Now showing items 1-20 of 488


      Nimtz, Manfred; Conradt, Harald S.; Department of Genetics and Cell Biology, GBF- Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg1, D-3300 Braunschweig, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Our group has elucidated the carbohydrate structures of a number of pharmaceutically relevant recombinant glycoproteins (IFN-8, IL-2, t-PA, AT II, EPO as well as mutant proteins thereof) expressed in several mammalian host cell lines (CHO, BHK, C127, Ltk) . After enzymatic liberation of the N-linked oligosaccharide chains by action of polypeptide:N-glycanase from the intact glycoprotein or tryptic fragments thereof, the individual oligosaccharides were separated by a combination of ion exchange chromatography and HPLC on NH,-phase. Oligosaccharide structures were elucidated using several analytical techniques: GC/MS (SIM-mode) for compositional and methylation analyses, FAB-MS for the determination of their molecular weight and the terminal substitution pattern as well as 600 MHz 7H-NMR spectrometry for the determination of anomeric configuration and linkage pattern of the monosaccharide building blocks. The recently introduced high-pH-anion-exchange-chromatography with pulsed amperometric detection (HPAE-PAD) was applied for comparison of differently charged oligosaccharide fractions after enzymatic desialylation, determination of oligosaccharide structures at individual glycosylation sites and for control of batch-to-batch consistency of biotechnologically produced glycoproteins.

      Stanley, Pamela; Albert Einstein College of Medicine, Bronx, New York USA 10461 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Most glycoproteins require their carbohydrates in order to be synthesized efficiently and in a biologically active, stable form. However it is clear that the actual structures of these carbohydrates can vary widely; severly truncated forms will often confer the desired characteristics. Minimizing carbohydrate heterogeneity may be advantageous for many reasons, especially in the production of recombinant glycocoproteins. To obtain glycoproteins with a limited set of carbohydrate structures glycosylation mutants with mutations in carbohydrate biosynthesis can be used. Chinese hamster ovary (CHO) glycosylation mutants that are missing an enzyme activity (e.g. a transferase or translocase) synthesize truncated carbohydrates with predictable structures. For purification or tissue-targeting purposes, it may be desirable to embellish carbohydrates with particular sugar residues. Dominant CHO mutants or CHO cells transfected with a cloned glycosyltransferase can be used for this purpose. Most of the CHO mutants that synthesize altered carbohydrates grow well in culture showing that a wide range of carbohydrate structures are compatible with viability. These lines can readily be used to engineer the carbohyrates of recombinant glycoproteins for a multitude of purposes.
    • Remodeling of the Carbohydrate Chains of hCG by Use of Sialyltransferases: Effects on the Biological Activity

      Nemansky, Martin; Eijnden, Dirk H. van den; Dedem, Gijs W. K. van; Mannaerts, Bernadette M. J. L.; Department of Medical Chemistry, Vrije Universiteit, Amsterdam and °Diosynth B.V./Organon B.V., Oss, The Netherlands (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Human chorionic gonadotropin (hCG) is a glycoprotein hormone which contains both N- and O-linked carbohydrate chains. It consists of two subunits, a and 8. The a-subunit contains two N-linked carbohydrate chains: a mono-antenna and a non-fucosylated bi-antenna. The $-subunit contains both (two) N and (four) O-linked carbohydrate structures. Both of the N-linked carbohydrate chainsare biantennary, one of which is fucosylated.

      Zettlmeißl, G.; Conradt, Harald S.; Nimtz, Manfred; Haigwood, N.; Paques, E. P.; 1) Behringwerke AG, Postfach 1140, 3550 Marburg, F.R.G. 2) GBF mbH, Mascheroder Weg 1, 3300 Braunschweig, F.R.G. 3) Chiron Corporation, 456 Horton St. Emeryville, CA 94608, USA (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Comparative analysis of the carbohydrate structure of plasma antithrombin III and recombinant antithrombin III synthesized in Chinese Hamster Ovary cells revealed differences in the linkage of NeuAc, the presence of higher than biantennary structures and the presence of proximal fucose. Treatment of the carbohydrate part of antithrombin III from both sources with glycopeptidase F or sialidase had a strong negative effect on the serum half-life. In order to analyze the effects of elimination of individual carbohydrate side chains on the pharmacokinetic and functional properties of AT III the four N-linked glycosylation sites of the recombinant molecule were altered individually or in combination by site directed mutagenesis of Asn to Gln. All mutants showed a shorter serum half-life compared to natural antithrombin II]. However molecules modified at residues Asn 135, Asn 155 and Asn 192 showed higher heparin affinity and/or maximal stimulation at lower heparin concentrations. As in the case of antithrombin III the three N-glycosylation sites of tissue plasminogen activator mutated individually or in combination. Whereas the specific activities of single glycosylation mutants were unaltered, simultaneous mutation of two (Asn 117 and Asn 184) or three Asn residues to Gln resulted in molecules with 2-3 fold higher specific activities.

