Day 1 :
Keynote Forum
Julian M Menter
Morehouse School of Medicine, USA
Keynote: Effects of temperature and sodium hyaluronate on fluorescence of type I calf skin solutions at physiological pH
Time : 10:05-10:35
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:
Mammalian collagens contain several age-related fluorescent chromophores, derived from tyrosyl residues that are unstable to solar UV wavelengths and to ground – state thermal autoxidation. Our preliminary studies on UV – induced photochemistry have shown that dityrosine, formed by UVC (mainly 254 nm), is unstable to longer – wavelength solar UV. Age – related tyrosine oxidation product(s) are also unstable to UV – wavelengths, and they also tend to destabilize the overall collagen supramolecular structure. Because dermal collagen is in close contact with the surrounding extracellular matrix (ECM), we wish to know whether the ECM provides extra chemical stability collagen fibrils in vivo. In vitro work with collagen - sodium hyaluronate (HA) mixtures (1:2) at pH 7.4 suggested that HA might have a slightly stabilizing effect, but the results were inconclusive. In this work, we investigated the effect of temperature and HA on fluorescence emission. Our results suggest that added HA practically no effect on either the intensities or the Arrhenius – like plots of the collagen fluorescence bands. This result suggests that there is little or no physical interaction between the collagen telopeptide (which contains the tyrosine residues) and the HA domains in vitro.
Keynote Forum
Myron R Szewczuk
Queen’s University, Canada
Keynote: Oseltamivir-conjugated polymeric micelles engineered as active smart drug delivery platforms for effective tumor targeting
Time : 10:35-11:05
Biography:
Dr. Szewczuk is Full Professor of Immunology and Medicine, Queen’s University, Kingston, Ontario Canada. Dr. Szewczuk’s current research has focused on the role of glycosylation in receptor activation with a particular focus on alternate new active tumor targeting drug delivery systems.
Abstract:
Traditional chemotherapy cancer agents are typically highly hydrophobic small molecules designed to work intracellularly, targeting rapidly dividing cells. Administration of these drugs alone, complicated by insolubility in biological fluids and tissues, and an incapacity to effectively localize in metastasized tumors, results in adverse toxic side effects and prevents potent selective targeting. Functionalized drug delivery systems using polymeric nanostructures are at the forefront of cancer research, engineered for safer, more efficient and effective use of chemotherapy. Here, we designed and engineered a new polymeric micelle delivery system for active tumor targeting followed by micelle–drug internalization via receptor-induced endocytosis. By decorating micelles with oseltamivir, we investigated whether they actively targeted human pancreatic PANC1 cancer cells. Amphiphilic block copolymers with oseltamivir conjugated at the hydrophilic end (oseltamivir-poly(polyethylene glycol methyl ether methacrylate)-block-poly(methyl methacrylate) were synthesized using reversible addition–fragmentation chain transfer (RAFT) living radical polymerization with self-assembling properties. Oseltamivir-micelles targeted pancreatic PANC1 cancer cells, reduced Neu1 sialidase activity and tumor cell viability with subsequent internalization of the micelles loaded with a fluorescent hydrophobic drug. Neu1 binding was shown to be a prerequisite step toward micelle internalization. The ability to both target and halt the growth of a tumor cell using a newly designed nanocarrier system, combined with the internalization of the micelle loaded with a cytotoxic chemotherapeutic represents the novel aspect of this work.
Keynote Forum
Rachel Chen
Georgia Institute of Technology, USA
Keynote: Metabolic engineering for oligo- and poly-saccharide synthesis: challenges and opportunities
Time : 11:20-11:50
Biography:
Rachel Chen received her PhD from California Institute of Technology in 1994 and subsequently worked as a research scientist in Bristol-Myers Squibb. She began her independent academic career in Virginia Commonwealth University and continues at Georgia Institute of Technology. Her research interfaces biology, chemistry, and engineering with major focuses on applying molecular engineering tools in the synthesis of molecules that are not attainable with conventional means. She has published ~80 peer-reviewed papers and has been serving as an associate editor for Microbial Cell Factories and on editorial boards of AIMS Bioengineering and AIMS Microbiology.
