPCR and matrix metalloproteinaser-9 (MMP-9) activation to induce Neu1. Activated Neu1 hydrolyzes α-2,3-sialyl residues linked to β-galactosides, which are distant from the ligand binding sites. These findings predict a prerequisite desialylation process by activated Neu1 enabling the removal of steric hindrance to receptor association. Here, we reasoned that there might exist a biased GPCR agonism as diffusible small molecules in the circulation involved in the activation of Neu1-mediated glycosylated receptor signaling platform contributing to type 2 diabetes mellitus, hypertension and cancer.

Biography:

Marc Botcherby obtained his PhD from University College London in the field of Molecular Genetics.  He has worked for the Medical Research Council in a number of virology and immunology groups during which time he led the core group of the UK Mouse Sequencing Consortium and then a microarray hotel dedicated to auto immune diseases. Subsequently, Marc joined innovative companies such as Molecular Devices, Cambridge Bluegnome and Ilumina.  He is now director of Eggsonchip Limited providing expertise in the field of pre implantation genetic screening as well as distributing and supporting Innopsys scanners in the UK and Ireland.

Abstract:

Microarrays offer miniaturised, highly parallel multiplexing options for glycobiology assays and as such are becoming standard tools in this field.  Applications range from Immunology (host-pathogen interaction studies, vaccine development and testing), Cell Biology (cell glycosylation profiling, ligand-receptor interaction studies), Cancer Research (tumour associated antigen screening, vaccine development and testing) to Virology (virus avidity assays, virus surveillance and screening).  Examples of these applications will be described.

Biography:

Eduardo Osinaga is Medical Doctor and completed his PhD at the Technological University of Compiègne, France. He is the director of the Laboratory of Glycobiology and Tumor Immunology at Pasteur Institut, Montevideo, and is the Head of the Department of Immunobiology, Faculty of Medicine, Montevideo, Uruguay. His major research interests is the glyco-immunology of cancer. He has published more than 75 papers in reputed journals. 

Abstract:

Lung cancer is currently the leading cause of cancer-related death worldwide, accounting for approximately a third of all cancer diagnoses and related-deaths. Non–small cell lung cancer (NSCLC) represents nearly 80% of lung tumors; the two most common NSCLC histological types are squamous cell carcinoma (SCC) and adenocarcinoma (ADCA). Several diagnosis procedures detecting altered glycosylation have been developed and incorporated as assistant procedures in clinical oncology.  The synthesis of mucin-type O-glycans is started in a reaction catalyzed by UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). This is a complex family of enzymes of at least 20 members in humans. We previously found that GALNT13, the gene encoding GalNAc-T13 isoenzyme, is a strong predictor of poor clinical outcome in neuroblastoma patients (Berois et al., Clin Chem. 2006; 52:1701-12). In the present study, we evaluated GalNAc-T13 expression in human NSCLC. We produced a monoclonal antibody (MAb T13.5) that was used to assess the expression profile of the GalNAc-T13 protein in a well-defined population of 443 surgically resected NSCLC patients with 7 years of follow-up. We found that ADCAs expressed higher levels of the enzyme than SCCs. GalNAc-T13 expression correlated significantly with worse overall survival in ADCA patients treated with neoadjuvant chemotherapy. These data suggest that GalNAc-T13 could be a novel marker associated to chemoresistance in lung adenocarcinomas.

Biography:

Joanne Blanchfield is a Teaching and Research Associate Professor in the School of Chemistry and Molecular Biosciences at the University of Queensland. Her research broadly concerns drug and vaccine development and delivery. Her research includes projects concerning the isolation and biological evaluation of the bioactive compounds from herbal extracts with emphasis on the oral bioavailability of compounds as estimated via the Caco-2 cell model of the GI tract. The group also explores the design and synthesis of antigen presenting constructs designed to mimic more complex antigen structures using simplified components on a rigid scaffold. We are currently studying the construction of carbohydrate and peptide antigens from Staphylococcus aureus, HIV and HPV.

Abstract:

Saponins are a class of natural product that are present in many herbal extracts. These compounds consist of a hydrophobic core, often a steroid or terpenoid derivative, decorated with varying numbers and types of sugar units attached at various positions. These compounds are often suggested to be the bioactive components of extracts but there is little known about the bioavailability of the compounds. Our group recently identified a new class of open-chain steroidal saponins from Chamaelirium luteum, an indigenous American herb marketed for “women’s issues”. We have fully characterised a series of these compounds and used the Caco-2 monolayer model of the GI tract to evaluate the potential bioavailability and metabolic vulnerability of eight of these compounds. The saponin, 6-Dehydrochamaeliroside A(1), was found to have good permeability and chamaeliroside A (2) was found to have moderate permeability. All bidesmodic saponins based on chiograsterol A and B cores exhibited low permeability. The aglycone steroids, chiograsterol B and helogenin, showed minimal bioavailability. None of the compounds appeared to be significantly metabolised by Caco-2 cell homogenate. Our results suggest an interesting structure activity relationship with the compounds with sugar units on one site of the core being absorbed while compounds with sugars at both ends of the core are not absorbed. We are further examining this relationship to determine if active transporters such as the GLUT transporters may be involved in the absorption of some saponins but not others. These results will also be presented in this talk.

Image

Figure 1: 6-Dehydrochamaeliroside A (1) X = O; Chamaeliroside A (2) X = H, OH.

References                                                                                                                                                                         

   1.     Challinor, V.L., Parsons, P.G., Chap S., White, E.F., Blanchfield, J.T., Lehmann, R.P., De Voss, J.J. “Steroidal saponins from the roots of Smilax sp.: Structure and bioactivity.” Steroids, 77 (5), 504-511, 2012. DOI:10.1016/j.steroids.2012.01.009

2.     Weaver, L.G.; Singh, Y.; Burn, P.L.; Blanchfield, J.T. “The synthesis and ring-opening metathesis polymerization of glycomonomers” RSC Advances, 2016, 6 (37), 31256-31264. DOI:10.1039/C5RA25732H

3.     Dewi, A.S., Cheney, K.L., Urquhart, H.H., Blanchfield, J.T. Garson, M.J., “The sequestration of oxy‑polybrominated diphenyl ethers in the nudibranchs Miamira magnifica and Miamira miamirana” Marine Drugs, 2016, 14(11), 198

4.     Weaver, L.G.; Singh, Y.; Vamvounis, G.; Wyatt, M.F.; Burn, P. L.; Blanchfield, J.T. –“Carbohydrate globules: molecular asterisk-cored dendrimers for carbohydrate presentation” Polymer Chemistry, 5(4), 1173-1179, 2014. DOI: 10.1039/ C3PY01123B.