      Parekh, R. B.; Oxford GlycoSystems Limited, Unit 4 Hitching Court, Blacklands Way, Abingdon, Oxon. OX14 1RG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      It is being increasingly recognised that many polypeptides of therapeutic interest, which in their native form are glycoproteins, need to be glycosylated in order to be of benefit in vivo..A consideration of polypeptide glycosylation therefore becomes relevant throughoutthe development and production of recombinant glycoproteins, principally for the following reasons. First, cell lines differ in their glycosylation characteristics, and the same polypeptide expressed in two different cell lines will generally be glycosylated differently. As a consequence, a recombinant glycoprotein is usually glycosylated differently to the native form, and such 'non-physiological' glycosylation can have profoundeffects on functional activity, physicochemical properties, and pharmacokinetic behaviour in vivo . A limited set of oligosaccharide determinants has been identified, the members of which influence the pharmacokinetic and immunogenic properties of a glycoprotein. It can therefore prove valuable to screen anycell line chosen for the production of a recombinant polypeptide, for expression of such determinants. Second, to ensurethat any changes in culture method (for example, during scale-up) are not associated with alterations in glycosylation, and that batch-to-batch uniformity is maintained during production,it is necessary to follow the glycosylation pattern of the secreted protein. Third, individual glycoformsof a polypeptide can differ with respect to functional properties. Identification of an improved product may, in some cases, involve nothing morethan isolation of a particular glycosylation variant. These and other aspects of the glycosylation of recombinant glycoproteins are discussed ir this paper.

      Egge, H.; Peter-Katalinic, J.; Klein, R. A. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Glycosylation is a crucial event in the post-translational modification of proteins. Numerous investigations have shown clearly that both the primary structure and the conformation of glycoproteins are intimately connected with their biological function and metabolic fate. Many laboratories are currently directing their efforts towards the development of more specific and sensitive methods for the determination of the primary and secondary structure of glycopeptides as a first step, towards the analysis of their biological function. In view of the complexity of the structures involved, it is evident that only a combination of different methods will yield the desired results. In this instance soft ionization mass spectrometry and high resolution NMR have proved to be expecially useful in determining such different structural parameters as, for example, the type and number of sugar components, the sequence of sugar residues, the sites of the glycosidic linkages and their anomeric confuguration, the sites of glycosylation on the peptide chain, and the 3D-conformation of these structures. Glycans linked N-glycosidically through a chitobiosyl-Asn linkage are usually released enzymatically from the peptide chain prior to analysis ‚whereas O-glycans, e.g., of the mucin type with a GalNAc (al-3)-Ser/Thr linkage, can be released by alkaline borohydride treatment. In this contribution special emphasis will be given to the potential of mass spectrometric methods in analysing the complex mixtures that are obtained after enzymatic release of N-glycans from a single glycosylation site, or after alkaline borohydride treatment of mucin O-glycans.

