Stem Cell Glycomics and Proteomics

Objectives

Mammalian cells are surrounded by a dense layer of glycans – the glycocalyx – that is unique for each cell type. Detailed structural analysis of the glycocalyx and understanding its functions represent major challenges. The goal of the RG Stem Cell Glycomics and Proteomics is to decipher the glycome (glycolipidome and glycoproteome) as well the glycan recognition potential of human pluripotent stem cells and to follow up changes in these structures during differentiation, reprogramming and disease development. Towards this goal we apply established technologies (e.g. mass spectrometry) and further develop physicochemical as well as array techniques.

Research Focus

Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide promising tools as disease models and bear the potential to be used in regenerative therapies. The identification, maintenance, and controlled propagation of specific cell stages is of utmost importance. The glycocalyx is the cellular structure with highest permutation potential and it is allowed to assume that each cellular stage is reflected in this structure. Our research focuses at the development and application of ultra-high sensitive glycoanalytical strategies, to correlate cellular stages and glycocalyx composition and thus provide a detailed and easy to access monitoring system. Because cardiac dysfunctions manifest with high frequency in a cluster of rare diseases summarized as congenital disorders of glycosylation (CDG), iPSCs derived from CDG patients are included to comparatively assess the glycome in health and disease. In summary our work shall help to advance protocols with which the generation and isolation of specific cell populations can be achieved and provide novel insight into the molecular underpinnings of cardiac (disease) development.

Collaborations

  • MHH, LEBAO, Dr. Robert Zweigerdt, REBIRTH Unit Mass Production Pluripotent Stem Cells and Derivates, Prof. Dr. Ulrich Martin, REBIRTH Unit iPSCs for Disease Modelling, Drug Screening and Cell Therapy: Proteomics and glycomics of hESCs and hiPSCs under different culture conditions and upon differentiation into cardiomyocytes. 
  • MHH, Department of Experimental Hematology, REBIRTH Unit Regenerative Gene Therapy, Prof. Dr. Dr. Axel Schambach, Dr. Dirk Hoffmann: Generation of patient-specific iPSCs. 
  • MHH, Molecular Cardiology, REBIRTH Unit Endogenous Regeneration Mechanisms of the Heart, Prof. Dr. Denise Hilfiker-Kleiner: Analysis of proteins secreted by patient-specific iPSC-derived cardiomyocytes.
  • MHH, HTTG, REBIRTH Unit Tissue Engineered Valves, Dr. Andres Hilfiker: Identification of immunogenic glyco-epitopes on xenogeneic heart valves.  
  • MHH, Institute for Toxicology Prof. Andreas Pich: Mass spectrometry-based proteomics. 
  • Leiden, The Netherlands, Dr. Manfred Wuhrer: Mass spectrometical analysis of stem cell glycans. 
  • Moscow, Russia, Prof. Nikolay Nifantiev: Synthesis of glycan structures for the analysis of enzymes involved in Notch glycosylation. 
  • Stony Brook, NY, USA, Prof. Robert Haltiwanger: Studies towards the function of Notch glycosylation 
  • University of Texas Health Science Center at Houston, Houston, Texas, USA, Dr. Hamed Jafar-Nejad: Notch xylosylation in Drosophila development. 

Further Research Projects

  • DFG-research group FOR2509 “The concert of dolichol-based glycosylation: from molecules to disease models”– project: “Involvement of C-mannosylation in protein processing in the endoplasmic reticulum and implications for specific target proteins”.
  • DFG-research group FOR2509 “The concert of dolichol-based glycosylation: from molecules to disease models”– project: “Characterization of induced pluripotent stem cell-based disease models for congenital disorders of glycosylation”.
  • DFG grant “(Glyco)proteomic alterations during cardiomyogenic differentiation of human pluripotent stem cells”.