Abstract:
As one of the four building blocks of life, sugar molecules permeate almost all aspects of life. They are also unique in being intimately associated with all major types of biopolymers (including DNA/RNA, proteins, lipids) meanwhile they stand alone as bioactive polysaccharides, or free soluble oligosaccharides. The widespread occurrence of glycosylation and its broad impact in biological processes underscores the importance of studying glycosylation. To study glycans and probe their roles in a biological system significant amount of pure molecules are needed. Besides basic research, there are a wide range of opportunities of utilizing oligosaccharides, polysaccharides, and glycoproteins and other glycoconjugates for diagnosis, vaccine development, as new drug entities, and many other medical applications. Unfortunately, these potential applications are all impeded by the lack of large scale synthesis technology for these molecules. Metabolic engineering, since its inception in late 80’s, has grown to be a field impactful in the synthesis of a variety of molecules of commercial and societal importance. Opportunities abound at the interface of glycosciences and metabolic engineering. In fact, all sugar moieties in biological components, small or big, free or bound, are important targets for metabolic engineering. Over the past decades, its use in the synthesis of sugar-containing molecules has gained significance. In this presentation, metabolic engineering challenges common to glycosyltransferase-catalyzed synthesis of oligosaccharides and polysaccharides are analyzed and successful examples from Chen and other labs are showcased to emphasize the power of metabolic engineering as an enabling technology.
- Track 1: Glycans in Diseases and Therapeutics
Track 2: Glycobiology: Its Role In Human Health
Track 3 : Glycobiology & Biotechnology
Track 4 : Glycomics & Bioinformatics
Location: Atlanta
Chair
Julian M Menter
Morehouse School of Medicine, USA
Co-Chair
Rachel Chen
Georgia Institute of Technology, USA
Session Introduction
Nahid Razi
AccuDava Inc., USA
Title: Glyco-Markers in Personalized Cancer Chemotherapy with Platinum-Drugs
Biography:
Dr. Razi is the founder of AccuDava Inc., a biomedical company for personalized cancer chemotherapy (http://www.accudava.com), based on the discovery of two Glycomarkers. Razi received her PhD in Medical Sciences from Uppsala University-Sweden, in 1995. In her post-doctoral program, at the University of California-San Diego, she discovered a novel glycan-mediated lymphocytes activation mechanism in the immune system. She then joined the Consortium for Functional Glycomics (CFG), at The-Scripps-Research-Institute, where her team successfully developed and launched the world’s largest mammalian glycan microarray in 2011. This unique platform that displays 611 defined glycans has since become available globally for studying glycan-mediated interactions.
Abstract:
Personalized chemotherapy is an unmet need in cancer treatments. A molecular test that can accurately predict the drug efficacy on a patient would be invaluable in selecting effective treatment strategy. We have identified two glycan structures, namely Glycomarker-1 and Glycomarker-2, whose expression levels on the cancer cell surface are associated with responses to chemotherapy with platinum drugs (US patent #7585503, and International pending patent). This finding introduces a new concept, linking glycan cell surface expressions with drug reactivity, and proposes a glycan-mediated mechanism for drug uptake. Our initial studies profiling the cell surface glycans, using Flow Cytometry with specific glycan-binding lectins, on three isogenic pairs of ovarian carcinoma cell-lines, consisting of chemosensitive and acquired chemoresistant phenotypes, revealed at least a ten-fold decrease in alpha2-6Sialyl-R motif (Glycomarker-1) on resistant phenotypes compared to sensitive cells. Further studies by fluorescent confocal microscopy, colony forming assay, sialidase treatments, and mass-spectrometry confirmed the association of Glycomarker-1 with drug uptake. Using Lectin histochemistry (LHC) on clinical samples proved a feasible assay for Glycomarker-1, tested on 64 human ovarian normal and cancerous tissue sections. The LHC on retrospective ovarian cancer specimens, with a known history of drug-response, correctly predicted drug-responses in 22 out of 27 (81.4%) patients. During the studies on the mechanism for drug response, another glycan structure, Glycomarker 2, was identified demonstrating a similar expression pattern to Glycomarker 1. Further studies on Glycomarker-2 suggest an association of the two glycomarkers that would put forward a glycan-mediated mechanism for platinum-drug uptake by cancer cells.