    5.     Weaver, Lucy G.; Singh, Yogendra; Blanchfield, Joanne T.; Burn, Paul L. "A simple iterative method for the synthesis of â-(1®6)-glucosamine oligosaccharides.” Carbohydrate Research, 371, 68-76, 2013. DOI:10.1016/j.carres.2013.01.008                                                                                                                                                                                              

Biography:

Dr. Menter received his PhD degree in Chemistry from the George Washington University in 1969. He completed a postdoctoral fellowship with Prof. Dr. Theodor Foerster at the Institut fuer physikalische Chemie der Universtiaet Stuttgart, Germany. Subsequently, he was at the University of Alabama, Birmingham, and the VA Medical Center (Atlanta) He currently serves as Research Professor of Biochemistry at Morehouse School of Medicine. Dr. Menter is recognized internationally for his work in the areas of collagen photochemistry and melanin photobiology as pertaining to redox reactivity

Abstract:

Dermal collagen and the surrounding extracellular matrix (ECM) have been described as the respective “bricks and mortar” of mammalian skin. Proteoglycans are glycosylated proteins with covalently attached anionic sulfated glycosaminoglycans (GAG). These in turn are attached to a hyaluronate (HA) backbone. Although the ECM macromolecules are best known for their architectural support of tissues, recent work indicates many other important cell functions. e.g. The ECM and collagen are both suscepible to environmental that can result in altered properties.

  We have arbitarily divided the ECM into two sections (1) the collagen – PG complex itself, including the external membrane – bound PG, (e.g. aggrecans and decorin) and (2) the perturbations caused by the effects of the surrounding internal and external environment. Examples are age, mechanical loading, ECM disruption, internal and external effects of UV, temperature, and glycation by abnormal amounts of simple sugars.

  We are interested in the effects of UV radiation on type I collagen. In simple in vitro work, UV causes collagen concomitant degradation and cross-linking that changes its basic properties and results in abnormal fibers, altered gelation and fluorescence properties, altered photochemical kinetics, and altered susceptibility to collagenase.

  More recently, we have commenced a study of the effect of hyaluronate on collagen photochemistry. Using a model in vitro system of collagen - HA 1:2 mixtures For T < Tm     ( ~36 C) HA retards the rate of photolysis by ~ 20 – 30 %. At T > Tm, where the coiled form predominates, there seems to be no such effect. Thus, stabilization of collagen helical structure seems to be one important function of the ECM. We envision further studies with added model proteoglycans. 

References

1.            JM Menter, L Freeman and O Edukuye (2015), Thermal and Photochemical Effects on the Fluorescence Properties of Type I Calf Skin Solutions at Physiological pH   Open Journal of Physical Chemistry, 5, 21-27

2.            JM Menter, AM Patta, RM Sayre, J Dowdy, and I Willis (2001).  Effect of UV irradiation on Type I Collagen Fibril Formation in Neutral Collagen Solutions.  Photodermatol. Photoimmunol. Photomed. 17:114-120.

3.            J.M.Menter, L.M. Cornelison, L Cannick, A.M. Patta, J.C.Dowdy, R.M. Sayre, I.K. Abukhalaf, N.S. Silvestrov, and I. Willis. (2003) Effect of UV on the Susceptibility of Acid – Soluble Skh – 1 Hairless Mouse Collagen to Collagenase. Photochem. Photoimmunol. Photomed: 19 : 28 – 34

4.            J. M. Menter (2006). Temperature Dependence of Collagen Fluorescence. Photochemical and Photobiological Sciences 5: 403 – 410.

5.            J.M. Menter, I.K. Abukhalaf , A.M. Patta,  N.A. Silvestrov, and I Willis (2007). Fluorescence of Putative Chromophores in Skh – 1 and Citrate – Soluble Calf Skin Collagens. Photochem. Photoimmunol. Photomed: 23 : 222 – 228. 

Biography:

Amelia Pilar Rauter has her expertise in biomolecular and medicinal chemistry focusing on disease prevention. Leader of the European Innovation Partnership FCUL consortium at the A3 group for disease prevention, and Secretary of the European Carbohydrate Organization and of the IUPAC Division (III) on Organic and Biomolecular Chemistry, she has dedicated her research to the innovation on functional ingredients for AD prevention by bridging nature with sustainable chemistry.

Abstract:

Alzheimer’s disease (AD) is a multifactorial pathology, with unknown etiology and, up to now, without cure. Solutions for early diagnosis and disease prevention are urgently needed. We present two case studies on the medicinal plants Salvia sclareoides and Genista tenera, the first one known for its effects on memory loss and the other one used to control diabetes, whose patients are at higher risk of developing AD than normal elderly. Methodologies are based on functional ingredient isolation and structure elucidation and synthesis, followed by the study of the mechanisms of action. The biological properties of S. sclareoides demonstrated that extracts are potent inhibitors of acetylcholinesterase (AChE), the enzyme degrading the neurotransmitter acetylcholine, whose inhibition is used to treat AD patients in early disease stages. Rosmarinic acid was identified as plant major component and a new binding site of AChE for this constituent was discovered [1], opening the way to new strategies for lead development. It was demonstrated that plant extracts prevent normal Prion protein to convert to Prion infectious isoform [2], and interact with AD toxic oligomers [3], removing amyloid fibrils to form amorphous aggregates. These findings clearly reinforce the potential of the plant to act on both amyloid and cholinergic events for AD prevention. Also diabetes is an amyloid disease and the scientific background of the traditional use of G. tenera has been unraveled. The active principles were mainly O- and C-glycosyl polyphenols, namely the potent antihyperglycemic 8-glucosylgenistein [4]. This discovery has encouraged the generation of a library of compounds structurally based on its precursors and analogues, and some of them demonstrated a potent antidiabetic activity [5]. The multiple mechanisms of action of the polyphenols studied were identified. α-Glucosidase and glucose 6-phosphatase inhibitory activity was shown by the polyphenols that also demonstrated UV cellular damaging protection and antioxidant activity, maintained after in vitro digestion with artificial gastric and pancreatic juices [6]. Dihydrochalcones demonstrated a selective and potent inhibition of sodium glucose co-transporter 2 [5]. Interestingly, suppression of islet amyloid polypeptide (IAPP) fibril formation was produced by 8-glucosylgenistein. Molecular recognition studies with IAPP and Aβ_1-42 confirmed the same binding mode for both amyloid peptides, supporting this molecular entity for intervention in amyloid events of both diabetes and the frequently associated Alzheimer’s disease [4]. C-glucosylation has also proven to efficiently increase the antidiabetic activity of the polyphenols studied and to prevent their behavior as PAINS (Pan-Assay Interference compounds).

