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Goncalves M.,New University of Lisbon | Tillack L.,GlycoThera GmbH | de Carvalho M.,Hospital Of Santa Maria | de Carvalho M.,University of Lisbon | And 4 more authors.
Clinica Chimica Acta | Year: 2015

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. © 2014 Elsevier B.V.


Gouveia R.,Institute Tecnologia Quimica e Biologica | Kandzia S.,GlycoThera GmbH | Conradt H.S.,GlycoThera GmbH | Costa J.,Institute Tecnologia Quimica e Biologica | Costa J.,Institute Biologia Experimental e Tecnologica
Journal of Biotechnology | Year: 2010

L1 is a cell adhesion molecule that is heavily glycosylated and is essential for normal development of the central nervous system. In this work, we compare the N-glycosylation of the L1 mutant that consists of immunoglobulin domains 5 and 6 (L1/Ig5-6), expressed in insect Spodoptera frugiperda Sf9 and Trichoplusia ni Tn cells, using the stable expression system. L1/Ig5-6 levels of 30 and 8 mg l-1 were obtained from the two cell lines, respectively. The N-glycans were characterized by high-performance anion-exchange-chromatography with pulsed-amperometric-detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-glycans from Sf9 cells were more homogeneous and consisted predominantly of the paucimannose-type structure Manα6(Manα3)Manβ4GlcNAcβ4(Fucα6)GlcNAc. On the other hand, the N-glycans from Tn cells were more heterogeneous and consisted of paucimannose-type structures with or without terminal N-acetylglucosamine. Allergenic proximal α3-linked fucose was only found in Tn cells. Dimethyl sulfoxide at 1.5% concentration has been found to increase the levels of L1/Ig5-6 and the L1 ectodomain in the Sf9 and Tn cells, without affecting cell viability nor protein integrity. Furthermore, the N-glycan composition of L1/Ig5-6 was not affected by dimethyl sulfoxide, with only a slight increase in the percentage of the minor high-mannose-type structures. © 2009 Elsevier B.V. All rights reserved.


PubMed | GlycoThera GmbH, University of Lisbon and New University of Lisbon
Type: | Journal: Clinica chimica acta; international journal of clinical chemistry | Year: 2014

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified.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.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 2,3- and 2,6-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).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.


MacHado E.,New University of Lisbon | Kandzia S.,GlycoThera GmbH | Carilho R.,New University of Lisbon | Altevogt P.,German Cancer Research Center | And 2 more authors.
Glycobiology | Year: 2011

Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma. © 2010 The Author.


Schmidbauer S.,CSL Behring GmbH | Witzel R.,CSL Behring GmbH | Robbel L.,CSL Behring GmbH | Sebastian P.,CSL Behring GmbH | And 3 more authors.
Thrombosis Research | Year: 2015

rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant β-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient. © 2015 Elsevier Ltd. All rights reserved.


Gouveia R.,New University of Lisbon | Schaffer L.,Scripps Research Institute | Papp S.,Scripps Research Institute | Grammel N.,GlycoThera GmbH | And 6 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2012

Background: Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. Methods: The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. Results and conclusions: One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewisx motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewisx structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewisx, as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. General Significance: These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis x motif plays a functional role in neurite outgrowth from human neurons. © 2012 Elsevier B.V.


Escrevente C.,New University of Lisbon | Grammel N.,GlycoThera GmbH | Kandzia S.,GlycoThera GmbH | Zeiser J.,GlycoThera GmbH | And 3 more authors.
PLoS ONE | Year: 2013

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. © 2013 Escrevente et al.


Ceaglio N.,National University of Santa | Etcheverrigaray M.,National University of Santa | Conradt H.S.,GlycoThera GmbH | Grammel N.,GlycoThera GmbH | And 2 more authors.
Journal of Biotechnology | Year: 2010

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. © 2010 Elsevier B.V. All rights reserved.


PubMed | GlycoThera GmbH and CSL Behring GmbH
Type: Journal Article | Journal: Thrombosis research | Year: 2015

rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant -sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient.

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