Myron R Szewczuk
Queen’s University, Canada
Title: Role of sialylation in 3D multicellular tumor spheroid formation and in vivo tumors
Time : 11:50-12:20
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. 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 of tumorigenesis.
Abstract:
Multicellular tumor spheroids are now at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is known to be highly influenced by aberrant cell surface specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate spheroid formation are not well defined. Here, matrix-free spheroids from human breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD induced self-assembly platform. Pretreatment with specific lectins, exogenous neuraminidase dose dependently reduced spheroid volume formation. Oseltamivir phosphate (OP) promoted cell aggregation and compaction into spheroid formation. PANC1 and triple negative breast MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significant higher levels of α-2,3-sialoglycan (SA) than α-2,6-SA. MCF-7 spheroids expressed higher α-2,3-SA to α-2,6-SA ratio. The relative levels of specific sialoglycan structures on the cell surface facilitate avascular 3D multicellular tumor spheroids in vitro as well as in vivo tumors.
Iva Turyan
Biogen, USA
Title: SAGM-RRBP- Selecting appropriate glycoanalysis methods to reduce risk in biopharmaceutical production
Time : 12:20-12:50
Biography:
Iva Turyan completed her PhD at the age of 27 years from St. Petersburg University and postdoctoral studies at The Hebrew University of Jerusalem. She is currently an Analytical Development Scientist at Biogen, Cambridge, MA. She has published more than 45 papers in reputed journals, and has been awarded 6 patents.
Abstract:
Glycosylation of therapeutic recombinant proteins is of particular importance due to its potential impact on solubility, bioactivity, pharmacokinetics and immunogenicity of glycoprotein pharmaceuticals. Detailed characterization of glycans present on recombinant glycoprotein remains an important challenge in the development and production of biotherapeutics. Analytical strategies for characterization of N- and O- glycosylation and monosaccharides analysis will be presented. These include comparison of HILIC-FLR, MALDI-TOF MS and CE-LIF for N-glycan analysis, choice of a method for quantitative and non-selective release of O-linked glycans, and selection of a method for monosaccharide composition analysis. Choosing appropriate glycoanalysis methods allowed detecting changes in glycosylation parameters. A case study will be presented that highlights glycoanalysis techniques useful for gaining understanding of the relationship between process inputs (raw materials) and product quality attributes. The findings confirm that the glycosylation profile of therapeutic antibodies needs to be monitored through development in order to ensure consistency, efficacy, and safety of therapeutic products.
Biography:
Maria Longas completed her PhD at New York University in 1978, and did her postdoctoral training at Columbia University School of Medicine with Dr. Karl Mayer. She has an MA in Organic Chemistry from New York University (1973) and a BA in Chemistry from Hunter College (1971). She is a full Professor of Chemistry at Purdue University Calumet in Hammond, IN. She has more than 24 papers published in reputable journals, and served as a reviewer for several journals.
Abstract:
Ultraviolet light B (UVB) is a technique used to investigate organic molecules. It was utilized in this study to analyze the content of vitamin D3 in face lifted female skin of subjects aged 43, 47, 51, 58, 60 and 66 years. The skin of these subjects had pigmentation of great similarity. It was kept at -40O C, defrosted to room temperature for 2 min and placed in an oven for another 2 min at 105o C. After these 2 min, we took the first UVB reading; a 2nd UVB run was done at room temperature. The amount of vitamin D3 was determined under standard conditions of 7-dehydrocholesterol (7-Dchol). The best temperature to measure D3 was when skin was at room temperature for 15 min. The D3 analysis of the UVB plots of human skin vs. the standard showed a linear decrease in human skin as compared to 7-Dchol. Because 7-Dchol is a direct precursor of vitamin D3, it appears from these results that as the skin ages, 7-Dchol concentration decreases which results in less production of vitamin D3. Conditions to increase vitamin D3 in the aged should be considered, to avoid problems of arthritis, tuberculosis and autoimmune disorders, etc.