Image

References

   1.            Marcelo F, Dias C, Martins A, Madeira PJ, Jorge J, Florêncio MH, Cañada FJ, Cabrita EJ, Jiménez-Barbero J, Rauter AP (2013) Molecular Recognition of Rosmarinic Acid from Salvia sclareoides Extracts by Acetylcholinesterase. A New Binding Site Detected by NMR. Chemistry A European Journal 19: 6641-6649.

2.            Rauter AP, Dias C, Martins A, Branco I,  Neng NR, Nogueira JM, Goulart M, Silva FVM, Justino J, Trevitt C, Waltho JP (2012) Non-toxic Salvia sclareoides Brot. extracts as a source of functional food ingredients: phenolic profile, antioxidant activity and prion binding properties. Food Chemistry 132: 1930-1935.

3.            Airoldi C, Sironi E, Dias C, Marcelo F, Martins A, Rauter AP, Nicotra F, Jimenez-Barbero J (2013) Natural compounds against Alzheimer’s Disease: Molecular recognition of Salvia sclareoides extract and its major component, rosmarinic acid, with Aß1-42 peptide, as investigated by NMR. Chemistry Asian Journal 8: 596-602. A. P. Rauter, et al, Food Chemistry 2012, 132(4), 1930

4.            Jesus R, Dias, C., Matos AM, Almeida RFM, Viana AS, Marcelo F, Ribeiro RT, Macedo MP, Airoldi C, Nicotra F, Martins A, Cabrita EJ, Jimenéz-Barbero J, Rauter AP (2014) Exploiting the Therapeutic Potential of 8-ß-D-Glucopyranosylgenistein: Synthesis, Antidiabetic Activity and Molecular Interaction with IAPP and Aß1-42. Journal of Medicinal Chemistry 57: 9463-9472.

Biography:

Daniel Salem received his B.S. in chemical engineering from the University at Buffalo and a Master's Degree in Chemical Engineering Practice from the Massachusetts Institute of Technology. He is currently a National Science Foundation Graduate Research Fellow under the guidance of Professor Michael Strano in the Department of Chemical Engineering at MIT. His research involves the design of carbon nanotube-based optical sensors for biopharmaceutical characterization, diagnostics, and food and water-borne contaminant detection.

Abstract:

It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble. As examples, we show that an array of near-infrared fluorescent single-walled carbon nanotube (SWNTs) sensors can estimate the mean and variance of the observed dissociation constant (KD), using three different examples of binding IgG with Protein A as the recognition site, including polyclonal human IgG, murine IgG and human IgG from CHO cells. In addition, we show that the observed dissociation constant is concentration-dependent, indicating multivalent interactions between the two binding partners. A bivalent binding mechanism is able to describe the concentration dependence of the effective dissociation constant, KD,eff, which varies from 100 pM to 1 μM for human IgG concentrations from 1 ng mL^-1 to 100 μ g mL^-1, respectively. We also demonstrate that an array of nanosensors can uniquely monitor weakly affined analyte interactions by monitoring the metabolically induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays. In addition to our glycan profiling work using lectin-functionalized nanosensors, we have designed synthetic molecular recognition sites for carbohydrates using Corona Phase Molecular Recognition; a detection technology invented by our lab. Herein we synthesized 14 variants of well-defined water-soluble boronic acid copolymers through RAFT polymerization and created 14 distinct corona phases by adsorbing them onto the nanotube surface. We screened them against a panel of saccharides and sugar alcohols, revealing a corona phase that recognizes specific saccharides with high selectivity. In response to D-Arabinose binding, SWNT fluorescence decreases by >60% at saturation, while showing less than 20% response to all other aldopentoses including L-Arabinose. Binding selectivity was tuned by varying the location of boronic acid from para to meta and was also observed through cryogenic-TEM.

Image

Biography:

Edita Sarukhanyan has obtained her Bachelor and Master of Science degrees in Physics (Biophysics) from Tbilisi State University in Georgia. In October 2013 she has received a PhD title in Chemistry (Computational Chemistry) from the joint International PhD program between University of Salerno in Italy and Jacobs University Bremen in Germany. Her thesis work was related to multiscale simulation studies of interactions of carbon nanotubes with biopolymers and lipid membranes. Since April 2015 to date she is a post-doctoral researcher at the University of Würzburg in Germany. Her current research is mainly focused on in silico drug design and development.

Abstract:

Zika virus belongs to the family of Flaviviruses. It evoked a worldwide attention after a large outbreak in Brazil in 2014. New drugs against Zika virus and flaviviridae in general are highly desirable. It has been observed by researchers that the attachment of the flaviviruses to the host cell occurs in the case when the glycosylation of the asparagine in the envelope protein of the virus takes place. However, the viral proteins are quite sensitive and can easily undergo mutation. Therefore, the focus was switched to the host target. Following this idea we have found in the recently published literature that the flavivirus internalization to the host cell is mediated by the Axl (one of the proteins from TAM family) receptor. In particular, when the dimerization of the last one with its ligand – Gas6 takes place. Gas6 binds to the glycosylated protein in the envelope of the virus, hence, facilitating the attachment and entry of the virus into the host cell. Herein, we hypothesize that the blocking of a host-factor, the Axl receptor, could be a best mode way to block Zika virus infection cycle. Instead of directly targeting the virus, we suggest improved compounds targeting Axl-Gas6 interaction site by in silico modification of the known Axl blockers - an anticoagulant drug warfarin and an anticancer drug R428. We have obtained better binding for both extending modifications, which was shown by docking simulation results.

Biography:

Rajagopal Chattopadhyaya (b.1957) completed his PhD (UCLA,1987) under Richard Dickerson, who as a postdoc solved the 2Å structure of myoglobin under John Kendrew.  Rajagopal did his postdoctoral work at UC Berkeley and Baylor College of Medicine. He has been a faculty member at Bose Institute since 1993, professor since 2006.  His work at Bose Institute is mentioned in the Encycl. Britannica. Book of the Year, 1996 and LexA model in Burton E. Tropp’s Molecular Biology : Genes to Proteins, 3rd edn, a continuation of David Freifelder’s classic undergraduate textbook.  Structural Biologist, biochemist, religious historian & author (as a hobby). 

Abstract:

The major tuber storage protein of Colocasia esculenta, is a monocot mannose-binding, widely used dietary lectin.  This tuber agglutinin contains two polypeptides of 12.0 and 12.4 kDa by MALDI-TOF analysis.  By both gel filtration and dynamic light scattering at pH 7.2 show the lectin has a α₂β₂ form; however, at pH 3, it converts to αβ form.  Our circular dichroism spectroscopy studies show that the lectin retains approximately 100% of its secondary structure between pH 2-8.  The fluorescence emission maxima of 346 to 350 nm for pH 4 to 10 show that the tryptophan residues are relatively exposed.  The unfolding is a simple two-state process, N₄ ↔ 4U, as seen in our denaturation scan profiles, when monitored by fluorescence, far-UV CD, and near-UV CD, are completely super imposable. Analyses of these profiles provide an estimate of several thermodynamic parameters at each guanidinium chloride concentration, including the melting temperature T_g, which is 346.9 K in 0 M, but lowers to 321.8 K in 3.6M.  Mannose-free mannose-bound lectin crystals were obtained by hanging-drop, vapor-diffusion method at room temperature and high-resolution X-ray diffraction data were collected using a home X-ray source.  The mannose-free structure (5D5G) and mannose-bound structure (5D9Z) are both available in the PDB, along with the X-ray data.  Some highlights of both structures will be presented.  Such high resolution structures obtained from a home X-ray source is rare among this class of lectins, and it has not been possible to crystallize simple mannose with such lectins before.

Biography:

Bettina Hesse has completed her Master of Science in Biochemistry and Molecularbiology in 2015 at the University of Kiel, Germany. Since 2016 she is working as PhD student at the University Hospital Muenster in the group of Professor Philipp Kümpers in cooperation with the group of Professor Kristina Kusche-Vihrog. Her current research focus is the molecular control of eGC damage and refurbishment in acute and chronic inflammatory diseases. 

Abstract:

The endothelial glycocalyx (eGC), a carbohydrate-rich layer mainly consisting of heparan sulfates (HS), lines the luminal side of the vascular endothelium. It provides a first vasoprotective barrier against vascular leakage and leukocyte adhesion in sepsis and vascular inflammation. The eGC is damaged in sepsis and thereby leads to defects in vascular permeability. Angiopoietin-2 (Angpt-2), an antagonist of the endothelium-stabilizing receptor tyrosine kinase Tie2 secreted by endothelial cells upon inflammatory stimulation, promotes vascular permeability through cellular contraction and junctional disintegration. Since the eGC damage and Angpt-2 are both involved in mechanisms influencing vascular permeability we hypothesize that Angpt-2 might also mediate the breakdown of the eGC. Using confocal and atomic force microscopy, we show that exogenous Angpt-2 induces a rapid loss of the eGC in endothelial cells and aortic endothelium in vitro. Glycocalyx deterioration involves the specific loss of its main constituent HS, paralleled by the secretion of the HS-specific heparanase. Corresponding in vivo experiments revealed that exogenous Angpt-2 leads to heparanase-dependent eGC breakdown, which contributes to plasma leakage and leukocyte recruitment in vivo. In summary, our data reveal a novel and crucial role for Angpt-2 in the regulation of the eGC. The results advance our understanding of the Angpt/Tie2 ligand-receptor system as a shared and concurrent gatekeeper of both layers of the vascular double barrier: the endothelial cell and the eGC. 

Biography:

Pierre van der Bruggen has a Ph.D. in Agronomical Sciences. In 1988, he joined the Ludwig Institute for Cancer Research and identified in 1991 the first human gene, MAGE-1, coding for a tumor antigen recognized by cytolytic T lymphocytes. He identified over the years several other tumor antigens, which have been used in clinical trials. His group has discovered a new type of anergy of human tumor-infiltrating lymphocytes, due to the presence of galectin-3, a lectin abundant in tumors. The group is further analyzing the mechanisms by which galectin antagonists reverse the impaired T cell functions.

Abstract:

We describe a new mechanism of dysfunction of human tumor-infiltrating lymphocytes (TILs). TILs failed to secrete cytokines and lytic enzymes upon stimulation, although they were normally activated and able to produce these effector molecules inside the cell. Surprisingly, these effector molecules remained trapped inside the cell. This defect is related to the presence of galectin-3 at the TIL surface and can be relieved by agents that detach galectin-3 from the TIL surface. The normal secretion process is blocked in dysfunctional TILs, due to impaired LFA-1 mobility and actin rearrangement at the secretory synapse. This is the first observation of uncoupling between cytokine production and cytokine secretion in TILs.

We also hypothesise that galectins lattices hanged on the tumor microenvironment may capture glycosylated immune factors, blocking their anti-tumor function. The presence of galectin-3 in the tumor microenvironment reduced IFNγ diffusion and ability to induce the chemokine gradient necessary to attract anti-tumor T lymphocytes. Galectin-3 captured in vitro glycosylated IFNγ, and reduces IFNγ diffusion through a collagen matrix. Inhibiting galectins enhanced the capacity of human tumor cells to express CXCL9/10 upon IFNγ treatment in vitro. In a humanized mouse model, human galectin-3 restricts the intratumor diffusion of IFNγ. Co-injection of IFNγ and galectin antagonists improved tumor infiltration by autologous CD8+ T cells injected intravenously and delayed tumor growth,  as compared with tumors injected with IFNγ alone . Our results contribute to explain why some human tumors can be considered as ‘cold’ as they are poorly infiltrated by anti-tumor T lymphocytes.

Biography:

Mirosława Ferens-Sieczkowska is an Associate Professor In the Department of Chemistry and Immunochemistry, Medical University of Wrocław, Poland. She graduated with honors from the Science Division, University of Wrocław, and received her PhD in Biochemistry from the same University. During her work in the Institute of Biochemistry and Molecular Biology, University of Wrocław, she focused her interest on plant biochemistry, especially the structure and function of plant lectins. Currently she is appointed in Medical University of Wrocław and her interests turned into the field of glycobiology, protein-carbohydrate interactions and the impact of altered and impaired glycosylation on disease etiology.

Abstract:

Conception problems affect about 15% of couples in developed countries. A male factor is considered crucial or co-existing in at least half of these cases, but evaluation of the reasons of male decreased reproductive potential is fairly not sufficient. Andrologists claim urgently for  reliable diagnostic and prognostic biomarkers, as well as molecular targets for potential treatments. Considering the importance of carbohydrate-protein interactions in different aspects of fertilization, also male infertility becomes a field of interest for glycobiology. Interestingly, both sperm and seminal plasma glycomes of fertile men contain substantial amounts of immunomodulatory glycoepitopes, usually rare in normal body fluids. These motifs include LeX, bisecting GlcNAc, and high-mannose type glycans.  It has been hypothesized that this unusual glycosylation profile is engaged in receiving maternal immune privilege during fertilization and pregnancy. If this hypothesis is true,  at least some cases of infertility may be related to incorrect glycosylation patterns within male reproductive tract. Our research is thus focused on the comparison of seminal plasma glycomes of fertile and infertile male subjects. Although we have found significant alterations in fucosylation, sialylation and high-mannose glycan content, these results are still far from diagnostically valuable conclusions. The main challenge is the  complexity of seminal plasma, consisting of  secretions of  testes, epididymis and different accessory glands. Some experimental data suggest that their glycosylation pathways are not necessarily identical. Regarding also extreme inter-individual variability, it seems that successful search for infertility glyco-biomarkers requires careful selection of candidate glycoproteins, representing particular parts of male reproductive tract.