      Peter-Katalinic, Jasna; Hanisch, Franz-Georg; Uhlenbruck, Gerhard; Egge, Heinz; 1) Institut für Physiologische Chemie der Universität Bonn, Nußallee 11, D-5300 Bonn 1, FRG 2) Institut für Immunbiologie der Universität Köln, Kerpener Straße 15, D-5000 Köln 41, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      The relative high abundance of O-linked oligosaccharides on the mucin-type glycoproteins has opened a favorable perspective for detailed studies of carbohydrate epitope structures, which may be involved in processes relevant for cellular differentiation in development and oncogenesis. Immunological experiments, in particular those with monoclonal antibodies raised against the cell surfaces, have been sucessfully performed in order to monitor qualitatively oncofetal carbohydrate antigens and the changes in their patterns during such processes (1). On the other hand the biosynthetic studies on glycosyltransferases responsible for assembly of complex carbohydrates bound to proteins and their substrates lead to the conclusion, that several enzymes compete for a common substrate of specific structure available in the respective cellular compartment at the certain phase of biosynthesis (2). Therefore some carbohydrate microheterogeneity arising from the presence of different terminal epitope structures as well as the different branch length and different branching patterns on the single glycan attachment site should be expected. The structures of the O-linked glycans, liberated from the mucin-type glycoproteins by reductive elimination can be determined by spectroscopic methods, using different techniques of nuclear magnetic resonance (NMR) (3) and mass spectrometry (4). By combining the immunological and spectroscopic approach numerous oncofetal carbohydrate antigen structures have been found not only on mucins of developing systems (5), but on mucins of human body fluids of normal individuals like in seminal plasma (6) and milk (7) as well. Oncofetal antigens, recognized by a number of monoclonal antibodies like C-50, NS 19-9, OC 125, Leu Ml, 49 H 8 and 115 C 2, are strongly expressed also in the mucine fraction of human amniotic fluid (8). These were analysed by fast atom bombardment mass spectrometry (FAB-MS) (9), in particular Suitable for the determination of carbohydrate sequences, their branching patterns and their molecular size in native and derivatized samples, even in complex mixtures.

      Chaplin, L. C.; Cockett, M. I.; Willenbrock, F.; Hipkiss, J. B.; Celltech Ltd, 216 Bath Road, Slough, SL1 4EN, U. K. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Tissue inhibitor of metalloproteinases (TIMP)is a glycoprotein of approximate M=30KDa, which has 2 Nlinked glycosylation sites giving rise to heterogeneous glycosylation, and characterised by a multiple bandpattern for the native protein on SDS-PAGE. The present work reports the production of TIMP with modified glycosylation by the use of a range of specific glycoprocessinginhibitors during cell culture. The inhibitors interfere with the normal trimming pathway during N-glycosylation of glycoproteins. Modified TIMPs showed greater homogeneity of glycosylation, higher iso-electric points, and similar inhibitory activity of metalloproteinases, compared with native TIMP. Theinhibitors could be used over a wide range of concentrations without having a significant effect on the production of TIMP,or on cell growth. This approach to modification of glycosylation may show significant advantagesin the production of glycoproteinsfor crystallization studies over more conventional methods such as enzymic deglycosylation, use of tunicamycin, or expression in coli,

      Curling, E. M. A.; Hayter, P.; Tong, J.; Jenkins, N.; Salmon, I.; Bull, A. T.; BIOLOGICAL LABORATORY, UNIVERSITY OF KENT, CANTERBURY, KENT, CT2 7NJ, ENGLAND (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      The production of recombinant human interferon-x (IFN- X¥ ) by Chinese hamster ovary (CHO) cells in serum-free medium was heterogeneous with up to twelve molecular weight variants secreted. Using the enzyme N-glycanase to remove all the (N-linked) oligosaccharides or tunicamycin to inhibit glycosylation, it was found that the variation was due to both variable asparagine residue occupation (Asnyg and/or Asn- 100) by complex oligosaccharide and to truncation of the IFN-$ polypeptide due to proteolytic cleavage at the carboxy-terminal end. Using neuraminidase treatment, terminal sialic acid expression was found to be invariant. When the CHD cells were grown in two litre batch suspension culture in serum-free medium further heterogeneity was discovered, seen as a reproducible shift towards the secretion of underglycosylated IFN-Y with time. This phenomenon was independent of glucose concentration in the medium. The IFN- ¥ was produced during the exponential growth phase and declined in line with the cell growth rate and glucose uptake rates. In contrast, during glucose-limited chemostat culture at a constant dilution rate of 0.015h”l, the relative levels of each major glycosylation variant did not change significantly, although the proportion of fully glycosylated IFN-¥ was still less than that seen at the start of batch culture while non-glycosylated IEN- ¥ represented 15% of the total IFN- ¥ secreted.