Myron R Szewczuk
Queen’s University, Canada
Title: Role of sialylation in 3D multicellular tumor spheroid formation and in vivo tumors
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. 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 of tumorigenesis.
Abstract:
Multicellular tumor spheroids are now at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is known to be highly influenced by aberrant cell surface specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate spheroid formation are not well defined. Here, matrix-free spheroids from human breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD induced self-assembly platform. Pretreatment with specific lectins, exogenous neuraminidase dose dependently reduced spheroid volume formation. Oseltamivir phosphate (OP) promoted cell aggregation and compaction into spheroid formation. PANC1 and triple negative breast MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significant higher levels of α-2,3-sialoglycan (SA) than α-2,6-SA. MCF-7 spheroids expressed higher α-2,3-SA to α-2,6-SA ratio. The relative levels of specific sialoglycan structures on the cell surface facilitate avascular 3D multicellular tumor spheroids in vitro as well as in vivo tumors.
Michael S Strano
Massachusetts Institute of Technology, USA
Title: Label-free lectin microarrays using fluorescent carbon nanotube sensors: Towards rapid glycan characterization and synthetic lectin design
Time : 12:50-13:20
Biography:
Michael Strano is currently the Carbon P. Dubbs Professor of Chemical Engineering at MIT. He received his B.S from Polytechnic University in Brooklyn, NY and Ph.D. from the University of Delaware, both in Chemical Engineering. He was a post doctoral research fellow at Rice University in the departments of Chemistry and Physics under the guidance of Nobel Laureate Richard E. Smalley. From 2003 to 2007, Michael was an Assistant Professor in the Department of Chemical and Biomolecular Engineering at the University of Illinois at Urbana-Champaign before moving to MIT. His research focuses on biomolecule/nanoparticle interactions and the surface chemistry of low dimensional systems, nano-electronics, nanoparticle separations, and applications of vibrational spectroscopy to nanotechnology.
Abstract:
Label-free lectin microarrays are a promising approach to rapidly characterize glycoprotein mixtures. However, to date, demonstrations of highly multiplexed label-free lectin microarrays have been limited. Our group uses near-infrared fluorescent single-walled carbon nanotubes to design glycan-responsive sensors capable of massive multiplexing and real-time detection for incorporation into a label-free lectin microarray. We employ two strategies for the design of our carbon nanotube sensors. The first design platform uses a His-tagged lectin that has been tethered to the nanotube via a Cu2+/NTA linker. We have demonstrated responsivity of these sensors to a variety of natural glycoproteins and to neoglycoproteins constructed from streptavidin and biotinylated sugars. Our second detection platform is based on Corona Phase Molecular Recognition (CoPhMoRe), a technology developed by our group at MIT whereby synthetic, non-biological recognition sites are created from the three-dimensional structure of a carbon nanotube and adsorbed heteropolymer. We have developed CoPhMoRe-based sensors for a variety of molecule types including carbohydrates, resulting in the creation of synthetic lectins capable of being incorporated into the label-free microarray. These sensors, along with a binding kinetic model that we developed, are capable of quantitatively characterizing glycoprotein mixtures at a much shorter time scale than existing characterization techniques. This technology has the potential to address longstanding problems in the fields of biopharmaceutical process analytics and medical diagnostics.
Joon Kim
Korea University, Korea
Title: Ribosomal protein S3 secreted from cancer cell lines is N-glycosylated
Time : 14:10-14:40
Biography:
Prof. Joon Kim has completed his BS and MS in Microbiology from Seoul National University, PhD in Biochemistry from the University of California at Berkeley and postdoctoral study from Harvard Medical School. He is a Professor in the Division of Life Sciences, and the Director of Radiation Safety and Management Center, Korea University, Seoul, Korea. He has published more than 150 papers in reputed journals
Abstract:
Ribosomal protein S3(rpS3) is a component of the 40S ribosomal small subunit but has multiple other extra-ribosomal functions like apoptosis, cell cycle control, DNA repair etc. It has a DNA repair endonuclease activity which is related with various cancers. Recently, we have discovered that this protein is secreted only from various cancer cell lines as a homodimer but not in normal cells. We also confirmed that rpS3 is secreted more into media from the more invasive cancer cell lines. Presently we confirmed that the secreted protein is glycosylated at the Asn 165 residue and point mutation on this site is defective for the secretion. The secretion pathway turned out to be a ER-Golgi dependent pathway. We propose that glysosylated rpS3 could be used as a useful cancer marker.