Biography:

Patricia Albanese is Professor in Cell Biology and Biochemistry in UPEC. She studies GAG effects on stem cells properties and biomaterials association for Tissular Engineering application in regenerative medicine. She had obtained numerous grants from french government and patient foundations, as well as institutional and industrial supports, as main coordinator of projects to develop associated matrix and cellular therapeutic products. She has 25 Publications in international journals, 1 patent, and experiences in management of researchers and students (MD, engineers, Post-Doc, Ph-D, Master). She collaborates with chemists and polymerists on GAG tools development (Pr. D. Papy-Garcia, Head of CRRET lab) and has strong collaborations with rheumatologist clinicians and academic teams that master pre-clinical murine models of degenerative pathologies (AP-HP Hospital and Inserm). CRRET lab is a 35 people unit that has all the facilities for characterization of polysaccharides on glycomic plateform, molecular interaction analysis, cell culture, flow cytometry, histology, animal models. 

Abstract:

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are very important macromolecules involved in homeostasis regulation of cell niches in numerous tissues. They are associated to core protein and constitute the superfamily of heparan sulfate proteoglycans (HSPGs) and CSPGs present on the cell surface and in the Extracellular Matrix (ECM). They are known to play fundamental roles on self-renew, clonogenicity, proliferation, differentiation, migration and survey of numerous cell types. Such functionality is linked to their ability to interact with heparin binding proteins (HBPs), including growth factors, cytokines, and chemokines. Interactions of sulfated GAGs with HBPs ensure the link to their high affinity receptors and then participate to crucial cell signaling pathways. The specificity of these interactions is linked to precise sulfation pattern of the disaccharides that constitute GAGs. According to the tools of our glycomic plateforms we are able to purify and characterize GAGs species, as well as their chemical signature and functionality on HBP and cells. Since many years, our team demonstrated that GAGs structural modifications are involved in regulation of physiological processes during aging and in regulation of pathological ones, such as vascular remodeling and inflammation, during degenerative diseases. The characterization of these glycanic targets permit also to propose innovative therapeutic strategies based on the use of matricial products to optimize current limiting steps of therapeutic uses of stem cells: mobilization for purification, clonogenicity for amplification and survey for engraftment in basically deleterious degenerative context. We propose to illustrate such strategies by examples of our results in brain, vascular and osteo-articular systems. 

Biography:

Raquel Gómez-Coca studied Pharmacy at the University of Seville (Spain). She did the experimental part of her PhD work at the University of Basel (Switzerland), thanks to which she obtained her European Doctor Degree. After several years in the private sector, in 2009 she started working as a Postdoctoral Researcher at the Spanish National Research Council. Presently she is part of the Department of Characterization and Quality of Lipids of the Instituto de la Grasa, where she is devoted to the development of analytical methods focused on olive oil quality, purity and fraud detection. 

Abstract:

Plant sterols have been used as cholesterol-lowering agents in humans for a very long time. Within this group of compounds, steryl glycosides (SG) – considered as non-nutrients plant-secondary metabolites- are separately categorised as glycolipids thanks to both the sugar molecule(s) bound to the 3-C atom of the sterol backbone and to their favourable effect in human health.

Different sources of SG have been identified, being olive oil (OO) the most recent one. To this respect some considerations should be taking into account. First of all, from the analytical point of view one must realise that the European Commission official method for sterol analysis in OO overlooks the presence of SG, causing underestimations when reporting on total phytosterol concentration. Furthermore, it is important to provide data also on the SG profile, since it has been pointed out that different species may have dissimilar biological effects. Under this perspective the development of a proper analytical approach prevailed and new procedures to determine these sterol derivatives in OO are been developed. Secondly, OO production and consumption is no longer restricted to the Mediterranean basin, which have resulted on an increasing competitiveness, lack of a centralised databank for validated methods of analysis, absence of harmonization, etc., driving to a significant weakness in the OO production and supply chain, which is nowadays being exploited by counterfeiters.

Our purpose here is to present the information available on the field of SG, giving an overview of what these molecules are and of what has been done in different fields of research. Furthermore, we will comment on one of our specific objectives in the European OLEUM project: the search for novel markers focused on the development and validation of innovative analytical solutions to solve part of the OO fraud problems presently under the microscope of the international community.

References

1.            Steryl glycosides in foods: an overview. R. B. Gómez-Coca, M. C. Pérez-Camino, W. Moreda. In: I. Boyd (Ed) Glucosides: Sources, Applications, and New Research, 23-62 (2016). Hauppauge, New York: Nova Science Publishers, Inc. LCCN: 2016030879, LCCN: 2016039140, ISBN: 9781634858410, ISBN 9781634858618.

     2.            Saturated hydrocarbon content in olive fruits and crude olive-pomace oils. R. B. Gómez-Coca, M. C. Pérez-Camino, W. Moreda. Food Addit. Contam. 33, 391-402 (2016)

     3.            Fatty Acid Ethyl Esters (FAEE) in extra virgin olive oil: a case study of a quality parameter. R. B. Gómez-Coca, G. D. Fernandes, M. C. Pérez-Camino, W. Moreda. LWT – Food Sci. Technol. 66, 378-383 (2016)

4.            Determination of saturated aliphatic hydrocarbons in vegetable oils. R. B. Gómez-Coca, R. Cert, M. C. Pérez-Camino, W. Moreda. Fats & Oils 67(2), 1-9 (2016)

5.            Neutral lipids: unsaponifiable. R. B. Gómez-Coca, M. C. Pérez-Camino, W. Moreda. In: L. M. Nollet, F. Toldrá (Eds) Handbook of Food Analysis 3^rd ed. Vol. I, 459-489 (2015). Marcel Dekker, Inc.: NY-Basel 2015. ISBN-10: 1466556546. ISBN-13: 9781466556546

Biography:

Isam Khalaila has completed his PhD on 2001 from Ben-Gurion University (BGU) and postdoctoral studies from Muenster University (Germany) and EPFL (Switzerland) on 2003 and 2005 respectively. On 2005 he established his Glycoproteomics lab in the department of Biotechnology engineering in BGU. He has published more than 50 papers in reputed journals and has been teaching grade and undergraduate students in the last 12 years. 

