Day 3 :
Keynote Forum
Konrad Sandhoff
LIMES Institut, Germany
Keynote: Membrane lipids regulate glycosphingolipid catabolism, their enzymes and lipid binding proteins
Time : 10:05-10.30
Biography:
Konrad Sandhoff completed his PhD in biochemistry in Munich. After research stays in Munich, Israel and the USA he became a full professor of biochemistry at the University of Bonn in 1979. Since 2007 he is a senior professor at the LIMES institute, Bonn. Major Research Interests: Molecular life sciences: analysis and pathobiochemistry of lysosomal (glyco-)sphingolipid storage diseases, structure and function of lysosomal enzymes and lipid binding proteins, topology of endocytosis and glycolipid metabolism, and regulation of glycolipid biosynthesis. He has published more than 480 peer-reviewed papers. Among many other prizes he also received the International Glycoconjugate Organization Award (2005).
Abstract:
Cholesterol and sphingolipids (SLs) are stabilizing compounds of eukaryotic plasma membranes. Together with phospholipids (PLs) they reach luminal vesicles of the endolysosomal compartment as platforms for membrane degradation. A maturation process removes lipids inhibiting lysosomal catabolism from the luminal vesicles. Sphingomyelin (SM) is hydrolyzed by acid sphingomyelinase, facilitating cholesterol export to the cytosol by NPC2 and NPC1. SM and cholesterol poor luminal vesicles then serve as platforms for glycosphingolipid degradation in the lysosomes employing soluble hydrolases, SAPs (sphingolipid activator proteins) and anionic PLs as stimulators. We reconstituted the catabolic proteins on liposomal surfaces, mimicking luminal vesicles of the lysosomes as platforms for SL degradation. Liposomes without anionic PLs and with no net surface charge generated only negligible and physiologically irrelevant catabolic rates even at lysosomal pH values. Incorporation of anionic PLs into the SL-carrying liposomes, however, stimulated the catabolic rate by up to more than an order of magnitude. We now found, that the incorporation of cholesterol or SM into the SL carrying liposomal membranes generated a strong inhibition of ganglioside GM2 hydrolysis and the transfer of membrane lipids between liposomal vesicles by SAPs, even in the presence of anionic phospholipids. Ongoing in vitro studies indicate that PM-stabilizing lipids, i.e. SM and cholesterol, inhibit several steps of lysosomal SL and glycosphingolipid catabolism and also lipid solubilisation as studied by Surface Plasmon Resonance and intervesicular (glyco-) lipid transfer activities of several SAPs and NPC2, even in the presence of activating anionic phospholipids.
- Track 1 : Glycans in Diseases and Therapeutics
Track 2: Glycomics and Bioinformatics
Track 3 : Glyco Immunology
Location: Independence C
Chair
Joseph J Barchi Jr
National Cancer Institute, USA
Co-Chair
Cecilia H. Marzabadi
Seton Hall University, USA
Session Introduction
Maria O. Longas
Purdue University Calumet, USA
Title: NaH Inhibits β-Amyloid Fibril Formation in Alzheimer’s Disease Rats
Time : 11:10-11:30
Biography:
Maria Longas has completed her PhD at New York University and done her postdoctoral training at Columbia University School of Medicine with Dr. Karl Meyer. 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 has served as a reviewer for several journals.
Abstract:
Alzheimer’s Disease Rats were developed by Taconic Biosciences, MPA; development took 5 weeks. After this time, each of the 6 Alzheimer’s Disease rats was injected with NaH (3 mg/3 mL sterilized phosphate buffer saline, SPBS); 3 grams of NaH/gram of Rat body mass was used for injection. The controls were normal Rats, 6 of which were injected with 3mL of SPBS per gram of Rat body mass. After 5 weeks, all Rats were guillotined; the heads were removed and stored at -35oC. Cells were then cultured following conditions described for human Alzheimer’s Disease cells (Longas, MO, et al, unpublished) and carried out in 6-well-tissue-culture-dishes, each containing 4 mL of the following media: Astrocyte Basal Medium from Lonza adjusted to 15% (w/w) fetal bovine serum and fortified with L-Glutamine (5.0 mL), Ascorbic Acid (0.5 mL), Epidermal Growth Factors (1.25 mL), Insulin (0.5mL) and Gentamicin Sulfate Amphotencin-ß (0.5 mL). Cells grew in these media at 37oC, under 5% CO2 for 7 days. They were then stained with Congo Red. Figure IIIR displays Alzheimer’s Disease Hippocampus cells which show plenty of β-Amyloid tangles, while Figure IVR has cells from the Normal Hippocampus with no tangle formation. NaH is, therefore, effective to inhibit β-Amyloid tangle formation in Alzheimer’s Disease.
George R. Bousfield
Wichita State University, USA
Title: Influence of glycans on biological activities of pituitary gonadotropins, follicle stimulating hormone and luteinizing hormone
Time : 11:30-11:50
Biography:
George Bousfield completed his PhD at Indiana University in 1981. He then pursued postdoctoral studies at the University of Texas, M.D. Anderson Cancer Center with Darrell Ward. He is currently the Dr. L.M. Jones Distinguished Professor in the Fairmount College of Liberal Arts and Sciences at Wichita State University, and director of the WSU Protein Core Facility. He has published more than 56 journal articles.
Abstract:
The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play central roles in the control of reproduction by connecting neuroendocrine hypothalamic/pituitary activities to gonadal function. LH and FSH receptor activation is an important part of assisted reproductive technologies, such as in vitro fertilization. Both FSH and LH are heterodimeric glycoproteins, each composed of a non-covalently associated, common ï¡- and a hormone-specific ï¢-subunit. Both subunits are N-glycosylated. Although high affinity binding of both hormones to their cognate receptors occurs exclusively by protein-protein interactions, full gonadotropin receptor activation involves N-linked glycans. However, the activation mechanism remains elusive. Part of the problem is the high degree of oligosaccharide microheterogeneity that challenges even contemporary analysis by high-resolution mass spectrometry. LH possesses three N-glycosylation sites that are always occupied by glycans and can be removed individually, permitting characterization in a site-specific manner. While FSH glycopeptides can be obtained from all 4 potential N-glycosylation sites, only 2 can be thoroughly analyzed by glycopeptide mass spectrometry. Even then, only about a third of the glycan population can be detected. While both FSHï¡ subunit glycosylation sites can be interrogated individually by sequential peptide-N-glycanase digestion, both FSHï¢ glycans are released simultaneously. This is unfortunate, because naturally occurring forms of FSH lacking either one of the 2 FSHï¢ N-glycans bind their receptors more rapidly, occupy more FSH binding sites, and provoke a more robust cellular response than fully-glycosylated FSH. Moreover, the ratio of FSH glycosylation variants may be under physiological regulation. Supported by NIH grant P01 AG029531.
Cecilia H. Marzabadi
Seton Hall University, USA
Title: Novel carbohydrate-based ligands with immunostimulatory properties
Time : 11:50-12:10
Biography:
Professor Marzabadi completed her PhD at the University of Missouri – St. Louis and carried out postdoctoral studies at Hunter College- CUNY. She has been at Seton Hall University for the past 15 years and has been a Visiting Professor at Harvard University Medical School and a Visiting Researcher at Memorial Sloan Kettering Cancer Center. She has published numerous papers in the carbohydrate literature and currently serves as a Member at Large for the ACS Division of Carbohydrate Chemistry.
Abstract:
We are interested in the synthesis and biological evaluation of new carbohydrate structures with the capacity to act as immunomodulators. Towards these ends we have synthesized molecules as mimics of capsular saccharides. These highly simplified saccharide structures have been shown to act as TLR4 agonists and to elicit the secretion of proinflammatory cytokines at concentrations lower than the known agonist, E. coli lipopolysaccharide. In this presentation, we will discuss the synthetic efforts towards a lead compound and the preliminary biological data obtained for this compound. Efforts to elucidate the mechanism of action of this molecule will be discussed.
Myron R Szewczuk
Queen’s University, Canada
Title: Therapeutic targeting neuraminidase-1 in multi-stage of tumorigenesis
Time : 12:10-12:30
Biography:
Myron Szewczuk has completed his PhD 1974 from University of Windsor in Biology and Immunochemistry and his postdoctoral studies from Cornell University Medical College, New York City, U.S.A. in Cellular immunology with Dr. Greg Siskind (1975-1978). He is presently Full Professor of Immunology and Associate Professor of Medicine, Queen’s University, Kingston, Ontario, Canada. He has published more than 100 papers in reputed journals and has been serving as an editorial board member of repute.
Abstract:
Various reports have suggested that receptor glycosylation modiï¬cation may in fact be the invisible link connecting ligand-binding and receptor dimerization. A novel organizational signaling platform linked to the glycosylated receptor tyrosine kinases (RTK) (e.g., EGFR, TrkA, insulin) and TOLL-like receptors induced receptor activation process, all of which are known to play major roles in tumorigenesis. This signaling paradigm proposes that ligand binding to its receptor on the cell surface induces a conformational change of the receptor to initiate 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 ï¬ndings predict a prerequisite desialylation process by activated Neu1 enabling the removal of steric hindrance to receptor association. The importance of these findings signify an innovative and promising entirely new targeted therapy for cancer. The role of mammalian neuraminidase-1 (Neu1) in complex with matrix metalloproteinase-9 and G-protein coupled receptor tethered to RTKs and TLRs is identified as a major target in the multi-stage of tumorigenesis. Preclinical studies support an entirely new cancer therapy targeting different growth factor receptors, tumor neovascularization, chemo-resistance of tumors, immune-mediated tumorigenesis and the development of tissue invasion and metastasis. Evidence exposing the link connecting growth factor-binding and immune-mediated tumorigenesis to this novel receptor-signaling paradigm will be presented in its current relationship to pancreatic, ovarian and triple-negative breast cancers.
Francis Jeshira Reynoso
University of Pennsylvania, USA
Title: The clinical phenotype of PIGN deficiency and consequences of defective GPI biogenesis
Time : 12:30-12:50
Biography:
Jeshira Reynoso has completed her Pediatrics Residency at Lincoln Medical Center; Bronx NY affiliated to Weill Cornell Medical College. She is currently a Clinical Genetics Fellow at the Children’s Hospital of Philadelphia and is conducting research under the mentorship of Miao He and her PhD is focused on Congenital Disorders of Glycosylation.
Abstract:
The PIGN gene (MIM606097) encodes glycosylphosphatidylinositol (GPI) ethanolamine phosphate transferase-1 which adds the ethanolamine phosphate to the first mannose on the GPI anchor. GPI anchoring of proteins is a highly conserved process present in most eukaryotic cells. GPI-anchored proteins perform a diverse set of functions including roles in signal transduction, cell adhesion and antigen presentation. To date mutations in 12 genes involved in GPI anchor synthesis have been associated with human disease. Maydan first described mutations in this gene in a consanguineous Israeli family of Palestinian origin; children presented with multiple congenital anomalies, severe neurological involvement and early death. Four additional cases have been reported since then. We report three unrelated patients with mutations in PIGN and diverse clinical characteristics. Patients 1 and 2 presented during their newborn period with hypotonia, intractable epilepsy, facial dysmorphism as well as gastrointestinal, genitourinary and skeletal anomalies including brachytelephalangy. Patient 1 had metopic suture craniosynostosis and left choanal atresia. Patient 2 had an imperforate anus, uterine didelphys and a small splenic cyst. Patient 3 with a milder phenotype manifested as hypotonia, developmental delay, dysmorphic facial features and early onset refractory epilepsy. Fluorescence activated cell sorting analysis was performed on cultured skin fibroblast lines from Patients 2 and 3 which demonstrated decreased cell surface expression of the GPI anchored protein CD59, consistent with deficient GPI anchor biogenesis in these cells. Mechanistic studies of PIGN deficiency are currently underway in our laboratory.