Joern Dalgaard Mikkelsen
The Technical University of Denmark, Denmark
Title: Synthesis of human milk oligosaccharides by trans-glycosidases
Time : 14:40-15:10
Biography:
JDM has completed his PhD at Carlsberg Laboratory, before he moved to Danisco, now Danisco/Dupont, where he worked 20 years as scientist and Research Manager and finally Director of Research in Danisco Biotechnology. He moved to the Technical University in 2008 with a Danisco grant to establish a group working on enzymes, hydrocolloids and prebiotic oligosaccharides. He has published 138 publications and 25 Patents.
Abstract:
Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote “reverse” catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This presentation will provide an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-L-fucosidases, β-N-acetylhexosaminidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of co-solvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure−function relationship of the enzymes is presently poorly understood.
Julian M Menter
Morehouse School of Medicine, USA
Title: Workshop on How the epidermis and dermal collagen/extracellular/matrix interact with each other
Time : 15:10-16:10
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:
Mammalian skin epidermis and dermis are usually considered as separate entities, owing to their different functions. However, there is much interaction between them, and it is even suspected that altered dermis can be a factor in epidermal carcinogenesis. In addition to epidermal carcinogenesis caused primarily by UVB (290 – 320 nm) radiation, UVA wavelengths (320 – 400 nm) can cause changes in dermal architecture and composition that causes a response analogous to wound healing. One hypothesis is that the latter changes may render the overall milieu more favorable for epidermal skin cancer formation. In cases of epidermal pigmentary incontinence, melanin pigment may play an additional role in dermal damages. In the present study, we use collagen’s intrinsic fluorescence properties to investigate the action of UV on two acid-soluble type I collagens in an in vitro system. These involve thermal (i.e. temperature dependence in ground state properties) and photochemical (involving excited state transformations). In further experiments, we are beginning a systematic study of the effect of surrounding extracellular matrix (ECM) on our present results. In a preliminary study (to be published) we have found, somewhat surprisingly, that addition of sodium hyaluronate (HA) has little or no effect on the thus far observed properties. We envision expanding the investigation to include the addition of proteoglycans or at least their component chondroitin sulfates.
Yves Durocher
National Research Council, Canada
Title: Production of sialylated monoclonal antibody in CHO cells
Time : 16:25-16:55
Biography:
Yves Durocher is a Research Officer at the National Research Council of Canada since 1995. He obtained his PhD in Biochemistry at the Université de Montréal in 1993. Yves manages the NRC’s Mammalian Cell Culture Section which is composed of 33 scientists involved in protein expression and CHO cell line development for internal projects and external clients. His research activities have been focused on the development the of large-scale transient gene expression (LSTGE) platforms using HEK293 and CHO cells for protein production and on the development of stable CHO pool and clonal cell line platforms for the manufacturing of recombinant therapeutic proteins. He also contributed to ~100 scientific publications in peer-reviewed journals.
Abstract:
IgGs that possess Fc-glycans with terminal sialic acid (SA) are thought to be responsible for the anti-inflammatory properties of intravenous immunoglobulins (IVIGs) through a mechanism that is still unclear. The impact of this sialylation on IgG’s effector functions (ADCC and CDC) also remains to be elucidated. To better understand the biological impact of IgG sialylation, there is a need to produce recombinant IgGs with well characterized and more homogeneous glycan structures. The type of SA (NANA or NGNA), the nature of its linkage with the galactose residue (alpha-2,3 or alpha-2,6) or the number of glycan antennae being sialylated, may vary according to the IgG subtype, the host cell in which it is expressed and the cell culture environment. In this study, we show that the a 2,6-sialylation of IgG1’s Fc domain can be efficiently achieved by the transient co-expression of the human beta 1,4-galactosyltransferase-1 (GT) and 2,6-sialyltransferase-1 (ST6) in CHO cells. The process allows for the production of milligram amounts of human-like sialylated monoclonal antibody within two weeks. The impact of this sialylation on IgG1 binding to FcγRIIIa is also presented.