      McDowell, William; Animashaun, Theresa; Hughes, R. Colin; MRC Collaborative Centre, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD and National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      The glycosylation of recombinant tissue plasminogen activator derived from transfected CHO cells was assessed using glycosyltransferases to probe for terminal Gal and GlcNAc residues and solid-state lectin binding assays. Analysis of the oligosaccharides released from each glycosylation site by hydrazinolysis by Con A affinity chromatography/ anion exchange HPLC/ BioGel P4 gel filtration corroborated the glycosyltransferase and lectin-binding assay data. Significant undersialylation of the oligosaccharides of t-PA and less than 1% of free terminal GlcNAc residues was indicated. The above assays could be useful in monitoring glycosylation status during quality control in recombinant glycoprotein production.

      Li, Shu-Ying; Arbeitsgruppe Zellkulturtechnik, Gesellschaft fiir Biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig, FRG. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Efficient recombinant protein production processes should optimize both productivity and yield. The fermentation process should also preserve the integrity and quality of the desired protein product. AT III (antithrombin III) is one of the most important serine protease inhibitors in plasma. It is an @,-glycoprotein of M, 60,000 to 64,000 containing three or four polysaccharide chains attached by N-glycosidic linkage to asparagine residues. The biological activity of AT III can be enhanced 2-3 orders of magnitude by binding with the cofactor heparin (1). Affinity chromatography of plasma-derived AT III on immobilized heparin resulted in two fractions of AT II which differ from each other in their affinity to heparin; their difference in affinity to heparin is due to different degrees of glycosylation (2). Concerns have been raised that if BHK-derived AT III exhibit the same behavior on purification as its plasma-derived counterpart. In the fermentation of AT III producing BHK (baby hamster kidney) cells, productivity was monitored and the possible influence of serum-free cultivation conditions on protein quality in termsof both integrity (against proteolytic activity) and affinity to heparin was investigated.
    • Carbohydrate Analysis of Glycoproteins on Blots Using the Immunological System: Digoxigenin/Anti-Digoxigenin Antibody

      Haselbeck, Anton; Hösel, Wolfgang; Boehringer Mannheim GmbH, Biochemica Research Center, 8132 Tutzing/Obb., FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Digoxigenin is bound to sugars either as digoxigenin-hydrazide after oxidation to aldehydes, or as lectin conjugates. Digoxigenin is then detected with a high-affinity anti-digoxigenin antibody, conjugated with alkaline phosphatase or peroxidase. Besides of the general labeling of the carbohydrates, the reaction with digoxigenin-hydrazide can further be made specific for the detection ofsialic acids or terminal galactose. By using digoxigenin labeled lectins with defined and narrow binding specificity for probing of the glycoproteins, it is possible to obtain substantial informations on the glycan structures. The high resolution power of electrophoretical separation systems is widely used for analyzing complex protein mixtures. The combination of these techniques with the sensitive digoxigenin based labeling and detection system allows the rapid analysis of small amounts of glycoproteins, which can be further refined by including exo- and endoglycosidase digestions as specific tools.
    • Micro-Scale Analysis of N-Acetylneuraminic Acid

      Hermentin, P.; Seidat, J.; Research Laboratories of Behringwerke AG, D-3550 Marburg, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      We have developed a micro-scale analytical method for the detection and quantification of Nacetylneuraminic acid (NANA)that does neither require any derivatization of NANAnoris it interfered by the presence of any monosaccharide component present on N-linked and O-linked carbohydrate chains of glycoproteins. The method involves acid hydrolysis of sub-nanomolar amounts of sialoglycoprotein (SGP) and subsequent neutralization, followed by high-pH anion-exchange chromatography (HPAE) with pulsed amperometric detection (PAD). The method wasvalidated, using a,-acid glycoprotein (AGP) (orosomucoid) as standard SGP. Six experimental series of decaplicates, each, were performed, varying i) the AGP concentration in PBS (i. e. 250, 500 and 750 ug/ml, respectively), ii) the sample amount used (i. e. 200, 100, 30, 15 and 10 ul, each, of defined AGP concentrations), iii) the mode of sample injection into the Dionex Bio-LC system (i. e. manual or automated injection by way of two different autoinjectors), iv) the sensitivity of the PAD detector(i. e. 1000 and 300 nAfull scale, respectively). Individual experimental series were repeated at different days and were occasionally performed by different persons. In each case, the NANA/AGP molar ratios were in the range of 15.0 +/- 0.4 mol/mol (corresponding with a standard deviation of <+/- 3 %), which is in excellent agreement with the NANA/AGP molar ratio described in theliterature.
    • Methods Suitable for Large Scale Quantitation of Sugar Residues in Microheterogenic Glycoproteins