Tony Velkov
Monash University, Australia
Title: Molecular characterization of the hemagglutinin receptor-binding specificity of avian influenza viruses: predicting the jump across the species barrier
Time : 13:35-13:55
Biography:
Dr Tony Velkov completed PhD in 2000 from Monash University. His anti-infective discovery research is at the leading edge globally. He was awarded a NHMRC Research Fellowships in 2006, 2011 and 2014. The quality and impact of his independent research was recognized by the NHMRC with an Excellence Award in 2011. He has published over 50 papers in high-caliber journals, 3 book chapters and 15 conference presentations. The dynamic team he leads consists of 3 postdocs, 3 RAs and 9 PhD students. Over the last 6 years, he has obtained >$9M funding from the NIH, NHMRC and foundations.
Abstract:
Influenza is a constant global burden to human health. Seasonal influenza results in significant infections and death, cycling through both hemispheres. On occasion, a novel avian influenza viruses crosses the species barrier from birds to humans resulting in an influenza pandemic. The threat of pandemic avian influenza continues with H5N1 and H7N9 consistently infecting humans. In order to evolve from its avian form and gain the pandemic potential for increased transmissibility between humans, the hemagglutinin (HA) of avian influenza viruses will need to undergo mutations in its receptor binding site (RBS) that bring about an avian to human receptor preference switch. In order to understand the major determinants of virus transmissibility and the pandemic potential of the novel avian influenza viruses we have determined the crystallographic structure of the novel avian influenza H10N7 A/Turkey/MN/3/79 to 1.96Å and mapped the RBS. The amino acid residues responsible for conferring receptor selectivity were identified by site-direct mutagenesis of recombinant H10 HA proteins. The receptor-binding selectivity of the HAs was determined using sialyl glycan binding assays. Docking models were constructed of the H10 HA in complex with α2,6-sialic acid (human) and α2,3-sialic acid (avian) pentasaccharide receptor analogs to ascertain the correlation between the binding assay data and the interactions within the receptor binding pocket. The presented findings provide a structure-recognition perspective for the receptor binding properties of the novel avian H10 influenza HA.
Albert M. Wu
Chang-Gung University, Taiwan
Title: Roles of Polyvalency in the Mechanism of Glyco Recognition. An Important Direction for the Future Glycosciences
Time : 13:55-14:15
Biography:
Dr. Wu obtained his Ph.D degree with W. Pigman, who is the pioneer in glycoproteins, at New York Medical College; and had his postdoctoral training at E.A. Kabat’s Lab for quantitative immunochemistry, Columbia University Medical Center, New York. He joined as a faculty position at Texas A&M University in 1982; promoted as a full professor at Chang-Gung University since 1989; and as Emeritus Professor in 2011. Dr. Wu published over 120 polyvalent glycotopes related papers. He is the chief editor for three volumes of Molecular Immunology of Complex Carbohydrates 1 to 3 in Adv. Exp. Med. Biol. 228, 451, 705 (Springer Publisher). His major interests are (i) Glycan purification and characterization; (ii) recognition factors of glycans; (iii) combining sites of lectins and antibodies. He received many Outstanding Research Awards (1997-2008) from government agents in Taiwan.
Abstract:
Lectins are an important class of proteins or glycoproteins that specifically or selectively bind to carbohydrates and play many critical roles in life processes. In order to characterize recognition roles of lectins, the following aspects have been taken into consideration: i) lectins affinity to monosaccharides; ii) expression of reactivities toward oligosaccharides (mammalian structural units/recognition units) and finding the most active ligand. However, it is not satisfied, because most lectins with the same mono- or oligosaccharide specificity may demonstrate different specificities in reaction with polyvalent forms – it has even shown a shift of binding specificity of lectin from one type of carbohydrate ligand to another when the density of the specific carbohydrate changed. Therefore, characterization of lectin specificity has been extended to: iii) simple oligovalent or cluster effect; and iv) complex multivalent or cluster effects. Simple oligovalent effect concerns the reactivity of lectins with oligomeric glycoconjugates (e.g. branched oligosaccharides carrying several active disaccharides, glycopeptides with several Tα or Tn glycotopes). A complex multivalent effect applies to interaction with high-molecular or aggregated molecules carrying multiple glycotopes recognized by a lectin. In this talk, we are focusing on the resulting intensities of three basic recognition factors ï€ (ii) essential mammalian structural units, (iii) their clusters and (iv) polyvalency in the recognition processes.
Julian M Menter
Morehouse School of Medicine, USA
Title: Effect of temperature and hyaluronate on photochemical changes in calf skin collagen solutions at physiological PH
Time : 14:15-14:35
Biography:
Dr. Menter received his Ph.D. 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 that are unstable to solar UV wavelengths. The consequences of the resulting collagen photo-degradation are not well known, since the direct collagen – UV interactions are poorly described at best. We are studying these interactions by following UV – induced changes in calf skin type I collagen fluorescence as functions of time, temperature, and hyaluronic acid (HA). We have observed that UVC radiation (mainly 254 nm) causes dimerization of tyrosyl residues to dityrosine and the disappearance of an age-related tyrosine oxidation product that is formed in the ground state. Dityrosine formation does not require oxygen and is relatively insensitive to changes in temperature and age of sample. The age-related oxidation product destabilizes the overall collagen supramolecular structure. Added sodium hyaluronate (2:1 ratio) shows little or no effect on these results. This latter result may indicate that there is little physical interaction between the collagen telopeptide and HA domains in vitro.
Xueli Li
University of Pennsylvania, USA
Title: Glycomics profile analysis by MALDI TOF/MS in human CSF
Time : 14:35-14:55
Biography:
Xueli Li has completed her PhD from Leipzig University, Germany. She is a Research Associate in The Michael J Palmieri Metabolic Laboratory, Children’s Hospital of Philadelphia, USA. She has more than 5 publications in the area of Glycomics recently.
Abstract:
Protein glycosylation is important for human brain development and function. The majority of the >100 known subtypes presents with intellectual disability. To explore possible defects in protein glycosylation in the brain, we developed comprehensive glycomics analysis of N-glycome, O-glycome and free glycome by MALDI TOF/MS in cerebrospinal fluid (CSF). 0.4 ml CSF was filtered to separate free oligosaccharides from glycoprotein. N-glycans were released via PNGase F digestion and O-glycans were released separately by reductive beta-elimination reaction. After purified, glycans were permethylated and analyzed by MALDI TOF/MS. Thirty eight different N-glycan species, 85 free oligosaccharide and 25 O-glycan species were identified in 10 control CSF samples. Compared with plasma glycomics data, CSF has much more O-glycan species including O-mannosylated glycans and polysialylated O-glycans. Comparing 166 CSF samples from patients with undiagnosed neurological disease from the NIH Undiagnosed Diseases Program (UDP) with 10 control CSF samples, about 20 UDP patients have CSF glycomics profiles significantly deviated from the profiles of control CSF samples. Four of the UDP patients have known genetic disorders that alter protein glycosylation in human brain, including one patient with putative CAD deficiency, a defect in uridine biosynthesis. Interestingly, one of the known patients has a completely normal plasma glycomics profile and urine oligosaccharide profile, while both his CSF N and O-glycomics profiles are abnormal. Our results suggest that CSF glycomics analysis could be a useful tool to discover new diseases or disease mechanism.
Sourav Sarkar
Lehigh University, USA
Title: MUC1 glycopeptide based anti-cancer vaccines
Time : 14:55-15:15
Biography:
Sourav Sarkar has completed his PhD from University of Toledo (research advisor: Prof. Steven J Sucheck) and postdoctoral studies from Complex Carbohydrate Research Center, University of Georgia (Prof. Geert-Jan Boons). Presently he is working as a Research Scientist III in the Department of Chemistry at Lehigh University. He has published numerous papers in reputed journals and is an inventor of the patent “Xeno-antigenic anti-tumor vaccines”. He is a member of the American Chemical Society and has served as a peer reviewer for Elsevier, Royal Society of Chemistry, Springer etc. journals.
Abstract:
MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesized that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anti-cancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface displayed L-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis we synthesized a 20-amino acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An L-Rha-cholesterol conjugate was prepared using tetraethylene glycol (TEG) as a linker. The liposome-based anti-cancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. The anti-MUC1-Tn antibodies in the vaccinated mice serum also recognized MUC1 on human leukemia U266 cells.
Michael S. Strano
Massachusetts Institute of Technology, USA
Title: New concepts in molecular and energy transport within carbon nanotubes and graphene: optical sensors, resonant ion channels and thermopower waves
Time : 15:30-15:50
Biography:
Professor Michael S. Strano is currently the Carbon P. Dubbs Professor of Chemical Engineering at the Massachusetts Institute of Technology. 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. Michael is the recipient of numerous awards for his work from 2005 to the present.
Abstract:
Our laboratory has been interested in how 1D electronic materials such as carbon nanotubes can be utilized to illustrate new concepts in molecular transport and energy transfer. In the first example, we predict and demonstrate the concept of thermopower waves for energy generation(1). Coupling an exothermic chemical reaction with a thermally conductive CNT creates a self-propagating reactive wave driven along its length. We realize such waves in MWNT and show that they produce concomitant electrical pulses of high specific power >7 kW/kg. Such waves of high power density may find uses as unique energy sources. In the second system, we fabricate and study SWNT ion channels for the first time(2) and show that the longest, highest aspect ratio, and smallest diameter synthetic nanopore examined to date, a 500 μm SWNT, demonstrates oscillations in electro-osmotic current at specific ranges of electric field, that are the signatures of coherence resonance, yielding self-generated rhythmic and frequency locked transport. The observed oscillations in the current occur due to a coupling between stochastic pore blocking and a diffusion limitation that develops at the pore mouth during proton transport. Lastly, our laboratory has been interested in how semiconducting single walled carbon nanotubes (SWNT) can be modified such that their fluorescent emission is modulated in response to specific molecules, hence creating a new class of sensor (3). Such sensors have important advantages, including enhanced optical penetration of tissues in the near infrared, reduced auto-fluorescence, infinite resistance to photobleaching and most recently, single molecule analyte sensitivity. This presentation will review our recent efforts in this space including new platforms for label free protein detection, nitric oxide, H2O2 and the interfacing of sensor arrays to living cells.
Ajiloseda Charles Adeniyi
Adyghe State University, Russia
Title: Cytokine gene polymorphism (IL-17A G197A, TNF-alpha G308A, IL-6 C174G) and human major histocompatibility complex (HLA DRB1) specificities control in bronchial asthma
Time : 15:50-16:10
Biography:
Ajiloseda Charles Adeniyi has completed a Specialist (MSc) and a Professional Education degree at the Adyghe State University, Russia and as a result of his ardent observation and understanding in the hybridoma culture, antibody purification and microscopic usage as well as his keen scientific interest was immediately inducted into the research group at the University’s immune-laboratory working with the lead Professor Tugus. He collaborated with others scientists to determine the importance of medicinal plant on health: A case study of Iris plantae. His personal publication includes but not limited to his recent work on the “Development and integrating of health sciences into foreign languagesâ€.
Abstract:
In the development of non-specific resistance in an immune system, important role belongs to mediators of the said immune system; the pro-inflammatory cytokines which in turn activates expression of class II HLA-DRB1 on antigen presenting cells (APC) that implement specific immunity. The pathologies of many infectious, autoimmune and malignant diseases are influenced by the profiles of cytokine production in pro-inflammatory (TH1) and anti-inflammatory (TH2) T cells. With TLR (Toll-like receptor) on dendritic cells recognizing PAMP (pathogen-associated molecular patterns); antigens that trigger a cascade of specific antibody and cytokine. Many studies have examined the relationship between cytokine gene polymorphism, cytokine gene expression in vitro and the susceptibility to and clinical severity of diseases. Our study here tends to and as extensively as possible expose the relationship of cytokines gene polymorphism and Human Major Histocompatibility complex with respect to bronchial asthma.
Yves St-Pierre
INRS-Institut Armand-Frappier, CANADA
Title: Targeting galectins as a new tumor microenvironment-based therapeutic strategy in cancer
Time : 16:10-16:30
Biography:
Dr. St-Pierre has received his Ph.D. in immunology at the University of Toronto and pursued his postdoctoral training Harvard Medical School, working on the molecular mechanisms controlling trafficking of immune and cancer cells. He was recruited as at the INRS-Institut-Armand-Frappier in 1994 where he has been ever since. He is currently pursuing research aimed at identifying novel molecular targets and markers of cancer using various immunological and molecular approaches.