Teaching

  • We organize a module Glycobiology and a module Biomembranes and participate in Pathobiochemistry in the master study Biochemistry and offer practical courses for the same study. For medical students we present the ‘Modul Chemische und Biochemische Grundlagen der Medizin’ in the 2nd and 3rd tertial.

Publications

2013 - ongoing

2017

Konze SA, Cajic S, Oberbeck A, Hennig R, Pich A, Rapp E, Buettner FFR. Quantitative Assessment of Sialo-Glycoproteins and N-Glycans during Cardiomyogenic Differentiation of Human Induced Pluripotent Stem Cells. Chembiochem. 2017/05/17 ed2017. p. 1317-31.

Somplatzki S, Muhlenhoff M, Kroger A, Gerardy-Schahn R, Boldicke T. Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells. BMC Biotechnol. 2017/05/14 ed2017. p. 42.

Ehrit J, Keys TG, Sutherland M, Wolf S, Meier C, Falconer RA, Gerardy-Schahn R. Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen. Chembiochem. 2017/05/05 ed2017. p. 1332-7.

Kommerein N, Stumpp SN, Musken M, Ehlert N, Winkel A, Haussler S, Behrens P, Buettner FF, Stiesch M. An oral multispecies biofilm model for high content screening applications. PLoS One. 2017/03/16 ed2017. p. e0173973.

Konze SA, Werneburg S, Oberbeck A, Olmer R, Kempf H, Jara-Avaca M, Pich A, Zweigerdt R, Buettner FF. Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms. J Proteome Res. 2017/02/01 ed2017. p. 1133-49.

Shcherbakova A, Tiemann B, Buettner FF, Bakker H. Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3. Proceedings of the National Academy of Sciences of the United States of America. 2017.

Boer U, Buettner FF, Schridde A, Klingenberg M, Sarikouch S, Haverich A, Wilhelmi M. Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and alphaGal epitopes and is reduced in healthy vegetarian subjects. Xenotransplantation. 2017.

2016

Werneburg S, Buettner FF, Erben L, Mathews M, Neumann H, Muhlenhoff M, Hildebrandt H. Polysialylation and lipopolysaccharide-induced shedding of E-selectin ligand-1 and neuropilin-2 by microglia and THP-1 macrophages. Glia. 2016;64(8):1314-30.

Thiesler CT, Cajic S, Hoffmann D, Thiel C, van Diepen L, Hennig R, Sgodda M, Weibetamann R, Reichl U, Steinemann D, Diekmann U, Huber NM, Oberbeck A, Cantz T, Kuss AW, Korner C, Schambach A, Rapp E, Buettner FF. Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG). Molecular & cellular proteomics : MCP. 2016;15(4):1435-52.

Kiermaier E, Moussion C, Veldkamp CT, Gerardy-Schahn R, de Vries I, Williams LG, Chaffee GR, Phillips AJ, Freiberger F, Imre R, Taleski D, Payne RJ, Braun A, Forster R, Mechtler K, Muhlenhoff M, Volkman BF, Sixt M. Polysialylation controls dendritic cell trafficking by regulating chemokine recognition. Science. 2016;351(6269):186-90.

Kempf H, Olmer R, Haase A, Franke A, Bolesani E, Schwanke K, Robles-Diaz D, Coffee M, Gohring G, Drager G, Potz O, Joos T, Martinez-Hackert E, Haverich A, Buettner FF, Martin U, Zweigerdt R. Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells. Nature communications. 2016;7:13602.

Kallolimath S, Castilho A, Strasser R, Grunwald-Gruber C, Altmann F, Strubl S, Galuska CE, Zlatina K, Galuska SP, Werner S, Thiesler H, Werneburg S, Hildebrandt H, Gerardy-Schahn R, Steinkellner H. Engineering of complex protein sialylation in plants. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(34):9498-503.

2015

Baumann AM, Bakkers MJ, Buettner FF, Hartmann M, Grove M, Langereis MA, de Groot RJ, Muhlenhoff M. 9-O-Acetylation of Sialic Acids Is Catalysed by Casd1 Via a Covalent Acetyl-Enzyme Intermediate. Nat Commun. 2015;6:7673. Epub 2015/07/15.