Abstract:

The Wnt/β-catenin signaling pathway and cadherin-mediated adhesion are implicated in epithelial-mesenchymal transition (EMT), a key cellular process in invasion and metastasis. O-GlcNAcylation, the addition of β-N-acetylglucosamine (O-GlcNAc) moiety to Ser/Thr residues is involved in cancer and tumorigenicity. The current study is aiming to investigate the effect of O-GlcNAcylation on β-catenin and E-cadherin expression and function and thus, on EMT, cell motility and cancer cell tumorigenicity. The enzyme machinery of O-GlcNAcylation was modulated either with chemical inhibitors or by gene silencing. When O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc, inhibited or silences, a global elevation of protein O-GlcNAcylation and increase in the expression of E-cadherin and b-catenin were noted. Concomitantly with enhanced O-GlcNAcylation, b-catenin import into the nucleus and its transcriptional activity were elevated. Additionally, fibroblast cell motility was enhanced. Consistent with the results obtained by OGA inhibition, OGT-silencing led to a significant reduction in b-catenin level. Murine orthotropic colorectal cancer model indicates that elevated O-GlcNAcylation leads to increased mortality rate. However, reduction in O-GlcNAcylation promoted survival and attenuation of metastases development. The results described herein provide circumstantial clues that O-GlcNAcylation deregulates β-catenin and E-cadherin expression and activity in fibroblast cell lines and this might impact EMT and cell motility, which may further influence tumorigenicity and metastasis.

Biography:

Nadine Legros has studied Molecular Biotechnology at the University of Bielefeld in Germany. She is currently working on her PhD thesis in the group of Prof. Dr. Johannes Müthing, Institute for Hygiene (Münster, Germany), headed by Prof. Dr. Dr. h.c. Helge Karch. Her project is part of the research consortium InfectControl 2020 (TFP, TV8, AS12) with the aim to develop new strategies for the early recognition, control and prevention of infectious diseases.

Abstract:

Human brain and kidney endothelial cells are important targets for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) and play key roles in the pathogenesis of life-threatening extraintestinal complications. The clinically important Stx1a and Stx2a subtypes bind to glycosphingolipids (GSLs) of the globo-series. Primary endothelial cells are fastidious and sensible cells, which are eminently suitable as an optimal native cell type for analyzing Stx-mediated cellular injury. Lipid rafts represent supramolecular membrane microdomains that are enriched in certain types of lipids such as cholesterol, sphingomyelin and GSLs. Detergent-resistant membranes (DRMs), commonly used as lipid raft-analogous structures, represent the ruling method to assign association with lipid rafts.

Here we present novel data on the composition of lipid rafts prepared from primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Most prominent receptor lipoforms of Stx-receptor GSLs of both types of endothelial cells were globotriaosylceramide (Galα4Galβ4Glcβ1Cer, Gb3Cer) and globotetraosylceramide (GalNAcβ3Galα4Galβ4Glcβ1Cer, Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0), determined by electrospray ionization mass spectrometry in combination with thin-layer chromatography (TLC) immunochemical detection. Gb3Cer and Gb4Cer were found to co-distribute with canonical lipid raft-markers cholesterol and sphingomyelin as well as flotillin-2 in DRMs, which represent the liquid-ordered membrane phase and indicate their association with lipid rafts. Figure 1 exemplarily shows the DRM and nonDRM distribution of Gb3Cer and Gb4Cer detected in sucrose density gradient fractions of pHBMECs using Stx1a- and Stx2a-TLC overlay assays. On the other hand, lyso-phosphatidylcholine was identified as a nonDRM marker phospholipid of the liquid-disordered membrane phase. Increasing knowledge on membranes of brain and kidney endothelial cells might help to develop new therapeutic strategies to fight EHEC infections.

Image

Fig. 1. Detection of Stx-binding GSLs in sucrose density gradient fractions of pHBMECs. (A) TLC overlay assay detection of Stx1a- and (B) Stx2a-binding GSLs using Stx1- and Stx2-specific antibody, respectively. GSL extracts correspond to       8 × 10⁵ cells and 2 µg of reference neutral GSLs from human erythrocytes (R1) were applied in each assay (Legros et al. 2017a). 

References

1.          Bauwens A, Betz J, Meisen I, Kemper B, Karch H, Müthing J (2013) Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature. Cell. Mol. Life Sci. 70: 425-457.

2.          Lee MS, Koo S, Tesh VL (2016) Shiga toxins as multi-functional proteins: induction of host cellular stress responses, role in pathogenesis and therapeutic applications. Toxins (Basel) 8:pii: E77.

3.          Legros N, Dusny S, Humpf HU, Pohlentz G, Karch H, Müthing J (2017a) Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood–brain barrier endothelial cells. 

4.          Legros N, Pohlentz G, Runde J, Dusny S, Humpf HU, Karch H, Müthing J (2017b) D-PDMP-induced changes of membrane microdomain lipids of primary human renal glomerular endothelial cells with focus on Shiga toxin glycosphingolipid receptors (submitted).

5.          Obrig TG, Karpman D (2012) Shiga toxin pathogenesis: kidney complications and renal failure. Curr. Top. Microbiol. Immunol. 357: 105-136.

6.          Trachtman H, Austin C, Lewinski M, Stahl RA (2012). Renal and neurological involvement in typical Shiga toxin-associated HUS. Nat. Rev. Nephrol. 8: 658-669.

Biography:

Nuno M. Xavier received a dual PhD degree in organic chemistry from the University of Lisbon and from the National Institute of Applied Sciences of Lyon in 2011 (advisors: Prof. Amélia P. Rauter and Dr. Yves Queneau, respectively). He was afterwards a postdoctoral research fellow at the University of Natural Resources and Life Sciences of Vienna in the group of Prof. Paul Kosma. He then returned to the University of Lisbon as postdoctoral member until the end of 2013, whereupon he was awarded an Investigator Starting Grant from the Portuguese Foundation for Science and Technology (FCT). Since then, he is Researcher (FCT Investigator) at the Faculty of Sciences, University of Lisbon. His research activities, reported in 25 publications, are focused on the synthesis of new carbohydrate-based molecules of therapeutic interest, for which he has been internationally recognized with various Young Scientist Awards. He is a member of the IUPAC Carbohydrate Nomenclature Task Group.

Abstract:

Nucleoside and nucleotide analogs or mimetics have attracted considerable attention as synthetic targets due to their ability to interfere with biological pathways in which their natural counterparts are involved, such as nucleic acid synthesis and cell division. Such physiological processes are deregulated in diseases such as cancer or viral infections and therefore these classes of compounds constitute potential anticancer or antiviral agents. Other biological effects that have been reported for nucleoside analogs include antimicrobial efficacies and cholinesterase inhibitory abilities. The access to structurally new analogs or mimetics of nucleosides and nucleotides and the exploitation of their biological profile remains of interest.

