Wouter Vervecken | Oxyrane - Academia.edu (original) (raw)

Papers by Wouter Vervecken

FEBS Letters, Jun 11, 1999

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La presente invention porte sur des procedes pour la production de sulfatases de type I activees,... more La presente invention porte sur des procedes pour la production de sulfatases de type I activees, ou de fragments fonctionnels de celles-ci, a l'aide d'enzymes produisant de la formylglycine (FGE). L'invention porte egalement sur des cellules fongiques recombinees (par exemple de Yarrowia lipolytica ) exprimant les FGE et, dans certains modes de realisation, des sulfatases de type I, ou des fragments fonctionnels de celles-ci, et/ou des enzymes accessoires supplementaires. L'invention porte egalement sur des sulfatases de type I activees ou des fragments fonctionnels de celles-ci, formees par les procedes selon l'invention et sur des procedes therapeutiques utilisant les sulfatases de type I activees ou des fragments fonctionnels de celles-ci.

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La presente invention a trait a des procedes et des souches de levure methylotrophique genetiquem... more La presente invention a trait a des procedes et des souches de levure methylotrophique genetiquement modifiees pour la production de glycoproteines assimilee a la glycosylation mammalienne. La presente invention a egalement trait a des vecteurs utiles pour la generation de souches de levure methylotrophique aptes a la production de glycoproteines assimilee a la glycosylation mammalienne. L'invention a trait en outre a des glycoproteines produites a partir de souches de levure methylotrophique genetiquement modifiees.

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desmanosilacao n-glycans phosphorylated. Methods for desmanosilacao n-phosphorylated glycans on a... more desmanosilacao n-glycans phosphorylated. Methods for desmanosilacao n-phosphorylated glycans on a glycoprotein are described which use a mannosidase capable of hydrolyzing an alpha-1,2-mannose terminal connection when the underlying is phosphorylated mannose.

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A method for the phosphorylated N- glycans using let manno capable of hydrolyzing the 1,2 mannose... more A method for the phosphorylated N- glycans using let manno capable of hydrolyzing the 1,2 mannose binding protein on or a part per the misfire talman furnace is started-terminal alpha when the mannose in the not be phosphorylated.

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Cytometry, 1999

It is unclear whether expression of newly described mitochondrial Apo2.7 molecules (7A6 antigen) ... more It is unclear whether expression of newly described mitochondrial Apo2.7 molecules (7A6 antigen) is specific for apoptosis or may also occur in necrosis. We incubated human lymphocytes with the apoptosis-inducing mistletoe lectin (ML) I and the cell membrane-permeabilizing viscotoxins (VT), and measured cell death-associated changes by flow cytometry. In ML I-treated lymphocytes, Apo2.7 expression and caspase-3 activation was recognized within 24 h. In VT-treated cells, we observed an Apo2.7 expression with low fluorescence level, while active caspase-3 and DNA fragments (TUNEL) were not detected within 24 h. In these cells, caspase-3 activation was recognized 48 h later. As a major subset of ML-treated cells expressing Apo2.7 molecules did not activated caspase-3, while all caspase-3(+) cells did express Apo2.7, one may suggest that the caspase pathway is activated secondarily to mitochondrial events. Expression of Apo2.7 is sensitive marker of cell death but may not be specific for apoptosis alone as it can be detected also in cells treated with cell membrane-permeabilizing toxins. On the other hand, this expression may be the consequence of an induction of distinct "death signals" resulting in apoptosis later on.

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Methods in Molecular Biology, 2007

Glycosylation is an important issue in heterologous protein production for therapeutic applicatio... more Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.

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The International Journal of Biochemistry & Cell Biology, 2000

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PLoS ONE, 2012

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Nature Protocols, 2008

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Nature Biotechnology, 2007

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Nature Biotechnology, 2012

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. ... more Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with α-mannosidase to produce in yeast a form of the Pompe disease enzyme α-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of α-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease.

