Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors

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Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the...

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Autores principales: Sartor, P.A., Agusti, R., Leguizamón, M.S., Campetella, O., de Lederkremer, R.M.
Formato: Artículo publishedVersion
Lenguaje:Inglés
Publicado: 2010
Materias:
Galactose oxidase
Inhibitors
Trans-sialidase
Trypanosoma cruzi
fetuin
sialic acid
sialidase
sialidase inhibitor
anion exchange chromatography
article
carbohydrate analysis
enzyme inhibition
enzyme substrate
glycosylation
nonhuman
priority journal
spectrophotometry
alpha-Fetoproteins
Enzyme Inhibitors
Galactose
Glycoproteins
Neuraminidase
Oligosaccharides
Sialic Acids
Staining and Labeling
Structure-Activity Relationship
Mammalia
Tetra
Acceso en línea:http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
id paperaa:paper_09596658_v20_n8_p982_Sartor
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spelling paperaa:paper_09596658_v20_n8_p982_Sartor2023-06-12T16:48:44Z Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors Glycobiology 2010;20(8):982-990 Sartor, P.A. Agusti, R. Leguizamón, M.S. Campetella, O. de Lederkremer, R.M. Galactose oxidase Inhibitors Trans-sialidase Trypanosoma cruzi fetuin sialic acid sialidase sialidase inhibitor anion exchange chromatography article carbohydrate analysis enzyme inhibition enzyme substrate glycosylation nonhuman priority journal spectrophotometry Trypanosoma cruzi alpha-Fetoproteins Enzyme Inhibitors Galactose Glycoproteins Neuraminidase Oligosaccharides Sialic Acids Staining and Labeling Structure-Activity Relationship Trypanosoma cruzi Mammalia Tetra Trypanosoma cruzi Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored transsialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3′-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl β-D-Fuc-(1→6)-α-D-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl α-D-GlcNAc with tetra-Obenzoyl-D-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced by trans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase and trans-sialylation activities by running the assay in the absence of acceptor 1. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org. Fil:Agusti, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Leguizamón, M.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Campetella, O. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:de Lederkremer, R.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2010 info:eu-repo/semantics/article info:ar-repo/semantics/artículo info:eu-repo/semantics/publishedVersion application/pdf eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-134
collection Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
language Inglés
orig_language_str_mv eng
topic Galactose oxidase
Inhibitors
Trans-sialidase
Trypanosoma cruzi
fetuin
sialic acid
sialidase
sialidase inhibitor
anion exchange chromatography
article
carbohydrate analysis
enzyme inhibition
enzyme substrate
glycosylation
nonhuman
priority journal
spectrophotometry
Trypanosoma cruzi
alpha-Fetoproteins
Enzyme Inhibitors
Galactose
Glycoproteins
Neuraminidase
Oligosaccharides
Sialic Acids
Staining and Labeling
Structure-Activity Relationship
Trypanosoma cruzi
Mammalia
Tetra
Trypanosoma cruzi
spellingShingle Galactose oxidase
Inhibitors
Trans-sialidase
Trypanosoma cruzi
fetuin
sialic acid
sialidase
sialidase inhibitor
anion exchange chromatography
article
carbohydrate analysis
enzyme inhibition
enzyme substrate
glycosylation
nonhuman
priority journal
spectrophotometry
Trypanosoma cruzi
alpha-Fetoproteins
Enzyme Inhibitors
Galactose
Glycoproteins
Neuraminidase
Oligosaccharides
Sialic Acids
Staining and Labeling
Structure-Activity Relationship
Trypanosoma cruzi
Mammalia
Tetra
Trypanosoma cruzi
Sartor, P.A.
Agusti, R.
Leguizamón, M.S.
Campetella, O.
de Lederkremer, R.M.
Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
topic_facet Galactose oxidase
Inhibitors
Trans-sialidase
Trypanosoma cruzi
fetuin
sialic acid
sialidase
sialidase inhibitor
anion exchange chromatography
article
carbohydrate analysis
enzyme inhibition
enzyme substrate
glycosylation
nonhuman
priority journal
spectrophotometry
Trypanosoma cruzi
alpha-Fetoproteins
Enzyme Inhibitors
Galactose
Glycoproteins
Neuraminidase
Oligosaccharides
Sialic Acids
Staining and Labeling
Structure-Activity Relationship
Trypanosoma cruzi
Mammalia
Tetra
Trypanosoma cruzi
description Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored transsialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3′-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl β-D-Fuc-(1→6)-α-D-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl α-D-GlcNAc with tetra-Obenzoyl-D-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced by trans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase and trans-sialylation activities by running the assay in the absence of acceptor 1. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.
format Artículo
Artículo
publishedVersion
author Sartor, P.A.
Agusti, R.
Leguizamón, M.S.
Campetella, O.
de Lederkremer, R.M.
author_facet Sartor, P.A.
Agusti, R.
Leguizamón, M.S.
Campetella, O.
de Lederkremer, R.M.
author_sort Sartor, P.A.
title Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
title_short Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
title_full Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
title_fullStr Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
title_full_unstemmed Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
title_sort continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
publishDate 2010
url http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor
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