Unit for Molecular Glycobiology

Gent, Belgium

Unit for Molecular Glycobiology

Gent, Belgium

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Vanderschaeghe D.,Unit for Molecular Glycobiology | Vanderschaeghe D.,Ghent University | Szekrenyes A.,Debrecen University | Wenz C.,Hewlett - Packard | And 8 more authors.
Analytical Chemistry | Year: 2010

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use. © 2010 American Chemical Society.


Jacobs P.P.,Unit for Molecular Glycobiology | Jacobs P.P.,Ghent University | Jacobs P.P.,Harvard University | Inan M.,University of Nebraska - Lincoln | And 11 more authors.
Microbial Cell Factories | Year: 2010

Background: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.Results: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man5GlcNAc2N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.Conclusions: Together, these data illustrate that the GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins. © 2010 Jacobs et al; licensee BioMed Central Ltd.


Debruyne E.N.,Ghent University | Vanderschaeghe D.,Ghent University | Van Vlierberghe H.,Ghent University | Vanhecke A.,Unit for Molecular Glycobiology | And 2 more authors.
Clinical Chemistry | Year: 2010

BACKGROUND: Hepatocellular carcinoma (HCC) is a common and rapidly fatal cancer. Current diagnostic methods for HCC have poor sensitivity and specificity, are invasive, and carry risk for complications. Newer markers are needed to overcome these problems and allow diagnosis of HCC at an earlier stage. In view of known associations between glycosylation changes and liver disease, we focused on the serum glycoprotein hemopexin and the specific characteristics of this liver-synthesized glycoprotein. METHODS: We studied 49 healthy volunteers and 81 patients divided into the categories of fibrosis, cirrhosis, and HCC with cirrhosis. Hemopexin was purified from study participants' serum by use of heme agarose beads. The hemopexin N-glycan profile was determined by use of the DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis technique. RESULTS: We found that branching α-1,3-fucosylated multiantennary glycans on hemopexin were increased in theHCCgroup compared with the cirrhosis without HCC, fibrosis, and healthy volunteer groups, whereas nonmodified biantennary glycans decreased progressively across groups from fibrosis to the cirrhosis and HCC groups. Summarization of this information in a new marker, called the hemopexin glycan marker, enabled distinction of patients with HCC and cirrhosis from healthy volunteers and patients with fibrosis or cirrhosis with a sensitivity and specificity of 79% and 93%, respectively. CONCLUSIONS: This study demonstrated hemopexin to be a model protein for studying liver-specific N-glycosylation. The hemopexin glycan marker could be a valuable complementary test to α-fetoprotein measurements for detection of HCC in patients with cirrhosis. Additional study of its utility for diagnosis and follow-up is recommended. © 2010 American Association for Clinical Chemistry.


Vanderschaeghe D.,Unit for Molecular Glycobiology | Vanderschaeghe D.,Ghent University | Festjens N.,Unit for Molecular Glycobiology | Festjens N.,Ghent University | And 3 more authors.
Biological Chemistry | Year: 2010

Glycomics research has become indispensable in many research fields such as immunity, signal transduction and development. Moreover, changes in the glycosylation of proteins and lipids have been reported in several diseases including cancer. The analysis of a complex post-translational modification such as glycosylation depends on the availability or development of appropriate analytical technologies. The research goal determines the sensitivity, resolution and throughput requirements and guides the choice of a particular technology. This review highlights the evolution of glycan profiling tools in the past 5 years. We focus on capillary electrophoresis, liquid chromatography, mass spectrometry and lectin microarrays. © 2010 by Walter de Gruyter Berlin New York.


Vanderschaeghe D.,Unit for Molecular Glycobiology | Guttman A.,Debrecen University | Callewaert N.,Ghent University
Methods in Molecular Biology | Year: 2013

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward. © 2013 Springer Science+Business Media, LLC.


Ryckaert S.,Unit for Molecular Glycobiology | Ryckaert S.,Ghent University | Pardon E.,Vlaams Institute for Biotechnology | Steyaert J.,Vlaams Institute for Biotechnology | And 2 more authors.
Journal of Biotechnology | Year: 2010

Yeast surface display is an efficient tool for isolating and engineering antibody fragments, both scFv and Fab. We describe the use of protein display on Pichia pastoris for the rapid selection of camelid antibodies composed only of heavy chains (nanobodies) from a library derived from a llama immunized with Green Fluorescent Protein. The library of nanobody-coding sequences was fused to the C-terminal part of the Saccharomyces cerevisiae alpha-agglutinin gene (SAG1) and expressed in glycoengineered P. pastoris. A high efficiency transformation protocol yielded a library of 5 × 10 7 clones. About 80% of the clones strongly expressed the nanobody fusion. Nanobody-displaying clones were rapidly enriched by fluorescence activated cell sorting (FACS), and GFP-specific nanobody-displaying clones were isolated and equilibrium dissociation constants (K d) determined. This technology for displaying protein libraries on P. pastoris enables the isolation and engineering of antibody-derived molecules in a robust eukaryotic expression host. © 2009 Elsevier B.V. All rights reserved.


Vanderschaeghe D.,Unit for Molecular Glycobiology
Methods in molecular biology (Clifton, N.J.) | Year: 2013

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward.


Vanderschaeghe D.,Unit for Molecular Glycobiology
Methods in molecular biology (Clifton, N.J.) | Year: 2013

Serum protein electrophoresis is widely used in clinical laboratories to measure the relative abundance of each obtained fraction. Moreover, we found that the migration time of the γ-globulin fraction can be reproducibly determined (CV = 1.1%). Immunoglobulins were purified from serum using protein L-agarose and their N-glycosylation was studied using CE on a DNA sequencer. Liver fibrosis patients showed a lower level of sialylation and this moderately correlates with the migration time of the γ-globulins (r = 0.2-0.4). This allowed us to differentiate healthy individuals from these patients with an acceptable diagnostic accuracy (area under the curve = 0.75). This glycomics approach could become a significant added value to a daily, routine clinical test.


PubMed | Unit for Molecular Glycobiology
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward.


PubMed | Unit for Molecular Glycobiology
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

Serum protein electrophoresis is widely used in clinical laboratories to measure the relative abundance of each obtained fraction. Moreover, we found that the migration time of the -globulin fraction can be reproducibly determined (CV = 1.1%). Immunoglobulins were purified from serum using protein L-agarose and their N-glycosylation was studied using CE on a DNA sequencer. Liver fibrosis patients showed a lower level of sialylation and this moderately correlates with the migration time of the -globulins (r = 0.2-0.4). This allowed us to differentiate healthy individuals from these patients with an acceptable diagnostic accuracy (area under the curve = 0.75). This glycomics approach could become a significant added value to a daily, routine clinical test.

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