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Microbial Cell Factories, 2012

Background Protein-based therapeutics represent the fastest growing class of compounds in the pha... more Background Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases Yl Och1p and Yl Mnn9p; the former inactivation yielded a strain produc...

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Journal of Gastroenterology and Hepatology, 2007

Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensiv... more Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensive evaluation. Animal model systems are often used for studying human liver disease and screening antifibrotic compounds. The aim of the present study was to investigate the potential use of serum N-glycan profiles to evaluate liver fibrosis in a rat model. Liver fibrosis and cirrhosis were induced in rats by oral administration of CCl(4). Liver injury was assessed biochemically (alanine aminotransferase [ALT] activity, aspartate aminotransferase [AST] activity and total bilirubin) and histologically. The N-glycan profile (GlycoTest) was performed using DNA sequencer-assisted-fluorophore-assisted carbohydrate electrophoresis technology. In parallel, the effect of cotreatment with antifibrotic interferon-gamma (IFN-gamma) was studied. The biopsy scoring system showed that CCl(4) induced early fibrosis (F < 1-2) in rats after 3 weeks of treatment, and cirrhosis (F4) after 12 weeks. Significant increases in ALT activity, AST activity and total bilirubin levels were detected only after 12 weeks of CCl(4) treatment. GlycoTest showed three glycans were significantly altered in the CCl(4)-goup. Peak 3 started at week 6, at an early stage in fibrosis development (F < 1-2), whereas peaks 4 and 5 occurred at week 9, at which time mild liver fibrosis (F = 1-2) had developed. The changes in the CCl(4)-IFN-gamma group were intermediate between the CCl(4)- and the control groups. The GlycoTest is much more sensitive than biochemical tests for evaluating liver fibrosis/cirrhosis in the rat model. The test can also be used as a non-invasive marker for screening and monitoring the antifibrotic activity of potential therapeutic compounds.

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FEBS Letters, 1999

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European Journal of Cancer, 2007

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FEBS Letters, Jun 11, 1999

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La presente invention porte sur des procedes pour la production de sulfatases de type I activees,... more La presente invention porte sur des procedes pour la production de sulfatases de type I activees, ou de fragments fonctionnels de celles-ci, a l'aide d'enzymes produisant de la formylglycine (FGE). L'invention porte egalement sur des cellules fongiques recombinees (par exemple de Yarrowia lipolytica ) exprimant les FGE et, dans certains modes de realisation, des sulfatases de type I, ou des fragments fonctionnels de celles-ci, et/ou des enzymes accessoires supplementaires. L'invention porte egalement sur des sulfatases de type I activees ou des fragments fonctionnels de celles-ci, formees par les procedes selon l'invention et sur des procedes therapeutiques utilisant les sulfatases de type I activees ou des fragments fonctionnels de celles-ci.

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La presente invention a trait a des procedes et des souches de levure methylotrophique genetiquem... more La presente invention a trait a des procedes et des souches de levure methylotrophique genetiquement modifiees pour la production de glycoproteines assimilee a la glycosylation mammalienne. La presente invention a egalement trait a des vecteurs utiles pour la generation de souches de levure methylotrophique aptes a la production de glycoproteines assimilee a la glycosylation mammalienne. L'invention a trait en outre a des glycoproteines produites a partir de souches de levure methylotrophique genetiquement modifiees.

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desmanosilacao n-glycans phosphorylated. Methods for desmanosilacao n-phosphorylated glycans on a... more desmanosilacao n-glycans phosphorylated. Methods for desmanosilacao n-phosphorylated glycans on a glycoprotein are described which use a mannosidase capable of hydrolyzing an alpha-1,2-mannose terminal connection when the underlying is phosphorylated mannose.