In this communication, the synthesis of glucuronic acid and glucuronamide-derived N-glycosyl compounds, including derivatives containing potential bioisostere groups for a phosphate functionality and nucleosides, will be presented.

Molecules of higher structural complexity intended to mimic nucleotides were also prepared, namely glucuronamide-based hybrids containing both a benzyltriazole moiety and an anomeric sulfonamide or a phosphoramidate function and pyranosyl/furanosyl nucleoside phosphoramidates.

The new compounds were subsequently evaluated for their cytotoxicity to cancer cells and for their inhibitory activities towards enzymes of therapeutic relevance, such as cyclin-dependent kinases, cholinesterases and carbonic anhydrase II. Some molecules were shown to be bioactive with inhibition constants or IC₅₀ values in the micromolar concentration range. The synthetic work and the findings of the bioactivity evaluation will be disclosed.

References

1.            Jordheim LP, Durantel D, Zoulim F, Dumontet C (2013) Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases.  Nature Reviews Drug Discovery 12:447–464.

 2.            Winn M, Goss RJM, Kimura K-i, Bugg T D H (2010) Antimicrobial nucleoside antibiotics targeting cell wall assembly: recent advances in structure-function studies and nucleoside biosynthesis. 

 3.            Xavier NM, Schwarz S, Vaz PD, Csuk R, Rauter AP (2014) Synthesis of Purine Nucleosides from D-Glucuronic Acid Derivatives and Evaluation of Their Cholinesterase-Inhibitory Activities. European Journal of Organic Chemistry 2014:2770–2779.

 4.            Batista D, Schwarz S, Loesche A, Csuk R, Costa PJ, Oliveira MC, Xavier NM (2016) Synthesis of glucopyranos-6′-yl purine and pyrimidine isonucleosides as potential cholinesterase inhibitors. Access to pyrimidine-linked pseudodisaccharides through Mitsunobu reaction. Pure and Applied Chemistry 88:363–379.

Biography:

Jonathan Cawley is currently a fourth year PhD student in the School of Biotechnology at Dublin City University. He received his B.Sc. in Biotechnology (1st class honours) from DCU in 2013. His research focuses on the glycosylation changes to the CHO cell surface by probing cells with recombinant lectins, which he has expressed and purified, and commercial lectins. Subsequently the cells are analysed with a fluorescent microscope and flow cytometer.

Abstract:

Chinese hamster ovary (CHO) cells are extensively used for the production of biopharmaceutical products. In 1985 the first recombinant proteins (i.e., monoclonal antibodies, blood factors, growth hormones and cytokines etc.) are produced in CHO cells. Proteins produced in CHO frequently undergo post-translational modifications (PTM). The most common PTM is glycosylation which can alter the function, stability, immunogenicity and efficacy of the resulting protein. Changes to the CHO glycocalyx, carbohydrate coat surrounding the cell membrane, may be informative to changes on the product being produced. It is therefore of great interest to understand how the host cell changes in different culture conditions. Fluorescent microscopy and flow cytometry are employed to perform glycoanalysis on CHO DP-12 cells. Recombinant eukaryotic lectins, purified using immobilized metal affinity chromatography (IMAC), and commercial plant and fungal lectins are used to probe CHO cells in order to establish a lectin binding profile.

Lectins from non-plant sources are perfect glycan probes as they are generally nontoxic but they are also superior to other binding proteins such as antibodies whose specificities for carbohydrate targets are ill-defined. In this work a recombinant eukaryotic lectin, Agrocybe aegerita lectin 2 (AAL-2), was expressed, purified and characterised. It was then successfully used to probe the CHO DP-12 surface, along with other lectins, demonstrating that a novel lectin produced recombinantly is the optimal choice of probe for investigating the cell surface glycosylation of live cells. 

Image

Figure 1: Fluorescent microscope images of CHO DP-12 cells probed with AAL-2. CHO DP- 12 cells were probed with the GlcNAc binding Agrocybe aegerita lectin 2 (AAL-2) for 30 min and viewed with a fluorescent microscope at 400x magnification.

A) Nuclear(Hoechst) stain. B) AAL-2:FITC stain. C) Merged image of A) and B). D) AAL-2:FITC stain + 200 mM GlcNAc - viewed in FITC filter.

References

Biography:

Tung-Kung Wu completed his PhD degree in Biophysics from the Johns Hopkins University and postdoctoral studies in Chemistry Department from Stanford University. He is appointed professor in the Department of Biological Science and Technology at the National Chiao Tung University. His research interests include interdisciplinary fields of bioorganic chemistry, chemical biology, and medicinal chemistry. His current research interests mainly focus on the structure-reactivity relationships of glycoenzymes and mode of action of steryl glycosides as drug candidates. 

Abstract:

Steryl glycosides consist of a sterol/steroid aglycone and one or more sugar moieties linked through an ether or ester bond. Diverse biological activities, including anti-inflammatory, anti-fungal, anti-microbial, anti-parasitic, and anti-tumor activities, and properties such as cell membrane modification, regulation of host defenses against pathogens, lipid metabolism, and developmental events, have been attributed to this family of natural products. Development of structural diverse steryl glycosides may hold potential to identify candidates for new drug development and pharmaceutical applications. In this study, we functionally express different glycosyltransferases for in vitro synthesis of steryl glycosides with a- or b-configuration specificity. The synthesized steryl glycosides were investigated for their anticancer activity. Interestingly, several steryl glycosides show dose-dependent depression of cell viability and enhanced drug effectiveness on MCF-7 breast cancer cells. The structure-reactivity relationships of these glycosyltransferases on substrate‒donor specificities and mode of action of steryl glycosides on anticancer activity will be discussed.

Biography:

Xue-Wei Liu, China Agricultural University (BSc & MSc 1996), University of Southern California (PhD 2000), Procter & Gamble (Research Scientist, 2000-2002), Chugai Pharma USA (Senior Research Scientist, 2002-2003), Caltech (Postdoc, 2003-2005). Currently Assoc/Professor at Nanyang Technological University, Singapore. Research field: carbohydrate chemistry, natural products, and glycoproteins in addressing problems of medicinal and biochemical significance.