Abstract:
Carbohydrates are traditionally considered to be an important source of energy for living organisms. In the field of biology, they are defined as organic compounds composed of carbon, hydrogen, and oxygen that are organized into ring structures. The analysis of these structures and their functions has led to a new field of biology called “glycobiology.†In the biomedical sciences, glycobiology is rapidly emerging as an integral part of complex biological processes. Evidence suggesting that the interactions between lectins and their ligands play a major role in the different steps of cancer progression has accumulated at a rapid pace and has gained the attention of several oncologists. This is particularly true for galectin family members because changes in their expression levels correlate with alterations in cancer cell growth, apoptosis, and, due to their cytokine-like properties, they are major activators or inhibitors of immune responses. In mammals, 19 galectins have been identified, of which 13 are expressed in human tissues. While they perform homeostatic functions inside normal cells, under pathological or stress conditions, cytosolic galectins are released either passively from dead cells or actively via non-classical secretion pathways. Once in the extracellular milieu, they bind all glycosylated growth receptors on the surface of normal and cancer cells to set their signaling threshold. Such properties enable galectins to kill of infiltrating immune cells while promoting growth of tumor cells. We hypothesize that galectins play an important role in the tumor microenvironment which undergo profound alterations during cancer. Such alterations remains one of the great obstacle in the successful treatment of cancer is therapeutic resistance and cancer recurrence. This is especially true for cancer with poor outcome, such as triple negative breast cancer (TNBC) or ovarian cancer, for which no therapeutic targets exist. Here, we will discuss the relevance of the galectinome in TNBCs and ovarian cancer.
Peng G. Wang
Georgia State University, USA
Title: Efficient chemoenzymatic synthesis of an n-glycan isomer library
Time : 16:30-16:50
Biography:
Peng George Wang obtained his B.S. Degree in Chemistry from Nankai University, China in 1984 and his PhD. Degree in organic chemistry from University of California, Berkeley in 1990. He then conducted postdoctoral research in Scripps Research Institute and became an Assistant Professor in 1994 in University of Miami. From 1997 to 2003, he was a faculty at Wayne State University. In 2003, he took a position in the Departments of Biochemistry and Chemistry at Ohio State University as Ohio Eminent Scholar in Macromolecular Structure and Function. In 2011 He became Professor and Georgia Research Eminent Scholar in Chemical Glycobiology in the Department of Chemistry at Georgia State University.
Abstract:
Quantification, characterization and biofunctional studies of N-glycans on proteins remain challenging tasks due to complexity, diversity and low abundance of these glycans. The availability of structurally defined N-glycans (especially isomers) libraries is essential to help on solving these tasks. We reported herein an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycan libraries. Starting with 5 chemically prepared building blocks, 8 N-glycan core structures containing one or two terminal N-acetyl-D-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in the convergent fragment coupling strategy. Each of these core structures was then extended to 5 to 15 N-glycan sequences by enzymatic reactions catalyzed by 4 robust glycosyltransferases. Success in synthesizing N-glycans with Neu5Gc and core-fucosylation further expanded the ability of enzymatic extension. High performance liquid chromatography (HPLC) with an amide column enabled rapid and efficient purification (>98% purity) of N-glycans in milligram scales. A total of 73 N-glycans (63 isomers) were successfully prepared and characterized by MS2 and NMR. The CSEE strategy provides a practical approach for “mass production†of structurally defined N-glycans, which are important standards and probes for glycoscience.
Shuxia Wang
University of Kentucky, USA
Title: Thrombospondin1 an important mediator of obesity associated inflammation and insulin resistance
Biography:
Dr. Wang has completed her PhD from Institute of Basic Medical Sciences at Chinese Academy of Medical Sciences and postdoctoral studies from University of Alabama at Birmingham. She is an Associate Professor in the Dept. of Pharmacology and Nutritional Sciences at University of Kentucky and a scientist at VA Medical Center in Lexington. She has published more than 35 papers in reputed journals and has been serving as an editorial board member for several journals.
Abstract:
Thrombospondin 1 (TSP1) is a multifunctional matricellular protein. It is highly expressed in visceral fat tissue (AT) from obese and insulin resistant humans or obese rodents. Recently, both human and rodent data from our lab and others suggest that TSP1 plays an important role in obesity-associated chronic inflammation and insulin resistance (IR). The positive association of adipose tissue TSP1 with AT inflammation and IR has been observed in obese human subjects. By using global TSP1 deficient mice, we revealed a novel role for TSP1 in stimulating macrophage accumulation and activation in AT that promotes inflammation and IR resulting from high fat diet-induced obesity (DIO). Specifically, we found that feeding a high fat diet to wild type and TSP1 deficient mice for 16 weeks caused similar obesity, but only mice with TSP1 deficiency remained insulin-sensitive. The protection of TSP1 deficient mice against IR was associated with reduced ATMs, decreased adipose and systemic inflammation, and reduced AT fibrosis. Moreover, TSP1 deficiency protected mice from obesity-induced hypertension and kidney damage. In vitro data demonstrated that TSP1 deficient monocyte/macrophages had decreased chemotactic activity and a reduced pro-inflammatory phenotype. TSP1 treatment stimulated macrophage migration. In addition, TSP1 stimulated macrophages to produce pro-inflammatory cytokines, which required TLR4 activation and was mediated by interaction between the type 1 repeats of TSP1 (TSR) and its receptor-CD36. Collectively, these data suggest that TSP1 acts as both a chemoattractant and proinflammatory activator for macrophages in inflamed AT, and promotes obesity-induced inflammation and IR.
Adam Kapelner
Queens College, USA
Title: Starvation of cancer via induced ketogenesis and severe hypoglycemia
Biography:
Adam Kapelner is an Assistant Professor of Mathematics at Queens College in New York City. He graduated with a Ph.D. in Statistics from Wharton Business School in 2014. He was previously a software engineer building web applications in San Francisco and a mathematics & computational science undergraduate at Stanford University. He has published in a number of fields: Statistical Methodology, Statistical Software, Economics, Natural Language Processing but has a special love for applications to medicine.
Abstract:
Neoplasms are highly dependent on glucose as their substrate for energy production and are generally not able to catabolize other fuel sources such as ketones and fatty acids. Thus, removing access to glucose has the potential to starve cancer cells and induce apoptosis. Unfortunately, other body tissues are also dependent on glucose for energy under normal conditions. However, in human starvation (or in the setting of diet-induced ketogenesis), the body ‘‘keto-adapts’’ and glucose requirements of most tissues drop to almost nil. Exceptions include the central nervous system (CNS) and various other tissues which have a small but obligatory requirement of glucose. Our hypothesized treatment takes keto-adaptation as a prerequisite. We then propose the induction of severe hypoglycemia by depressing gluconeogenesis while administering glucose to the brain. Although severe hypoglycemia normally produces adverse effects such as seizure and coma, it is relatively safe following keto-adaptation. We hypothesize that our therapeutic hypoglycemia treatment has potential to rapidly induce tumor cell necrosis. This is joint work
Diwakar Jain
New York Medical College, USA
Title: Radiolabeled glucose analogues for myocardial ischemia imaging and for diagnosing coronary artery disease
Biography:
Dr Jain is a Professor of Medicine (Cardiovascular Medicine) and director of nuclear cardiology laboratory at New York Medical College. He attended Medical College, Rohtak (India) and underwent residency and fellowship training in Medicine and Cardiology at Post Graduate Institute of Medical Education and Research, Chandigarh (India). He underwent further training in cardiology at Northwick Park Hospital and Clinical Research Center, Harrow (UK) and Yale University School of Medicine. He has conducted research in the fields of molecular imaging, cardiotoxicity of cancer chemotherapy, and behavior, mental stress and heart disease. He has published over 150 scientific papers, book-chapters, editorials and review articles.
Abstract:
Glucose and free fatty acids (FFA) are the predominant metabolic substrates in heart. Their relative uptake in normal hearts varies with the metabolic milieu (fed/fasting state), plasma levels and insulin levels. Myocardial ischemia results in a profound upregulation of glucose and downregulation of FFA metabolism. Fluorine-18 labeled deoxyglucose (FDG), a glucose analogue is used extensively in clinical practice for imaging glucose metabolism in heart, tumors and other organs. Alternations in glucose metabolism accompanying several disease conditions can be used to diagnose these conditions. Exercise results in ischemia and upregulates glucose uptake in regions perfused by diseased coronary arteries. This can be used for developing a non-invasive diagnostic imaging test for coronary artery disease (CAD). We evaluated the diagnostic potential of exercise FDG imaging for CAD and compared it with exercise-rest myocardial perfusion imaging (MPI), an established and routinely used test. FDG imaging had higher diagnostic sensitivity compared to MPI. Furthermore, increased regional FDG uptake is observed only on exercise images. Persistence of FDG uptake 24 hours later is seen in less that one third of cases and is indicative of more severe CAD. Cardiac imaging using radiolabeled sugars is a highly promising new diagnostic test for CAD and may also provide a powerful tool for studying the pathophysiology of myocardial ischemia.
Yi-Pin Lin
Tufts University School of Medicine, USA
Title: Glycosaminoglycan binding adhesins of Lyme disease spirochetes contributes to tissue colonization and disease manifestations
Biography:
Yi-Pin Lin has completed his Ph.D. from Cornell University currently is a research assistant professor in the laboratory of Dr. John Leong at Tufts University School of Medicine. His research is emphasis on virulence mechanisms of bacterial pathogens that cause infectious diseases in humans and animals and host immunity to bacterial infection. His specific expertise is in pathogenesis of the Lyme Disease agent Borrelia burgdorferi, particularly in the bacterial-cell adhesion and immune evasion mechanisms that promote colonization and disease.
Abstract:
Borrelia burgdorferi is a bacterial spirochete that causes Lyme disease, the most common vector-borne disease in U.S. B. burgdorferi can infect the skin at the site of the tick bite, and subsequently colonize the heart, joints, or brain. The establishment of B. burgdorferi infection at diverse sites may be partly dictated by the attachment of spirochetes to target tissues. Indeed, B. burgdorferi encodes several adhesins that recognize components of the extracellular matrix such as proteoglycans and is often localized in connective tissue during infection. Proteoglycans consist of a core protein and one or more covalently linked long repeating disaccharides glycosaminoglycan (GAG). Upon adaptation to the mammalian host, B. burgdorferi produces surface-localized GAG-binding proteins as known as adhesins. The GAG-binding activity of these adhesins was speculated to mediate the tissues colonization and disease. Recently, spirochetes deficient in specific GAG-binding adhesins, such as DbpA or BBK32, have been shown to display reduced infectivity in a murine model, indicating a critical role for GAG-binding during mammalian infection. We further demonstrated that the GAG-binding activity of DbpA promotes colonization and disease manifestation at all of the tissues while this activity of BBK32 specifically promotes joint colonization. Therefore, GAG-binding activities may play roles in tissue colonization and the clinical manifestations of Lyme disease.
Biography:
Dr. Rosa Peracaula completed her PhD at the University Ramon Llull, Barcelona in 1995. Since 1995 she joined to the group of Biochemistry at the University of Girona and performed postdoctoral stays at MS Anderson Cancer Center in Houston (1997) and at the University of Oxford (2001 and 2002). Since 2003, she is associate Professor of Biochemistry and Molecular Biology at the University of Girona and leads the research group of Glycobiology and Cancer with projects related to find new biomarkers associated to changes of glycosylation in serum proteins and aimed to understand the role of the altered glycosylation in the tumorigenic process. She has three patents and has published over 25 scientific articles in international journals, some of them with more than 150 citations. She has directed five Doctoral Theses and has also given several presentations at national and international conferences. The research performed over these years has a strong translational component, with important collaborations with medical doctors and several national and international groups.
Abstract:
Altered glycosylation is one of the hallmarks of tumor cells and it is involved in each and every aspect of tumor progression. It affects cell surface carbohydrates and cellular and secreted glycoproteins, some of which may reach the bloodstream and be used as tumor markers 1. Our group has focused on the altered glycosylation of serum proteins in prostate and pancreatic cancer as potential tumor markers. We have described glycosylation changes of Prostate Specific Antigen (PSA) glycans, related to sialylation and fucosylation, in prostate cancer compared to Benign Prostate Hyperplasia and seminal plasma from healthy controls 2-5. In pancreatic cancer, we have described glycosylation changes on human pancreatic ribonuclease (RNase 1) and acute-phase proteins. An increase in core fucosylated structures in the N-glycan chains of RNase 1 6 and an increase of sialyl-Lewis x (SLex) and fucosylation of the acute-phase proteins ceruloplasmin and alpha-1-acid glycoprotein, respectively, were described and were found in advanced pancreatic cancer patients 7-9. These tumor associated glycan changes are currently being investigated in larger cohort of patients as cancer diagnostic or prognostic tools. The expression of the glycosyltransferases responsible for the synthesis of the tumor associated carbohydrate antigens such as SLex has been found deregulated in cancer. In particular, our group has focused on the study of sialyltransferases, which have received much attention recently as they are frequently up-regulated in cancer cells. We have described using in vitro and in vivo models the involvement of the α2,3-sialyltransferases ST3Gal III, and ST3Gal IV in key steps of pancreatic tumor progression processes, and have found that they are highly expressed in most pancreatic adenocarcinoma tissues10-12.
Achuth Padmanabhan
Yale University, USA
Title: Tracking cancer progression through monitoring the dynamic serum phospho-proteome
Biography:
Achuth Padmanabhan completed his PhD from University of Maryland Baltimore County and is currently pursuing his postdoctoral studies at Yale University, School of Medicine. His research has helped identify novel signaling modules in prostate cancer that regulates the levels of the key oncogene MYC and the prostate tumor suppressor NKX3.1. These discoveries have opened up new avenues that can be potentially used to target these proteins in prostate cancer. He has published several papers in reputed journals and has been serving as an editorial board member of repute.
Abstract:
Serum proteome has been shown to reflect pathological changes in tissues. Changes in serum proteomic pattern during carcinogenesis have been exploited successfully to serve as diagnostic and prognostic markers. Protein Kinase A catalytic subunit α has been shown to be secreted into the serum as ecto-kinase and the ecto-PKA level is significantly higher in the serum of prostate cancer patients. Here, I describe the utility of the Reverse In-gel Kinase Assay coupled with LC-MS/MS to profile potential serum substrates of PKA and in following their phosphorylation status during disease progression (1-4). The sensitivity and linearity of this assay will help identify potential biomarkers, which can be used to track the progression of prostate cancer. This strategy can be adapted and extended to track the progression of other cancers and pathological conditions.
Robin Polt
University of Arizona, USA
Title: Glycolipids and Glycopeptides-minimally competent lewis acid catalysis produce surfactants for use ex vivo and in vivo
Biography:
Robin Polt developed methods for the synthesis of amino acids in Prof. Martin O’Donnell’s lab at IUPUI. He was then trained in the art of Organic Synthesis in the laboratories of Professors Gilbert J. Stork at Columbia University in New York and Dieter Seebach at the ETH in Zürich. He has continued the development of useful synthetic methods for amino acids, amino alcohols, glycosides and glycopeptides. Polt has mentored undergraduates, Ph.D. students from the USA, Czech Republic, China, India, Iran, Ireland, Kenya, Korea, Mexico and Sri Lanka, as well as post-doctoral students who have taken positions in academia, industry and government.
Abstract:
Use of minimally competent main group Lewis acids such as InBr3 and Bi(OSO2CF3)3 permit the formation of glycosides in high yield and purity from simple sugar per-acetates at room temperature or above with remarkable α/β-selectivity. Classical reactions rely on very reactive glycosyl donors in conjunction with metal promoters (Hg++, Ag+, etc). Newer methods have used trichloroacetimidates and strong Brønsted or Lewis acids, or thioglycosides in conjunction with oxidative or thiophilic activators. Use of the more stable glycosylperacetates in conjunction with stoichiometric amounts of strong Lewis acids (BF3•Et2O, FeBr3, etc) has been explored with limited success. The use of “minimally competent†Lewis acids has allowed us to perform glycosidation reactions with catalytic amounts of InBr3 or Bi(OTfl)3 well above room temperature and without significant decomposition of the glycoside products. We will discuss the exploitation of these methods to produce glycolipid surfactants from renewable resources, as well as glycopeptide drugs that penetrate the blood-brain barrier (BBB). These “biousian glycopeptides†are not subject to “Lipinski’s rules†that would otherwise eliminate them as CNS drug candidates. Glycosylated amphipathic helices, or “address segments†have been used to target G-protein coupled receptors (GPCRs) in the brain after intravenous administration. The addition of glycosides to endogenous peptide neurotransmitters and peptide hormones imparts favorable pharmacokinetic and pharmacodynamics (PK/PD) properties, and enables penetration of the BBB, which is not constrained by molecular weight.
- Track 4 : Glycobiology: Its Role In Human Health
Track 5 : Discovery and Classification of Glycan Binding Proteins
Track 6 : Glycan Catabolism and Systemic Physiology
Track 7 : Evolution of Glycan Diversity
Location: Independence C
Chair
Neil P.J. Price
National Center for Agricultural Utilization Research, USA
Co-Chair
Myron R Szewczuk
Queen’s University, Canada
Session Introduction
Neil P. J. Price
National Center for Agricultural Utilization Research, U.S.A
Title: Liamocins, sophorolipids and frost grape polysaccharides. New carbohydrate research from the USDA’s NCAUR national laboratory
Time : 11-10-11:30
Biography:
Neil Price has a Ph.D. from the University of London, and has postdoctoral experience at the Univ. of Geneva in Switzerland, Paul-Sabatier University in Toulouse, and the Complex Carbohydrate Research Center in Athens, GA. He held a faculty position the U. of Rochester, NY before moving to the NCAUR, Peoria. His research includes carbohydrate chemistry and biochemistry, mass spectrometry, and microbial metabolism. He currently serves on the ARS National Chemical Patent committee, and has over 90 peer-reviewed research publications.
Abstract:
The USDA’s National Center for Agricultural Utilization Research has a prominent history in carbohydrate research, including the development of xanthan gum, ‘super slurper’ polysaccharides, beta-dextrans, alternan, and beta lactamase antibiotics (penicillins), as well as analytical tools such as aldononitrile acetates. This presentation will focus on three more recent innovations: sophorolipid bio-surfactants; liamocin anti-microbial agents; and frost grape polysaccharide. Sophorolipids (SLs) can be produced by fermentation, often in high yield (50 – 400 g/L), by yeasts of the Starmarella clade. The SLs are composed of a sophorose sugar head group (Glc-beta-1,2-Glc) glycosidically attached to a hydroxyl fatty acid. In a MALDI-TOF MS-based screen of Starmarella yeast we found that Candida kuoi NRRL Y-27208 produces non-lactone type SLs in which the sophorose is O-linked to ω-hydroxy fatty acids. Unlike the more common lactone-type SLs, these novel SL’s have an open chain structure that confers low-foaming bio-surfactant properties. The liamocins are novel polyol-lipids from a black yeast-like fungus, Aureobasidium pullulans. Four liamocins have been characterized from Aureobasidium NRRL 50380 that contain a mannitol head-group linked to several polyester-linked 3,5-dihydroxydecanoate acyl chains. Two of the liamocins are also 3’-O-acetylated, and all four have pronounced antimicrobial properties. A new, high molecular weight polysaccharide has also been found to be produced by the frost grape (Vitis riparia), and the structural characterization and properties of this will also be presented and discussed.
Pi-Wan Cheng
University of Nebraska Medical Center, USA
Title: Disregulation of golgi localization of glycosyltransferases alters mucin o-glycosylation and survival or metastatic properties of cancer cells
Time : 11.30-11.50
Biography:
Pi-Wan Cheng received his PhD degree in Biochemistry from Case Western Reserve University in 1975 and is currently a Professor of Biochemistry and Molecular Biology at the University of Nebraska Medical Center. He has published more than 95 papers mostly in Glycobiology field and has served in grant review panels of many funding agencies, including NIH, states and nonprofit organizations. He is a member of the editorial boards of the Journal of Glycobiology and the American Journal of Respiratory Cell and Molecular Biology.
Abstract:
Glycosylation is a posttranslational, template-independent process. Mucin O-glycosylation is catalyzed by glycosyltransferases (GTs) localized at various Golgi stacks according to the glycosylation steps they participate in. We have identified three different Golgi targeting sites for GTs. Giantin is the exclusive site for core 2 N-acetylglucosaminyltransferases (C2GnTs) and the primary site for Galβ3GalNAc:α2-3sialyltransferase 1 (ST3Gal1), GM130-GRASP65 is the primary site for core 1 synthase (C1GalT1) and the secondary site for ST3Gal1, and GM130-giantin is the secondary site for C1GalT1. Defective giantin in aggressive prostate cancer cells prevents C2GnTs but not other GTs from targeting the Golgi. As a result, core 2-associated glycans, such as polylactosamine, cannot be formed but sialyl-T level is elevated. Inhibition or knockdown of non-muscle myosin IIA restores giantin structure, normal Golgi targeting of GTs and core 2-associated polylactosamine, which renders these cancer cells susceptible to galectin 1-induced apoptosis. The result demonstrates that aggressive prostate cancer cells acquire survival advantage by altering Golgi targeting of GTs. This process does not require any change in the expression of GT genes. Also, we have identified proteins that help retain C2GnTs in the Golgi; Golgi phosphoprotein 3 (GOLPH3) for C2GnT-L and keratin 1 for C2GnT-M. Loss of keratin 1 prevents Golgi localization of C2GnT-M and increases sialyl-T level. Loss of GOLPH3 prevents Golgi retention of C2GnT-L, causes loss of selectin ligand sialyl Lewis x and decreases selectin-mediated metastatic properties. In conclusion, disregulation of Golgi targeting or retention of GTs can alter mucin O-glycosylation and survival or metastatic properties of cancer cells.
Ying Qing Yu
Waters Corporation, USA
Title: N-linked glycan characterization and profiling: combining the power of a novel labeling reagent and a streamlined analytical platform for confident glycan assignments
Time : 11:50-12:10
Biography:
Ying Qing is a Principal Scientist in the Pharmaceutical group at Waters Corporation. She joined Waters Corporation since 2001, shortly after she received her Ph.D. from the Analytical Chemistry Department at Purdue University. She is a group leader in the Biopharmaceutical lab unit at Waters. Her group’s focus is on Protein Biotherapeutics characterization using UPLC/QTOF MS platform. The projects she is currently working on range from glycan profiling, peptide mapping and lately the Hydrogen Deuterium Exchange Mass Spectrometry for protein higher order structure analysis. She has extensive experience in Mass Spectrometry, Gas-Phase Ion Chemistry and Liquid Chromatography Separation Techniques.
Abstract:
UPLC/FLR/MS(MS) analysis of released N-glycans labeled with a fluorescent tag has made routine with high performance LC and MS instrumentations. Glycans labeled with conventional fluorescent tags, such as 2-AB and 2-AA, can be detected by Fluorescent (FLR) detector with ultra-high sensitivity. Unlike FLR detector, mass spectrometry is known to be less sensitive to detect native or tagged glycans, especially the low abundant ones, due to their poor ESI performance. The limited dynamic range of this approach has been restricting the utilization of this combined automated workflow for glycan characterization. In order to overcome the low MS ionization efficiency associated with the conventional labels and confidently assign lower level glycans, a novel tag, RapiFluor-MSTM (RFMS) developed by Waters Corporation, has been employed. RFMS contains a rapid tagging reactive group, an efficient fluorophore, and a functional group that imparts high ionization efficiency. Complete tagging of glycans can be achieved in less than 5 minutes using this novel reagent. Initial results with the new glycan label show significant enhancement in both the FLR and MS(MS) signals compared to 2-AB. The increased sensitivity enables the detection and identification of very low level glycans at (0.1%) with sufficient MS signals. In this study, we demonstrate the benefits of combining the novel labeling reagent RFMS with an integrated UPLC/FLR/Xevo G2-XS QTOF MS system for detailed characterization of the minor glycoforms from therapeutical proteins.