      Cervén, Erik; Inst. Medical and Physiological Chemistry Biomedical Centre, University of Uppsala Uppsala, Sweden (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Three methods suitable for the purpose of large scale quantitation of sugar residues in samples of microheterogenic glycoproteins are presented, named by the generic names 1) Marked-Lectin Immunoassay (MALIA), 2) Oxidative-Reductive Immunoassay (Redox-IA), or 3) relying on incorporation of labeled sugars from precursors using serum glycosyltransferases. All three methods can be used in the presence of serum and comprise only simple incubations and washings that would be possible to perform with standard or slightly modified RIA or ELISA equipment. 1) In MALIA, the glycoproteins are bound to an immunoadsorbentandthis is used in combination with an incubation with marked lectin. The utility of the method is restricted only by the specificity of the lectin and its affinity for the solid phase antigen during washing. When thelectin has low affinity for the sugar residues, they can be measured in the washing supernatants with a similar sensitivity of method. 2) In Redox-IA, the contentofsialic acid in a glycoprotein bound from a body fluid onto an immunoadsorbent is measured using the increased Susceptibility of sialic acid residues to oxidation which are a) oxidized with prefered specificity and b) reduced using a labeled reductant followed by c) isolation of the labeled species. The utility of the method is restricted to measurementsof averagesialic acid content in glycoproteins whose identity is specified by their affinity to the immunoadsorbent. 3) Using the endogenous serum glycosyltransferases and labeled nucleotide-linked sugars, crude information can be obtained about the stage of glycosylation of the serum components. The previously predominant method to precipitate the labeled species with acid has been replaced by a method using solid phase lectins onto which the labeled product is collected with preserved immunospecificity.

      Eijnden, Dirk H. van den; Nemansky, Martin; Schiphorst, Wietske E. C. M.; Dedem, Gijs W. K. van; Mannaerts, Bernadette M. J. L.; *Department of Medical Chemistry, Vrije Universiteit, Amsterdam and °Diosynth B.V./Organon B.V., Oss, The Netherlands. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      In order to apply sialyltransferases in the remodelling of the carbohydrate chains on biologically active glycoproteins, it is a prerequisite to know the fine specificity of these enzymes. In this report the specificity of several sialyltransferses involved in the sialylation of O- and N-linked oligosaccharide chains is reviewed. Also novel results on the branch specificity of a3- and a6-sialyltransferase are reported. The potential application of these enzymes in carbohydrate remodelling was studied using human chorionic gonadotropin (hCG) as a model glycoprotein. Differently sialylated preparations of this hormone were obtained and tested for their stimulatory effect on steroidogenesis in Leydig cells in vitro. Asialo-hCcG appeared to be only 45% as effective as native hCG. a3-Resialylation of the O-linked chains on the ß-subunit of this hormone did not restore the biological activity to a higher level. By contrast, 55% a6- resialylation of the N-linked chains yielded a preparation which was almost as active as native hCG. Interestingly, further sialylation by the a6-sialyltransferase resulted in a decrease of the bio-activity to levels lower than obtained with asialo-hcG. It is concluded that the lectin-carbohydrate binding, which is part of the process that triggers the biological respons of the target cell can be mimicked by N-linked chains carrying a6-linked sialic acid. However, too high a density of such residues interferes with this interaction.