Stefan Kubick
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Germany
Title: Cell-free synthesis of glycoproteins
Time : 16:55-17:25
Biography:
Dr. Kubick is head of the department “Cell-free Bioproduction” at the Fraunhofer IZI-BB. His laboratory exploits cell-free protein synthesis as a versatile tool for membrane protein and glycoprotein synthesis, as well as chip-based protein synthesis and translational regulation. In collaboration with Qiagen GmbH he developed novel eukaryotic in vitro translation systems. Dr. Kubick is also a lecturer at the Free University of Berlin and the University of Potsdam. He is an affiliate of the Technical University of Berlin and Lecturer at the University of Applied Sciences, Berlin, Germany.
Abstract:
Membrane proteins and glycoproteins have become an important focus of the current efforts in structural and functional genomics. In contrast, the molecular analysis of posttranslationally modified proteins lags far behind that of non-modified, cytosolic and soluble proteins. Preparing high quality samples of functionally folded proteins represents a major bottleneck that restricts further structural and functional studies. Cell-free protein synthesis systems, in particular those of eukaryotic origin, have recently been developed as promising tools for the rapid and efficient production of a wide variety of posttranslationally modified proteins. This is mainly due to the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications such as the addition of N- and O- linked oligosaccharides, but also palmitoylation, myristylation, and phosphorylation. Based on these versatile properties of cultured cell lines, we have developed a technique for the standardized production of translationally active eukaryotic cell lysates. Our homogenization procedure avoids any serious breakdown of membrane vesicles already existing in the cytoplasm of the prepared eukaryotic cells. We have demonstrated the functional integrity of these subcellular components by showing signal peptide cleavage as well as glycosylation of in vitro produced proteins. Moreover, we have expanded our cell-free protein synthesis system by the insertion of orthogonal tRNA/synthetase pairs to facilitate the cotranslational and site directed incorporation of non-canonical building blocks. These fluorescently labeled and chemoselective moieties enable the site-specific modification of de novo synthesized glycoproteins.
Cheng Ma
Georgia State University, USA
Title: Improvement of core-fucosylated glycoproteome coverage via alternating HCD and ETD fragmentation
Time : 17:25-17:55
Biography:
Cheng Ma has completed his PhD from Beijing Proteome Research Center (BPRC). His work as a research scientist in Georgia State University focused on development of novel techniques to analyze the sequence of glycopeptides through the fragment ions of MS/MS. He has a broad background in proteomics, glycoprotemics, glycomics, as well as bioinformatics. Meanwhile, He has more than eight years’ experience in mass spectrometry operation.
Abstract:
Core-fucosylation (CF) plays important roles in regulating biological processes in eukaryotes. Alterations of CF-glycosites or CF-glycans in bodily fluids correlate with cancer development. Therefore, global research of protein core-fucosylation with an emphasis on proteomics can explain pathogenic and metastasis mechanisms and aid in the discovery of new potential biomarkers for early clinical diagnosis. In this study, a precise and high throughput method was established to identify CF-glycosites from human plasma. We found that alternating HCD and ETD fragmentation (AHEF) can provide a complementary method to discover CF-glycosites. A total of 407 CF-glycosites among 267 CF-glycoproteins were identified in a mixed sample made from six normal human plasma samples. Among the 407 CF-glycosites, 10 are without the N-X-S/T/C consensus motif, representing 2.5% of the total number identified. All identified CF-glycopeptide results from HCD and ETD fragmentation were filtered with neutral loss peaks and characteristic ions of GlcNAc from HCD spectra, which assured the credibility of the results. This study provides an effective method for CF-glycosites identification and a valuable biomarker reference for clinical research.
Wanyi Guan
Georgia State University, USA
Title: Identification of the binding roles of terminal and internal epitopes via enzymatic synthesized N-glycans with tandem epitopes
Time : 17:55-18:25
Biography:
Wanyi Guan has completed her PhD from Shandong University, China. She is currently a postdoc in Dr. Peng George Wang’s group in Department of Chemistry in Georgia State University. Her research focuses on enzymatic and chemo-enzymatic synthesis of sugar nucleotide, glycans and their analogs.