Thomas J. Boltje
Radboud University, The Netherlands
Title: New tools to study and perturb the glycocalyx
Time : 12:10-12:30
Biography:
Thomas J. Boltje obtained his PhD degree in 2011 from the University of Georgia under the supervision of Prof. Dr. G.J. Boons. After a short postdoctoral period at the Radboud University Nijmegen (The Netherlands), he started his current position as a tenure track assistant professor of chemical biology in 2013 at the same university.
Abstract:
Our research is focused on identifying the specific structure and function of sugar molecules present on the surface of cells. To achieve this we develop tools to block the biosynthesis of these molecules and metabolic precursors to follow and perturb the function of sugars. In addition we assemble oligosaccharides by chemical synthesis to construct a screening platform that can also be used to do SAR studies. The preparation of oligosaccharides is challenging and hence we develop new chemical methodologies to make the process of glycosylation faster and more stereoselective. The most important application of these technologies is to enable the design and synthesis of glycomedicine that target aberrant glycosylation patterns on cancer cells and pathogenic bacteria either by small molecule glycomimetics or oligosaccharide glycovaccines.
Fiona Haxho
Queen’s University, Canada
Title: Role of G protein-coupled receptors in insulin receptor glycosylation and activation
Time : 12:30-12:50
Biography:
F. Haxho is a graduate trainee in Dr. Myron Szewczuk’s laboratory, Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada. She holds the Queen’s Graduate Award (QGA), Graduate Entrance Tuition Award (GETA) and the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS M).
Abstract:
The insulin receptor (IR) is a transmembrane tyrosine kinase receptor (RTK) that is activated by insulin and insulin growth factors-I and II. Metabolically, insulin-induced IR activation plays a key role in the regulation of glucose homeostasis. A dysfunctional process of insulin-induced IR activation may result in a range of clinical manifestations including insulin resistance, type 2 diabetes mellitus, obesity, cancer, hypertension, and cardiovascular disorders. Although signal transduction pathways for many tyrosine kinase receptors, including IR, are generally well characterized, parameters controlling the activation of these receptors have remained poorly defined. Our recent reports describe a novel G-protein coupled receptor (GPCR)-signaling platform to potentiate mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) cross-talk in regulating RTKs, and specifically IR. Central to this regulatory signaling axis is that Neu1, MMP9 and neuromedin-B GPCR form a complex with the IRβ-subunit on the cell surface. This signaling paradigm proposes that insulin binding to its receptor on the cell surface induces a conformational change of the receptor to initiate GPCR Giα-signaling and MMP9 activation to induce Neu1. Activated Neu1 hydrolyzes α-2,3-sialyl residues linked to β-galactosides, which are distant from the insulin binding sites. These findings support a prerequisite desialylation process by activated Neu1 enabling the removal of steric hindrance to IRβ subunit association and the activation of tyrosine kinases. These reports uncover an unprecedented mode of control for insulin-induced IR activation and present an innovative platform for targeting hormone signaling by the modification of IR glycosylation.
Agata Steenackers
Lille 1 University, France
Title: Expression of OGT correlates with migration and proliferation of colon cells lines
Time : 13:35-13:55
Biography:
Agata Steenackers defended her PhD thesis in the field of Biology and Biotechnologies in November 2013 at the Lille 1 University (France). During her PhD, she developed a project around the expression of GD3 synthase and gangliosides in breast cancer cells lines. She is now on a post-doctoral position in Tony Lefebvre's team (UGSF) where she is studying the role of O-GlcNAcylation in colon cancer development.
Abstract:
The O-GlcNAc transferase (OGT) is a key regulator of the post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) onto Ser/Thr residues. OGT uses the end product of the hexosamine biosynthetic pathway (HBP), UDP-GlcNAc, as a donor for O-GlcNAcylation processes. It is reported that OGT and O-GlcNAcylation levels are increased in cancers. We showed that in the colorectal cancers (CRC) cell lines (HT29, HCT116) the expression of OGT and O-GlcNAcylation level were elevated, and that O-GlcNAcylation directly interfered with β-catenin stability and proliferation of cells. Previous studies showed that oncogenic factors such as p53, MYC or β-catenin are O-GlcNAcylated. The Wnt/β-catenin pathway is modified in most CRC by genetic alteration of β-catenin or one member of the destruction complex. Consequently, β-catenin is protected from proteasomal degradation and therefore induces cell proliferation. A similar observation was made when HBP flux was increased by culturing cells in high glucose medium. In these conditions, ï¢-catenin was protected against the degradation thus accelerating cell proliferation. In a recent study, we identified four O-GlcNAcylation sites at the N-terminus of β-catenin, one of those (T41) localized in the destruction box is crucial for the control of β-catenin degradation. In that context we studied the effect of OGT silencing in CRC cell lines and non-cancer cell line CCD841CoN. We reported that silencing of OGT halved proliferative and migratory capacities of cancer cells. OGT knock-down also diminished cell adhesion corroborating previous observations that inhibiting O-GlcNAcylation decreases β-catenin/α-catenin interactions necessary for mucosa integrity, which suggests that O-GlcNAcylation also affects localization of ï¢-catenin at adherens junction level.
Steffi Baldini
Lille 1 University, France
Title: Regulation of hepatic fatty acid synthase properties by O-glcnacylation in vivo and ex vivo
Time : 13:55-14:15
Biography:
Steffi Baldini is PhD student in the field of Biology and Biotechnologies since September 2013 at the Lille 1 University (France). During her PhD, she develops a project around the regulation of hepatic Fatty Acid Synthase expression by O-GlcNAcylation.
Abstract:
During meal intake, two metabolic pathways are activated in the liver, the glycolysis and the lipogenesis, to drive the production of fatty acids. The Hexosamine Biosynthesis Pathway (HBP), which end product is UDP-GlcNAc the substrate of OGT (O-GlcNAc Transferase) to O-GlcNAcylate proteins, is also activated. O-GlcNAcylation is a dynamic post translational modification (PTM) that controlled a plethora of protein properties. Disturbance in the O-GlcNAcylation dynamism is implicated in several pathologies. Numerous studies link metabolic disorders emergence to O-GlcNAcylation mechanisms deregulation. Knowing that there is a close relationship between glucose, O-GlcNAcylation levels and activation of the glucido-lipid metabolism, a link between the activation of the glycolytic and the lipogenic enzymes and O-GlcNAcylation should exist. More precisely we focused on Fatty Acid Synthase, FAS which produces fatty acids. In this study, O-GlcNAcylation levels and FAS expression were analyzed in liver of C57BL6 mice fed a Chow Diet (CD) or High Carbohydrate Diet (HCD), in liver of mice harboring an inhibition of OGA and in primary hepatocytes of mice cultured in different O-GlcNAcylation levels. Co-immunoprecipitation experiments showed that OGT and FAS interacted physically but this interaction did not lead to FAS O-GlcNAcylation. However, a correlation between FAS expression and O-GlcNAcylation level was shown and an increase of O-GlcNAcylation levels paralleled the protection of FAS against this degradation. Moreover FAS activity was increased in fasted HCD mice compared to fasted CD mice. Taken together, our results suggest that O-GlcNAcylation may represent indirectly a new regulation of FAS protein content and activity in liver under both physiological and physiopathological conditions.
Ivan U. Kouzel
University of Münster, Germany
Title: Glycosphingolipid-mediated interaction of shiga toxin with the human endothelium: status quo of receptor research
Time : 14:15-14:35
Biography:
Dr. Ivan U. Kouzel has received his PhD in 2014 at the age of 28 years from the University of Münster. He is currently postdoctoral researcher in the group of Prof. Dr. Johannes Müthing, Institute for Hygiene, Münster, Germany, headed by Prof. Dr. Dr. h.c. Helge Karch. Ivan is involved in 5 publications in reputed journals, where he holds the first authorship in 2 publications.
Abstract:
Certain pathogenic Escherichia coli strains belong to the group of Shiga toxin (Stx)-producing E. coli (STEC), whereof the subgroup of enterohemorrhagic E. coli (EHEC) may cause epidemics like the 2011 European E. coli O104:H4 outbreak [1]. EHEC are implicated in a wide range of clinical complications in humans such as the potentially lethal hemolytic uremic syndrome (HUS). Once released from the gut into the blood circulation, Stxs are transported via highly debated mechanisms to the target endothelium with putative involvement of leukocytes and/or lipoproteins as delivery vehicles [2, 3]. Microvascular endothelial cells of human kidney and brain are the preferential targets [4, 5], injury of which is key in the development of HUS and damage of the blood-brain barrier [6]. Stxs specifically bind to the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), followed by internalization into the cell and eventual cell death. Lipid raft association of GSL receptors in the plasma membrane [5] is supposed to play a crucial role in binding, uptake, retrograde transport and cytotoxicity of Stxs. In this presentation the current knowledge on the molecular mechanisms of GSL-mediated interaction of Stxs with the human endothelium will be presented and discussed.
Cheorl-Ho Kim
SungKyunKwan University, Korea
Title: Cellular roles of ganglioside GM3 biosynthesis in human cancer cells
Time : 14:35-14:55
Biography:
Cheorl-Ho Kim has completed his Ph.D at the age of 28 years from The University of Tokyo and was positioned as a senior scientist from Korea Research Institute of Bioscience and Biotechnology. He is a professor of Molecular Glycobiology, SungKyunKwan University, Korea, leading organization of Korea, which is cooperated with the SamSung Group. He has published more than 337 papers in reputed journals and serving as an editorial board member, executive editor and editor-in chief of the international journals. His work was contributed to the mechanisms of glycan-mediated Hepatis B viral oncogenesis and invasion, sialoglycan-mediated leukemic differentiation and vascular biology. He is being serves as an Editor-in-Chief of Journal of Glycobiology, Editor-in-Chief of Journal of Microbial and Biochemical Technology, Executive Editor of Journal of Glycomics and Lipidomics and Editor of eCAM.
Abstract:
Ganglioside GM3, sialic acid (NeuAc)-containing glycosphingolipid, is the first and the simplest of the gangliosides and are found on the outer leaflet of the plasma membrane in vertebrates. It plays important roles in a large variety of biological processes, such as cellular interactions, differentiation, oncogenesis, adhesion, cell growth, and receptor function in various cell systems. Ganglioside GM3 is synthesized by lactosylceramide α-2,3-sialyltransferase (hST3Gal V, EC 2.4.99.9) which catalyzes the transfer of NeuAc from CMP-NeuAc to the non-reducing terminal galactose of lactosylceramide in human. The amount of ganglioside GM3 increases with a concomitant increase of hST3Gal V activity during megakaryocytic differentiation of K562 cells treated with PMA that is a megakaryocytic differentiation inducer, but not with an erythrocyte differentiation inducer, hemin. Ganglioside GM3 may play an important role as a trigger in differentiation induction of K562 cells. hST3Gal V is a key regulatory enzyme for ganglioside biosynthesis because it catalyzes the first committed step in the synthesis of nearly all gangliosides. Differentiation of K562 cells requires Erk1/2 activation and p38 MAPK inhibition for the transcriptional activity of lactosylceramide α-2,3-sialyltransferase (hST3Gal V) and synthesis of ganglioside GM3. The expression of hST3Gal V mRNA induces expression of the megakaryocytic markers and differentiation of K562 cells. Ganglioside GM3 mediates megakaryocytic differentiation of human chronic myelogenous cells and apoptosis of many human cancer cells.
Rune Thorbjørn Nordvang
Technical University of Denmark, Denmark
Title: A rational approach to identification of wild type trans-sialidases for the production of human milk oligosaccharides
Time : 14:55-15:15
Biography:
Rune Thorbjørn Nordvang is in the final stages of his PhD at the Technical University of Denmark (DTU). In the BioEng group (at DTU) he has worked with all aspects of enzymatic production of human milk oligo-saccharides from dairy side stream products and has published 6 articles on the topic.
