Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line.

Conradt, Harald S.; Egge, Heinz; Peter‐Katalinić, Jasna; Reiser, Walter; Siklosi, T; Schaper, K

doi:10.1016/s0021-9258(18)47838-6

Cited by 104 publications

(11 citation statements)

References 23 publications

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“…To confirm this hypothesis, we synthesized another glycoprotein, interferon (IFN)-β (Figure B, M9-IFN-β), having the same helical bundle structure as EPO, and used it for confirming substrate specificity of UGGT. IFN-β has the same number of amino acid residues, 166, and forms five helices structure . We synthesized IFN-β bearing an M9-glycan at the native glycosylation position, Asn80, using a three segment coupling strategy (synthesis is shown in SI, Figures S63–S70).…”

Section: Resultsmentioning

confidence: 99%

“…IFN-β has the same number of amino acid residues, 166, and forms five helices structure. 29 We synthesized IFN-β bearing an M9glycan at the native glycosylation position, Asn80, using a three segment coupling strategy (synthesis is shown in SI, Figures S63−S70). 30 However, UGGT did not transfer a glucose residue onto this homogeneous native folded IFN-β (Figure 3A, right lane).…”

Section: Journal Of the American Chemical Societymentioning

confidence: 94%

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Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins

et al. 2018

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The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDPglucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-β (IFN-β), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

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Section: Resultsmentioning

confidence: 99%

Section: Journal Of the American Chemical Societymentioning

confidence: 94%

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins

et al. 2018

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“…Recombinant human interferon beta (IFNβ), a cytokine with anti-inflammatory and tumor-suppressor functions, is considered as the first choice treatment of multiple sclerosis, a severe neurodegenerative autoimmune disease . Interferon beta-1a (IFNβ1a), in contrast to interferon beta-1b (IFNβ1b), is glycosylated at asparagine 80 carrying mainly biantennary and triantennary glycan structures. , IFNβ1a has an average molecular weight of about 22.5 kDa, contains 166 amino acid residues, and is produced in Chinese hamster ovarian (CHO) cells . As described by Karpusas et al, the protein contains five alpha helices, an intramolecular disulfide bridge between cysteines in positions 31 and 141, and a free cysteine (Cys) in position 17.…”

Section: Introductionmentioning

confidence: 99%

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

et al. 2013

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Oxidation via Cu2+/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu2+/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl) benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan and Tyr residues were identified throughout the primary sequence. Covalent crosslinks via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.

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“…Among other things, the application of mammalian cell culture has made it feasible to produce recombinant glycoproteins in quantities sufficient for pharmaceutical use. Mammalian-cell-expressed recombinant glycoproteins that are approved or under development as pharmaceutical agents include tissue plasminogen activator (2, 3), erythropoietin (4,5), soluble CD4 (6), factor VIII (7), and /3-interferon 0003-2700/90/0362-1714$02.50/0 (8,9). Of these, recombinant tissue plasminogen activator (rt-PA) and recombinant erythropoietin have been approved by the U.S. Food and Drug Administration for therapeutic use in humans, while most of the others are in clinical trials.…”

Section: Introductionmentioning

confidence: 99%

Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest

Spellman¹

1990

Anal. Chem.

118

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Carbohydrate characterization of recombinant glycoproteins entails determination of the primary structures and points of attachment of the oligosaccharide moieties. This article reviews several methods for oligosaccharide- and glycosylation-site characterization. A major recent advance in carbohydrate analysis has been the use of high-pH anion exchange (HPAE) chromatography for separation of glycoprotein-derived oligosaccharides. These separations are sensitive to molecular size, carbohydrate composition, linkage positions, and anomeric configurations. As a result, HPAE chromatography is a powerful technique for glycoprotein characterization and can serve as the basis of an "oligosaccharide map." Characterization of potential N-glycosylation sites involves determining whether each potential site is glycosylated, the extent of oligosaccharide processing at each site, and ideally, a detailed description of the distribution of oligosaccharides at each site. Several approaches to characterizing glycosylation sites are described, including peptide mapping and mass spectrometry. Treatment of a glycoprotein with endo-beta-N-acetylglucosaminidase H (endo H) followed by peptide:N-glycosidase F (PNGase F) can be used to distinguish sites that are not glycosylated from those carrying high-mannose structures and from those containing attached complex oligosaccharides. After individual glycosylation sites have been labeled by this series of reaction, the resulting peptides are characterized by automated Edman degradation. This technique is particularly valuable for characterizing peptides that contain more than one potential N-glycosylation site. An example is also given in which HPAE chromatography is used in conjunction with reversed-phase high-performance liquid chromatography tryptic mapping to obtain detailed information on the distribution of oligosaccharides at individual glycosylation sites.

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Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line.

Cited by 104 publications

References 23 publications

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest

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