Identification of the human Lewisa carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.)

This paper reports for the first time the presence of the human Lewisa type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxCl from Vaccinium myrtillus L. by HPLC/ESI-MS. The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis.info:eu-repo/semantics/publishedVersio

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.EU Joint Programme JPND/0003/2011, FCT grant: Pest-OE/EQB/LA0004/2011, FCT PhD fellowship

Carbohydrate structures of a human tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary cells

AbstractThe carbohydrate structures of a genetically engineered human tissue plasminogen activator variant bearing a single N-glycosylation site at Asn 448 are reported. After isolation of the tryptic glycopeptide and liberation of the N-linked carbohydrates by polypeptide:N-glycosidase F, 6 major oligosaccharide fractions were separated by HPLC on NH2-bonded phase. Their structures were determined by compositional and methylation analyses combined with fast atom bombardment mass spectrometry. Seventy percent of the carbohydrates were of the biantennary complex type with fucose at the proximal GlcNAc and zero, one or two α2-3 linked NeuAc. The remainder were triantennary structures with one, two or three NeuAc

Title, Preface, Content, List of authors

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

OLIGOSACCHARIDE STRUCTURES OF GLYCOPROTEINS FROM RECOMBINANT MAMMALIAN CELL LINES

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

INFLUENCE OF GLYCOSYLATION ON THE FUNCTIONAL PROPERTIES OF HUMAN THERAPEUTIC PLASMA PROTEINS

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

Phosphoneurofilament heavy chain and N-glycomics from the cerebrospinal fluid in amyotrophic lateral sclerosis

© 2014 Elsevier B.V. All rights reserved.Background: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. Methods: We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. Results: pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). Conclusions: Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker.This is an EU Joint Programme — Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organisations under the aegis of JPND — www.jpnd.eu: France, Agence Nationale de la Recherche; Germany, Bundesministerium für Bildung und Forschung; Ireland, Health Research Board; Italy, Ministero della Salute; The Netherlands, The Netherlands Organisation for Health Research and Development; Poland, Narodowe Centrum Badań i Rozwoju; Portugal, Fundação para a Ciência e a Tecnologia (JPND/0002/2011; JPND/0003/2011); Spain, Ministerio de Ciencia e Innovación; Switzerland, Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung; and the Turkey, Tübitak; United Kingdom, Medical Research Council. We also acknowledge funding from Fundação para a Ciência e a Tecnologia, Pest-OE/EQB/LA0004/2011, and ENMed/0001/2013, Portugal.info:eu-repo/semantics/publishedVersio

Highly glycosylated human alpha interferon: An insight into a new therapeutic candidate

The type I human interferon alpha (hIFN-α) family consists of small proteins that exert a multiplicity of biological actions including antiviral, antiproliferative and immunomodulatory effects. However, though administration of recombinant hIFN-α2b is the current treatment for chronic hepatitis B and C and for some types of cancers, therapy outcomes have not been completely satisfactory. The short serum half-life and rapid clearance of the cytokine accounts for its low in vivo biological activity. Here we describe and characterize a long-acting rhIFN-α2b mutein, 4N-IFN, which has been created by introducing four N-glycosylation sites via site-directed mutagenesis. The hyperglycosylated protein was found to have a 25-fold longer plasma half-life than the non-glycosylated rhIFN-α2b, even greater than the commercial pegylated derivative Intron-A PEG. In addition, glycosylation increased the in vitro stability of the mutein against serum protease inactivation. Interestingly, despite its lower in vitro activity, 4N-IFN showed a markedly enhanced in vivo antitumor activity in human prostate carcinoma implanted in nude mice. MALDI-TOF MS and HPAEC-PAD carbohydrate analyses revealed the presence of high amounts of tetrasialylated (40%) and trisialylated (28%) N-glycan structures, which are consequently responsible for the improved characteristics of the cytokine, making 4N-IFN a new therapeutic candidate for viral and malignant diseases.Fil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Conradt, Harald S.. GlycoThera GmbH; AlemaniaFil: Grammel, Nicolás. GlycoThera GmbH; AlemaniaFil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Oggero Eberhardt, Marcos Rafael. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin