This is the institutional Repository of the Helmholtz Centre for Infection Research in Braunschweig/Germany (HZI), the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken/Germany, the TWINCORE Zentrum für Exprerimentelle und Klinische Infektionsforschung, Hannover/Germany,Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI), Würzburg/Germany, Braunschweig Integrated Centre for Systems biology (BRICS), Centre for Structural Systems Biology (CSSB) the Study Centre Hannover, Hannover/Germany and the Centre for Individualised Infection Medicine (CiiM).

 

  • Processing and Re-Processing of Asparagine-linked Oligosaccharides

    Hughes, R. C.; National Institute for Medical Research Mill Hill, London NW7 1AA (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    The assembly of asparagine-linked oligosaccharides in glycoprotein biosynthesis is cell specific, polypeptide specific and glycosylation site specific. Recombinant glycoproteins produced in nonhomologous cells are likely to be glycosylated abnormally and the consequences on proteinstability, conformation and biological activity need to be considered. Although the major pathways of assembly of asparagine-linked oligosaccharides are identified, their regulation during biosynthesis is not understood. The early events in oligosaccharide processing catalyzed by glucosidases I and II and specific mannosidases are particularly complex. Experiments using various inhibitors of processing glucosidases and mannosidases as well as structural analysis of processing intermediates, show that different processing pathways. are selected for assembly of glycans substituted at specific sites in glycoproteins. New mannosidases are being described that participate in these diverse pathways. A novel mannosidase of rat liver is concentrated in endosomes as well as the cis Golgi compartment and may play an additional role in remodelling of glycoproteins that occurs during internalisation and recycling of cell surface glycoproteins.
  • Title, Preface, Content, List of authors

    Conradt, Harald S. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Glycoproteins (i.e. proteins containing covalently bound carbohydrate) are ubiquitous constituents of all living organisms including bacteria. The posttranslational modification of polypeptides with carbohydrate groups is very commonforsecretory as well as integral membrane proteins of higher organisms which may function as enzymes, antibodies, hormones, structural or carrier proteins and receptors. Overthe past two decades theprincipal biosynthetic pathwaysleadingto thefinal carbohydrate structures of glycoproteins have been elucidated. The introduction of improved techniquessuchas high-resolution NMRandfast atom bombardment mass spectrometryas well as the introduction of novel chromatographic techniques for oligosaccharides over the past decade have expanded our knowledge of the enormous microheterogeneity of oligosaccharide structures that can be present at even a single glycosylation domain. The biological significance ofthis structural diversity seen in glycoproteinsis unclear. Recombinant DNAtechnology has permitted the efficient production of manybiologically important glycoproteins (membranereceptorsas well as their ligands) by expressionin heterologous mammaliancell lines. By using defined glycosylation mutantcell lines as hosts as have been derived from CHOand BHKcells (see paper byP. Stanley, this volume)it should be possible to obtain pure glycoproteinsof defined carbohydratestructures. Thestudyof the biological functionality of these glycosylation formswill considerably increase our understanding of the role of protein linked oligosaccharides. Pharmaceutical companies’ interest in the productionof clinically important humanproteins (many of which are glycoproteins) by biotechnological means, will undoubtedly have an impact on the developmentof glycoprotein biochemistry in the near future. The efforts of the pharmaceutical industry are directed toward human medicine, and manyclinically useful glycoproteins (immune-modulators, differentiation factors, glycoprotein hormones and receptors) are now available from recombinant sources. They shouldbe usedto develop our understandingofbiological phenomenaassociated with protein linked carbohydrates. However, only a multidisciplinary approachincluding molecular structure research, computer graphic model building as well as genetic engineering andcell biologyis likely to be successful. The present volume evolved from a workshopheld at the GBF in Braunschweigin June 1990 with the aim ofbringing together a balanced mixture of people from university settings whose interest runsfrom basicscience to the possible practical application of their research, i.e. including researchers from industrial laboratories with strong biotechnological interest. I thank the GBF administration, especially Sabine Peters, for help in running the workshop. Myspecial thank goesto all speakers, chairpersons and contributors to the book. The professionalhelp of Dr. J.-H. Walsdorff in editing this volumeis gratefully acknowledged.
  • GLYCOSYLATION OF THE INFLUENZA VIRUS HEMAGGLUTININ IN INSECT CELLS