Abstract:
In this study, sequence analysis and 3D alignment were used for the identification of a novel trans-sialidase, namely the trans-sialidase of Haemophilus parasuis. The H. parasuis trans-sialidase was one of four candidate enzymes selected from a database of 2909 protein sequences. It is the first time that a sequence analysis approach has been successful in identifying a trans-sialidase and additionally all remaining candidates (the sialidases of Pasteurella multosida, Actinomyces Oris and Manheimia Haemolytica) exhibited trans-activity, however they were ultimately not defined as trans-sialidases due to the comprehensive definition of a trans-silalidase. A trans-sialidase can be defined as a sialidase which, under a specific set of conditions, prefers the transfer of a sialic acid residue from a donor to an acceptor molecule over the hydrolysis of the donor. Trans-sialidases are sought after because they can be applied for the enzymatic production of human milk oligosaccharides (for addition to infant formula) from dairy side-stream products. So far, the only native trans-sialidase that has been found is the trans-sialidase of the human pathogen Trypanosomas cruzi. However, an additional trans-sialidase has been engineered through directed evolution of the sialidase of Trypanosomas rangeli. Rational inspection of the 3D structure of these known trans-sialidases was the basis for this study, and it is hoped that attributes of the newly identified H. parasuis trans-sialidase can be the basis of further trans-sialidase discovery.
Ivan Martinez Duncker
Cell Dynamics Research Center, MEXICO
Title: Sialobiology of T Cd4+ Cells
Time : 15:30-15:50
Biography:
Dr. Martinez Duncker is 37 years old and born in Mexico City. He earned his MD degree in the Military Medical School in Mexico after which he went to Paris, France to earn his PhD degree in the Ecole Pratique des Hautes Etudes. He is Director of the Cell Dynamics Research Center of the Morelos State Autonomous University in Cuernavaca, Mexico where he leads the Glycobiology and Human Genetics Program. He also is founder and President of the Latin American Society of Glycobiology.
Abstract:
T cells are probably one of the more dynamic models of cell glycosylation, particularly T CD4+ cells (Th). After activation and stimulation with specific cytokines, Th cells can differentiate into different subsets that include Th1, Th2, Th9, Th17 and T regulators (Treg), each one with different cytokine secretion profiles and effector functions. In the last few years it has been recognized that the presence and type of linkage in surface glycans of the negatively charged monosaccharide Sia, is different between this subsets and is involved in their distinct susceptibility to Galectin 1 mediated apoptosis. In this work we report the dynamics of sialylation during anti-CD3/anti-CD28 mediated activation of human CD4+ T helper lymphocytes (Th), including sialyltransferase gene expression changes and metabolic flux of sialic acid. The identification of novel sialoproteins and sialolipids through this approach sheds light into novel functions of sialic acids during Th activation.
Ezat Khosravi
Durham University, UK
Title: Functionalization and grafting of carbohydrates and synthesis of temperature responsive polymers
Biography:
Dr. Khosravi obtained his Ph.D. in polymer science from Sussex University, UK, 1982. He joined the polymer group at Chemistry Department, Durham University, UK, in 1987 where he is now a Reader in Polymer Chemistry. His research interests ranges from well-defined polymerisation chemistry to novel polymers and biopolymers. He has given 88 invited lectures worldwide, has been chairman in 16 international conferences, published 59 papers; has written 15 chapters for books, has edited 4 books and has filed 7 patent applications. He is an FRSC (Fellow of the Royal Society of Chemistry) and a member of the American Chemical Society.
Abstract:
The lecture describes a novel and versatile method for the modification of 2-hydroxyethyl cellulose (HEC) to prepare biocompatible and biodegradable materials to be used as personal products. The process of Click reactions involving azide-alkyne cycloaddition was used to impart neutral (ester) and ionic (carboxylic acid and 1ry amine) functionalities on HEC. Sequential Click reactions were also used to successfully synthesize polydimethylsiloxane (PDMS) grafted HEC containing neutral (ester) and ionic (carboxylic acid and 1ry amine) functionalities. Furthermore, The Click Coupling technique was utilized for grafting onto HEC; PLA (as hydrophobic segments) and PEG (as hydrophilic segments). The temperature responsive materials with low critical solution temperatures (LCST) in the physiological range (30-40 oC) attract much attention due to their potential biomedical and drug delivery applications. The lecture will also discuss the synthesis and characterization of a novel temperature responsive water-soluble glycopolymer based on trehalose via copper wire-catalyzed click-polymerization. The investigation of the cloud point of the aqueous solution of glycopolymer by optical microscopy and UV-Vis spectroscopy will also be discussed. The LCST of the glycopolymer was found to be within physiological range of about 39 oC, known as fever temperature. The full characterization of all the products as well as the intermediates by NMR, MS, IR, SEC, TGA and DSC will be presented.
Claudia Koch-Brandt
J.-Gutenberg-University, Germany
Title: Clusterin/ApoJ - How glycosylation modulates the function of this apolipoprotein
Biography:
Having worked in 1977/78 as a guest investigator in G. Blobel’s laboratory at Rockefeller University, New York, Dr. Koch-Brandt completed her PhD in 1980 from the Goethe University Frankfurt/Main, Germany. After five years as postdoc and staff scientist at the EMBL, Heidelberg, she became assistant professor at the Goethe–University and in 1991 full professor at Mainz University. She has been serving as acting department director and reviewer for international journals and science funding organisations. In 1984 she received the Heinz-Maier-Leibnitz-Award of the German Research Foundation, in 1998 she was the German nominee for the UNESCO-L’Oréal- then Women in Science-Award).
Abstract:
Clusterin (CLU), also known as ApolipoproteinJ (ApoJ) is a highly glycosylated extracellular chaperone. In humans it is expressed in a broad spectrum of tissues and related to a plethora of pathophysiological processes, such as M. Alzheimer, atherosclerosis and cancer, where the protein exerts a cytoprotective role. In its dominant form it is expressed as a secretory protein (sCLU) which during maturation is N-glycosylated and cleaved intracellularly into an α- and a β-chain connected by five symmetrical disulfide bonds. In early studies we examined the role of the carbohydrate moieties in the vectorial secretion of ApoJ at the apical surface of polarized epithelial cells. If N-glycosylation is inhibited by tunicamycin treatment the protein is secreted in equal amounts at both cell surfaces, demonstrating that the carbohydrates are dispensible for the acquisition of a transport competent conformation, however indicating a role of the carbohydrate moieties in the vectorial transport of this protein. Recently, it has been demonstrated that besides the predominant sCLU, rare intracellular CLU forms are expressed in stressed cells. Since these isoforms do not enter nor complete the secretory pathway, they display either no or only core glycosylation and are not proteolytically processed. Due to their sparsity, these intracellular forms are functionally poorly characterized. To evaluate the function(s) of these stress-induced intracellular forms, we first examined whether these isoforms display chaperone activity, to then investigate the impact of glycosylation and proteolytic maturation on this activity.
William L Holland
The University of Texas Southwestern Medical Center, USA
Title: Ceramides and glucosylceramides in diabetes
Biography:
Will Holland completed his dissertation work with Scott Summers at the University of Utah in 2007. Together, they produced seminal work establishing the role of ceramide in insulin resistance. During his subsequent postdoctoral work with Philipp Scherer, he uncovered the roles of adiponectin and FGF21 as potent regulators of sphingolipid metabolism. He is currently an Assistant Professor in the Touchstone Diabetes Center, focusing efforts on the roles of ceramide catabolic enzymes on glucose and lipid metabolism.
Abstract:
Insulin promotes the uptake and storage of carbohydrates, as well as other nutrients in skeletal muscle and adipose. It simultaneously represses secretion of glucagon (insulin’s counter-regulatory hormone) and subsequent glucose efflux from the liver. The hepatic glucose production stimulated by glucagon has been thought to play a significant role in the development of hyperglycemia. Just as insulin insufficiency can lead to elevated glucagon secretion, impaired insulin and leptin actions upon the alpha cell can also promote hyperglucagonemia and hyperglycemia. Sphingolipids, such as ceramides and glucosylceramides, are important bioactive lipid metabolites which can impair Akt-mediated signal transduction. We have demonstrated that ceramide is sufficient to impair insulin-induced suppression of glucagon from cultured glucagon-producing alpha-cells. Here, we elucidate the role of sphingolipid accumulation in aberrant glucagon production in vivo, we have used novel mouse models to drive expression of acid ceramidase under the control of a pre-proglucagon promoter. The local overexpression of acid ceramidase within the alpha cell is sufficient to improve insulin- or leptin-mediated Akt signaling within the alpha cell and prevent aberrant glucagon production. Induction of the acid ceramidase transgene is sufficient to reverse hyperglycemia within 48 hours in type 2 diabetic mice. Moreover, it is sufficient to prevent the onset of diabetes in ob/ob mice. Similarly, inducing the cre-lox mediated excision of glucosylceramide synthase within the alpha cell reverses glucagon overproduction and restores glycemia. Collectively, these data suggest that aberrant accumulation of ceramides or glucosylceramides within the alpha cell may be a causal link between insulin resistance and frank diabetes.
Biography:
Dr. Samira has completed his PhD at the age of 28 years from University of Malaya (151 in the world university ranking) and postdoctoral studies from University of Malaya, Nanotechnology & Catalysis Research Centre (NANOCAT). Her main research interests are in the areas of carbon nanomaterials, such as carbon nanotubes, graphene oxide and graphene nanosheets, metal oxide nanocomposites advance smart nanohybrids, especially apply in electrochemical sensors, supercapacitors, fuel cells and biomedical. She has published more than 45 papers in reputed journals and has been serving as an editorial board member of repute.
Abstract:
Carbohydrates are the main component of the extracellular matrix (ECM) where they associate with proteins to form glycoproteins or proteoglycans, or exist as long-chain dis-accharides. All ECM proteins except elastin have associated sugar, and, in some cases, ECM proteins require proper glycosylation to achieve the full biological activity. It is also now clear that many ECM proteins have carbohydrate-binding domains that specifically recognize and interact with glycoconjugates with other matrix components and on the cell surface. Carbohydrates have been implicated in a wide variety of processes, ranging from cell adhesion and migration to matrix assembly, growth factor sequestration and regulation, involvement in many aspects of immune function, binding of plasma proteins, and control of thrombogenesis. This contribution is a method of immobilizing and processing functional multi-component structures of the ECM, comprising the following successive process steps, including covalent binding of an adhesion promoter layer to cell culture carriers; culturing cells of a desired type on the adhesion promoter layer and thus immobilizing the ECM secreted by the cells by secretion and binding to the adhesion promoter layer; and application of a de-cellurization protocol so as to detach matrix-secreting cells from the surface while simultaneously retaining the structure and functionality of the immobilized ECM, which is connected to the adhesion promoter.
E. Ada Cavalcanti-Adam
University of Heidelberg, Germany
Title: Cell-matrix interactions at the nanoscale
Biography:
E. Ada Cavalcanti-Adam has completed her PhD in Biology from University of Heidelberg (Germany) and postdoctoral training at the Max Planck Institute for Metals Research in Stuttgart. She is research group leader at the University of Heidelberg Institute of Physical Chemistry and head of the Central Scientific Facility “Biomaterials and Molecular Biology†at the Max Planck of Intelligent Systems. She is also visiting Professor at the University of Pennsylvania. In recognition of her work she has received a number of awards, most notably the UNESCO-L’Orèal Prize “for women in scienceâ€.
Abstract:
In tissues of mesenchymal origin, cell-extracellular matrix interactions are necessary for adhesion and migration and rely on the assembly of focal adhesions, micrometer-sized structures comprising transmembrane and intracellular protein clusters. Over the past two decades these structures have been extensively studied to elucidate their organization, assembly, and molecular composition, as well as to determine their functional role. Synthetic materials decorated with biological molecules, such as adhesive molecules and growth factors, are widely used to mimic the extracellular environment and to induce specific cellular responses dependent on cell adhesion. Nanotechnology provides tools to mimic and investigate such responses at single molecule resolution. This lecture focuses on cell interactions with nanopatterned surfaces biofunctionalized with adhesive peptides recognized by integrins, as well as on surfaces decorated with bone morphogenetic protein 2. Results on cell adhesion and adhesion-mediated signaling induced by surface immobilization and spatial distribution of the ligands will be presented. Surface patterning strategies for presenting on the same platform different chemical cues of the extracellular space will be also discussed.