Niemann MC, Bartrina I, Ashikov A, Weber H, Novak O, Spichal L, Strnad M, Strasser R, Bakker H, Schmulling T, Werner T. Arabidopsis Rock1 Transports Udp-Glcnac/Udp-Galnac and Regulates Er Protein Quality Control and Cytokinin Activity. Proc Natl Acad Sci U S A. 2015;112(1):291-6.

Kruger AT, Engel J, Buettner FF, Routier FH. Aspergillus Fumigatus Cap59-Like Protein a Is Involved in Alpha1,3-Mannosylation of Gpi-Anchors. Glycobiology. 2015. Epub 2015/09/16.

Engel J, Schmalhorst PS, Kruger AT, Muller CT, Buettner FF, Routier FH. Characterization of an N-Acetylglucosaminyltransferase Involved in Aspergillus Fumigatus Zwitterionic Glycoinositolphosphoceramide Biosynthesis. Glycobiology. 2015.

Karsten CB, Buettner FF, Cajic S, Nehlmeier I, Neumann B, Klippert A, Sauermann U, Reichl U, Gerardy-Schahn R, Rapp E, Stahl-Hennig C, Pohlmann S. Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques. J Virol. 2015;89(22):11727-33. Epub 2015/09/12.

Damerow S, Hoppe C, Bandini G, Zarnovican P, Buettner FF, Ferguson MA, Routier FH. Leishmania Major Udp-Sugar Pyrophosphorylase Salvages Galactose for Glycoconjugate Biosynthesis. Int J Parasitol. 2015;45(12):783-90.

Yu H, Takeuchi M, LeBarron J, Kantharia J, London E, Bakker H, Haltiwanger RS, Li H, Takeuchi H. Notch-Modifying Xylosyltransferase Structures Support an Sni-Like Retaining Mechanism. Nat Chem Biol. 2015;11(11):847-54.

Werneburg S, Buettner FF, Muhlenhoff M, Hildebrandt H. Polysialic Acid Modification of the Synaptic Cell Adhesion Molecule Syncam 1 in Human Embryonic Stem Cell-Derived Oligodendrocyte Precursor Cells. Stem Cell Res. 2015;14(3):339-46.

Schwarzer D, Browning C, Stummeyer K, Oberbeck A, Muhlenhoff M, Gerardy-Schahn R, Leiman PG. Structure and Biochemical Characterization of Bacteriophage Phi92 Endosialidase. Virology. 2015;477:133-43. Epub 2014/12/06.

2014

Tsuchiya A, Lu WY, Weinhold B, Boulter L, Stutchfield BM, Williams MJ, Guest RV, Minnis-Lyons SE, MacKinnon AC, Schwarzer D, Ichida T, Nomoto M, Aoyagi Y, Gerardy-Schahn R, Forbes SJ. Polysialic Acid/Neural Cell Adhesion Molecule Modulates the Formation of Ductular Reactions in Liver Injury. Hepatology. 2014;60(5):1727-40. Epub 2014/03/04.

Romanow A, Keys TG, Stummeyer K, Freiberger F, Henrissat B, Gerardy-Schahn R. Dissection of Hexosyl- and Sialyltransferase Domains in the Bifunctional Capsule Polymerases from Neisseria Meningitidis W and Y Defines a New Sialyltransferase Family. J Biol Chem. 2014;289(49):33945-57. Epub 2014/10/25.

Kröcher T, Röckle I, Diederichs U, Weinhold B, Burkhardt H, Yanagawa Y, Gerardy-Schahn R, Hildebrandt H. A Crucial Role for Polysialic Acid in Developmental Interneuron Migration and the Establishment of Interneuron Densities in the Mouse Prefrontal Cortex. Development. 2014;141(15):3022-32. Epub 2014/07/06.