Abstract:

Glycopeptides and glycoproteins play vital roles in a wide array of biological activities including cell-cell adhesion, cell differentiation, cell growth and tumour metastasis. In addition, a host of major illnesses such as autoimmune diseases, infectious diseases and cancer are caused by aberrant protein glycosylation. Consequently, the development of new glycopeptides-based vaccines, diagnostics and therapeutics has gained significant interests, stimulating the study of glycopeptide synthesis. Here, a practical approach towards N-glycopeptide synthesis using an auxiliary-mediated dual native chemical ligation (NCL) is presented. The first NCL connects an N-linked glycosyl auxiliary to the thioester side chain of an N-terminal aspartate oligopeptide. This intermediate undergoes a second NCL with a C-terminal thioester peptide. Mild deprotection provides the desired N-glycopeptide. The flexibility and systematic control of our method clearly have significant advantages over preceding ones and present a major step forward in glycoscience. Taking into consideration of the previous approaches, our method offers an attractive and practical procedure to N-glycopeptide that does not require cysteine residue, allowing for straightforward glycosylation and ligation at aspartic acid terminal. The auxiliary-linked glycosyl donor can be obtained from readily available glycosyl azide. Our method can leverage modern techniques such as intein-mediated ligation or recombinant expression to gain access to peptides of virtually any length and order. In addition, quick access to polymeric peptidoglycans will also be presented.

Biography:

She received her BSC degree in Biochemistry, Faculty of Science from Ege University in 2006. She is currently doing her master thesis about “Glycan Analysis of Monoclonal Antibodies” and she will be graduated at 2017 summer. At the same time she is working as a scientific researcher at the Center for Drug Research & Development and Pharmacokinetic Applications (ARGEFAR) since 2008. Her professional experience includes usage of HPLC, LC MS, MALDI TOF MS techniques. Her research interest covers physicochemical characterization of biosimilar drugs and biopharmaceutics.

Abstract:

Monoclonal antibodies (MAbs) are increasingly being used for the treatment of cancer or other autoimmune diseases. Correct glycan structure of MAbs is essential for functional and biological activity of the related Mab such as identification of antigens triggered by immune system cells, regulation of the signaling activities and physicochemical properties of produced therapeutics etc. Therefore, rapid monitoring of glycan structure is very critical for therapeutic drug production, biosimilar drug production and their quality control processes.

The principal of this study is the characterization of glycan structure of MAbs. In our study, glycan structure of trastuzumab, used as a drug for the treatment of breast cancer, was characterized. N-glycans were enzymatically released from Trastuzumab protein structure by trypsin digestion and PNGase F (Peptide:N-Glycosidase F) cleavage.  Moreover, special buffer exchange and Solid Phase extraction procedures were used for the clean-up purposes prior to MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization-Time of Flight- Mass Spectrometry) detection.

After designing appropriate sample treatment and optimization, GO, GOF, G1, G1F, GOF-GN  glycans were clearly detected by MALDI-Q-IT-TOF MS without any labelling or using any additional chromatographic separation methods.

The current talk deals with the theoretical and experimental aspects of MALDI-Q-IT-TOF MS for the study of glycans on monoclonal antibodies. Sample preparation procedures and tips for MALDI-TOF-MS measurements will be addressed and the original data obtained from MALDI-Q-IT-TOF MS will be presented.

Biography:

Minghua Hu is R&D Lead Engineer of Infinitus (China) Company Ltd that is engaged in Chinese herbal plantations,R&D, production, sales and service of TCM health products. She is also the principle members of Joint Laboratory for the Research of Chinese Herbal Polysaccharides-Chinese Academy of Science Shanghai Institute of Materia Medica and Infinitus. She has published more than 20 papers in reputed journals

Abstract:

Fungal polysaccharides have been shown broad spectrum of biological activities, including anti-inflammatory, ant-oxidative and improve immunity. However, oral administration of fungal polysaccharides for rendering the conventional vaccine against influenza virus has been reported rarely. Here, we investigated the potential of fungal polysaccharides enhancing the influenza vaccine efficacy in a mouse model. Mice were immunized with inactivated H1N1 (A/PR8/1934) influenza vaccine combined with oral polysaccharides lentinan, tremellan, pachymaran, and a mixture of the three. The results showed that mice in the polysaccharides/vaccine groups had reduced morbidity, improved viral clearance, and recovered faster than the mice receiving the conventional vaccine only after infection. This effect could be attributed to the increased levels of virus-specific serum antibody IgG and decreased levels of inflammatory cytokine IFN-γ in the lung tissue. Our finding suggests that taking fungal polysaccharides orally might be useful for improving the efficacy of conventional inactive influenza vaccines.

References

1.            Luo Xia,Minghua Hu ,et(2016)Different impacts of polysaccharides from Chinese materia medica on T-cell subsets in immuno-suppressed mice . Prac J Med&Pharm．2016,07:621-624.

2.            Xiangliang Deng, Zhonghua Ma, Xiaoping Lai, Minghua Hu,et(2016)Rhizoma dioscoreae polysaccharide promotes M1 macrophage polarization in mice.IMMUNOLOGICAL JOURNAL 2016,12:1019-1023.

3.            LUO Xia,MA Zhonghua,HU Minghua,et(2016)Effects of compound polysaccharide on cellular immunity in mice.CHINESE JOUï¼²NAL OF FOOD HYGIENE 2016,02:186-191.

4.            REN Wenkang,HU Minghua,YIN Xiquan,et(2016)Effect of Cyclocarya paliurus（Batal.）Ijinsk Aqueous Extracts on Improving Traditional Chinese Medical Symptoms and Immune Function of Type 2 Diabetic Mellitus Rats with Yin Deficiency Generating Intrinsic Heat Syndrome.Journal of Guangzhou University of Traditional Chinese Medicine 2016,06:851-855.

5.            Minghua Hu,et(2012)Comparison of Content of the Water-Soluble Polysaccharide Included in Poria Cocos from Different Habitat and Specification.China Pharmaceuticals 2012,07:10-12.

Biography:

Ting Ting Zhao has completed her PhD at the age of 27 years from University of Chinese Academy of Sciences. She has published 19 papers in the international journals.

Abstract:

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes. Energy metabolism is altered under diabetic conditions. Decorin is a multifunctional small leucine-rich proteoglycan involved in the autophagy and energetic homeostasis. We investigated the role of decorin and its glycosaminoglycan chain in DN. Both decorin and phosphoralated AMPK were highly expressed in the kidney of an accelerated type 1 DN induced by streptozotocin in uninephrectomized Wistar rats at week 20. In high glucose cultured HK2 cell, glycosaminoglycan-free decorin, generated by mutating Ser4 of the mature protein core into Ala (DCN-S4A), showed more significant role in promoting of AMPK phosphoralation and LC3 II compared with the decorin proteoglycan. These data provides clues that the glycosaminoglycan chain of decorin has a reducing effect on activation of AMPK signaling pathway in DN.