Regina Célia Teixeira Gomes
Federal University of São Paulo, Brazil
Title: Metoclopramide-induced hyperprolactinemia changed on murine uterine
Biography:
Gomes, RCT. has completed his PhD at the age of 42 years from Federal University of Sao Paulo, Brazil and postdoctoral studies from the same University. Since 2004 performs experimental research with hyperprolactinemia. Currently receives scholarship by the Foundation for Research Support of the State of São Paulo - FAPESP. Published several articles related to hyperprolactinemia and extracellular matrix.
Abstract:
Initially our group found out that the metoclopramide-induced hyperprolactinemia may negatively affect the endometrial morphology. And, in later studies we found out that the hyperprolactinemia caused a decrease in pinopode numbers and embryo implantation in female mice, thus interfering with the fertility and in ovarian function. In order to show the effectiveness of treatment with 200 µg metoclopramide for 50 consecutive days, we measured the serum prolactin levels and also analyzed the pituitary of animals. We proved that the hyperprolactinemia caused by metoclopramide in mice is due to an increase in the number and activity of lactotrophs. The deepening of the research led us to several questions. What are the biochemical changes that were occurring in the endometrial stroma (cells and extracellular matrix) of these animals? Finally, our results showed that the elevation of prolactin may lead to changes in the amounts of glycosaminoglycans, which are important for embryo implantation, in an animal model of hyperprolactinemia accompanied by a regular estrous cycle. Recently, we have researched the gene expression of small leucine-rich proteoglycans (SLRPs) on the murine uterus non pregnant and pregnant with hyperprolactinemia induced metoclopramide. The interactions between the production and degradation of these substances with steroids and hyperprolactinemia is complex and difficult to explain, because the signaling pathways involving those hormones may influence the cell-cell and cell-extracellular matrix interactions in the endometrial stroma, as well as they may interfere with the appropriate preparation of the endometrium to receive the embryo.
- Track 8: Synthesis and Biological Role of Glycans
Location: Independence C
Chair
Konrad Sandhoff
LIMES Institut, Germany
Co-Chair
Barbara Klajnert-Maculewicz
University of Lodz, Poland
Session Introduction
John F. Robyt
Iowa State University, USA
Title: Recent advances for the mechanisms involved in glycan biosynthesis
Time : 10:30-10.50
Biography:
Prof. John F. Robyt, Short Bio-sketch: John Robyt received a B.S. in chemistry from St. Louis University in 1958. In the summer of 1957, he received an Internship at the USDA Laboratory in Peoria, IL. He then went to Iowa State University in 1958 to study for a Ph.D., joining Prof. Dexter French’s group in Carbohydrate Chemistry and Enzymology, where he received a Ph.D. in 1962, publishing four papers: on the Action Pattern and Mechanism of Bacillus amyloliquefaciens α-amylase, Bacillus polymyxa ß-amylase, porcine pancreatic α-amylase, the separation and large-scale purification of maltodextrins by charcoal column chromatography, and the development of ascending and descending paper chromatography of carbohydrates. He then went to Louisiana State University in Baton Rouge, LA on a Teaching Postdoctoral Fellowship and taught seven courses in chemistry and biochemistry; then the next year (1963) he received a NIH, Research Fellowship, to work and study at the Lister Institute of Preventive Medicine in London, England, under Prof. William J. Whelan, and published 4 papers. In September 1964, he returned to Iowa State University, as an Instructor and Research Associate with Prof. Dexter French. He was appointed an Assistant Professor in 1967, Associate Professor in 1974, and a Professor in 1982 in the Department of Biochemistry, Biophysics, and Molecular Biology. Besides working on Starch Chemistry and Enzymology, he pursued studies on Dextran Chemistry, and Enzymology, and the formation and prevention of dental plaque, supported by the NIH. He has pursued the use of TLC of carbohydrates, use of NMR to study enzyme mechanisms, application of the use of radioisotopes, and analytical methods of carbohydrates and enzymes, such as reducing-value methods, total carbohydrates by phenol sulfuric acid, and several chromatographic methods, such as ascending and descending paper chromatography, TLC, and column chromatographic methods for separating carbohydrates and enzymes.
Abstract:
Glycan biosynthesis has recently undergone a revolutionary change as to the understanding of how both α- and ß-linked glycans are biosynthesized. Back in the 1940’s C. S. Hanes observed that potato phosphorylase transferred 2-3 glucose units from α-D-Glc-1-P to the nonreducing ends of starch chains. This observation set the stage that starch biosynthesis required a primer. In the 1960’s Luis Leloir found that starch was biosynthesized from ADPGlc. In the late 1960’s, Robbins et al. using pulse and chase techniques found that Salmonella O-antigen was biosynthesized by the transfer of a tetra-saccharide from bactoprenol pyrophosphate to the reducing-ends of a growing chain. Four years later Ward and Perkins showed that the bacterial cell wall, murein, was also biosynthesized by the addition of the NAG-NAM-pentaphosphate to the reducing-ends of growing chains. A year later Robyt, et al. using pulse and chase experiments showed that dextran was biosynthesized by the addition of glucose from sucrose to the reducing ends of a growing dextran chains. Some years later (2007) they showed a two-site insertion mechanism for dextran biosynthesis in which glucose is added to the reducing-ends of growing dextran chains. In 2012, Mukerjea & Robyt showed that the de novo biosynthesis of starch chains by potato starch-synthase adds glucose from ADPGlc to the reducing-ends of growing starch chains. Mukerjea, McIntyre, and Robyt also found that Tris-buffers were potent inhibitors of starch-synthase and has been responsible for the perpetuation of the primer myth for starch biosynthesis, as the putative primers partially reverse the Tris-buffer inhibition.
Konrad Sandhoff
LIMES Institut, Germany
Title: Lysosomal & extracellular GlcCer degradation: Protein & lipid modifiers
Time : 10:50-11:10
Biography:
Konrad Sandhoff completed his PhD in biochemistry in Munich. After research stays in Munich, Israel and the USA he became a full professor of biochemistry at the University of Bonn in 1979. Since 2007 he is a senior professor at the LIMES institute, Bonn. Major Research Interests: Molecular life sciences: analysis and pathobiochemistry of lysosomal (glyco-)sphingolipid storage diseases, structure and function of lysosomal enzymes and lipid binding proteins, topology of endocytosis and glycolipid metabolism, and regulation of glycolipid biosynthesis. He has published more than 480 peer-reviewed papers. Among many other prizes he also received the International Glycoconjugate Organization Award (2005).
Abstract:
Lysosomal sphingolipid degradation requires the presence of water-soluble hydrolases, SAPs, anionic phospholipids like BMP, and an acidic pH value. Inherited defects of catabolic hydrolases or SAPs cause various sphingolipidoses. SAPs are membrane-perturbing proteins which facilitate glycolipid digestion by presenting insoluble lipid molecules to soluble catabolic enzymes. SAPs (the GM2-activator and saposins A-D) bind to lipid bilayers and mobilize lipids out of them at acidic pH values. As demonstrated by plasmon resonance studies for saposins A and B, low cholesterol levels and increasing concentrations of BMP favour lipid extraction and membrane disintegration. Variant saposins as identified in patients with Krabbe disease and metachromatic leukodystrophy, respectively, are deficient in mobilizing membrane lipids. The inherited absence of all four saposins (A-D) causes a severe membrane and sphingolipid storage disease, also disrupting the water permeability barrier of the skin. Saposins and glucosylceramidase are also involved in the extracellular catabolism of ultralongchain acylglucosylceramides, key components for the generation of the extracellular lipid layers forming the immune and the water permeability barrier in the stratum corneum of the mammalian skin. Their complete functional deficiency causes perinatal fatal diseases of the collodion baby type. Ongoing in vitro studies indicate that PM-stabilizing lipids, i.e. SM and cholesterol, inhibit several steps of lysosomal SL and glycosphingolipid catabolism, and also lipid solubilisation as studied by Surface Plasmon Resonance and intervesicular (glyco-) lipid transfer activities of several SAPs and NPC2, even in the presence of activating anionic PLs.
Jiahua Jay Xie
North Carolina Central University, USA
Title: Cytoprotective properties of plant-produced asialoerythropoietin (asialo-rhuepo)
Time : 11:25-11:45
Biography:
Jiahua (Jay) Xie has completed his PhD in Biophysics at the age of 31 from Zhejiang University and postdoctoral studies from Departments of Genetics and Horticultural Science, North Carolina State University. He served as a Senior Scientist at the Vector Research Inc. (a subsidiary of Vector Group Ltd) for five years. Currently, he is an Associate Professor in the Department of Pharmaceutical Sciences and the PI of the BRITE, NCCU. He has published more than 50 papers in reputed journals and has been serving as an editorial board member of Journal of Zhejiang University (Agriculture and Life Sciences).
Abstract:
Asialo-rhuEPO, a non-hematopoietic recombinant human erythropoietin (EPO) derivative lacking sialic acid, has been reported to display broad tissue-protective effects against damage triggered by ischemia/reperfusion, hypoxia or cytotoxic agents in the brain, the heart, the kidneys and the liver. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. It is known that plants can synthesize complex N-glycans similar to mammals, but lack sialylating capacity. In our lab, we generated stable transgenic tobacco lines co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes to produce asialo-rhuEPO (asialo-rhuEPOP) lacking sialic acid, but bearing mammalian-type β1,4-galactose residues. We also developed an efficient purification system for isolating asialo-rhuEPOP from leaf tissues both at small scale (grams) and medium scale (kilograms). Purified asialo-rhuEPOP from transgenic tobacco leaves was found to have superior cytoprotective effect than rhuEPOM in protecting neuronal-like mouse neuroblastoma cells, murine HL-1 cardiomyocytes and pancreatic β-cells from staurosporine-induced cell death. These milestone studies have set the stage for the future investigations on its tissue-protective effects and action mechanisms in various animal models of tissue injury.
Guijun Wang
Old Dominion University, USA
Title: Design, synthesis and characterization of glycolipids and glycoclusters
Time : 11:45-12:05
Biography:
Guijun Wang has completed her PhD in 1999 from Michigan State University and postdoctoral research at Yale University in 2002. She started her independent career as an assistant professor at the University of New Orleans in 2002. In 2012, she relocated to Old Dominion University (ODU), currently she is a professor of Chemistry and Biochemistry at ODU. Her research interests include synthetic methodology development; asymmetric synthesis; and the synthesis and study of carbohydrate based self-assembling systems. She has co-authored 50 peer reviewed papers and is the co-inventor on 20 international and US patents.
Abstract:
Glycolipids and glycoclusters that are able to form self-assembled supramolecular structures are interesting compounds with potential applications as new materials for biomedical research. Our group has a long interest in the design, synthesis and study of various monosaccharide derivatives and we have discovered several new classes of glycolipids based low molecular weight gelators (LMWGs). These compounds form unique soft materials such as organogels or hydrogels that may be useful in biomedical research. They can be used as advanced functional materials for controlled release drug delivery and enzyme immobilization. Using naproxen as a model, we have shown that acid sensitive glucosamine derivatives are effective compounds for controlled release of naproxen from the gel matrix. By incorporating photosensitive functional groups, we have also synthesized and characterized polymerizable diacetylene containing organogels. The resulting polydiacetylenes gels are useful as stimuli-responsive soft materials. Besides these systems, several branched molecules with sugar moieties at the periphery have also been designed and synthesized. These compounds have accurate molecular weight and can form interesting molecular assemblies. The dendritic glycolipids have shown enhanced self-assembling tendencies, which mimic the multivalency effect. In this presentation, our recent studies on glycolipids that can form supramolecular gels and self-assembling glycoconjugates will be discussed.
David Ben-Menahem
Ben-Gurion University of the Negev, Israel
Title: O-glycosylation and protein evolution: the case of the LHï¢ to CGï¢ development
Time : 12:05-12:25
Biography:
David Ben-Menahem has completed his PhD at Tel-Aviv University in Tel-Aviv Israel, and did his postdoctorate studies at Washington University Medical School, in St. Louis Missouri, USA. He is at the department of Clinical Biochemistry and Pharmacology at Ben-Gurion University of the Negev in Beer-Sheva, Israel. His major research focus is related to structure-function studies of the gonadotropins which are members of the glycoprotein hormone family.