      Svensson, Eric C.; Lee, Eryn Ujita; Livingston, Brian; Wen, Xiao-Hong; Weinstein, Jasminder; Paulson, James C.; Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90024 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      Mammalian cell lines used for production of recombinant glycoproteins elaborate terminal glycosylation structures on N-linked and O-linked carbohydrate groupsthat are determined by the glycosyltransferases expressed by these cells. As many as twelve glycosyltransferase cDNAs have now been cloned by a variety of strategies (1). By expressing these glycosyltransferase cDNAs incells not normally expressing them, it is now possible to alter the cellular glycosylation machinery to produce new terminal glycosylation sequences (2,3). This principle was demonstrated by expressing the rat B-galactoside 02,6 sialyltransferase (a2,6ST) cDNA in CHOcells, which are known notto express the product of this sialyltransferase. After selection for stable expression, these cells were shown to produce Nlinked carbohydrate groups with terminal 02,6 linked sialic acid, demonstrating an altered glycosylation machinery (2).

      Brossmer, R.; Groß, H. J.; Rose, U.; Sticher, U.; Mirelis, P.; Gradel, F.; Imhof, A.; Kovac, A.; Institut für Biochemie II der Universität, Im Neuenheimer Feld 328, D-6900 Heidelberg (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      To complete their biosynthesis many soluble or membrane bound glycoconjugates require a final sialylation step which is catalyzed by distinct sialyltransferases differing in specificity with respect to the glycan acceptor structure and the linkage formed. Important biological functions of glycoconjugates depend on the degree of terminal sialylation. Replacement of naturally occurring sialic acids by synthetic analogues endowed with special properties can serve to influence certain biological functions of soluble or membrane-bound sialoglycoconjugates. In particular, those sialic acid analogues that deserve special interest are sialidase-resistant or O-acetylesteraseresistant, or they are radiolabeled, fluorescently labeled, or carry a photoreactive functional group, or they may differ in charge or hydrophobicity from the parent sialic acid. It has been shown that CMP sialate synthase has a low substrate specificity with respect to the C-5 and C-9 positions of sialic acid. As a consequence many CMPactivated analogues of sialic acid can be obtained on a preparative scale. Detailed studies have revealed that different sialyltransferases accept a variety of CMP-activated sialic acid analogues as donor substrates. This finding opens the way for preparation by enzymatic synthesis on a large scale of glycoproteins differently modified in the sialic acid moiety. Membrane-bound receptors can be modified and the biological consequences studied. With accumulating knowledge of the donor substrate specificity of CMP-sialic acid synthase and sialyltransferase, analogues can be synthesized that are tailored for special applications or for use as inhibitors of enzymes of sialoglycoconjugate biosynthesis. Furthermore, glycoproteins can be prepared that resist biological degradation by sialidases.

      Hammel, M.; Schneider, R.; Berger, E. G.; Gygax, D.; 1) Central Research Laboratories, Ciba-Geigy Ltd, 4002 Basel, Switzerland 2) Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      N-linked glycoprotein glycans were modified by combined use of glycosidases and glycosyltransferases. Human milk 81,4 galactosyltransferase andrat liver 02,6 sialyltransferase were immobilized by covalent or affinity binding methods. These immobilized glycosyltransferases were used in a slurry reactor for continuous glycosylation of endoglycosidase H-treated invertase or r-tPA. Theresulting sialylated glycan chains mimicking the structureof outer complex N-glycansconferred extended plasma half-life time compared to galactosylated glycoprotein.

      Joziasse, David H.; Shaper, Nancy L.; Salyer, Linda S.; Eijnden, Dirk H. van den; Spoel, Aarnoud C. van der; Shaper, Joel H.; Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands, and ?Cell Structure and Function Laboratory, The Oncology Center and ®Department of Pharmacology and Molecular Sciences, School of Medicine, The Johns Hopkins University, Baltimore, USA (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
      We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDPGal: Galß1>4G1cNAc «1>3-galactosyltransferase (Joziasse, D.H. et al., (1989) J. Biol. Chem. 264, 14290-14297). Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Sf9 insect cells with recombinant virus, resulted in high-level expression of enzymatically active al>3-galactosyltransferase. The recombinant &1>3-galactosyltransferase could be readily detergent solubilized and subsequently purified by affinity-chromatography on UDP-hexanolamine-Sepharose. The recombinant «1>3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (GalB1>4G1cNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus a1 >3- galactosyltransferase.
    • 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.