Abstract:
In nature, glycans are displayed universally at the surface of living cells. They play important roles in numerous biological events through binding with corresponding glycan binding proteins (GBPs). Usually, the carbohydrate moiety involved in these bindings is composed of the sugar residues at the non-reducing terminal by linked to each other in certain manners and forming specific epitope determinants. Nevertheless, internal epitopes may also participate in the binding and initiate subsequent signaling process. To investigate whether the internal epitope involved in the glycan binding process, an array of symmetric bi-antennary N-glycans were synthesized with tandem epitopes, including LacNAc (Galβ1,4-GlcNAc), sialyl LacNAc (Siaα2,3-LacNAc), 6-sialyl LacNAc (Siaα2,6-LacNAc), Lewis x (Galβ1,4-(Fucα1,3-)GlcNAc), sialyl Lewis x (Siaα2,3-Galβ1,4-(Fucα1,3-)GlcNAc), α-Gal (Galα1,3-LacNAc), and disialic acid (Siaα2,8-Sia), by enzymatic extension of N-glycan in the glycopeptide isolated from chicken egg yolk. For rapid production of glycans, one-pot multiple enzyme (OPME) strategy was employed, and the produced glycans were separated by high performance liquid chromatography monitored with UV detector. In total, 36 glycans were prepared to milligram scale and over 98% purity. Their binding profile to selected GBPs and viruses showed that internal glycan epitopes and modification of terminal epitopes exhibited obvious, but diverse effects to the binding of terminal epitopes.
Clifford A Lingwood
University of Toronto, Canada
Title: Glycosphingolipid synthesis is remodeled by statin-mediated reduction of Rab prenylation
Time : 18:25-18:55
Biography:
Clifford Lingwood completed his PhD at the University of London at 25 years of age, and postdoctoral studies at the Universities of Washington and Toronto. He has been a professor at the University of Toronto since 1997 and is a senior scientist within the Molecular Structure and Function program of the Research Institute at the Hospital for Sick Children, Toronto He has published more than 200 papers in reputed journals.
Abstract:
Glycosphingolipids (GSLs) accumulate together with cholesterol in membrane lipid rafts, dynamic domains of increased order, which provide foci for transmembrane signaling, membrane trafficking and portals for microbial pathogens. Membrane cholesterol can also complex with GSLs to alter the carbohydrate from a membrane perpendicular to parallel conformation and thereby restrict ligand access to GSLs. Cholesterol ligand access is also restricted in the GSL complex. Since statins are widely used to decrease serum cholesterol levels, we question whether cholesterol depletion affected GSL synthesis. While we found that statins are unable to reduce cholesterol in serum cultured cells, statins nevertheless had a remarkable effect on cell GSL synthesis in that glucosyl ceramide was markedly increased and downstream GSLs increased according to cell line. The enzyme glucosyl ceramide synthase was mislocalized within statin treated cells. This correlated with a loss of cholesterol accumulation in the transGolgi network. The trihexoside, lactotriaosyl ceramide, was induced in 70% of cell lines. This effect was duplicated by the Rab prenylation inhibitor, 3-PEPHC and reversed by addition of the isoprenoid precursor, geranylgeranyl pyrophosphate, a downstream product of HMG Co-A reductase, which is inhibited by statins. These results are consistent with statin induced depletion of Rab prenylation, subsequent aberrant Golgi vesicular traffic, mislocalization of GSL anabolic enzymes and remodeling of GSL synthesis. This process also inhibits the normal retrograde transport of GSLs and the internalization of cholera and Shiga toxins, which hijack GSL retrograde transport for endoplasmic reticulum targeting, are also mislocalized intracellular, thereby protecting statin treated cells.