Abstract:
The glycoprotein hormones LH, FSH and CG are non-covalent heterodimers composed of the common ï¡ and hormone specific ï¢ subunit. The subunits contain N-linked glycans, which are important for the folding, heterodimer assembly and bioactivity of the hormone. In addition, the carboxy-terminal region of the CGï¢ subunit is O-glycosylated, and this unique domain (known as the CTP) extends the circulatory survival of CG relative to the other glycoprotein hormones. While the genes encoding the ï¡, LHï¢ and FSHï¢ subunits are generic to vertebrates, the CGï¢ gene is restricted to primates and equids. This is curious because the CGï¢ gene presumably evolved from the ancestral LHï¢ gene following only a small set of mutations, and the resulting O-glycosylated CTP confers new hormonal properties to CG relative to LH that seems advantageous to maintain early gestation. To address this restricted evolution, we combined bioinformatics, in-vitro and in-vivo experiments that suggest a) the potential of the LHï¢ to CGï¢ transformation is present in several animal phyla, and b) the ability of a CTP domain to have the clustered O-glycans is important for the CGï¢ development. Additional studies with the equine CTP-extended ï¢ subunit suggest that this subunit, which is expressed in both in the pituitary and placenta of equids integrates intracellular properties that diverged in the LHï¢ and CGï¢ subunits of primates that are expressed in different tissues. Our studies demonstrate a potential role for the CTP O-glycosylation in the LHï¢ to CGï¢ evolution, and a link between tissue expression and subunit characteristics.
Shigeomi Horito
Tokyo University of Science, Japan
Title: Reconstruction of a transmembrane protein tetraspanin (CD9) into lipid bilayer by interaction of ganglioside GM3 and tetraspanin
Time : 12:25-12:45
Biography:
Dr. Horito has completed his PhD from Tokyo Institute of Technology in organic synthesis of carbohydrates and postdoctoral studies from Hamburg University of Germany as Humboldt researcher. He is an Associate Professor in the Dept. of Biological Science & Technology at Tokyo University of Science. He has published more than 25 papers in reputed journals.
Abstract:
Tetraspanin is four times transmembrane protein which constructs a super molecular complex with ganglioside GM3 and CD81 to regulate cell proliferation. Cancer proliferation is also regulate this super molecular complex. It is exciting challenge to elucidate the role of ganglioside GM3 in the super molecular complex. Guofei Son et al reported reconstruction of transmembrane protein aquaporine Z into lipid bilayer by interaction of nickel chelate and histidine tag. We tried to reconstruct tetraspanin by interaction of only natural compounds. GM3/dipalmitoylphosphatigylcholine/dioreoilphosphatigylcholine(1/9/9) lipid-monolayer indicate 2 types domains (0.9 nm high and 2.1 nm high). Hetero-bilayer indicates 2 types domains (6.7 nm high and 12.5 nm high), which is given tetraspanin to indicate 3 types domains (6.1 nm high, 11.7 nm high and 15.6 nm). The highest domain indicates the reconstruction of tetraspanin into the lipid-bilayer.
Barbara Klajnert-Maculewicz
University of Lodz, Poland
Title: Sugar-modified PPI dendrimers as carriers of anti-leukemia drugs
Time : 13:30-13:50
Biography:
Barbara Klajnert-Maculewicz has completed her Ph.D in 2002 from the University of Lodz, Poland and postdoctoral studies from the McMaster University, Ontario, Canada. She is a professor at the University of Lodz, Poland and an external scientific member at Leibniz IPF in Dresden, Germany. She is a co-author of 2 books and 9 chapters in monographs. She has published more than 90 papers in reputed journals (h-index 23). In the years 2009-2012 she was the Management Committee Chair of COST Action TD0802 “Dendrimers in biomedical applications” that gathered 24 countries. She has been awarded L’Oréal-UNESCO Fellowship for Women in Science.
Abstract:
Anticancer drugs such as cytarabine (araC) belong to nucleoside analogues (NAs). NAs are commonly used in the treatment of acute myeloid leukemia, acute lymphocytic leukemia, and lymphomas. AraC acts by interfering with newly synthesized nucleic acids or by modifying physiological nucleosides metabolism. Like most nucleoside analogs, araC is administered as an inactive prodrug and requires specialized nucleoside transporters to cross plasma membranes. Inside a cell, araC is activated to cytotoxic 5’-triphosphates form (araCTP) by intracellular kinases. Unfortunately, a therapy based on cytarabine has its limitations due to several primary and acquired resistance mechanisms that arise during prodrug activation steps. It may lead to inefficient concentration of the therapeutics in cancer cells. Carrier systems that would deliver active forms of NAs are currently seeking. It has been demonstrated that polypropylene imine (PPI) dendrimers with a partially modified surface by maltose residues (PPI-m) easily form complexes with negatively charged 5’-triphosphates of nucleoside analogues. It happens due to the presence of protonated primary and tertiary amino groups. PPI-m dendrimers are non-toxic and highly biocompatible. Moreover, PPI-m dendrimers protect bound drug molecules from enzymatic degradation. Complexes of araCTP and PPI-m dendrimers show enhanced cytotoxic activity against an acute myeloid lukemia cell line 1301 in comparison with free cytarabine and 5’-triphosphate of cytarabine. Thus, PPI-m dendrimers improve stability of NAs and efficiently deliver the active drug forms directly to cancer cells. To sum up, maltose-modified polypropylene imine dendrimers are attractive systems as anticancer drug carriers, especially with a vision to apply them when drug-resistance occurs.
Jan Willem Kok
University of Groningen, Netherlands
Title: Glycosphingolipids enhance transfection efficiency in gm95 cells
Time : 13:50-14:10
Biography:
Jan Willem Kok has completed his PhD at the age of 31 from Groningen University. He is associate professor at the Cell Biology Department of the University Medical Center Groningen, University of Groningen in The Netherlands. He has published more than 70 peer-reviewed papers in reputed journals. His research interest includes sphingolipid biology, lipid rafts, and ABC transporters.
Abstract:
We investigated whether glycosphingolipids in the plasma membrane of cells affect transfection efficiency mediated by cationic lipoplexes. The ideal model cell line GM95 was used, which lacks glycosphingolipids due to mutated glucosylceramide synthase. GM95-GCS cells were generated, which stably express active glucosylceramide synthase. These cells display enhanced transfection efficiency of the reporter gene GFP and enhanced binding/uptake of lipoplexes compared to mock-transfected GM95 cells. The latter effect was mimicked by loading mock-transfected GM95 cells with GM3, but not in case of GM1. We conclude that glycosphingolipids enhance transfection efficiency of cationic lipoplexes and this likely is the result of increased binding/uptake of lipoplexes mediated selectively by GM3.
Miki Hara-Yokoyama
Tokyo Medical and Dental University, Japan
Title: Glycosylation regulates CD38 assembly on the cell surface
Time : 14:10-14:30
Biography:
Miki Yokoyama received her Ph.D. in 1986 at the Dept. of Biophysics and Biochemistry, Faculty of Science, Tokyo University. She worked at the department of physiology in Nihon University School of Dentistry at Matsudo as a research associate (1986-1995) and as a lecturer (1995-2001). Then, she moved to the department of biochemistry in Tokyo Medical and Dental University (TMDU) and worked as a lecturer (2001-2004). Currently, she is an associate professor of the department. Her research interests include regulation of protein assembly on the membrane by glycosylation or lipid-environment.
Abstract:
Many proteins have their functions on the cell membranes or organelle membranes. To understand the function on the membranes, it is important to elucidate the cell-surface assembly. The leukocyte cell-surface antigen CD38 is a type II transmembrane glycoprotein and has four N-glycosylation sites. CD38 is the major NAD+ glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 also acts as a lipid raft-dependent signaling molecule to promote cell proliferation or death. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38, by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane ï¡-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers underlies the catalytic activity and the localization of CD38 in lipid rafts. The N-linked glycosylation sites are found to be located in strategic positions to prevent further self-association of the tetramer. Accordingly, the glycosylation is likely to ensure the function of CD38, by regulating the cell-surface assembly.
Chitra Mandal
CSIR-Indian Institute of Chemical Biology, India
Title: Sialylation of outer membrane porin protein D impedes β-lactam antibiotic entrance in Pseudomonas aeruginosa
Time : 14:30-14:50
Biography:
Prof. Chitra Mandal completed her Ph.D. at the age of 28 years from Indian Institute of Science, Bangalore and post doctoral studies at University of Pennsylvania. She is the Director of CSIR-Indian Institute of Chemical Biology, Kolkata, India and Sir J.C. Bose National Fellow, Head CSIR-Innovation Complex. She has published 150 research papers and holds 10 patents, guided 26 Ph.D students and transferred three technologies. She is an elected fellow of 'The World Academy of Sciences, main four Academies in India. Main theme of her group is to understand the mystery of glycosylation of biomolecules and their potential applications in health and diseases.
Abstract:
SIalic acids (Sias) are typically present as terminal sugars in oligosaccharide moieties attached to glycoproteins. Pseudomonas aeruginosa (PA), a gram-negative bacterium infects immuno-suppressed patients. We have established the presence of linkage-specific Sias on PA. Sialic acid-binding immunoglobulin-type lectins (Siglecs) are present on all immune cells. Sialylated PA (PA+Sias) binds to neutrophils through Sias-siglecs interactions. We have observed reduced oxidative burst, release of elastase and decrease NETs formation thus indicating subversion of host innate immunity. Next, we have affinity purified and sequenced twenty six sialoglycoproteins from PA+Sias. One such identified sialoglycoprotein is outer membrane porin protein D (OprD), a beta-barrel shaped channel-forming protein. To establish the role of Sias on OprD proteins, they are purified from sialylated (PA+Sias) and non-sialylated (PA-Sias) and their sialylation status are established. Profiling of glycan structures reveals the presence of sialylated N- and O-glycans in OprD+Sias. Bioinformatics studies reveal that amongst four N-glycosylation sites of OprD, Asn311 is present in the extracellular loop region having high solvent accessibility for its proper glycosylation. Core glycan moieties can properly fit into Asn311 site with no spatial overlaps with suitable glycosidic conformations. Molecular modeling studies suggest the presence of glycan structure with terminal bulky sialic acid hinders the channel passage of OprD towards β-lactam antibiotic permeabilization. This might be one of the new mechanisms for β-lactam antibiotic resistance of PA and thereby facilitating their survival in host. Our findings might help to open new avenue to design latestdrug which can enter cells freely even in presence of sialic acids.
Eliot T. Smith
East Tennessee State University, USA
Title: Expression of recombinant human mast cell chymase with asn-linked glycans in glycoengineered pichia pastoris
Time : 14:50-15:10
Biography:
Eliot T. Smith completed his PhD at the James H. Quillen College of Medicine at East Tennessee State University in 2013. He currently works as a postdoctoral researcher at the University of Pennsylvania, where he studies NDR kinases in the laboratory of Francis Luca.
Abstract:
Human mast cell chymase is a potent serine protease with roles in inflammation and allergy response. Chymase has received attention for its ability to generate angiotensin II from angiotensin I or angiotensin (1-12). The primary natural source of human chymase is skin tissue and recombinant expression provides a safe and abundant alternative for chymase research. One drawback of many expression systems, however, is the inability to generate proteins with human glycosylation patterns. To generate recombinant human chymase (rhChymase) with a glycosylation pattern that more closely resembles the natural enzyme, rhChymase was expressed and secreted in active form with (Man)5(GlcNAc)2 Asn-linked glycans using the SuperMan5 strain of GlycoSwitch® Pichia pastoris (BioGrammatics). Five milligrams of active enzyme were recovered from one liter of fermentation medium by cation exchange and heparin affinity chromatography. Purified rhChymase glycoprotein appeared as a single band migrating at an apparent molecular weight of 30 kDa on SDS-PAGE and treatment to remove glycosylation reduced the apparent molecular weight to 25 kDa, consistent with properties of the native enzyme. Western blotting with antibodies against human chymase labeled rhChymase. Active site titration with the potent chymase inhibitor Eglin C followed by kinetic analyses with peptide synthetic substrates demonstrated that glycosylated rhChymase possesses enzymatic activity that closely resembles its native counterpart. This work provides a source of active rhChymase with glycosylation similar to the as-yet unidentified human chymase glycan pattern(s) and offers a foundation for future production of chymase with true human glycosylation.