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A method for the phosphorylated N- glycans using let manno capable of hydrolyzing the 1,2 mannose... more A method for the phosphorylated N- glycans using let manno capable of hydrolyzing the 1,2 mannose binding protein on or a part per the misfire talman furnace is started-terminal alpha when the mannose in the not be phosphorylated.

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Cytometry, 1999

It is unclear whether expression of newly described mitochondrial Apo2.7 molecules (7A6 antigen) ... more It is unclear whether expression of newly described mitochondrial Apo2.7 molecules (7A6 antigen) is specific for apoptosis or may also occur in necrosis. We incubated human lymphocytes with the apoptosis-inducing mistletoe lectin (ML) I and the cell membrane-permeabilizing viscotoxins (VT), and measured cell death-associated changes by flow cytometry. In ML I-treated lymphocytes, Apo2.7 expression and caspase-3 activation was recognized within 24 h. In VT-treated cells, we observed an Apo2.7 expression with low fluorescence level, while active caspase-3 and DNA fragments (TUNEL) were not detected within 24 h. In these cells, caspase-3 activation was recognized 48 h later. As a major subset of ML-treated cells expressing Apo2.7 molecules did not activated caspase-3, while all caspase-3(+) cells did express Apo2.7, one may suggest that the caspase pathway is activated secondarily to mitochondrial events. Expression of Apo2.7 is sensitive marker of cell death but may not be specific for apoptosis alone as it can be detected also in cells treated with cell membrane-permeabilizing toxins. On the other hand, this expression may be the consequence of an induction of distinct "death signals" resulting in apoptosis later on.

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Methods in Molecular Biology, 2007

Glycosylation is an important issue in heterologous protein production for therapeutic applicatio... more Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.

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The International Journal of Biochemistry & Cell Biology, 2000

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PLoS ONE, 2012

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Nature Protocols, 2008

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Nature Biotechnology, 2007

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Nature Biotechnology, 2012

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. ... more Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with α-mannosidase to produce in yeast a form of the Pompe disease enzyme α-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of α-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease.

Bookmarks Related papers MentionsView impact

Microbial Cell Factories, 2012

Background Protein-based therapeutics represent the fastest growing class of compounds in the pha... more Background Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases Yl Och1p and Yl Mnn9p; the former inactivation yielded a strain produc...

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Journal of Gastroenterology and Hepatology, 2007

Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensiv... more Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensive evaluation. Animal model systems are often used for studying human liver disease and screening antifibrotic compounds. The aim of the present study was to investigate the potential use of serum N-glycan profiles to evaluate liver fibrosis in a rat model. Liver fibrosis and cirrhosis were induced in rats by oral administration of CCl(4). Liver injury was assessed biochemically (alanine aminotransferase [ALT] activity, aspartate aminotransferase [AST] activity and total bilirubin) and histologically. The N-glycan profile (GlycoTest) was performed using DNA sequencer-assisted-fluorophore-assisted carbohydrate electrophoresis technology. In parallel, the effect of cotreatment with antifibrotic interferon-gamma (IFN-gamma) was studied. The biopsy scoring system showed that CCl(4) induced early fibrosis (F < 1-2) in rats after 3 weeks of treatment, and cirrhosis (F4) after 12 weeks. Significant increases in ALT activity, AST activity and total bilirubin levels were detected only after 12 weeks of CCl(4) treatment. GlycoTest showed three glycans were significantly altered in the CCl(4)-goup. Peak 3 started at week 6, at an early stage in fibrosis development (F < 1-2), whereas peaks 4 and 5 occurred at week 9, at which time mild liver fibrosis (F = 1-2) had developed. The changes in the CCl(4)-IFN-gamma group were intermediate between the CCl(4)- and the control groups. The GlycoTest is much more sensitive than biochemical tests for evaluating liver fibrosis/cirrhosis in the rat model. The test can also be used as a non-invasive marker for screening and monitoring the antifibrotic activity of potential therapeutic compounds.

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FEBS Letters, 1999

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European Journal of Cancer, 2007

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