Konze SA, van Diepen L, Schroder A, Olmer R, Moller H, Pich A, Weissmann R, Kuss AW, Zweigerdt R, Buettner FF. Cleavage of E-Cadherin and Beta-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells. Molecular & Cellular Proteomics. 2014;13(4):990-1007. Epub 2014/02/01.

Keys TG, Fuchs HL, Ehrit J, Alves J, Freiberger F, Gerardy-Schahn R. Engineering the Product Profile of a Polysialyltransferase. Nature Chemical Biology. 2014;10(6):437-42. Epub 2014/04/15.

Fiebig T, Berti F, Freiberger F, Pinto V, Claus H, Romano MR, Proietti D, Brogioni B, Stummeyer K, Berger M, Vogel U, Costantino P, Gerardy-Schahn R. Functional Expression of the Capsule Polymerase of Neisseria Meningitidis Serogroup X: A New Perspective for Vaccine Development. Glycobiology. 2014;24(2):150-8. Epub 2013/11/22.

Boeer U, Buettner FF, Klingenberg M, Antonopoulos GC, Meyer H, Haverich A, Wilhelmi M. Immunogenicity of Intensively Decellularized Equine Carotid Arteries Is Conferred by the Extracellular Matrix Protein Collagen Type Vi. PLoS One. 2014;9(8):e105964. Epub 2014/08/27.

Bartel J, Feuerstacke C, Galuska CE, Weinhold B, Gerardy-Schahn R, Geyer R, Munster-Kuhnel A, Middendorff R, Galuska SP. Laser Microdissection of Paraffin Embedded Tissue as a Tool to Estimate the Sialylation Status of Selected Cell Populations. Anal Chem. 2014;86(5):2326-31. Epub 2014/02/05.

2013

Sethi MK, Buettner FF, Ashikov A, Bakker H. In Vitro Assays of Orphan Glycosyltransferases and Their Application to Identify Notch Xylosyltransferases. Immunotherapy of Cancer: Methods and Protocols. 2013;1022:307-20. Epub 2013/06/15.

Rollenhagen M, Buettner FF, Reismann M, Jirmo AC, Grove M, Behrens GM, Gerardy-Schahn R, Hanisch FG, Muhlenhoff M. Polysialic Acid on Neuropilin-2 Is Exclusively Synthesized by the Polysialyltransferase St8siaiv and Attached to Mucin-Type O-Glycans Located between the B2 and C Domain. J Biol Chem. 2013;288(32):22880-92. Epub 2013/06/27.

Lee TV, Sethi MK, Leonardi J, Rana NA, Buettner FF, Haltiwanger RS, Bakker H, Jafar-Nejad H. Negative Regulation of Notch Signaling by Xylose. PLoS Genet. 2013;9(6):e1003547. Epub 2013/06/12.

Freiberger F, Böhm R, Schwarzer D, Gerardy-Schahn R, Haselhorst T, von Itzstein M. Defining a Substrate-Binding Model of a Polysialyltransferase. Chembiochem. 2013;14(15):1949-53. Epub 2013/09/07.

Elschami M, Scherr M, Philippens B, Gerardy-Schahn R. Reduction of Stat3 Expression Induces Mitochondrial Dysfunction and Autophagy in Cardiac Hl-1 Cells. Eur J Cell Biol. 2013;92(1):21-9. Epub 2012/10/30.

Buettner FF, Ashikov A, Tiemann B, Lehle L, Bakker H. C. Elegans Dpy-19 Is a C-Mannosyltransferase Glycosylating Thrombospondin Repeats. Mol Cell. 2013;50(2):295-302. Epub 2013/04/09.

Ashikov A, Buettner FF, Tiemann B, Gerardy-Schahn R, Bakker H. Large2 Generates the Same Xylose- and Glucuronic Acid-Containing Glycan Structures as Large. Glycobiology. 2013;23(3):303-9. Epub 2012/11/09.