Amelia Pilar Rauter
University of Lisbon, Portugal
Title: Glycan synthesis towards new glycolipid antibiotics with new mechanisms of action
Biography:
Amélia Pilar Rauter has made her Habilitation at Faculty of Sciences, University of Lisbon (FCUL, 2002) and completed her Ph.D. at Technische Universitaet Graz, Austria (1982). She is the Head of the Carbohydrate Chemistry Group - Center of Chemistry and Biochemistry (FCUL), and has been serving as Editor (RSC Carbohydrate Chemistry book series), Associated Editor (Mediterranean Journal of Chemistry), and member of journals advisory board in Organic/Carbohydrate Chemistry. She is Secretary of the European Carbohydrate Organisation and Secretary of the IUPAC Division on Organic and Biomolecular Chemistry, has published more than 130 papers and book chapters, and authored 12 patents.
Abstract:
The search for new antimicrobial drugs is currently one of the major ongoing research areas due to the spread of multidrug-resistance, thus encouraging research on antibacterial agents with new mechanisms of action. A new family of antibiotics, structurally based on glycolipids, with a potent antimicrobial activity against Bacillus species will be disclosed. We present our recent findings that cover synthetic approaches to new antimicrobial molecular entities differing in the glycon structure, based on alkyl deoxy pento- and hexopyranosides belonging to the D- or L-series. Stereochemistry, in particular the anomeric configuration, and the deoxygenation pattern are modified in order to evaluate their contribution to the structure/activity relationship. Also the lipophilic chain is changed for the recognition of the key structural features for the bioactivity/selectivity for Bacillus species. The surface properties of the most active compounds, in terms of adsorption and aggregation parameters, are also presented. Although surface activity is required for the bioactivity, this property is not sufficient to account for bioactivity, as shown by some promising surface active glycosides, that were not active. In order to have insights into the mechanism of action of this family of compounds, expected to target bacterial membranes, a multidisciplinary approach covering biophysical and biological methods was followed. The elucidation of the new mechanisms of action of this family of antibiotics will be disclosed and discussed, demonstrating the relevance of the glycan structure for the bactericidal activity.
Francesco Peri
University of Milano Bicocca, Italy
Title: Molecular and in vivo studies on new small-molecule therapeutics targeting TLR4 by an original mechanism of action
Biography:
Francesco Peri (age: 47) is Professor of Organic and Medicinal Chemistry at the University of Milano Bicocca. He has also a permanent professorship in Medicinal Chemistry at the Ecole Normale Superieure (ENS) in Lyon, France. He coordinates the H2020-funded Marie Curie ETN project TOLLerant: “Toll-Like Receptor 4 activation and function in diseases: an integrated chemical-biology approach” (4 years project, 2015-2019, 8 academic and 2 industrial partners, 13 enrolled PhDs). He has published more than 87 papers in reputed journals, H-index: 24; H-index from 2010: 20.
Abstract:
Toll-like Receptors (TLRs) activation by pathogen associated molecular patterns (PAMPs) is a pivotal molecular event in inflammation and innate immunity, and TLRs and their agonists are responsible for the efficacy of almost every vaccine. Converserly, TLRs hyperactivation by endogenous factors such as oxidized phopsholipids or heat shock proteins is the main cause of many inflammatory and autoimmune diseases. Activating or inhibiting specifically TLRs provides access to a new generation of therapeutics. We developed synthetic molecules able to modulate TLR4 activation and signalling and we studied the mechanism of action (MOA) of these non toxic, drug-like compounds. Positively or negatively charged synthetic glycolipids are active in blocking TLR4 activation by specifical targeting the CD14 co-receptor. These molecules are very efficient in inhibiting TLR4 activation in cells and in contrasting diseases related to TLR4 hyperactivation by infectious and endogenous agents in animal models. We investigated at a molecular level the MOA of these molecules by binding experiments with purified CD14, MD-2 and TLR4 receptors, and experiments on dendritic cells, macrophages and HEK-TLR4 cells. The unique MOA of these molecules is based on the capacity to dissociate CD14 and TLR4 endocytosis, thus creating an inducible CD14 deficiency at the cell surface. These conditions are expected to antagonize TLR4 signaling more effectively than simply competing with LPS for CD14 and TLR4. Very promising results have been obtained at a preclinical level using these drug hits, and recent data on animal models of atherosclerosis, neuroinflammation, and amyotrophic lateral sclerosis will be reported.