    Klenk, Hans-Dieter; Kuroda, Kazumichi; Gröner, Albrecht; Doerfler, Walter; Geyer, Hildegard; Geyer, Rudolf; Institut für Virologie, Philipps-Universität Marburgl, Behringwerke AG, MarburgZ, Institut für Genetik, Universität zu Köln’, Biochemisches Institut, Justus-Liebig-Universität Gießen‘, Germany (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    The hemagglutinin of influenza virus grown in chick embryo cells has N-glycosidic carbohydrate side chains which are of the complex type at most and of the oligomannosidic type at a few attachmentsites. When the hemagglutinin was expressed in insect cells using a baculovirus vector, it was found that these cells have the capacity to trim N-glycans to ManzGlcNAc, cores and to further process these by the addition of fucose to Manz[Fuc]GleNAc,. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains. The effects of the truncated oligosaccharides on maturation of the hemagglutinin and onits biological functions will be discussed.
  • SMALL MOLECULES AS PROBES FOR THE SECRETORY PATHWAY

    Wieland, Felix; Institut für Biochemie I der Universitat Heidelberg Im Neuenheimer Feld 328, D-6900 Heidelberg (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Markers for the unsignalled vesicular flow, the "bulk flow", from the Endoplasmic Reticulum (ER) and the Golgi apparatus were generated according to the following concept: A substrate analog that is able to diffuse through biological membranes is added to living cells. This substrate can be converted into a derivative that is unable to permeate membranes by a corresponding enzyme that is uniquely located to the organelle from which vesicular bulk flow is to be measured. If the non diffusable derivative reaches the medium of the cell culture, it must have been transported in the luminal side of vesicles, thus serving as a bulk phase marker. By use of diffusable substrate analogues that are radioactively labeled the kinetics of secretion of such bulk phase markers can easily be quantitated. Two examples for this approach are described.
  • STUDIES ON THE STRUCTURE OF CARBOHYDRATE CHAINS OF GLYCOPROTEINS

    Vliegenthart, J. F. G.; Hard, K.; de Waard, P.; Kamerling, J. P.; Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TB Utrecht, The Netherlands (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    To obtain detailed information on the primary structures ofthe carbohydrate chains of glycoproteins,it is still necessary to degradethe glycoprotein to partial structures each representing only one glycosylation position. The partial structures suitable for analysis, can be oligosaccharides, oligosaccharide-alditols or glycopeptides. In our approachfor the analysis of N,O-glycoproteins we cleavefirst the N-linked chains with the aid of PNGaseF. Theextentofthis reaction has to be checked carefully, in order to makesure that in case the reaction doesnot lead to complete removal of the carbohydrate chains, the chainsthat are still linked to the protein do not have a specific structure. Forthis purpose several methodscan be applied. The pool of N-glycans is separated from the O-glycoprotein, whichis then isolated and subjected to alkaline borohydride treatment. The pools correspondingto the N- and O-linked chains, respectively, are fractionated to homogeneous compounds asfar as possible. Analysis of the compoundsis carried out by 500-MHz !H-NMR spectroscopy. The occurrence of non-carbohydrate substituentslike alkyl, acyl, sulfate and phosphate groups may giverise to serious complications. Often the contents of substituents are far below molar equivalents in a compound,thereby enhancingthe (micro)heterogeneity. It should be noted that information about the type andposition of the non-carbohydrate substituents can be obtained by 1D- or 2D-1H-NMRspectroscopy. For the analysis of phosphate-containing oligosaccharides we developed a 1H(31P} relayed spin-echo difference spectroscopy technique to characterize the residue to whichthe phosphateis attached and to detect the C-atom that is substituted. By meansof this analytical procedure we were able to determine the structure of glycoprotein-derived carbohydrate chains for quite a numberof glycoproteins.

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