2006 - 2012

2012

Weinhold B, Sellmeier M, Schaper W, Blume L, Philippens B, Kats E, Bernard U, Galuska SP, Geyer H, Geyer R, Worthmann K, Schiffer M, Groos S, Gerardy-Schahn R, Munster-Kuhnel AK. Deficits in Sialylation Impair Podocyte Maturation. Journal of the American Society of Nephrology. 2012;23(8):1319-28. Epub 2012/06/30.

Sethi MK, Buettner FF, Ashikov A, Krylov VB, Takeuchi H, Nifantiev NE, Haltiwanger RS, Gerardy-Schahn R, Bakker H. Molecular Cloning of a Xylosyltransferase That Transfers the Second Xylose to O-Glucosylated Epidermal Growth Factor Repeats of Notch. J Biol Chem. 2012;287(4):2739-48. Epub 2011/11/26.

Schaper W, Bentrop J, Ustinova J, Blume L, Kats E, Tiralongo J, Weinhold B, Bastmeyer M, Munster-Kuhnel AK. Identification and Biochemical Characterization of Two Functional Cmp-Sialic Acid Synthetases in Danio Rerio. J Biol Chem. 2012;287(16):13239-48. Epub 2012/02/22.

2011

Buettner FF, Konze SA, Maas A, Gerlach GF. Proteomic and immunoproteomic characterization of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae. Proteome Sci. 2011;9(1):23. Epub 2011/04/22.

Eggers K, Werneburg S, Schertzinger A, Abeln M, Schiff M, Scharenberg MA, Burkhardt H, Muhlenhoff M, Hildebrandt H. Polysialic acid controls NCAM signals at cell-cell contacts to regulate focal adhesion independent from FGF receptor activity. J Cell Sci. 2011;124(Pt 19):3279-91. Epub 2011/09/24.

2010

Galuska SP, Geyer H, Bleckmann C, Rohrich RC, Maass K, Bergfeld AK, Muhlenhoff M, Geyer R. Mass spectrometric fragmentation analysis of oligosialic and polysialic acids. Anal Chem. 2010;82(5):2059-66. Epub 2010/02/04. 

Galuska SP, Rollenhagen M, Kaup M, Eggers K, Oltmann-Norden I, Schiff M, Hartmann M, Weinhold B, Hildebrandt H, Geyer R, Muhlenhoff M, Geyer H. Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain. Proc Natl Acad Sci U S A. 2010;107(22):10250-5. Epub 2010/05/19. 

Schulz EC, Dickmanns A, Urlaub H, Schmitt A, Muhlenhoff M, Stummeyer K, Schwarzer D, Gerardy-Schahn R, Ficner R. Crystal structure of an intramolecular chaperone mediating triple-beta-helix folding. Nat Struct Mol Biol. 2010;17(2):210-5. Epub 2010/02/02. 

Schulz EC, Schwarzer D, Frank M, Stummeyer K, Muhlenhoff M, Dickmanns A, Gerardy-Schahn R, Ficner R. Structural basis for the recognition and cleavage of polysialic acid by the bacteriophage K1F tailspike protein EndoNF. J Mol Biol. 2010;397(1):341-51. Epub 2010/01/26. 

Sethi MK, Buettner FF, Krylov VB, Takeuchi H, Nifantiev NE, Haltiwanger RS, Gerardy-Schahn R, Bakker H. Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem. 2010;285(3):1582-6. Epub 2009/11/27. 

2009

Bakker H, Oka T, Ashikov A, Yadav A, Berger M, Rana NA, Bai X, Jigami Y, Haltiwanger RS, Esko JD, Gerardy-Schahn R. Functional UDP-xylose transport across the endoplasmic reticulum/Golgi membrane in a Chinese hamster ovary cell mutant defective in UDP-xylose Synthase. J Biol Chem. 2009;284(4):2576-83. Epub 2008/11/26. 

Boztug K, Appaswamy G, Ashikov A, Schaffer AA, Salzer U, Diestelhorst J, Germeshausen M, Brandes G, Lee-Gossler J, Noyan F, Gatzke AK, Minkov M, Greil J, Kratz C, Petropoulou T, Pellier I, Bellanne-Chantelot C, Rezaei N, Monkemoller K, Irani-Hakimeh N, Bakker H, Gerardy-Schahn R, Zeidler C, Grimbacher B, Welte K, Klein C. A syndrome with congenital neutropenia and mutations in G6PC3. N Engl J Med. 2009;360(1):32-43. Epub 2009/01/02. 

Buettner FF, Bendalla IM, Bosse JT, Meens J, Nash JH, Hartig E, Langford PR, Gerlach GF. Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor. Proteomics. 2009;9(9):2383-98. Epub 2009/04/04. 

Johswich A, Kraft B, Wuhrer M, Berger M, Deelder AM, Hokke CH, Gerardy-Schahn R, Bakker H. Golgi targeting of Drosophila melanogaster beta4GalNAcTB requires a DHHC protein family-related protein as a pilot. J Cell Biol. 2009;184(1):173-83. Epub 2009/01/14. 

Kahlisch D, Buettner FF, Naim HY, Gerlach GF. Glycoprotein analysis of porcine bronchoalveolar lavage fluid reveals potential biomarkers corresponding to resistance to Actinobacillus pleuropneumoniae infection. Vet Res. 2009;40(6):60. Epub 2009/07/30. 

Schwarzer D, Stummeyer K, Haselhorst T, Freiberger F, Rode B, Grove M, Scheper T, von Itzstein M, Muhlenhoff M, Gerardy-Schahn R. Proteolytic release of the intramolecular chaperone domain confers processivity to endosialidase F. J Biol Chem. 2009;284(14):9465-74. Epub 2009/02/05. 

2008

Galuska SP, Geyer R, Gerardy-Schahn R, Muhlenhoff M, Geyer H. Enzyme-dependent variations in the polysialylation of the neural cell adhesion molecule (NCAM) in vivo. J Biol Chem. 2008;283(1):17-28. Epub 2007/11/08. 

Oltmann-Norden I, Galuska SP, Hildebrandt H, Geyer R, Gerardy-Schahn R, Geyer H, Muhlenhoff M. Impact of the polysialyltransferases ST8SiaII and ST8SiaIV on polysialic acid synthesis during postnatal mouse brain development. J Biol Chem. 2008;283(3):1463-71. Epub 2007/11/30. 

Stolz A, Haines N, Pich A, Irvine KD, Hokke CH, Deelder AM, Gerardy-Schahn R, Wuhrer M, Bakker H. Distinct contributions of beta 4GalNAcTA and beta 4GalNAcTB to Drosophila glycosphingolipid biosynthesis. Glycoconj J. 2008;25(2):167-75. Epub 2007/09/19. 

2007

Curreli S, Arany Z, Gerardy-Schahn R, Mann D, Stamatos NM. Polysialylated neuropilin-2 is expressed on the surface of human dendritic cells and modulates dendritic cell-T lymphocyte interactions. J Biol Chem. 2007;282(42):30346-56. Epub 2007/08/19. 

Galuska SP, Geyer R, Muhlenhoff M, Geyer H. Characterization of oligo- and polysialic acids by MALDI-TOF-MS. Anal Chem. 2007;79(18):7161-9. Epub 2007/08/21. 

Krylov V, Ustyuzhanina N, Grachev A, Bakker H, Nifantiev N. Stereoselective synthesis of the 3-aminopropyl glycosides of alpha-D-Xyl-(1 -> 3)beta-D-Glc and alpha-D-Xyl-(1 -> 3)-alpha-D-Xyl-(1 -> 3)-beta-D-Glc and of their corresponding N-octanoyl derivatives. Synthesis-Stuttgart. 2007(20):3147-54. 

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